JP2001521744A - Reagents and methods useful for detecting breast disease - Google Patents

Abstract

  (57) [Summary] A set of contiguous or partially overlapping RNA sequences, termed BS106, and the polypeptide encoded therein and transcribed from breast tissue are described. These sequences are useful for detecting, diagnosing, staging, monitoring, prognosing, preventing or treating or predisposing to a breast disease or condition, such as breast cancer, in an individual. Also, antibodies that specifically bind to a polypeptide or protein encoded by BS106, which are molecules useful for treating breast disease, tumors or metastases, and agonists or inhibitors that interfere with the action of tissue-specific BS106 polypeptides I will provide a.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]Background of the Invention  The present invention relates generally to the detection of breast disease. In particular, the present invention
Reagents such as otide sequences, polypeptide sequences encoded by them, and
It relates to a method using an array. These are tests for breast diseases or conditions, such as breast cancer.
Prognosis, diagnosis, staging, monitoring, prognosis, prevention or treatment or predisposition
Useful for

[0002] Breast cancer is the most common form of cancer that occurs in women in the United States. With respect to the development of breast cancer in the United States, 180,200 cases have been diagnosed and 43,900 related deaths have been estimated to have occurred in 1997 (American Cance).
r Society statistics). Globally, the incidence of breast cancer was 70 in 1985.
From 000 to 900,000 in 1990 (GNH)
Ortobagyi et al., CA Cancer J Clin 45: 199-2.
26 (1995)).

[0003] Methods for detecting, diagnosing, staging, monitoring, prognosing, preventing or treating or predisposing to breast diseases or conditions, such as breast cancer, are very important to the outcome of the patient. For example, patients diagnosed with early stage breast cancer have a 5-year relative survival rate of greater than 90%, while patients diagnosed with distant metastatic breast cancer have a survival rate of about 20% (A
American Cancer Society statistics). At present, the best early indicators of early breast cancer are breast screening and mammography (JR Harr).
is et al., Cancer: Principles and Practice of Oncology , 4th ed. 1264-1332, Philade
lphia, PA: J / B. Lippincott Co. (1993)). Mammography can detect breast cancer before it is detected by screening, but it has its limitations. For example, the predicted value of a mammogram depends on the skill of the observer and the quality of the mammogram. Also, 80-93% of suspected mammograms are false positives and 10-15% of women with breast cancer have false negative mammograms (CJ Wri).
ght et al., Lancet 346: 29-32 (1995)). Clearly, there is a need for new diagnostic methods that are more sensitive and specific for detecting early stage breast cancer.

[0004] Breast cancer patients are closely monitored after initial therapy and during adjuvant therapy to determine response to therapy and detect persistent or recurrent disease or early distant metastasis. Current diagnostic methods for monitoring breast cancer include mammography, bone scans, chest radiographs, liver function tests and tests for serum markers. The most commonly used serum tumor markers to monitor patients are carcinoembryonic antigen (CEA) and CA 15-3. The limitations of CEA include the absence of elevated serum levels in about 40% of women with metastatic disease. Furthermore, elevated CEA during adjuvant therapy was not associated with relapse,
It may be related to other factors that are not clinically significant. Also, CA 15-
3 can be negative in a significant number of patients with progressive disease and is therefore not predictive of metastasis. Both CEA and CA 15-3 can be elevated in non-malignant benign conditions and can give false positive results. Therefore,
Finding breast cancer-related markers that are more sensitive and specific in detecting cancer recurrence would be clinically beneficial (JR Harris et al., Supra; MK Schwa).
rtz et al., Cancer: Principles and Practice of Oncology, Vol. 1 , 4th edition, pp. 531-542, Phi
ladelphia, PA: J / B. Lippincott Co. (1993
)).

[0005] Another important step in the treatment of breast cancer is to determine the stage of the patient. Because it has potential prognostic value and provides a basis for the design of optimal therapy. At present, pathological staging of breast cancer is preferred over clinical staging. Because the former gives a more accurate prognosis (JR Harris et al., Supra). On the other hand, if clinical staging is at least as accurate as pathological staging, then clinical staging may be preferred. Because it does not rely on invasive methods to obtain tissue for pathological evaluation. Detecting new markers in serum or urine that could distinguish different stages of invasion could improve breast cancer staging. Such markers can be mRNA or protein markers derived from primary breast cancer but expressed by cells present in the blood, bone marrow or lymph nodes, and are sensitive to metastasis to these distal organs May be an indicator of For example, specific protein antigens and mRNA associated with breast epithelial cells
They have been detected in bone marrow, lymph nodes and blood of breast cancer patients by immunohistochemical techniques and RT-PCR, respectively, suggesting metastasis (K. Pantel et al., Onkologie 18: 394-401 (1995)). .

[0006] Such methods are based on the appearance of various disease markers in test samples such as blood, plasma, serum, urine, etc., which are detectable by immunological methods and obtained by methods which are as invasive as possible. Assays could be included. These methods will provide information to help physicians treat patients with breast disease at a light burden on the patient. There are markers such as prostate specific antigen (PSA) and human chorionic gonadotropin (hCG), and such markers have been used clinically to screen patients for prostate and testicular cancers, respectively. For example, PSA is normally secreted at high levels from the prostate into semen, but is present at very low levels in the blood of men with normal prostate. Elevated levels of serum PSA protein can be used in the early detection of prostate cancer or disease in asymptomatic men. For example, G. E. FIG. Ha
nks et al., Cancer: Principles and Practice.
of Oncology, Vol. 1, 4th edition, pp. 1073-1111, P
hildelphia, PA: J. B. Lippincott Co. 199
3, M.P. K. Schwarz et al., Cancer: Principles an.
d Practice of Oncology, Vol. 1, 4th edition, pp.
531-542, Philadelphia, PA: J. Mol. B. Lippinco
tt Co. See 1993. Similarly, it is normally expressed in the breast,
By utilizing novel markers found in elevated amounts in inappropriate body fractions as a result of breast disease, it would be possible to improve the treatment of breast disease.

[0007] Also, novel markers that can predict the biological behavior of early breast cancer would be invaluable. Early breast cancer that threatens or threatens a patient's life is more clinically important than one that does or does not threaten it (GE Hanks,
Supra). To predict in which patients with histologically negative lymph nodes the cancer will recur, and to predict which cases of ductal carcinoma in situ will progress to invasive breast cancer, Such markers are needed. A more accurate prognostic marker would allow a physician to accurately identify early breast cancer that progresses and metastasizes if not actively treated. In addition, the ability to indicate that a marker for aggressive cancer is absent in a patient would prevent the patient from receiving expensive and useless treatments (JR Harris et al., Supra, ER. Frykbe
rg et al., Cancer 74: 350-361 (1994)).

It would be advantageous to provide specific methods and reagents for the detection, diagnosis, staging, monitoring, prognosis, prevention or treatment or predisposition of breast disease or conditions. Such a method would involve assaying a test sample for the product of a gene that is overexpressed in breast-related diseases and conditions (eg, cancer). Also, such methods may be used to treat breast related diseases or conditions (
(Eg, cancer). Further, such methods may include assaying a test sample for a product of a gene whose distribution in various tissues and fractions of the body has been altered by a breast-related disease or condition (eg, cancer). . Such a method involves preparing cDNA from mRNA in a test sample and, optionally,
Amplifying a portion of the cDNA corresponding to the gene or a fragment thereof, detecting a cDNA product as an indicator of the presence of a disease or condition such as cancer,
It would involve detecting the translation product of NA as an indicator of the presence of the disease. Useful reagents include polynucleotides or fragments thereof that can be used in diagnostic methods such as reverse transcription-polymerase chain reaction (RT-PCR), PCR or hybridization assays of mRNA extracted from biopsy tissue, blood or other test samples. Or proteins that are translation products of such mRNAs; or antibodies to these proteins. Such assays would include methods for assaying a sample for the product of the gene and detecting the product as an indicator of breast disease. Drug or gene therapy for breast diseases and conditions (eg, cancer) can be based on these identified gene sequences or their expressed proteins and monitor the efficacy of any particular therapy It is possible to Further, it would be beneficial if alternative non-surgical diagnostic methods were available that could detect early stage breast disease (eg, cancer).

[0009]Summary of the Invention  The present invention relates to a method for detecting a target BS106 polynucleotide in a test sample.
Wherein said test sample comprises at least one BS106-specific polynucleotide.
And the presence of the target BS106 polynucleotide in the test sample.
Providing a method comprising detecting. BS106-specific polynucleotide
Are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and
Less than a polynucleotide selected from the group consisting of fragments or complements thereof.
Both have 50% identity. Prior to performing the method, a BS106 specific
It is also possible to attach the oligonucleotide to a solid phase.

[0010] The present invention also provides a method for detecting BS106 mRNA in a test sample, wherein the cDNA is obtained by performing reverse transcription (RT) with at least one primer, and the obtained cDNA is subjected to a BS106 oligonucleotide. Amplify using primers and antisense primers to obtain BS106 amplicon,
Detecting the presence of the BS106 amplicon as an indicator of the presence of BS106 mRNA in the test sample, wherein the BS106 oligonucleotide comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and at least 50% identity to a sequence selected from the group consisting of: Amplification can be performed by the polymerase chain reaction. Also, the test sample can be reacted with a solid phase prior to performing the method, prior to amplification or prior to detection. This reaction can be a direct or indirect reaction. Further, the detecting step involves the use of a detectable label capable of producing a measurable signal. This detectable label is
It is possible to bind to a solid phase.

The present invention further provides a method of detecting a target BS106 polynucleotide in a test sample suspected of containing the target BS106 polynucleotide, comprising: (a) using the test sample with at least one BS106 oligo as a sense primer At least one BS106 as nucleotide and antisense primer
Contacting with an oligonucleotide and amplifying it to obtain a first stage reaction product, (b)
Contacting the first-stage reaction product with at least one other BS106 oligonucleotide to obtain a second-stage reaction product, wherein the other BS106 oligonucleotide is the BS106 oligonucleotide used in step (a); And is complementary to the first step reaction product), (c) target B in the test sample
There is provided a method comprising detecting the second stage reaction product as an indicator of the presence of an S106 oligonucleotide. BS106 selected as a reagent in the method
The oligonucleotide has at least 50% identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and fragments or complements thereof. Amplification can be performed by the polymerase chain reaction. It is also possible to react the test sample directly or indirectly with the solid phase before performing the method, or before amplification or before detection.
The detection step also involves the use of a detectable label capable of producing a measurable signal. Further, the detectable label can be bound to a solid phase. Also, a test kit useful for detecting a target BS106 polynucleotide in a test sample, comprising a kit comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and fragments or complements thereof. A test kit comprising a container containing at least one BS106-specific polynucleotide selected from the group consisting of: These test kits further include a container having means useful for collecting test samples (eg, blood, urine, saliva, and feces). Such means include a lancet and absorbent paper or cloth for collecting and stabilizing blood, a swab for collecting and stabilizing saliva, and a cup for collecting and stabilizing a urine or fecal sample. It is. To avoid denaturation or irreversible adsorption of the sample, it is possible to treat the collected material, such as paper, cloth, swab, cup, etc., if desired. Also, the collected material may be treated with a preservative, stabilizing agent, or antimicrobial to aid in maintaining the integrity of the sample, or the collected material may contain a preservative, stabilizer, or antimicrobial It is possible to do.

[0012] The present invention provides a purified polynucleotide or a fragment thereof derived from the BS106 gene. The purified polynucleotide has the ability to selectively hybridize to the nucleic acid of the BS106 gene or its complement. The polynucleotide has at least 50% identity to a polynucleotide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and fragments or complements thereof. . Further, the purified polynucleotide can be produced by recombinant and / or synthetic techniques. The purified recombinant polynucleotide can be contained in a recombinant vector. The invention further includes a host cell transfected with the vector.

[0013] The present invention further provides a recombinant expression system comprising a nucleic acid sequence comprising an open reading frame derived from BS106. The nucleic acid sequence has at least 50% identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and fragments or complements thereof. The nucleic acid sequence is operably linked to control sequences compatible with the desired host. Also provided are cells transfected with the recombinant expression system.

[0014] The present invention also provides a polypeptide encoded by BS106. The polypeptides can be produced by recombinant techniques and provided in purified form, or can be produced by synthetic techniques. The polypeptide has SEQ ID NO: 1.
6, at least 50 relative to an amino acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and fragments of SEQ ID NOs: 17 to 20.
% Amino acid sequences.

[0015] Also provided are antibodies that specifically bind to at least one BS106 epitope. The antibodies can be polyclonal or monoclonal. The epitope is derived from an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and fragments thereof. An assay kit for determining the presence of a BS106 antigen or anti-BS106 antibody in a test sample is also included. In one embodiment, the assay kit comprises at least 50% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and fragments thereof. A container containing at least one BS106 polypeptide having a sexual activity. In addition, the test kit includes a container having means useful for collecting test samples (eg, blood, urine, saliva, and feces). Such means include a lancet and absorbent paper or cloth for collecting and stabilizing blood, a swab for collecting and stabilizing saliva, and a cup for collecting and stabilizing a urine or fecal sample. It is. To avoid denaturation or irreversible adsorption of the sample, it is possible to treat the collected material, such as paper, cloth, swab, cup, etc., if desired. Also, to aid in maintaining the integrity of the sample, these collected materials may be treated with preservatives, stabilizers, or antimicrobial agents, or preservatives, stabilizers, or antimicrobial agents may be added to the collected materials. It can be contained. Further, the polypeptide can be bound to a solid phase.

[0016] Another assay kit for determining the presence of a BS106 antigen or anti-BS106 antibody in a test sample comprises a container containing an antibody that specifically binds to the BS106 antigen, wherein the BS106 antigen comprises: It is characterized by including at least one epitope encoded by BS106. The BS106 antigen has at least 60% of the sequence of an antigen encoded by BS106 selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and fragments thereof. Have similarities. In addition, the test kit includes a container having means useful for collecting test samples (eg, blood, urine, saliva, and feces). Such means include a lancet and absorbent paper or cloth for collecting and stabilizing blood, a swab for collecting and stabilizing saliva, and a cup for collecting and stabilizing a urine or fecal sample. It is. To avoid denaturation or irreversible adsorption of the sample, it is possible to treat the collected material, such as paper, cloth, swab, cup, etc., if desired. These materials may be treated with preservatives, stabilizers or antimicrobial agents to help maintain sample integrity,
The collection material can contain preservatives, stabilizers or antimicrobial agents. Further, the antibody can be bound to a solid phase.

[0017] A method for producing a polypeptide containing at least one of the epitopes of BS106, comprising incubating a host cell transfected with the expression vector is provided. This vector comprises an amino acid sequence having at least 50% identity to a BS106 amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and fragments thereof. Includes a polynucleotide sequence that encodes the containing polypeptide.

[0018] Also provided is a method for detecting a BS106 antigen in a test sample suspected of containing the BS106 antigen. The method comprises contacting an antibody or fragment thereof that specifically binds at least one of the epitopes of the BS106 antigen with the test sample for a time and under conditions sufficient for the formation of an antibody / antigen complex. Detecting the presence of such a complex in the test sample as an indicator of the presence of the BS106 antigen. The antibody can be bound to a solid phase and can be a monoclonal or polyclonal antibody. Further, the antibody specifically binds to at least one BS106 antigen selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and fragments thereof.

Another method is provided for detecting antibodies that specifically bind to the BS106 antigen in a test sample suspected of containing these antibodies. The method comprises the steps of
At least 5 amino acids or a fragment thereof encoded by the polynucleotide;
Contacting said test sample with a polypeptide containing at least one BS106 epitope comprising an amino acid sequence having 0% identity. Contacting is performed for a time and under conditions sufficient to allow formation of the antigen / antibody complex.
The method further includes detecting a complex containing the polypeptide. The polypeptide can be bound to a solid phase. Further, the polypeptide has at least 50 amino acids relative to an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and fragments thereof.
It can be a recombinant protein or a synthetic peptide with a% identity.

The present invention provides cells transfected with a BS106 nucleic acid sequence encoding at least one epitope of a BS106 antigen or a fragment thereof. The nucleic acid sequence is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and fragments or complements thereof.

Also, a method of producing an antibody against the BS106 antigen, comprising administering to the individual an isolated immunogenic polypeptide or fragment thereof containing at least one BS106 epitope in an amount sufficient to elicit an immune response. A manufacturing method comprising: The isolated immunogenic polypeptide has SEQ ID NO: 16, SEQ ID NO: 17,
It includes an amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and fragments thereof.

Another method for producing an antibody that specifically binds to a BS106 antigen, is a method selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and fragments thereof. At least one BS derived from the amino acid sequence
Disclosed is a production method comprising administering to a mammal a plasmid containing a nucleic acid sequence encoding the 106 epitope.

In addition, it has at least 50% identity to a polynucleotide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and fragments or complements thereof. BS1 of at least about 10-12 nucleotides
A composition comprising the 06 polynucleotide is provided. A BS106 polynucleotide encodes an amino acid sequence having at least one BS106 epitope. Another composition provided by the present invention comprises a BS106 of about 8-10 amino acids.
Includes polypeptides having at least one of the epitopes. The polypeptide is
An amino acid sequence having at least 50% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and fragments of SEQ ID NOs: 17 to 20 Including. Also, a gene encoding a BS106 polypeptide having at least 50% identity to SEQ ID NO: 16 or a fragment thereof, and at least 5% to SEQ ID NO: 4 or SEQ ID NO: 5.
A gene or a fragment thereof comprising DNA having 0% identity is provided.

[0024]Detailed description of the invention  The present invention relates to a BS1 having at least about 50% identity to SEQ ID NO: 16.
A gene encoding the 06 polypeptide or a fragment thereof. The present invention
Has at least about 50% identity to SEQ ID NO: 4 or SEQ ID NO: 5
BS106 gene comprising DNA or a fragment thereof.

The present invention is a method for assaying a test sample for the product of a breast tissue gene, referred to as BS106, comprising the steps of:
And detecting the cDNA as an indicator of the presence of the breast tissue gene BS106. The method includes an amplification step that amplifies one or more mRNA portions corresponding to the gene or a fragment thereof derived from BS106. Also provided are methods for assaying for the translation product of BS106. Test samples that can be assayed by the methods provided herein include tissues, cells, body fluids, and secretions. The present invention also provides reagents such as oligonucleotide primers, polypeptides, etc., useful for performing these methods.

Some of the nucleic acid sequences disclosed herein may be used for reverse transcription of RNA or for cD
As a primer for amplification of NA or in an mRNA in a test sample
Useful as a probe to determine the presence of a sequence. Nucleic acid sequences that enable the production of encoded polypeptide sequences that are useful as standards or reagents in diagnostic immunoassays, as targets in pharmaceutical screening assays, and / or as components or target sites for various therapies. Disclose. Monoclonal and polyclonal antibodies directed against at least one epitope contained within these polypeptide sequences are useful as vehicles for therapeutic agents, are useful in diagnostic tests, and are useful for diseases or conditions associated with BS106, particularly It is useful for screening for breast cancer). Isolation of sequences for other portions of the gene of interest can be performed using probes or PCR primers derived from these nucleic acid sequences. This allows for additional probes of the mRNA or cDNA of interest to be confirmed and the corresponding encoded polypeptide sequence. These additional molecules may be used for the detection, diagnosis, staging, monitoring, prognosis, prevention or treatment or predisposition of breast diseases and conditions (eg, breast cancer) characterized by the BS106 disclosed herein. Useful for

Techniques for determining “similarity” of amino acid sequences are well known in the art. In general, "similarity" refers to the strictness of two or more polypeptides at appropriate positions if the amino acids are identical or have similar chemical and / or physical properties (eg, charge or hydrophobicity). Means a comparison of different amino acids versus amino acids. Thus, a so-called "percent similarity (%)" can be determined between the compared polypeptide sequences. Techniques for determining the identity of nucleic acid and amino acid sequences are also well known in the art, and determine the nucleotide sequence of the mRNA for that gene (usually via a cDNA intermediate) and encode it. Determining an amino acid sequence and comparing it to another amino acid sequence. In general, "identity" means an exact nucleotide-to-nucleotide or amino acid-to-amino acid match of two polynucleotides or polypeptide sequences, respectively. Two or more polynucleotide sequences can be compared by determining their "% identity". Similarly, two or more amino acid sequences can be compared by determining their “percent identity”. Wisconsin Sequence Analysis P
package, Version 8 (Genetics Computer)
(Available from Group, Madison, Wis.), Such as the GAP program, provides the ability to calculate both the identity between two polynucleotides and the identity and similarity between two polypeptide sequences. Have. Other programs for calculating identity or similarity between sequences are known in the art.

The compositions and methods described herein allow for the identification of certain markers as indicators of disease or condition of breast tissue, and the information obtained therefrom can be used to identify diseases and conditions associated with BS106, particularly breast cancer. ) Will aid in the detection, diagnosis, staging, monitoring, prognosis, prevention or treatment or predisposition. Testing methods include:
For example, probe assays that can utilize nucleic acid amplification methods utilizing the sequences provided herein, such as polymerase chain reaction (PCR), ligase chain reaction (LCR), and hybridization, are included. Also, the nucleotide sequence provided in the present invention contains an open reading frame in which an immunogenic epitope can be found. This epitope is believed to be unique to the pathology or condition associated with BS106. Also, polynucleotides or polypeptides and proteins encoded by the BS106 gene are believed to be useful as markers. This marker is elevated in a disease such as breast cancer, is altered in a disease such as breast cancer, or is present as a normal protein but appears in inappropriate body fractions. The uniqueness of the epitope can be measured by its immunoreactivity and specificity for antibodies to the proteins and polypeptides encoded by the BS106 gene, and (ii) its non-reactivity to any other tissue marker. Methods for measuring an immune reaction are well known and include, for example, a radioimmunoassay (RIA) and an enzyme-linked immunosorbent assay (ELIS).
A), hemagglutination (HA), fluorescence polarization immunoassay (FPIA), chemiluminescence immunoassay (CLIA) and the like, but are not limited thereto. Some examples of suitable methods are described herein.

Unless otherwise indicated, the following terms have the following meanings:

A polynucleotide “derived from” or “specific for” a given sequence is at least about 6 nucleotides corresponding to (ie, matched or complementary to) the region of the given nucleotide sequence, Preferably at least about 8 nucleotides,
More preferably, it means a polynucleotide sequence comprising a contiguous sequence of at least about 10-12 nucleotides, even more preferably at least about 15-20 nucleotides. The sequence can be complementary or identical to a sequence specific to a particular polynucleotide sequence, as determined by techniques known in the art. As a method for determining the uniqueness of the indicated sequence, for example, comparison with a sequence in a data bank can be used. Regions from which the sequence is derived include regions encoding specific epitopes, and untranslated and / or non-transcribed regions,
It is not limited to these.

The derived polynucleotide is not always physically derived from the nucleotide sequence of interest under study, but may be chemically synthesized, replicated based on information obtained from the base sequence in the region from which the polynucleotide is derived. Could be generated by any method, including, but not limited to, reverse transcription or transcription. In such cases, it can correspond to the sense or antisense orientation of the original polynucleotide. In addition, combinations of regions corresponding to those of the indicated sequence can be modified in a manner known in the art to be compatible with the intended use.

A “fragment” of the identified polynucleotide is at least about 6 nucleotides, preferably at least about 8 nucleotides, corresponding to the region of the nucleotide sequence shown (ie, matched or complementary), and more Preferably, it means a polynucleotide sequence comprising a contiguous sequence of at least about 10-12 nucleotides, even more preferably at least about 15-20 nucleotides.

[0033] The term "primer" refers to a specific oligonucleotide sequence that is complementary to and used to hybridize to a target nucleotide sequence. Primers serve as a starting point for the polymerization of nucleotides catalyzed by DNA polymerase, RNA polymerase or reverse transcriptase.

The term “probe” refers to a nucleic acid segment (or nucleotide analog segment, for example, as defined below) that can be used to identify a specific polynucleotide present in a sample containing the complementary sequence. PNA).

“Encoded” means that the polypeptide sequence is encoded by a nucleic acid sequence (the polypeptide or a portion thereof is at least 3 amino acids from the polypeptide encoded by the nucleic acid sequence). ~ 5 amino acids, more preferably at least 8-1
0 amino acids, even more preferably containing an amino acid sequence of at least 15-20 amino acids). Also included are polypeptide sequences that are immunologically identifiable with the polypeptide encoded by the sequence. Thus, a “polypeptide”, “protein” or “amino acid” sequence has at least about 5 amino acids relative to the BS106 amino acid sequence.
It has 0% identity, preferably about 60% identity, more preferably about 75-85% identity, and most preferably about 90-95% or more identity. In addition, BS1
06 "polypeptide", "protein" or "amino acid"
At least about 60% similarity, preferably at least about 75% similarity, more preferably about 85% similarity, and most preferably about 95% or more similarity to a polypeptide or amino acid sequence of Is possible. This amino acid sequence can be selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and fragments thereof.

The terms “recombinant polypeptide”, “recombinant protein” or “recombinantly produced polypeptide”, which may be used interchangeably herein, are inherently or by manipulation. And / or a polypeptide that is not bound to all or a portion of the polypeptide bound to and / or is bound to a polypeptide other than the naturally bound polypeptide. A recombinant or encoded polypeptide or protein is not necessarily translated from the indicated nucleic acid sequence. It can also be obtained by any method, including chemical synthesis or expression in a recombinant expression system.

As used herein, the term “synthetic peptide” refers to a polymeric form of amino acids of any length that can be chemically synthesized by methods well known to those skilled in the art.
These synthetic peptides are useful in various applications.

As used herein, the term “polynucleotide” refers to a polymeric form of ribonucleotides or deoxyribonucleotides of nucleotides of any length. This term refers only to the primary structure of the molecule. Thus, the term includes double- and single-stranded DNA and double- and single-stranded RNA. It also includes modified and unmodified forms of the polynucleotide, such as methylated or capped forms. “Polynucleotide”, “oligomer”, “oligonucleotide” and “oligo” are used interchangeably herein.

“Sequence corresponding to cDNA” means that the sequence contains a polynucleotide sequence that is identical or complementary to a sequence in the indicated DNA. cDN
The degree of identity or complementarity (or percentage (%)) to A is about 50% or more;
Preferably it will be at least about 70% or more, more preferably at least about 90% or more. The sequence corresponding to the identified cDNA will be at least about 50 nucleotides in length, preferably at least about 60 nucleotides in length, more preferably at least about 70 nucleotides in length. Gene or gene fragment of interest and said cD
The degree of correspondence with NA can be determined by a method known in the art,
For example, it involves direct comparison of the sequenced material with the described cDNA, or hybridization, and digestion with single-stranded nuclease, followed by measurement of the size of the digested fragment.

“Purified (purified) polynucleotide” is substantially free of the protein to which the polynucleotide is naturally associated (eg, about 50% or more, preferably about 70% or more, more preferably (Contains no more than about 90%). Techniques for purifying polynucleotides of interest are well known in the art, for example, disrupting cells containing polynucleotides with chaotropic reagents, and ion-exchange chromatography, affinity chromatography and density chromatography. Separation of polynucleotides and proteins by sedimentation in response to

“Purified (purified) polypeptide” or “purified (purified) protein” is substantially free of the cellular component to which the polypeptide of interest is naturally associated (eg, about 50% thereof). (Preferably containing no more than about 70%, more preferably no more than about 90%) means a polypeptide or fragment thereof of interest. Methods for purifying polypeptides of interest are known in the art.

The term “isolated” means that the substance has been removed from its original environment (eg, the natural environment if it is naturally occurring).
For example, a naturally occurring polynucleotide or polypeptide present in a living animal is the same polynucleotide or DNA or polypeptide that has not been isolated but has been separated from some or all of the coexisting materials in the natural system. Has been isolated. Such a polynucleotide can be part of a vector,
And / or such polynucleotides or polypeptides can be part of a composition, which has also been isolated because the vector or composition is not part of its natural environment.

“Polypeptide” and “protein” are used interchangeably herein and indicate at least one of a molecular chain of amino acids covalently and / or non-covalently linked. These terms do not imply a particular length of the product. Thus, the definition of polypeptide includes peptides, oligopeptides and proteins. These terms include post-translational modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation, and the like. In addition, a protein fragment, an analog, a mutant or mutant protein, a fusion protein and the like are included in the meaning of the polypeptide.

A “fragment” of a specified polypeptide is at least about 3-5 amino acids, more preferably at least about 8-10, derived from the specified polypeptide.
Amino acid, even more preferably an amino acid sequence comprising at least about 15-20 amino acids.

“Recombinant host cell”, “host cell”, “cell”, “cell line”, “cell culture” and other such terms referring to microorganisms or higher eukaryotic cell lines cultured as single cell bodies Refers to cells that can be or have been used as recipients of recombinant vectors or other introduced DNA, including the original progeny of the original cells transfected.

As used herein, “replicon” refers to any genetic element, such as a plasmid, chromosome, or virus, that behaves as an autonomous unit of polynucleotide replication in a cell.

A “vector” is a replicon to which another polynucleotide segment has been linked, such as to cause replication and / or expression of the linked segment.

The term “control sequence” refers to a polynucleotide sequence necessary to cause expression of a coding sequence to which it is attached. The nature of such control sequences will vary depending on the host organism. In prokaryotes, such control sequences generally include a promoter, a ribosome binding site and a terminator; in eukaryotes, such control sequences generally include a promoter, a terminator, and optionally an enhancer. Thus, the term "control sequence" is intended to include, at a minimum, all components that need to be present for expression, and to include additional components that are advantageous if present (eg, leader sequences). Is possible.

“Operably linked” refers to a situation in which the recited components are in a relationship that allows them to function in their intended manner. Thus, for example, a "control sequence" that is "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence.

“Open reading frame” or “ORF” refers to a region of a polynucleotide sequence that encodes a polypeptide. This region corresponds to part or all of the coding sequence.

“Coding sequence” is a polynucleotide sequence that is transcribed into mRNA and translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are defined by a translation start codon at the 5 'end and a translation stop codon at the 3' end. A coding sequence can include, but is not limited to, mRNA, cDNA, and recombinant polynucleotide sequences.

The term “immunologically identifiable” refers to the presence of epitopes and polypeptides present in and unique to the indicated polypeptide. Immunological identity is
It can be determined by antibody binding and / or competition in binding. These techniques are known to those skilled in the art and are also described herein. Also, the uniqueness of the epitope can be determined by GenB with respect to the polynucleotide sequence encoding the epitope.
It can be determined by computer search of known data banks such as ank, and by comparison of amino acid sequences with other known proteins.

“Epitope” as used herein refers to an antigenic determinant of a polypeptide or protein. It is believed that an epitope can include three amino acids in a spatial conformation unique to the epitope. Generally, an epitope consists of at least 5 such amino acids, and usually it consists of at least 8-10 amino acids. Methods for examining spatial conformation are known in the art and include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance.

A “conformational epitope” is an epitope that comprises a specific juxtaposition of amino acids of an immunologically recognizable structure, wherein such amino acids are in the same polypeptide in a continuous or discontinuous order. Present on a different polypeptide.

A polypeptide is “immunoreactive” with an antibody if the polypeptide binds to the antibody through antibody recognition of a specific epitope contained within the polypeptide. Immunoreactivity can be measured by antibody binding, more particularly by the kinetics of antibody binding, and / or by competition in binding using a known polypeptide containing an epitope for the antibody as a competitor. Methods for determining whether a polypeptide is immunoreactive with an antibody are known in the art.

As used herein, an “immunogenic polypeptide containing an epitope of interest” refers to a naturally occurring polypeptide of interest or a fragment thereof, and other means (eg, chemically synthesized, or in a recombinant organism). Expression of the polypeptide in the above).

The term “transfection” refers to the introduction of an exogenous polynucleotide into a prokaryotic or eukaryotic host cell, regardless of the method used for the introduction. The term "transfection" refers to both stable and transient introduction of a polynucleotide, and includes direct uptake of a polynucleotide, transformation, transduction, and f-conjugation. An exogenous polynucleotide, once introduced into a host cell, can be maintained as a non-integrated replicon (eg, a plasmid) or can be integrated into the host genome.

“Treatment” refers to prevention and / or therapy.

The term “individual” as used in the present invention refers to vertebrates, particularly members of the mammalian species, and includes, but is not limited to, livestock, sport animals, primates and humans. The term particularly refers to humans.

As used herein, the term “sense strand” or “plus strand” (or “+”)
A nucleic acid containing a sequence encoding the polypeptide is meant. "Antisense strand" or "minus strand" (or "-") means a nucleic acid that contains a sequence complementary to the sequence of the "plus" strand.

The term “test sample” refers to a component of the individual's body that is the source of a subject (eg, an antibody or antigen of interest). These components are well known in the art. The test sample is typically one that is suspected of containing the target sequence. Test samples are prepared by methods well known in the art.
For example, it can be prepared by obtaining a sample from an individual and, if necessary, destroying any cells contained therein to release the target nucleic acid. These test samples include biological samples that can be tested by the methods of the invention described herein and include human and animal body fluids, such as whole blood, serum, plasma, cerebrospinal fluid, Sputum, bronchial washings, bronchial aspirate, urine, lymph, and various exocrine secretions of the respiratory tract, intestinal tract and genitourinary tract, tears, saliva, milk, leukocytes, myeloma, etc .; biological fluids, eg, on cell culture Clear; fixable tissue samples; and fixable cell samples.

“Purified (purified) product” refers to the product that is isolated from the cellular components to which the product normally binds and from other types of cells that may be present in the sample of interest. Means a preparation.

“PNA” means a “peptide nucleic acid analog” that can be used in a method such as the assays described herein to determine the presence of a target. “MA” means “morpholino analog” that can be used in a method such as the assays described herein to determine the presence of a target. See, for example, U.S. Patent No. 5,378,841 which is incorporated herein by reference. A PNA is a neutrally charged moiety that can be directed to an RNA target or DNA. For example, a PNA probe used in an assay instead of the DNA probe of the present invention provides advantages that cannot be achieved using a DNA probe. These advantages include manufacturability, large-scale labeling, reproducibility, stability, insensitivity to changes in ionic strength, and DNA or RNA
Resistance to enzymatic degradation present in methods using These P
The NA can be labeled ("bound") with a signal-generating compound such as fluorescein, radionucleotide, chemiluminescent compound, and the like. Thus, instead of DNA or RNA, PNA or other nucleic acid analogs, such as MA, can be used in the assay method. Although an assay using a DNA probe is described herein, it is understood by those skilled in the art to use PNA or MA instead of RNA or DNA and to make appropriate changes in assay reagents as necessary. Included in the range of skills.

The “analyte” used in the present invention is a substance to be detected which may be present in a test sample. The analyte can be a substance for a naturally occurring specific binding member (eg, an antibody) or any substance that can provide a specific binding member. Thus, an analyte is a substance that can bind to one or more specific binding members in an assay. "Subject" also includes any antigenic substance, hapten, antibody, and combinations thereof. Using naturally occurring specific binding partners (pairs) (eg, using intrinsic factor protein as a member of a specific binding pair to measure vitamin B12, or using folate binding protein to measure folate) Alternatively, or using lectin as a member of a specific binding pair to measure carbohydrates), the analyte can be detected as a member of a specific binding pair. The subject can include proteins, polypeptides, amino acids, nucleotide targets, and the like.

As used herein, “breast disease” or “breast disease” or “breast condition”
It refers to any disease or condition of the breast, including but not limited to atypical hyperplasia, fibroadenoma, cystic mammary gland and cancer.

As used herein, “breast cancer” includes ductal carcinoma in situ, lobular carcinoma in situ, invasive ductal carcinoma,
It refers to any malignant disease of the breast, including, but not limited to, medullary, tubular, mucinous, invasive lobular, invasive comedone and inflammatory-like cancers.

“Expressed tag sequence” or “EST” refers to mRNA extracted from tissue
And the subsequent sequence of the cDNA insert produced by reverse transcription of the cDNA and subsequent insertion into a vector.

“Transcript image” refers to a table or list showing the quantitative distribution of ESTs in a library and represents genes active in the tissue from which the library is derived.

The present invention provides assays that use specific binding members. A “specific binding member” as used in the present invention is a member of a specific binding pair. That is, 2
One of the two different molecules specifically binds to the other molecule by chemical or physical means. Thus, in addition to antigen and antibody specific binding pairs for common immunoassays, other specific binding pairs include biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules, cofactors and enzymes, Enzyme inhibitors and enzymes may be included.
Further, a specific binding pair can include members that are analogs (eg, analyte analogs) of the original specific binding member. Immunoreactive specific binding members include antigens, antigen fragments, antibodies and antibody fragments (both monoclonal and polyclonal and complexes thereof), including those formed by recombinant DNA molecules.

As used herein, the term “hapten” refers to a partial antigen or non-protein binding member that has the ability to bind an antibody, but does not have the ability to elicit antibody formation unless bound to a carrier protein.

As used herein, a “capture reagent” is specific for the analyte in a sandwich assay, or an indicator reagent in a competition assay, or an auxiliary specific binding member (eg, By itself, it refers to an unlabeled specific binding member specific to the analyte). Prior to or during the performance of the assay, the capture reagent can be directly or indirectly attached to a solid phase material to allow for separation of the immobilized complex from the test sample.

An “indicating reagent” is a “signal” capable of producing a measurable signal conjugated (“bound”) to a specific binding member and detectable by external means. Product compound "(" label "). The indicator reagent may be a hapten-antihapten system (e.g., biotin or antibiotin, avidin or biotin, carbohydrate or lectin, a complementary nucleotide sequence, an effector or receptor molecule, in addition to being an antibody member of a specific binding pair. , Enzyme cofactors and enzymes, enzyme inhibitors or enzymes, etc.). The immunoreactive specific binding member is an antibody, antigen or antibody capable of binding to either the polypeptide of interest in a sandwich assay, or a capture reagent in a competition assay, or an auxiliary specific binding member in an indirect assay. / Antigen complex. When describing probes and probe assays, the term "reporter molecule" may be used. The reporter molecule is a signal-generating compound as described above (eg, carbazole or adamantane) bound to a specific binding member of a specific binding pair.
including.

The various “signal generating compounds” (labels) contemplated include chromogenic substances (chr
omagens), catalysts (eg, enzymes), luminescent compounds (eg, fluorescein and rhodamine), chemiluminescent compounds (eg, dioxetane, acridinium, phenanthridinium and luminol), radioactive elements and direct vision (d
direct visual) labels. Enzymes include, for example, alkaline phosphatase, horseradish peroxidase, β-galactosidase and the like. The choice of the particular label is not critical, but may be
It must be capable of generating a signal with the additional substances.

A “solid phase” (“solid support”) is well known to those skilled in the art, and includes well walls of reaction trays, test tubes, polystyrene beads, magnetic or non-magnetic beads, nitrocellulose particles, membranes, microparticles (eg, , Latex particles), sheep (or other animal) erythrocytes and Duracytes® (Abbott Laborator)
ies, Abbott Park, Ill., erythrocytes "fixed" with pyruvate aldehyde and formaldehyde). The "solid phase" is not critical and can be selected by one skilled in the art. For example,
Latex particles, microparticles, magnetic or non-magnetic beads, membranes, plastic tubes, microtiter well walls, glass or silicon chips, sheep (or other suitable animal) erythrocytes and Duracytes® are all suitable materials It is an example. Suitable methods for immobilizing peptides on a solid phase include ionic,
Includes hydrophobicity, covalent interactions, and the like. As used herein, "solid phase" means any material that is insoluble or can be made insoluble in subsequent reactions. The solid phase can be selected for its inherent ability to attract and immobilize the capture reagent. Alternatively, the solid phase can carry additional receptors capable of attracting and immobilizing the capture reagent. The additional receptor can include a charged substance that is charged opposite to the capture reagent itself or opposite to the charged substance bound to the capture reagent. Alternatively, the receptor molecule can be any specific binding member that has the ability to immobilize a capture reagent by a specific binding reaction and is immobilized (bound) on a solid phase. The receptor molecule allows the capture reagent to bind indirectly to the solid phase material before or during the performance of the assay. Thus, the solid phase can be plastic, derived plastic, magnetic or non-magnetic metal, glass or silicon surfaces in test tubes, microtiter wells, sheets, beads, microparticles, chips, sheep (or other suitable animal) erythrocytes, Durac
It can be in ytes® and other forms known to those skilled in the art.

The solid phase may comprise any suitable porous material having sufficient porosity to permit access of the detection antibody and appropriate surface affinity to bind the antigen. It is contemplated and such cases are included within the scope of the present invention. While a porous structure is generally preferred, materials having a hydrated gel structure can also be used. Such useful solid supports include, but are not limited to, nitrocellulose and nylon. Such a porous solid support described herein is preferably intended to be in the form of a sheet having a thickness of about 0.01-0.5 mm, preferably about 0.1 mm. The pore size can take a wide range of values, preferably from about 0.025 to 15 microns, particularly preferably from about 0.15 to 15 microns. The surface of such a support can be activated by a chemical method that causes the covalent attachment of an antigen or antibody to the support. However, in general, irreversible binding of antigens or antibodies is obtained by adsorption on porous materials by poorly understood hydrophobic forces. Other suitable solid supports are known in the art.

[0076]reagent  The present invention relates to a polynucleotide, designated BS106, derived from the breast tissue of interest.
Sequences, the polypeptides encoded by them, and the specificity of these polypeptides.
Provide body and other reagents. The invention also relates to the disclosed polynucleotides and polynucleotides.
And oligonucleotides derived from nucleic acid sequences complementary to these polynucleotides
Provide reagents such as fragments. A polynucleotide, polypeptide or anti-
The body can be used to detect, diagnose, and stage breast diseases or conditions (eg, cancer).
Obtain information to monitor, determine prognosis, prevent or treat or predispose
be able to. The sequences disclosed herein have specific profiles of gene transcriptional activity
And represent unique polynucleotides that can be used in assays. So
Such an assay is described in EP 0 732, hereby incorporated by reference.
03B1 and International Publication WO 95/11995.

A selected BS106-derived polynucleotide can be used in the methods described herein for the detection of normal or altered gene expression. In such methods, it is possible to use BS106 polynucleotides or oligonucleotides, fragments or derivatives thereof, or nucleic acid sequences complementary thereto.

The polynucleotides disclosed herein, their complementary sequences, or fragments thereof, may be used to convert genes, nucleic acids, cDNs associated with diseases or conditions of breast tissue.
It can be used in assays for the detection, amplification or quantification of A or mRNA. Also, they can be used to identify all or part of the coding region of a BS106 polypeptide. In addition, they can be provided in individual containers in the form of a kit for the assay, or as individual compositions. When provided in an assay kit, other suitable reagents such as buffers, conjugates, etc., can be included.

The polynucleotide can be in the form of RNA or DNA.
Polynucleotides in the form of DNA, cDNA, genomic DNA, nucleic acid analogs and synthetic DNA are included within the scope of the present invention. The DNA can be double-stranded or single-stranded, and if single-stranded can be the coding (sense) or non-coding (antisense) strand. The coding sequence encoding the polypeptide can be identical to the coding sequence provided in the present invention, and encodes the same polypeptide as the DNA provided in the present invention as a result of duplication or degeneracy of the genetic code. Different coding sequences are possible.

The polynucleotide may comprise only the coding sequence of the polypeptide,
Alternatively, it comprises a coding sequence for the polypeptide and an additional coding sequence (eg, a leader or secretory sequence or a proprotein sequence) or a non-coding sequence for the polypeptide (and optionally, an additional coding sequence). Coding sequence (eg, a non-coding sequence 5 'and / or 3' to the coding sequence of the polypeptide).

The present invention also includes a mutant polynucleotide containing a modification such as a deletion, substitution or addition of a polynucleotide, and any modified polynucleotide resulting from the mutant polynucleotide sequence. Also, a polynucleotide of the invention can have a coding sequence that is a naturally occurring allelic variant of a coding sequence provided herein.

In addition, the coding sequence of the polypeptide may be a polynucleotide sequence that assists expression and secretion of the polypeptide in a host cell (eg, a leader sequence that functions as a secretory sequence for controlling transport of the polypeptide from the cell). ) Can be fused with the same reading frame. A polypeptide having a leader sequence is a preprotein, which can have a leader sequence that is cleaved by a host cell to form the polypeptide. Also, the polynucleotide can encode a proprotein that includes the protein and additional 5 'amino acid residues. A protein having a prosequence is a proprotein and, in some cases, can be an inactive form of the protein.
Cleavage of the prosequence leaves an active protein. Accordingly, the polynucleotide of the present invention can encode a protein, encode a protein having a prosequence, or encode a protein having a presequence (leader sequence) and a prosequence.

The polynucleotides of the present invention can also have a coding sequence fused in frame with a marker sequence that allows for purification of the polypeptide of the present invention. The marker sequence can be a hexahistidine tag supplied by the pQE-9 vector to effect purification of the polypeptide fused to the marker, in the case of a bacterial host, or for example, a mammalian host. Host (eg, CO
When using the S-7 cell line), the marker sequence can be a hemagglutinin (HA) tag. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein. For example, I. Wilson et al., Cell 3
7: 767 (1984).

[0084] If there is at least 50%, preferably at least 70%, more preferably at least 90% identity between a polynucleotide and a sequence provided herein, the polynucleotide is considered to hybridize to the sequence. It is intended to be.

The present invention also provides an antibody produced using a purified BS106 polypeptide, wherein at least a portion of the polypeptide encodes a BS106 polynucleotide selected from the polynucleotides provided by the present invention. An antibody is provided. These antibodies can be used in the methods provided herein for the detection of BS106 antigen in a test sample. BS10 in test sample
The presence of the six antigens indicates the presence of a breast disease or condition. The antibodies can also be used for therapeutic purposes, for example, in neutralizing the activity of a BS106 polypeptide in conditions associated with altered or aberrant expression.

The present invention further relates to BS106 polypeptides having the deduced amino acid sequences provided herein, as well as fragments, analogs and derivatives of such polypeptides. The polypeptide of the present invention can be a recombinant polypeptide, a naturally occurring purified polypeptide or a synthetic polypeptide. A fragment, derivative, or analog of a BS106 polypeptide can be one in which one or more of the amino acid residues has been replaced with a conservative or non-conservative amino acid residue (preferably, a conservative amino acid residue). It is. Such substituted amino acid residues may or may not be encoded by the genetic code. Alternatively, it can be one in which one or more of the amino acid residues contains a substituent. Alternatively, it can be that the polypeptide is fused to another compound (eg, a compound that increases the half-life of the polypeptide, such as polyethylene glycol). Alternatively, it can be one in which additional amino acids (eg, a leader or secretory sequence, or a sequence used for purification of the polypeptide, or a proprotein sequence) are fused to the polypeptide. . Such fragments, derivatives and analogs are included within the scope of the present invention. The polypeptides and polynucleotides of the present invention are preferably provided in an isolated (preferably purified) form.

Thus, the polypeptides of the present invention can have the same amino acid sequence as the naturally occurring polypeptide or have a different amino acid sequence due to minor mutations due to the substitution of one or more amino acids. The mutation can typically be a "conservative change" in the range of about 1-5 amino acids, wherein the replacement amino acid has similar structural or chemical properties (e.g., Substitution of leucine for isoleucine or threonine for serine). In contrast, mutations can include non-conservative changes (eg, replacement of glycine with tryptophan). Also, similar small mutations can include amino acid deletions or insertions, or both. Guidance in determining the type and number of amino acids to substitute, insert or delete without causing a change in biological or immunological activity can be found in computer programs well known in the art, such as DN.
ASTAR software (DNASTAR Inc., Madison WI)
Can be obtained.

Probes constructed according to the polynucleotide sequences of the present invention can be used in various assay methods to perform various types of analysis. For example, such probes can be used in fluorescence in situ hybridization (FISH) techniques to perform chromosome analysis, and can also be detected from chromosomal spread or by PCR production and / or allele-specific Oligonucleotide probes can be used to identify cancer-specific structural alterations in the chromosome, such as deletions or translocations that can be detected using allele-specific amplification or by direct sequencing. In addition, the probe can be labeled with a radioisotope, a directly or indirectly detectable hapten, or a fluorescent molecule to evaluate mRNA expression of the polynucleotide-containing gene in a tissue sample or cell. It can be used in in situ hybridization studies.

The present invention also provides teachings relating to the production of the polynucleotides and polypeptides provided herein.

[0090]Probe assay  To obtain probes that can be used in assays for the detection of nucleic acids in test samples
For this purpose, the sequences provided in the present invention can be used. The probe is of interest
From the conserved nucleotide region of the polynucleotide or the polynucleotide of interest.
It can be designed from non-conserved nucleotide regions of the otide. Assay optimization
The design of such probes for is within the skill of the art. Generally
If maximum specificity is desired, nucleic acid probes can be generated from non-conserved or unique regions.
That are closely related to different members of the multigene family
The leotide region or nucleotide regions in related species such as mouse and human
When assaying for nucleic acids, a nucleic acid probe is obtained from the conserved region.

[0091] Polymerase chain reaction (PCR) is a technique for amplifying a desired nucleic acid sequence (target) contained in a nucleic acid or a mixture thereof. In PCR, an excess of a pair of primers is used to hybridize with the complementary strand of the target nucleic acid. Each of the primers is extended by a polymerase using the target nucleic acid as a template. The extension product itself becomes a target sequence after dissociating from the original target strand. The new primer is then hybridized, extended by polymerase, and the cycle repeated to geometrically increase the number of target sequence molecules. PC
R is disclosed in U.S. Patent Nos. 4,683,195 and 4,683,202, which are incorporated herein by reference.

The ligase chain reaction (LCR) is another method for amplifying nucleic acids. LCR uses a probe pair that includes two primary (first and second) probes and two secondary (third and fourth) probes, all of which are used in molar excess relative to the target. I do. The first probe hybridizes to a first segment of the target strand, and the second probe hybridizes to a second segment of the target strand. The first is such that the primary probes are adjacent to each other in a 5 ′ phosphate-3 ′ hydroxyl relationship and that the ligase is capable of covalently linking or linking the two probes to the fusion product. The segment and the second segment are adjacent. In addition, the third (secondary) probe can hybridize to a portion of the first probe, and the fourth (secondary) probe hybridizes to a portion of the second probe in a similar adjacent manner. It is possible. Of course, if the target is initially double-stranded, the secondary probe will also hybridize to the complement of the original target. When the ligated strand of the primary probe separates from the target strand, it becomes
And a fourth probe, which can be ligated to form a complementary secondary ligation product. It is important to recognize that the ligation product is functionally equivalent to either the target or its complement. By repeated cycles of hybridization and ligation, amplification of the target sequence is achieved. This technology is disclosed in K. K., published on June 16, 1989. B
ackman EP-A-320 308 and K. K., published July 31, 1991. It is described in more detail in EP-A-439 182 to Backman et al. These are both incorporated herein by reference.

[0093] In the case of mRNA amplification, the mRNA is reverse transcribed into cDNA followed by polymerase chain reaction (RT-PCR), or US Pat. No. 5,322,770, which is incorporated herein by reference. The use of a single enzyme in both steps, as described in R. et al. L
. Marshall et al., PCR Methods and Applicati ons 4: 80-84 as described in (1994), reverse transcribed mRNA to cDNA and then performing the asymmetric gap ligase chain reaction, (RT-AGL
CR) are included within the scope of the present invention.

Other known amplification methods that can be used in the present invention include those described in C Guatelli et al.P NAS USA 87: 1874-1878 (1990) and J Compt.
on,Nature 350 (No. 6313): 91-92 (1991)
So-called "NASBA" or "3SR" technology, European Patent Application Publication (EPA)
No. 4544610, Q-β amplification and strand displacement amplification (strand disp).
placement amplification) (GT Walker et al., Clin. Chem . 42: 9-13 (1996) and European Patent Application No. 68.
No. 4315) and the target mediators described in WO 93/22461.
Includes target mediated amplification
However, the present invention is not limited to these.

Detection of BS 106 can be performed using any suitable detection method, including those detection methods now well known in the art and those that may appear in the future.
The above examples of known detection methods are incorporated herein by reference. For example, Caskey
U.S. Patent No. 5,582,989 and Gelfand et al.
See No. 210,015. Such detection methods include, for example, target amplification methods and signal amplification techniques. Currently known detection methods include, for example, P
Nucleic acid amplification techniques referred to as CR, LCR, NASBA, SDA, RCR and TMA will be included. See, for example, Caskey et al., US Patent No. 5,582,98.
No. 9 and Gelfand et al., US Pat. No. 5,210,015. All prior art is incorporated herein by reference. Also, Sitman
Detection can be performed using signal amplification as disclosed in US Pat. No. 5,273,882. Although amplification of the target or signal is presently preferred, it is contemplated that ultrasensitive detection methods that do not require amplification may be used with the present invention and are included within the scope of the present invention.

Various amplified or non-amplified forms of detection can be performed (in combination) using various heterogeneous or homogeneous detection modes. A specific example of the heterogeneous detection format is Snit
man et al., U.S. Patent No. 5,273,882; Albarella et al., EP-8.
411441.9, Urdea et al., US Pat. No. 5,124,246, Ull.
Man et al., U.S. Pat. No. 5,185,243 and Kourisky et al., U.S. Pat. No. 4,581,333. All prior art is incorporated herein by reference. Specific examples of uniform detection modalities are disclosed in Caskey et al., US Pat. No. 5,582,989, Gelfand et al., US Pat. No. 5,210,015, which is incorporated herein by reference. Also, the use of multiple probes in a hybridization assay, the use of which improves the sensitivity and amplification of the BS106 signal, is contemplated and within the scope of the present invention. See, for example, Caskey et al., US Patent No. 5,5, incorporated herein by reference.
No. 82,989 and Gelfand et al., US Pat. No. 5,210,015.

In one embodiment, the invention generally relates to an amplification reaction comprising a test sample suspected of containing a target polynucleotide sequence and a detection probe and an amplification primer capable of hybridizing to an internal region of the amplicon sequence. Contacting with a reagent. Probes and primers used in accordance with the methods provided herein are labeled with capture and detection labels, where the probe is labeled with one type of label and the primer is labeled with another type of label. In addition, primers and probes are selected such that the probe sequence has a lower melting temperature than the primer sequence. The amplification reagent, the detection reagent and the test sample are placed under amplification conditions that produce a copy (amplicon) of the target sequence in the presence of the target sequence. Usually, the amplicon is double-stranded, since a primer is provided to amplify the target sequence and its complementary strand. Next, the double-stranded amplicon is heat-denatured to obtain a single-stranded amplicon member. Once the single-stranded amplicon member has formed, the mixture is cooled to allow for the formation of a complex between the probe and the single-stranded amplicon member.

[0098] As the single-stranded amplicon sequence and the probe sequence cool, the probe sequence preferentially binds to the single-stranded amplicon member. This finding is counter-intuitive given that the probe sequence is generally chosen to be shorter than the primer sequence, and thus has a lower melting temperature than the primer. Therefore, the melting temperature of the amplicon produced by the primer should also have a higher melting temperature than the probe. Thus, as the mixture cools, reformation of the double-stranded amplicon would be expected. However,
As mentioned above, that is not the case in this case. The probe has been found to bind preferentially to the single-stranded amplicon member. Furthermore, such a preference for probe / single-stranded amplicon binding also exists when the primer sequence is added in excess to the probe.

After the probe / single-stranded amplicon member hybrids have formed, they are detected. Standard heterogeneous assay formats are suitable for detecting the hybrid using the detection and capture labels present on the primer and probe. The hybrid can be bound to a solid phase reagent by the capture label and detected by the detection label. If the detection label is directly detectable, it is possible to detect the presence of the hybrid on the solid phase by causing the label to generate a detectable signal and, if necessary, detecting the signal. it can. If the label is not directly detected, it is possible to contact the capture hybrid with a conjugate generally comprising a binding member bound to a directly detectable label.
The conjugate becomes bound to the complex and the presence of the conjugate on the complex can be detected with its directly detectable label. In this way, the presence of the hybrid on the solid phase reagent can be determined. One skilled in the art will recognize that a washing step may be performed to wash away unhybridized amplicons or probes and unbound conjugate.

Although the target sequence is described as single stranded, the target sequence is actually double stranded and the target sequence is separated from its complement prior to hybridization with the amplification primer sequence It is intended to include cases where only In this way PC
When R is used, the end of the target sequence is usually known. When using LCR or a variant thereof in a preferred method, usually all target sequences are known. Typically, the target sequence is a nucleic acid sequence such as RNA, DNA and the like.

The methods provided herein can be used in well-known amplification reactions involving thermocycling reaction mixtures, particularly in PCR and gap LCR (GLCR). Amplification reactions typically use primers to repeatedly produce copies of a target nucleic acid sequence, which is usually a small region of a larger nucleic acid sequence. The primer itself is a nucleic acid sequence that is complementary to a region of the target sequence. Under amplification conditions, these primers hybridize or bind to complementary regions of the target sequence. A copy of the target sequence is typically obtained by primer extension and / or ligation using a polymerase or an enzyme with ligase activity, separately or in combination, to add nucleotides to the hybridization primer and / or ligate adjacent probe pairs. It is caused by the process of
Nucleotides added to the primer or probe as monomers or preformed oligomers are also complementary to the target sequence. If the primer or probe
Once fully extended and / or ligated, they are separated from the target sequence by, for example, heating the reaction mixture to the "melting temperature" at which the complementary nucleic acid strands dissociate. In this way, a sequence complementary to the target sequence is formed.

[0102] Any double-stranded sequences can then be separated to allow the primers or probes to hybridize to their respective targets, and the hybridized primers or probes can be extended and / or ligated and re-separated. By doing so, a new amplification cycle can be performed to further amplify the number of target sequences. The complementary sequence produced by the amplification cycle can serve as a template to extend the primer to further amplify the number of target sequences or to fill the gap between the two probes. Typically, the reaction mixture is subjected to 20-100 cycles. More typically, the reaction mixture is subjected to 25-50 cycles. The determination of the number of cycles is possible for a person skilled in the art. In this way, multiple copies of the target sequence and its complementary sequence are obtained. Thus, if a primer is present under amplification conditions, it will initiate amplification of the target sequence.

In general, two primers complementary to a part of a target strand and its complement are used in PCR. LCR generally uses four probes, two of which are complementary to the target sequence and the other two are also complementary to the complement of the target. In addition to the primer sets and enzymes described above, the nucleic acid amplification reaction mixture can also include other well-known reagents, including enzyme cofactors (eg, manganese), magnesium salts, nicotinamide These include, but are not limited to, adenine dinucleotide (NAD), and deoxynucleotide triphosphate (dNTP) (eg, deoxyadenine triphosphate, deoxyguanine triphosphate, deoxycytosine triphosphate and deoxythymine triphosphate). It is not limited.

The amplification primer initiates amplification of the target sequence, while the detection (or hybridization) probe does not participate in the amplification. Detection probes are generally nucleic acid sequences or uncharged nucleic acid analogs, such as the peptide nucleic acids disclosed in WO 92/20702, US Pat. Nos. 5,185,444, 5,034,506.
And the morpholino analogs described in US Pat. No. 5,142,047. Depending on the type of label that the probe carries, the probe is used to capture or detect amplicons generated by the amplification reaction. The probe does not participate in the amplification of the target sequence, and thus it may be necessary to make the probe "non-extendable" in that no additional dNTPs can be added to the probe. Usually, the analog itself is inherently inextensible. The nucleic acid probe is 3 'of the probe.
By modifying the terminus so that the hydroxyl group can no longer participate in the extension, it can be rendered non-extendable. For example, the 3 'end of the probe is functionalized with a capture or detection label to consume or protect the hydroxyl group. Alternatively, it is possible to simply cleave, replace or modify the 3 'hydroxyl group. US patent application Ser. No. 07 / 049,061, filed Apr. 19, 1993, which is incorporated herein by reference, describes modifications that can be used to render a probe non-extendable.

The ratio of primer to probe is not critical. Thus, it is possible to add either the probe or the primer in excess to the reaction mixture so that one concentration is greater than the other. Alternatively, primers and probes can be used at equivalent concentrations. However, preferably, an excess of primer to probe is added to the reaction mixture. Thus, the ratio of primer to probe is, for example, 5: 1, preferably 20: 1.

The length of the primers and probes can vary, but the probe sequence is chosen such that the probe sequence has a lower melting temperature than the primer sequence. Thus, a primer sequence is generally longer than a probe sequence. Typically, primer sequences range from 20 to 50 nucleotides in length, more typically from 20 to 30 nucleotides in length. Typical probes are 10-25
Range of nucleotide length.

[0107] Various methods for synthesizing primers and probes are well known in the art. Similarly, methods for attaching labels to primers or probes are well known in the art. See, for example, conventional nucleotide phosphoramidite chemistry and Applied Biosystems, Inc. (Fo
Ster City, CA), DuPont (Wilmington, DE)
Alternatively, it is conventional to synthesize the desired nucleic acid primers or probes using an instrument available from Milligen (Bedford MA).
Numerous methods for labeling oligonucleotides, such as primers, probes, etc. of the present invention have been described. Enzo Biochemical (New
Both York, NY) and Clontech (Palo Alto, CA) describe and commercialize probe labeling technology. For example, 3'-Amin-O
Using NCPG ^™ (Clontech, Palo Alto, CA)
Primary amines can be attached to the 3 'oligo terminus. Similarly, Amino
Modifier II® (Clontech) can be used to attach a primary amine to the 5 ′ oligo terminus. The amine can be reacted with various haptens using conventional activation and conjugation chemistry. Also, co-pending U.S. Patent Application No. 625,566, filed December 11, 1990 and 630,908, filed December 20, 1990, each of which is incorporated herein by reference, are 5 ' And a method for labeling the probe at the 3 'end. International Publication WO92 / 1 published on June 25, 1992
WO 05/11388 published 0505 and July 9, 1992 teaches methods for labeling probes at the 5 'and 3' ends, respectively. In one known method for labeling oligonucleotides, a label-phosphoramidite reagent is prepared and used to add the label to the oligonucleotide during its synthesis. For example, N. T Thong et al . , Tet. Letters 29 (46)
: 5905-5908 (1988) or J. Org. S. Cohen et al., Published U.S. patent application Ser. No. 07 / 246,688 (NTIS ORDER No. PAT-AP).
PL-7-246, 688) (1989). Preferably, the probe is labeled at its 3 'and 5' ends.

[0108] The capture label is attached to a primer or probe. The capture label can be a specific binding member that forms a binding pair with a specific binding member of the solid phase reagent. It will be appreciated that the primer or probe itself can function as a capture label. For example, when the binding member of the solid phase reagent is a nucleic acid sequence,
It can be selected to bind to the complementary part of the primer or probe to immobilize the primer or probe on a solid phase. If the probe itself functions as a binding member, one of skill in the art will recognize that the probe contains a sequence or "tail" that is not complementary to the single-stranded amplicon member. If the primer itself functions as a capture label, at least a portion of the primer will be able to freely hybridize to the nucleic acid on the solid phase.
This is because the probe is chosen so that it is not completely complementary to the primer sequence.

In general, the techniques commonly used to perform heterogeneous immunoassays can be used to detect probe / single-stranded amplicon member complexes. Preferably, in this embodiment, the commercially available Abbott LCx® (Abbott Laboratories, Abbott Park,
IL) Detection is performed according to the protocol used in the apparatus.

The primers and probes disclosed herein are useful in typical PCR assays in which a test sample is contacted with a pair of primers, amplified, a hybridization probe is added, and detection is performed.

Another method provided by the present invention includes contacting a test sample with a plurality of polynucleotides, wherein at least one polynucleotide is a BS106 molecule described herein, and contacting the test sample with a plurality of polynucleotides. Hybridizing to the polynucleotide and detecting the hybridization complex. Hybridization complexes are identified and quantified to gather a profile indicative of disease of the breast tissue (eg, breast cancer). In addition, the expressed RNA sequence can be detected by reverse transcription and amplification of the DNA by methods well known in the art, such as the polymerase chain reaction (PCR).

[0112]Drug screening and gene therapy  The present invention also relates to such polynucleotides or oligonucleotides as the present invention.
Antisense BS106-derived molecule for disease or condition of breast tissue (particularly breast cancer)
Introduce into a patient with a condition associated with aberrant expression of a related polynucleotide
The use of gene therapy methods to Antisense RNA and DNA fragments
And these molecules, including ribozymes, suppress translation of BS106-mRNA
Designed to modify or aberrantly express BS106 polynucleotides
It can be used therapeutically in the treatment of related conditions.

Alternatively, the oligonucleotide may be delivered to a cell by methods known in the art to express the antisense RNA or DNA in vivo and suppress the production of BS106 polypeptide as described above. It is possible. Thus, antisense constructs against a BS106 polynucleotide reverse the effects of BS106 transcripts and can be used to treat breast tissue conditions (eg, breast cancer). Also, these antisense constructs can be used to treat tumor metastasis.

The present invention also provides a method of screening a plurality of compounds for specific binding to a BS106 polypeptide or any fragment thereof, to identify at least one compound that specifically binds to the BS106 polypeptide. . Such methods include providing at least one compound, combining the BS106 polypeptide with each compound under appropriate conditions for a time sufficient to permit binding, and detecting the BS106 polypeptide binding to each compound. The step of performing

The polypeptide or peptide fragment used in such a test may be free in solution, immobilized on a solid phase, carried on a cell surface, or It is also possible to be localized within. One screening method uses a eukaryotic or prokaryotic host cell stably transfected with a recombinant nucleic acid capable of expressing the polypeptide or peptide fragment. Such transfected cells can be screened for drugs, compounds or any other substances in a competitive binding assay. For example, the formation of a complex between the polypeptide and the analyte can be measured in either viable or fixed cells.

Thus, the present invention provides methods of screening for drugs, compounds or any other substances that can be used to treat a disease associated with BS106. These methods include contacting the substance with a polypeptide or fragment thereof and assaying for the presence of a complex of the substance and the polypeptide or for the presence of a complex of the polypeptide and the cell. . In a competitive binding assay, the polypeptide is typically labeled. After a suitable incubation,
The free (or uncomplexed) polypeptide or fragment thereof is separated from the polypeptide or fragment thereof present in bound form, and the amount of free or uncomplexed label is determined by the amount of the individual substance bound to the polypeptide. Or as a measure of the ability to interfere with the polypeptide / cell complex.

The present invention also includes the use of competitive screening assays in which neutralizing antibodies capable of binding to a polypeptide specifically compete with a test agent for binding to the polypeptide or a fragment thereof. Thus, the BS1 provided by the present invention
The antibody can be used to detect the presence of any polypeptide in the test sample that shares one or more antigenic determinants with the 06 polypeptide.

Another method for screening substances provides for high-throughput screening for compounds having suitable binding affinity for at least one polypeptide of BS106 disclosed herein. Briefly, a number of different small peptide test compounds are synthesized on a solid phase, eg, a plastic pin or some other surface. The peptide test compound is reacted with the polypeptide and washed. Polypeptides so bound to the solid phase are detected by methods well known in the art. Also, purified polypeptides can be directly coated on a plate for use in the screening methods described herein.
In addition, non-neutralizing antibodies can be used to capture the polypeptide and immobilize it on a solid phase. For example, September 13, 1984, incorporated herein by reference.
See EP 84/03564, published dated.

The goal of rational drug design is to obtain structural analogs of the biologically active polypeptides of interest or small molecules, including agonists, antagonists or inhibitors with which they interact. is there. Also, such structural analogs can be used to design drugs that are more active or stable forms of the polypeptide or that enhance or inhibit the function of the polypeptide in vivo (herein incorporated by reference herein). Incorporated J. Hodgson, Bio / Technology 9: 19-21 (
1991)).

For example, in one approach, the three-dimensional structure of a polypeptide or polypeptide-inhibitor complex is determined by X-ray crystallography, computer modeling, or most commonly, a combination of the two approaches. In order to elucidate the structure of the polypeptide and determine its active site, both the shape and charge of the polypeptide must be confirmed. Very little useful information about the structure of the polypeptide can be obtained by modeling based on the structure of the homologous protein. In both cases, relevant structural information is used to design similar polypeptide-like molecules or to identify efficient inhibitors.

Useful specific examples of rational drug design include S.D. Braxton et al, Bioche mistry 31: 7796-7801 As shown in (1992),
Molecules with improved activity or stability, or S. elegans, incorporated herein by reference. B. P. Athauda et al . Biochem. (Tokyo) 113 (
6): 742-746 (1993), which may include molecules that act as inhibitors, agonists or antagonists of the native peptide.

It is also possible to isolate the target-specific antibody selected by the above assay and then determine its crystal structure. In principle, this approach provides a pharmacophore that can be the basis for subsequent drug design. Furthermore, by producing anti-idiotypic antibodies ("anti-ids") to functional pharmacologically active antibodies, it is possible to dispense with crystallographic analysis of the protein at all. The anti-id binding site will be an analog of the original receptor as a mirror image of the mirror image. Thus, anti-ids can be used to identify and isolate peptides from banks of chemically or biologically obtained peptides.
The isolated peptide is then used as a pharmacophore (ie, prototype).
function).

A recombinant polypeptide of the invention may be available in an amount sufficient to perform an analytical study, such as X-ray crystallography. Further, knowledge of the amino acid sequence of the polypeptide that can be derived from the nucleic acid sequences provided herein will provide guidance to those using computer modeling techniques instead of or in addition to X-ray crystallography.

In addition, antibodies specific for the BS106 polypeptide (eg, anti-BS106 antibodies) can be used to inhibit the biological effect of the polypeptide by binding to the polypeptide. In this way, the antibodies can be used in therapy to treat breast tissue diseases including, for example, breast cancer and its metastases.

In addition, such antibodies can detect the presence or absence of a BS106 polypeptide in a test sample, and thus as a diagnostic marker for the diagnosis of a disease or condition of breast tissue, especially breast cancer. Useful. In addition, such antibodies can function as diagnostic markers for breast tissue pathologies (eg, breast cancer). The present invention also relates to antagonists and inhibitors of the polypeptide of the present invention. The antagonists and inhibitors inhibit or eliminate the function of the polypeptide. Thus, for example, an antagonist may bind to a polypeptide of the invention and inhibit or eliminate its function. The antagonist is, for example, B
It could be an antibody to the polypeptide that removes the activity of the BS106 polypeptide by binding to the S106 polypeptide. In some cases,
The antagonist can be an oligonucleotide. Small molecule inhibitors include, but are not limited to, for example, small peptides or peptide-like molecules.

The antagonists and inhibitors include saline, buffered saline, dextrose,
It can be used as a composition with a pharmaceutically acceptable carrier, including, but not limited to, water, glycerol, ethanol and combinations thereof. Administration of the BS106 polypeptide inhibitor is preferably systemic. The invention also provides antibodies that inhibit the action of such polypeptides.

Antisense technology can be used to reduce gene expression by triple helix formation or antisense DNA or RNA,
Based on polynucleotide binding to NA or RNA. For example, using the 5 'coding portion of a polynucleotide sequence encoding a polypeptide of the invention,
Design antisense RNA oligonucleotides from 0 to 40 base pairs in length. DNA
Oligonucleotides are designed to interfere with transcription and production of BS106 polypeptide by being complementary to the region of the gene involved in transcription. Regarding triple helices, see, for example, Lee et al . , Nuc. Acids Res , 6: 3073 (
1979); Cooney et al., Science 241: 456 (1988) and Dervan et al., Science 251: 1360 (1991). The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into a BS106 polypeptide. For antisense, see, for example, Okano, J. et al . Neurochem ,
56: 560 (1991) and "Oligodeoxynucleotides."
sas Antisense Inhibitors of Gene Ex
press ", CRC Press, Boca Raton, Fla.
1988). Antisense oligonucleotides work more potently when they have been modified to contain artificial internucleotide linkages that render the molecule resistant to nucleotide cleavage. Such artificial internucleotide linkages include, but are not limited to, methylphosphonate, phosphorothiolate and phosphoramidate internucleotide linkages.

[0128]Recombinant technology  The present invention provides a host cell comprising the BS106 polynucleotide of the present invention;
An expression vector and a method for producing the polypeptide encoded thereby are provided. That
Such a production method can be performed under conditions suitable for the expression of the BS106 polynucleotide.
Culturing the cells and recovering the BS106 polynucleotide from the cell culture.
.

The invention also provides a vector comprising a BS106 polynucleotide of the invention, a host cell genetically engineered with a vector of the invention, and the production of a polypeptide of the invention by recombinant techniques.

[0130] Host cells are genetically engineered (transfected, transduced or transformed) with the vectors of the present invention, which can be a cloning vector or an expression vector. The vector can be in the form of a plasmid, virus particle, phage, and the like. The engineered host cells can be cultured in a conventional nutrient medium, optionally modified to activate the promoter, select for transfected cells, or amplify the BS106 gene. Culture conditions such as temperature, pH, etc. are those already used in the host cell selected for expression and will be apparent to those skilled in the art.

[0131] The polynucleotides of the present invention can be used for the production of polypeptides by recombinant techniques. Thus, it is possible to include the polynucleotide sequence in any one of a variety of expression vehicles (particularly vectors or plasmids) for expressing the polypeptide. Such vectors include chromosomal, non-chromosomal and synthetic DNA sequences such as derivatives of SV40, bacterial plasmids, phage DNA, yeast plasmids, vectors derived from combinations of plasmid and phage DNA, viral DNA (eg, vaccinia, adenovirus). Virus, fowlpox virus and pseudorabies virus). However, any other plasmid or vector can be used as long as they can be propagated and viable in the host.

[0132] The appropriate DNA sequence can be inserted into the vector by a variety of methods.
Generally, the DNA sequence is inserted into a suitable restriction endonuclease site by methods known in the art. Such and other methods are deemed to be within the skill of those in the art. The DNA sequence in the expression vector is an mRNA
It is operably linked to a suitable expression control sequence (promoter) that directs synthesis. Representative examples of such promoters include LTR or SV40 promoter, E. coli lac or trp, phage λP subL promoter,
And other promoters known to control the expression of the gene in prokaryotic or eukaryotic cells or their viruses. Expression vectors also contain a ribosome binding site for translation initiation and a transcription terminator. The vector can also include appropriate sequences for sequence amplification. Further, the expression vector is preferably a gene that confers a phenotypic trait for selection of transfected host cells (eg,
Dihydrofolate reductase or neomycin resistance in eukaryotic cell culture or tetracycline or ampicillin resistance in E. coli)
It contains.

The vector containing the appropriate DNA sequence and an appropriate promoter or control sequence can be used to transfect an appropriate host so that the host can express the protein. is there. Representative examples of suitable hosts include bacterial cells such as E. coli, Salmonella typhimurium (Sal).
monella typhimurium); Streptomyces sp.
Streptomyces sp); fungal cells such as yeast; insect cells such as Drosophila and Sf9; animal cells such as CHO, COS or Bow.
es melanoma; plant cells and the like. The selection of a suitable host is considered to be within the skill of those in the art based on the teachings herein.

More specifically, the present invention also includes a recombinant construct comprising one or more of the sequences widely described above. The construct includes a vector (eg, a plasmid or viral vector) into which the sequences of the invention have been inserted in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises a regulatory sequence operably linked to the sequence (eg, a promoter, etc.). Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are exemplified: Bacterial: pINCY (Incyte Pharmaceutical)
als Inc. , Palo Alto, CA), pSPORT1 (Life
Technologies, Gaithersburg, MD), pQE70,
pQE60, pQE-9 (Qiagen) pBs, phasescript, p
siX174, pBluescript SK, pBsKS, pNH8a, pN
H16a, pNH18a, pNH46a (Stratagene); pTrc9
9A, pKK223-3, pKK233-3, pDR540, pRIT5 (Ph5
eukaryotic: pWLneo, pSV2cat, pOG44, pX
T1, pSG (Stratagene) pSVK3, pBPV, pMSG, pS
VL (Pharmacia). However, any other plasmid or vector can be used as long as it is replicable and viable in the host.

[0135] Plasmid pINCY1 is generally the same as plasmid pSPORT1 (Lif
e Technologies, available from Gaithersburg, MD). These modifications are that (1) it lacks a HindIII restriction site, and (2) that its EcoRI restriction site is at a different position. pSP
PINCY is made from pSPORT1 by cutting ORT1 with both HindIII and EcoRI and replacing the excised fragment of the polylinker with synthetic DNA fragments (SEQ ID NO: 6 and SEQ ID NO: 7). This substitution can be made by any method known to those skilled in the art. For example, their two nucleotide sequences (SEQ ID NO: 6 and SEQ ID NO: 7) are generated synthetically with a 5 'terminal phosphate, mixed together, and then p-cleaved with HindIII and EcoRI.
Ligation can be performed in the SPORT1 plasmid under standard conditions for performing cohesive end ligation. Then a suitable host cell (eg,
E. coli (DH5∝ cells) are transfected with the ligated DNA and recombinant clones are selected for ampicillin resistance. Next, plasmid DNA was prepared from individual clones and analyzed by restriction enzyme analysis or DNA analysis.
Sequencing ensures that the inserted sequence is present in the proper orientation. Other cloning methods known to those skilled in the art can also be used.

The promoter region can be selected from any desired gene using a CAT (chloramphenicol transferase) vector or other vector with a selectable marker. As two suitable vectors, pKK232-8
And pCM7. Individual bacterial promoters that may be mentioned include la
cI, lacZ, T3, SP6, T7, gpt, λP sub R, P sub
L, and trp. Eukaryotic promoters include cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein I. The selection of an appropriate vector and promoter is well within the skill of those in the art.

[0137] In another embodiment, the invention provides a host cell containing the construct. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower animal cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. is there. Introduction of the construct into the host cell can be by calcium phosphate transfection, DEAE-dextran mediated transfection or electroporation (L. Davis et al., "Basic Me
things in Molecular Biology ", 2nd edition, Appl
eton and Lang, Paramount Publishing, E
Ast Norwalk, CT (1994)).

The construct in a host cell can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptide of the present invention comprises
It can be produced synthetically by a usual peptide synthesizer.

[0139] Recombinant proteins can be expressed in mammalian cells, yeast, bacteria or other cells under the control of appropriate promoters. Alternatively, a cell-free translation system can be used to produce such a protein using RNA derived from the DNA construct of the present invention. Suitable cloning and expression vectors for use with prokaryotic or eukaryotic hosts are described in Sambr, incorporated herein by reference.
OK et al., Molecular Cloning: A Laboratory Manual , 2nd edition (Cold Spring Harbor, NY, 1).
989).

[0140] Transcription of a DNA encoding a polypeptide of the invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements, usually about 10 to 300 bp, that act on a promoter to enhance its transcription. Specific examples include the SV40 enhancer (late stage of replication origin, b
p100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer (late side of the replication origin) and the adenovirus enhancer.

In general, recombinant expression vectors contain an origin of replication, a selectable marker that allows transfection of the host cell (eg, the ampicillin resistance gene of E. coli and the S. cerevisiae TRP1 gene). ), And promoters from highly expressed genes to direct transcription of the downstream structural sequences. Such a promoter can be derived from an operon encoding a glycolytic enzyme such as 3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase or heat shock protein, among others. The heterologous structural sequence is combined at an appropriate stage with a translation initiation and termination sequence and, preferably, a leader sequence capable of directing secretion of the translated protein into the periplasmic or extracellular medium. If desired, the heterologous sequence can encode a fusion protein containing an N-terminal identifying peptide that confers the desired property (eg, stabilization or convenient purification of the expressed recombinant product).

Useful expression vectors for use in bacteria are constructed by inserting a structural DNA sequence encoding the desired protein along with the appropriate translation initiation and termination signals, with an operable readout consistent with a functional promoter. I do. The vector will include one or more trait selectable markers and an origin of replication to ensure maintenance of the vector and, if desired, effect amplification in the host. Suitable prokaryotic hosts for transfection include E. coli, Bacillus subtilis, Salmonella typhimurium, and Pseudomonas (
Pseudomonas sp., Streptomyces
) And various species within the genus Staphylococcus, but other prokaryotic hosts can be used as usual selections.

Useful expression vectors for use in bacteria include selectable markers and bacterial origins of replication (the well-known cloning vector pBR322 (ATCC37017).
) Which are derived from a plasmid containing the genetic element). Other vectors include
PKK223-3 (Pharmacia Fine Chemicals, Up
psala, Sweden, and GEM1 (Promega Biotec,
Madison, WI), but is not limited to these. These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.

After transfection into a suitable host and growing the host to a suitable cell density, the selected promoter is replaced by suitable means (eg, temperature changes or chemical induction).
And the cells are further cultured for a certain period of time. The cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is stored for further purification. The microbial cells used in protein expression were:
Disruption can be by any convenient method, including freeze-thaw cycles, sonication, mechanical disruption, or use of cell lysing agents. Such methods are well-known to those skilled in the art.

A variety of mammalian cell culture systems can be used to express the recombinant protein. Mammalian expression systems include, for example, Gluzman, Cell 2
3: 175 (1981) and the COS-7 line of monkey kidney fibroblasts and other cell lines capable of expressing compatible vectors (eg, C127, HEK-2
93, 3T3, CHO, HeLa and BHK cell lines). A mammalian expression vector will include an origin of replication, a suitable promoter and enhancer, and any necessary ribosome binding, polyadenylation, splice donor and acceptor, transcription termination and 5 'flanking non-transcribed sequences. SV4
DNA sequences (eg, SV40 origin, early promoter, enhancer, splice and polyadenylation sites) from the 0 viral genome can be used to obtain the necessary non-transcribed genetic elements. Representative useful vectors include pR
c / CMV and pcDNA3 (Invitrogen, San Diego,
(Available from CA).

Affinity chromatography, ammonium sulfate precipitation, ethanol precipitation, acid extraction,
Ion or cation exchange chromatography, phosphocellulose chromatography
Fee, hydrophobic interaction chromatography, hydroxyapatite chromatography
By known methods such as luffy or lectin chromatography, BS106
The polypeptide is recovered from the recombinant cell culture and purified. Calci present during purification
It is preferred to use low concentrations (about 0.1-5 mM) of the calcium ions (Price et al., J. Biol. Chem . 244: 917 (1969)). The polypeptide
When refining the configuration of the protein, refold the protein as necessary.
A printing step. Finally, a high-performance liquid
Chromatography (HPLC) can be performed.

Thus, the polypeptides of the present invention can be obtained from naturally purified products expressed from highly expressing cell lines, or by chemical methods or from prokaryotic or eukaryotic hosts (eg, bacteria, yeasts in culture). (By higher plant, insect and mammalian cells). Depending on the host used in the recombinant production process, the polypeptides of the present invention can be glycosylated with mammalian or other eukaryotic carbohydrates, or in non-glycosylated form. Also,
The polypeptides of the invention can include an initial methionine amino acid residue.

The starting plasmid can be constructed from available plasmids according to published known methods. Furthermore, plasmids equivalent to those described are
Known in the art and will be apparent to those skilled in the art.

The following is a general method for the isolation and analysis of cDNA clones. In certain embodiments disclosed herein, mRNA is isolated from breast tissue,
Used for construction of cDNA library. Breast tissue was obtained by surgical resection from the patient, which was classified by the pathologist as tumor or non-tumor tissue.

The cDNA insert from the random isolate of the breast tissue library was partially sequenced and analyzed in detail as described in the Examples. It is SEQ ID NO: 1,
These are disclosed in the sequence listing as SEQ ID NO: 2 and SEQ ID NO: 3. The consensus sequence of these inserts is set forth as SEQ ID NO: 4. These polynucleotides can contain the entire open reading frame with or without the associated regulatory sequences for a particular gene, or they may encode only a portion of the gene of interest. It is possible. This is because current genes are often hundreds of bases in length, and sometimes thousands of bases in length, and due to vector constraints, incomplete reverse transcription of the first strand or incomplete replication of the second strand, Due to the fact that it cannot be cloned in its entirety. Successive secondary clones containing additional nucleotide sequences can be obtained using various methods known to those skilled in the art.

[0151] Methods of DNA sequencing are well known in the art. In a typical enzymatic method, a DNA polymerase, Klenow fragment, Seq is used to extend a DNA strand from an oligonucleotide primer annealed to the DNA template of interest.
uenase (US Biochemical Corp., Cleveland
OH) or Taq polymerase. Methods have been developed for using both single-stranded and double-stranded templates. The chain termination reaction product can be electrophoresed on a urea / polyacrylamide gel and detected by autoradiography (for radiolabeled precursors) or fluorescence (for fluorescently labeled precursors). Mechanized reaction preparation using fluorescence detection methods,
Recent improvements in sequencing and analysis include the Applied Biosystem.
ms 377 DNA Sequencers (Applied Biosys)
(Tem, Foster City, Calif.) enabled the expansion of the number of sequences that can be measured per day.

[0152] The reading frame of a nucleotide sequence can be confirmed by several types of analysis. First, the reading frame contained within the coding sequence can be analyzed for the presence of the start codon ATG and the stop codon TGA, TAA or TAG. Typically, one reading frame is c
Continuing over the main portion of the DNA sequence, other reading frames tend to contain multiple stop codons. In such a case, the determination of the reading frame is straightforward. In other, more difficult cases, further analysis is required.

Analyze the frequency of occurrence of individual nucleotide bases in each putative codon triplet
An algorithm has been created to do this. For example, J. W. Fickett, Nuc Acids Res 10: 5303 (1982). Special
The coding DNA for certain organisms (bacteria, plants and animals) is a triplet
They tend to contain certain nucleotides within the periodicity (eg, a pin at the third codon position).
The priority of limidine is significantly recognized). These priorities depend on the given DNA extension.
Determine the coding potential (and frame) of the
Incorporated into widely available software that can be used to determine Argo
Use rhythm-derived information along with start / stop codon information to get the proper frame
Can be determined with high certainty. And this is an accurate reading
Facilitates cloning of sequences within the gene into a suitable expression vector
.

The nucleic acid sequences disclosed herein can be linked to various other polynucleotide sequences and vectors of interest by well-established recombinant DNA techniques. See J Sambrook et al., Supra. Vectors of interest include cloning vectors such as plasmids, cosmids, phage derivatives, phagemids, and the like, sequencing, replication and expression vectors, and the like. Generally, such vectors contain an origin of replication functional in at least one organism, a convenient restriction endonuclease digestion site, and a selection marker appropriate for the particular host cell. The vector can be introduced into an appropriate host cell that produces the desired DNA, RNA or polypeptide by various means known to those skilled in the art.

Sometimes, errors in sequencing or random reverse transcription mask the presence of an appropriate open reading frame or regulatory element. In such cases, it is possible to determine the correct reading frame by attempting to express the polypeptide and determining the amino acid sequence by standard peptide mapping and sequencing techniques. F. M. Ausubel et al., Current Protocols in Molecular Biology , John Wile.
See y & Sons, New York, NY (1989). In addition, the actual reading frame of a given nucleotide sequence can be a total of 3
This can be determined by transfecting the host cell with a vector containing a number of potential reading frames. Only cells with the correct reading frame nucleotide sequence will produce peptides of the expected length.

The nucleotide sequences provided by the present invention have been produced by the current state of the art automated methods and may themselves contain unidentified nucleotides. This will not raise a problem for those skilled in the art who wish to practice the present invention. Missing sequence information can be completed using several methods using standard recombination techniques described in J Sambrook et al. (Supra) or its periodic updates. The same techniques used to obtain the full-length sequence described herein can be used to obtain the nucleotide sequence.

[0157] Expression of a specific cDNA can be achieved by subcloning the cDNA into an appropriate expression vector and transfecting the vector into an appropriate expression host. The cloning vector used to create the breast tissue cDNA library can be used to transcribe the mRNA of a specific cDNA, the promoter for β-galactosidase, the amino-terminal met and the subsequent 7 amino acid residues of β-galactosidase. Containing groups. Immediately after these eight residues, an engineered bacteriophage promoter useful for artificial priming and transcription and a number of unique restriction sites for cloning (eg, Ec
oRI). The vector can be transfected into a suitable host strain of E. coli.

The isolated bacterial strain was purified by standard methods using isopropylthiogalactoside (IP
Induction with TG) will result in a fusion protein containing the first 7 residues of β-galactosidase, about 15 residues of the linker, and the peptide encoded in the cDNA. Since the insert of a cDNA clone is made in a substantially random manner, the chance that the contained cDNA is present in the correct frame for proper translation is one in three. If the cDNA is not in the proper reading frame, in vitro mutagenesis, exonuclease III
Alternatively, the correct frame can be obtained by deletion or insertion of the appropriate number of bases by well-known methods, such as digestion with mung bean nuclease or incorporation of an oligonucleotide linker.

The cDNA can be shuttled to other vectors known to be useful for expression of the protein in a particular host. Oligonucleotide primers containing a cloning site and a DNA segment sufficient to hybridize to extensions at both ends of the target DNA can be chemically synthesized by standard methods. These primers can then be used to amplify the desired gene segment by PCR. The resulting new gene segment can be digested with appropriate restriction enzymes under standard conditions and isolated by gel electrophoresis. Alternatively, by digesting the cDNA with appropriate restriction enzymes and filling in the missing gene segments with chemically synthesized oligonucleotides,
Similar gene segments can be obtained. Segments of the coding sequence from multiple genes can be ligated together and cloned into a suitable vector to optimize expression of the recombinant sequence.

Suitable expression hosts for such chimeric molecules include mammalian cells such as Chinese hamster ovary (CHO), human embryonic kidney (HEK) 293 cells, Sf9
Insect cells such as cells, Saccharomyces
yeast cells such as S. cerevisiae and bacteria such as E. coli. For each of these cell lines, useful expression vectors include an origin of replication that allows for growth in bacteria and a selectable marker that allows for selection in bacteria (eg, a β-lactamase antibiotic resistance gene). It is also possible. In addition, the vector may contain another selectable marker (e.g.,
Neomycin phosphotransferase gene). For vectors for use in eukaryotic expression hosts, the addition of a 3 'polyA tail may be necessary if the sequence of interest lacks polyA.

Further, the vector may contain a promoter or enhancer that enhances gene expression. Such promoters are host-specific, such as the MMTV, SV40 or metallothionein promoters for CHO cells, the trp, lac or T7 promoters for bacterial hosts, or the α-factor, alcohol oxidase for yeast. Or containing the PGH promoter,
It is not limited to these. Adenoviral vectors with or without a transcription enhancer (eg, the Rous sarcoma virus (RSV) enhancer) can be used to drive expression of the protein in mammalian cell lines.
Once a homogeneous culture of the recombinant cells has been obtained, large amounts of the recombinantly produced protein can be recovered from the conditioned media and analyzed by chromatographic methods well known in the art. Another method for producing large amounts of secreted protein involves transfection of mammalian embryos and recovery of the recombinant protein from milk produced by transgenic cows, goats, sheep and the like. Polypeptides, and closely related molecules, can be expressed recombinantly in such a way as to facilitate protein purification. One approach involves the expression of a chimeric protein that contains one or more additional polypeptide domains that are not naturally present on the human polypeptide. Such domains that facilitate purification include metal-chelating peptides (eg, histidine-tryptophan domains) that allow for purification on immobilized metals, and allow for purification on immobilized immunoglobulins. Protein A
Domain and FLAGS extension / affinity purification system (Immunex C
orp, Seattle, WA). Inclusion of a cleavable linker sequence, such as factor XA or enterokinase (available from Invitrogen, San Diego, CA), between the polypeptide sequence and the purification domain may be useful for recovery of the polypeptide. Absent.

[0162]Immunoassay  BS106 polypeptide (including fragments, derivatives and analogs thereof) or its
Cells expressing such polypeptides can be used as seeds for the detection of antibodies to breast tissue.
For use in various assays, many of which are described herein.
it can. They can also be used as immunogens to produce antibodies
. These antibodies include, for example, polyclonal or monoclonal antibodies,
La, single-chain and humanized antibodies and Fab fragments, or Fab expression libraries
Can be the product of For the production of such antibodies and fragments,
Various methods known in the art can be used.

For example, antibodies raised against a polypeptide comprising a sequence of the present invention can be obtained by direct injection of the polypeptide into an animal, or by administering the polypeptide to an animal (eg, mouse, rabbit, goat or human). )). Mice, rabbits or goats are preferred. The polypeptide has SEQ ID NO: 16,
It is selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and fragments thereof. Subsequently, the antibody thus obtained will bind to the polypeptide itself. Thus, even a sequence encoding only a fragment of the polypeptide can be used to raise antibodies that bind to the native polypeptide. Such polypeptides can then be used to isolate the polypeptide from a test sample, such as a tissue suspected of containing the polypeptide. For production of monoclonal antibodies, any technique which provides antibodies produced by continuous cell culture can be used. Specific examples include Kohler and Mil
Stein, Nature 256: 495-497 (1975), hybridoma technology, trioma technology, Kozbor et al . , Immun. Today
4:72 (1983), and the human B cell hybridoma technology described in Cole.
Et al., Monoclonal Antibodies and Cancer Therapy , Alan R. et al. Liss, Inc, New York, NY, p
p. 77-96 (1985), including EBV-hybridoma technology for producing human monoclonal antibodies. The techniques described for the production of single-chain antibodies can be applied to produce single-chain antibodies to the immunogenic polypeptide products of the invention. See, for example, U.S. Patent No. 4,946,778, which is incorporated herein by reference.

The antibodies of the present invention can be used in various assay formats, including “sandwich” immunoassays and probe assays. For example, the antibodies or fragments thereof of the invention can be used in various assay systems to determine the presence of the BS106 antigen in a test sample. For example, in a first assay format, a polyclonal or monoclonal antibody or fragment thereof, or a combination of these antibodies, coated on a solid phase, is contacted with a test sample to form a first mixture. The first mixture is incubated for a time and under conditions sufficient to form an antigen / antibody complex. An indicator reagent comprising a monoclonal or polyclonal antibody or fragment thereof to which the signal producing compound is bound, or a combination of these antibodies, is then contacted with the antigen / antibody complex to form a second mixture. This second mixture is then incubated for a time and under conditions sufficient to form an antibody / antigen / antibody complex. By detecting a measurable signal generated by the signal generating compound, the presence of the BS106 antigen captured on the solid phase and included in the test sample is determined. B present in the test sample
The amount of S106 antigen is proportional to the signal generated.

In another assay format, (1) a polyclonal antibody, monoclonal antibody or fragment thereof that specifically binds to the BS106 antigen, or a combination of such antibodies bound to a solid support, (2) a test sample, And (3) contacting the mixture with an indicator reagent comprising a monoclonal antibody, polyclonal antibody or a fragment thereof (or a combination of these antibodies) that specifically binds to a different BS106 antigen to which the signal-generating compound is bound. obtain. The mixture is incubated for a time and under conditions sufficient to form an antibody / antigen / antibody complex. By detecting a measurable signal generated by the signal generating compound,
The presence of the BS106 antigen present in the test sample and captured on the solid phase is determined.
The amount of BS106 antigen present in the test sample is proportional to the signal generated.

In another assay format, one or a combination of at least two monoclonal antibodies of the invention can be used as a competitive probe for the detection of antibodies to the BS106 antigen. For example, a BS106 polypeptide (eg, a recombinant antigen disclosed herein), alone or in combination, is coated on a solid phase. The test sample suspected of containing an antibody to the BS106 antigen is then combined with an indicator reagent comprising a signal-generating compound and at least one monoclonal antibody of the invention together with the test sample and the indicator reagent or solid phase bound to the solid phase. Incubate for a time and under conditions sufficient to form an antigen / antibody complex for any of the indicator reagents bound to the. The decrease in binding of the monoclonal antibody to the solid phase can be quantitatively measured.

In yet another method of detection, each of the monoclonal or polyclonal antibodies of the present invention can be used to detect the presence of BS in tissue sections or cells by immunohistochemical analysis.
106 antigen can be used. These antibodies can be directly labeled (eg, labeled with fluorescein, colloidal gold, horseradish peroxidase, alkaline phosphatase, etc.) or labeled using a secondary labeled anti-species antibody (various examples illustrated herein). Labeling) and cytochemical analysis to track the histopathology of the disease are also included within the scope of the present invention.

In addition, these monoclonal antibodies can be bound to matrices similar to CNBr-activated Sepharose to generate specific BS from cell culture or biological tissue.
Affinity purification of polypeptides (eg, recombinant and native BS106)
Protein purification).

The monoclonal antibodies of the present invention can also be used for producing chimeric antibodies for therapeutic or other similar uses.

[0170] Monoclonal antibodies or fragments thereof can be provided individually for detection of the BS106 antigen. In addition, the combination of the monoclonal antibodies (and fragments thereof) provided by the present invention can be combined with at least one BS10 of the present invention together with antibodies that specifically bind to other BS106 regions (each antibody having a different binding specificity).
It can be used together as a component in a mixture or "cocktail" of six antibodies. Thus, the cocktail can include a monoclonal antibody of the invention to a BS106 polypeptide disclosed herein and another monoclonal antibody specific for the BS106 antigen or other antigenic determinant of another related protein. It is.

A polyclonal antibody or fragment thereof that can be used in the assay format is BS106.
It should specifically bind to the polypeptide or other BS106 polypeptides additionally used in the assay. Preferably used polyclonal antibodies are BS
106 derived from a mammal, such as a human, goat, rabbit or sheep polyclonal antibody that binds to the polypeptide. Most preferably, the polyclonal antibody is from a rabbit. The polyclonal antibodies used in the assay can be used alone or as a cocktail of polyclonal antibodies. Since the cocktails used in the assay format contain monoclonal or polyclonal antibodies with different binding affinities for the BS106 polypeptide, they can be used to detect, diagnose, staging, monitor, detect breast diseases or conditions, such as breast cancer. It is useful for prognosis, prevention or treatment, or predisposition.

The use of recombinant antigens or the use of synthetic or purified peptides, which comprise the amino acid sequence of BS106, allows the assay of B
It is intended to allow detection of the S106 antigen, which is included within the scope of the present invention. The amino acid sequence of such a polypeptide is SEQ ID NO: 16, SEQ ID NO: 17
, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and fragments thereof. Also, different synthetic, recombinant or purified peptides defining different epitopes of BS106 may be used for the detection, diagnosis, staging, monitoring, prognosis, prevention or treatment or predisposition of breast diseases or conditions such as breast cancer. Co-use in assays is also within the scope of the invention. In this case, all of these peptides may be coated on a solid phase, or each separated peptide may be coated on a separate solid phase (eg, microparticles) to form a peptide mixture that can be used later in an assay. It is possible to combine as follows. Further, the use of multiple peptides defining epitopes from different antigens for the detection, diagnosis, staging, monitoring, prognosis, prevention or treatment or predisposition of a breast disease or condition, such as breast cancer, may be employed. It is intended to be possible. The peptide, coated on a solid phase or labeled with a detectable label, then competes with the peptide present (if present) in the patient sample for an amount of antibody. A decrease in the binding of the synthetic, recombinant or purified peptide to the antibody is indicative of the presence of the BS106 antigen in the patient's sample. The presence of the BS106 antigen indicates the presence of disease of the breast tissue in the patient, especially breast cancer. Variations on the assay format are known to those of skill in the art and will be discussed later herein.

In another assay format, the presence of anti-BS106 antibody and / or BS106 antigen can be detected simultaneously, as follows. The test sample is simultaneously treated with a first analyte capture reagent (the capture reagent includes a first binding member specific for the first analyte bound to a solid phase) and a second analyte capture reagent (the capture reagent). The reagent is the second
(Including a first binding member for a second analyte bound to a solid phase) to obtain a mixture. The mixture is incubated for a time and under conditions sufficient to form the capture reagent / first analyte and capture reagent / second analyte complex. The complexes thus formed are then combined with a member of a binding pair specific for the first analyte labeled with the signal generating compound and bound specifically to the second analyte labeled with the signal generating compound. A second mixture is obtained by contacting with an indicator reagent comprising a member of the pair. The second mixture is incubated for a time and under conditions sufficient to form a capture reagent / first analyte / indicating reagent complex and a capture reagent / second analyte / indicating reagent complex. The presence of one or more analytes is determined by detecting the signal generated for the complex formed on either or both solid phases as an indicator of the presence of one or more analytes in the test sample. This assay format can use recombinant antigens derived from the expression systems disclosed herein, and monoclonal antibodies produced from proteins derived from the expression systems disclosed herein. For example, in this assay system, the BS106 antigen can be used as the first analyte. Such an assay system is described in more detail in EP 0 473 065.

In yet another assay format, the polypeptides disclosed herein can be used to detect the presence of antibodies to the BS106 antigen in a test sample. For example, a test sample is incubated with a solid phase to which at least one polypeptide (eg, a recombinant protein or a synthetic peptide) is bound. The polypeptide is selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and fragments thereof. These are reacted for a time and under conditions sufficient to form an antigen / antibody complex. After incubation, the antigen / antibody complex is detected. Depending on the assay system chosen, indicator reagents can be used to facilitate detection. In another assay format, a test sample is contacted with a solid phase to which a recombinant protein produced as described herein is bound, and is preferably labeled with an indicator reagent.
Contact with a monoclonal or polyclonal antibody specific for the protein. After incubation for a time and under conditions sufficient for the formation of the antibody / antigen complex, the solid phase is separated from the free phase and the label is used in the solid phase or in the free phase as an indicator of the presence of antibody to BS106 antigen. To detect. Other assay formats using the recombinant antigens disclosed herein are contemplated. These contact the test sample with a solid phase to which at least one antigen from the first source is bound, and combine the solid phase with the test sample for a time and under conditions sufficient to form an antigen / antibody complex. Incubating and then contacting the solid phase with a labeled antigen, wherein the antigen is from a second source different from the first source. For example, a recombinant protein from a first source, such as E. coli, is used as a capture antigen on a solid phase, and a test sample is added to the solid phase so prepared, and standard After extensive incubation and washing steps, recombinant proteins from different sources (ie, non-E. Coli) are used as part of the indicator reagent to be detected later. Similarly, a combination of a recombinant antigen on a solid phase with a synthetic peptide (in the indicator phase) is also possible.
Any assay format that uses an antigen as a capture antigen specific to BS106 produced or derived from a first source and an antigen specific to BS106 from a different second source is contemplated. Thus, various combinations of recombinant antigens and the use of synthetic peptides, purified proteins, etc. are included within the scope of the present invention. This and other assays enjoy the same proprietary rights as the present application and are described in US Pat. No. 5,254,458, which is incorporated herein by reference.

Other embodiments using various other solid phases are also contemplated and are within the scope of the present invention. For example, in order to perform a fast liquid phase immunochemical reaction, an ion trapping method for immobilizing an immobilizable reaction complex with a negatively charged polymer (EP Publication No. 03)
26100 and EP Publication No. 0406473) can be used in the present invention. The immobilizable immune complex is separated from the remainder of the reaction mixture by ionic interactions between the negatively charged poly-anion / immune complex and a pretreated positively charged porous matrix. The detection is performed using various signal generation systems already described (for example, those described in the measurement of chemiluminescence signal described in EPO Publication No. 0273,115).

It is also possible to adapt the method of the invention for use in systems using microparticle technology in which the solid phase includes microparticles (magnetic or non-magnetic) (eg, automated and semi-automated systems). Such systems include, for example, the published EPO application EP 0
425 633 and the systems described in EP 0 424 634, respectively.

Scanning probe microscopy in immunoassays (scanning p
The use of probe microscopy (SPM) is another technique that can be easily adapted to the monoclonal antibodies of the present invention. In scanning probe microscopy, particularly atomic force microscopy, a capture phase, for example, at least one of the monoclonal antibodies of the present invention, is attached to a solid phase and the scanning phase is used to probe the solid phase. Detect antigen / antibody complexes that may be present on the surface. Normal,
In many immunoassay systems, labels must be used to detect the antigen / antibody complex, but the use of scanning tunneling microscopy obviates the need for such labels. The use of SPM to monitor specific binding reactions
It can be implemented in a number of ways. In one embodiment, one of the members of the specific binding partner (the analyte-specific substance that is a monoclonal antibody of the invention) is bound to a surface suitable for scanning. The binding of the analyte-specific substance can be by adsorption to a test strip containing a solid phase on a plastic or metal surface, performed according to methods known to those skilled in the art. Alternatively, the covalent attachment of a specific binding partner (analyte-specific substance) to a test strip comprising a solid phase of derivatized plastic, metal, silicon (silicon) or glass can be used. Covalent attachment methods are known to those skilled in the art and include various means for irreversibly binding a specific binding partner to a test strip. If the test piece is silicon or glass, the surface needs to be activated before binding the specific binding partner. Also, in order to immobilize the specific binding partner on the surface of the test piece by a chemical method, it is necessary to use a polyelectrolyte interac.
) can be used. The preferred method of attachment is by covalent means. After binding of the specific binding member, the surface can be further treated with substances such as serum, proteins or other blocking agents to minimize non-specific binding. Also, the surface can be scanned at the site of manufacture or at the point of use to confirm suitability for the purpose of the assay. The scanning process would not alter the specific binding properties of the test strip.

Although the present invention is disclosed primarily with reference to the use of solid phases, it is contemplated that the reagents, such as antibodies, proteins, peptides, etc., of the present invention can be used in non-solid phase assay systems. These assay systems are known to those of skill in the art and are considered to be within the scope of the present invention.

It is contemplated that the reagents used in the assays can be provided in the form of a test kit having one or more containers, such as vials, bottles, and the like. Each container contains a separate reagent, such as a probe, primer, monoclonal antibody, or a cocktail of monoclonal antibodies, or a polypeptide (eg, recombinantly, synthetically produced or purified) for use in the assay. I do. The polypeptide is selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and fragments thereof. Other components known to those of skill in the art (eg, buffers, controls, etc.) can be included in such test kits. It is also intended to provide a test kit having means for collecting a test sample containing available body fluids (eg, blood, urine, saliva, and feces). Such means useful for collection ("collection material") include a lancet and absorbent paper or cloth for collecting and stabilizing blood, and a swab, urine or fecal sample for collecting and stabilizing saliva. A stabilizing cup is included. To avoid denaturation or irreversible adsorption of the sample, it is possible to treat the collected material, paper, cloth, swab, cup, etc., if desired. Also, the collected material may be treated with a preservative, stabilizing agent, or antimicrobial to aid in maintaining the integrity of the sample, or the collected material may contain a preservative, stabilizer, or antimicrobial It is possible to do. Also useful are test kits designed for the collection, stabilization, and storage of test samples obtained by surgery or needle biopsy. All kits have two components that can be provided separately, one for sample collection and transport and the other for sample analysis. It is intended to be able to be configured.
For example, a collected component can be provided to open market consumers, while an analytical component can be provided to a researcher or the like for determining the presence, absence or amount of an analyte. It is. In addition, kits for collecting, stabilizing, and storing test samples can be configured for use by unskilled persons, for analysis of the test samples after use at home. Available on the open market with the intention of being transported to the laboratory.

[0180] E. coli (clone 1668855) is based on the provisions of the Budapest Treaty and is based on the American Type Culture Collection.
on (ATCCC), 12301 Parklawn Drive, Ro
CKVILLE, Maryland 20852, deposited 11/20/96 and will be kept for 30 years from the date of the deposit or 5 years after the final request for a deposit or until the latest in the life of the US patent. The deposit and any other deposited materials described herein are provided for convenience only and it is not required that the present invention be practiced in light of the teachings herein. The cDNA sequences in all of these deposited materials are incorporated herein by reference.
Clone 166885 includes A. T. C. C. Accession No. 98256 has been granted.

Next, the present invention will be described with reference to examples. However, these examples are merely examples,
It does not limit the scope of the invention.

ExamplesExample 1: Identification of a breast tissue library BS106 gene-specific clone  A. Library of expressed tag sequences (ESTs) or transcript images Comparison  Partial sequence of cDNA clone insert, so-called “expressing tag sequence” (
ESTs) can be used for breast tumor tissue, breast non-tumor tissue and many other tissues (tumor and
Gene library from both non-tumor cDNA libraries
A database (Incyte Pharmaceuticals,
LIFESEQ available from Palo Alto, CA^TMDatabase)
I input it. See International Publication WO 95/20681 (transfer image is
EST for each of the representative genes in a given tissue library
It is a list of numbers. ESTs that share regions of mutual sequence overlap are classified into clusters.
It is. Clusters are assigned clone numbers from a representative 5 'EST
. Often, clusters of interest are identified by their consensus sequence and automation class.
Extension by comparison with other EST sequences that did not satisfy the
And it is possible. All available clusters and single EST alignments are
, Represents the contig from which the consensus sequence is derived). Next, the transfer image was evaluated.
To identify EST clusters that were primarily representative of the breast tissue library
. These target clusters are then reduced to their abundance in the target library (
Frequency of EST members) and those in the background library
Was ranked based on the absence of Having a low background frequency,
Higher abundance clusters were given higher study priority. Duplicate clone
1668852 (SEQ ID NO: 1), 896988 (SEQ ID NO: 2) and 12098
14 (SEQ ID NO: 3) was identified for further study. These are referred to herein as
BS106 from which the described consensus sequence (SEQ ID NO: 4) was derived
The minimum number of clones required to form a contig was represented.

[0183]B. Creating a consensus sequence  EST clones 1662885 (SEQ ID NO: 1), 89988 (SEQ ID NO: 2)
And 1209814 (SEQ ID NO: 3)
er^TMProgram (Gene Codes Corporation, An
n Arbor, MI) and enter the nucleotide alignment (
Contig map) and then their consensus sequence (SEQ ID NO: 4)
. FIG. 1 shows the nucleotide sequence alignment of these clones and the resulting
3 shows their nucleotide consensus sequence (SEQ ID NO: 4). Figure 2 shows the BS
The clone 1662885 (SEQ ID NO: 1) forming an overlapping region of 106 genes;
894988 (SEQ ID NO: 2) and 1209814 (SEQ ID NO: 3)
And the consensus nucleotide sequences obtained from these clones.
Column number 4). After this, on the consensus sequence (SEQ ID NO: 4)
Performed 3 frame translation. The first forward frame contains 90 amino acids
Was found to have an open reading frame encoding the sequence,
This is shown as SEQ ID NO: 16. FIG. 1 shows the G / T polymorphism at position 54 of the consensus sequence.
As shown (indicated as "K"), each two nucleotides are represented by LIF
ESEQ^TMIt was found almost equally frequently in the database. Methionine residue
The polymorphism at position 54 located 10 nucleotides upstream of the group (potential translation initiation site)
It was thought that it might be involved in regulating the expression level of this protein.
FIG. 1 shows the C / T polymorphism at position 312 of the consensus sequence ("Y").
Shown). LIFESEQ^TM312 recognized in the database
The ratio of C to T at the position was 3: 1.

[0184]C. Specificity of EST expression corresponding to consensus sequence  Using the BLAST search tool, the consensus sequence obtained in section B was updated to the latest LI
FESEQ^TMCompared to the entire database (September 1997). The outlet
The EST corresponding to the suspension sequence was 85.7% of the breast library (24 out of 28 breast libraries).
), And 0.2% (1 of 455) of non-breast libraries. I
Thus, the consensus sequence or a fragment thereof is present in non-breast tissue in breast tissue.
Were found at a frequency of 389 times or more as compared to

[0185]Example 2: Sequencing of BS106 EST specific clone  Clone 16628 containing the 5'-most EST of the BS106 gene contig
No. 85 DNA sequence (SEQ ID NO: 5) was synthesized using a dye terminator according to a known method.
Dideoxy termination sequencing method used (F Sanger et al.)
,PNAS U.S. S. A.74: 5463 (1977)). In SEQ ID NO: 5
The A / G polymorphism at position 543 is shown as "R".

The pINCY vector (Life Technologies, Gaithe)
rsburg, MD) contains a priming site immediately adjacent to the 3 ′ and 5 ′ ligation junctions of the insert, and thus is a universal primer (SEQ ID NO: 8).
And SEQ ID NO: 9) (New England Biolabs, B, respectively)
everly, MA and Applied Biosystems Inc, F
About 300 bases of the insert were sequenced in both directions using an Oster City, CA). The sequencing reaction was performed on a polyacrylamide denaturing gel and
Applied Biosystems 377 Sequencer (Appl
(available from ied Biosystems, Foster City, CA)
Alternatively, the sequence was determined by another sequencing device. Additional sequencing primer BS106. F1 and BS106. R1 (SEQ ID NO: 10 and SEQ ID NO: 11, respectively) was designed from the sequence determined by the initial sequencing reaction near the 3 'end of its two DNA strands. Then, using these primers,
As previously described, the remaining D of the cloned insert from each DNA strand
The NA sequence was determined.

[0187]Example 3: Preparation of nucleic acid  A. Extraction of RNA from tissue  Total RNA was isolated from solid breast tissue or cells and non-breast tissue. The technology
The lithium chloride / urea method known in the art and described in the literature (Kato et al.,J. Virol. 61: 21-2191, (1987)), Ultraspec.^{T M} (Biotecx Laboratories, Inc., Houston
Texas) and TRIzol^TM(Life Technologies,
Inc. , Gaithersburg, MD) including but not limited to
Not) various methods were used.

For Northern blot analysis, the tissues were placed in sterile conical tubes on ice and 10-1
Five volumes of 3 M LiCl, 6 M urea, 5 mM EDTA, 0.1 M β-mercaptoethanol, 50 mM Tris-HCl (pH 7.5) were added. The tissue is placed on a Polytron® homogenizer (Brinkman) on ice.
Instruments, Inc. , Westbury, NY) for 30-50 seconds. The solution was transferred to a 15 ml plastic centrifuge tube and left at -20 ° C overnight. The tubes were centrifuged at 9,000 × g at 0-4 ° C. for 90 minutes, and the supernatant was immediately decanted. Then, 10 ml of 3M LiCl was added, the tube was vortexed for 5 seconds and centrifuged at 11,000 xg, 0-4 ° C for 45 minutes. These decants, resuspension in LiCl and centrifugation were repeated. Air dry the final pellet, 2
ml of 1 mM EDTA, 0.5% SDS, 10 mM Tris (pH 7.5)
). Then, 20 μl of proteinase K (20 mg / ml) was added and the resulting solution was incubated at 37 ° C. for 30 minutes with occasional mixing.
One-tenth volume (0.22 to 025 ml) of 3M NaCl was added, the solution was vortexed and then transferred to another tube containing 2 ml of phenol / chloroform / isoamyl alcohol (PCI). Vortex the tube for 1-3 seconds,
Centrifugation was performed at 000 × g and 10 ° C. for 20 minutes. The PCI extraction was repeated twice more, followed by two similar extractions with chloroform / isoamyl alcohol. The final aqueous solution was transferred to a pre-cooled 15 ml Corex glass tube containing 6 ml of 100% absolute ethanol, the tube was covered with paraffin and left at -20 ° C overnight. The tubes were centrifuged at 10,000 xg at 0-4 ° C for 30 minutes and the ethanol supernatant was immediately decanted. The RNA pellet was washed four times with 10 ml of 75% ice-cold ethanol and after each centrifugation at 10,000 xg for 10 minutes. Final pellet at room temperature
Air dried for minutes. The RNA was added to 0.5 ml of 10 mM Tris (pH 7.6), 1
It was suspended in mM EDTA and its concentration was measured spectrophotometrically. RNA samples were aliquoted and stored at -70 ° C as ethanol precipitate.

The amount of the RNA was determined by agarose gel electrophoresis (see Example 5) and staining with 0.5 μg / ml ethidium bromide for 1 hour. Intact 28
RNA samples that did not contain S / 18S rRNA were excluded from the study.

Alternatively, for RT-PCR analysis, 1 ml of Ultraspec RNA reagent was added to 120 mg of milled tissue in a 2.0 ml polypropylene microcentrifuge tube and P
olytron® homogenizer (Brinkman Instru
ments, Inc. , Westbury, NY) for 50 seconds.
Leave on ice for 5 minutes. Then 0.2 ml of chloroform was added to each sample,
Then vortexed for 15 seconds. The sample was left on ice for a further 5 minutes and then centrifuged at 12,000 xg for 15 minutes at 4 ° C. The upper layer was collected and transferred to another 2.0 ml microcentrifuge tube without RNase. An equal volume of isopropanol was added to each sample and the solution was left on ice for 10 minutes. 12,000 ×
g, and centrifuged at 4 ° C. for 10 minutes, and the supernatant was discarded. The remaining pellet was washed twice with cold 75% ethanol, resuspended by vortexing and the resuspended material was then
The pellet was re-pelleted by centrifugation at 7,500 × g at 4 ° C. for 5 minutes. Finally, the R
The NA pellet was dried in a speedvac for at least 5 minutes and reconstituted in RNase-free water.

[0191]B. RNA extraction from blood mononuclear cells  Mononuclear cells are centrifuged using Ficoll-Hypaque as follows
To isolate from a blood sample from the patient. Equal volume of 10 ml whole blood
RPMI medium (Life Technologies, Gaitherbu)
rg, MD). Then, under the mixture, 10 ml of Ficoll-H
ypaque (Pharmacia, Piscataway, NJ)
Centrifuge at 200 × g for 30 minutes. Take out the buffy coat containing the mononuclear cells
And Dulbecco PBS (Life Technologies, Gai)
ther., MD) to 50 ml and dilute the mixture with 200 xg to 10 ml.
Centrifuge for minutes. After two washes, the resulting pellet was washed with Dulbecco PBS.
To a final volume of 1 ml.

RNA is prepared from isolated mononuclear cells as described by Kato et al. (Supra). Briefly, the pelleted mononuclear cells were brought to a final volume of 1 ml, then resuspended in 250 μl of PBS, 2.5 ml of 3 M LiCl, 6 M urea, 5 mM
M EDTA, 0.1 M 2-mercaptoethanol, 50 mM Tris-H
Mix with Cl 2 (pH 7.5). The resulting mixture is homogenized and at -20 ° C.
And incubate overnight. The homogenate was purified using Beckman J2-21M
Centrifuge in a rotor at 8,000 RPM, 0-4 ° C for 90 minutes. The pellet,
Resuspend in 10 ml of 3M LiCl by vortexing, then
Centrifuge at 10,000 RPM, 0-4 ° C for 45 minutes in a ckman J2-21M rotor centrifuge. Then, the resuspension step and the pelletizing step are repeated. The pellet is vortexed with 2 ml of 1 mM EDTA, 0.5% SDS, 1 ml.
Resuspend in 0 mM Tris (pH 7.5) and 400 μg Proteinase K, then incubate it at 37 ° C. for 30 minutes with shaking. One-tenth volume of 3M NaCl is then added and the mixture is vortexed. The protein is removed by two cycles of extraction with phenol / chloroform / isoamyl alcohol, followed by a single extraction with chloroform / isoamyl alcohol. The RNA is precipitated by adding 6 ml of ethanol and then incubating at -20 ° C overnight. After collecting the precipitated RNA by centrifugation,
The pellet is washed four times in 75% ethanol. The pelleted RNA is then dissolved in 1 mM EDTA, 10 mM Tris-HCl (pH 7.5).

[0193] Non-breast tissue is used as a negative control. Furthermore, oligo dT cellulose spin columns (Pharmacia, Uppsal) for the isolation of polyadenylated RNA
The mRNA can be purified from total RNA using a commercially available kit, such as a, RediCol ^™ from Sweden. Total RNA or mRNA is lysed in lysis buffer (for analysis in a ribonuclease protection assay).
5M guanidine thiocyanate, 0.1 M EDTA, pH 7.0).

[0194]C. RNA extraction from polysomes  The tissue is minced at 4 ° C. in saline, and 6 mM 2-mercaptoethanol is added.
TK contained₁₅₀M (150 mM KCl, 5 mM MgCl₂, 50 mM
Tris-HCl, pH 7.4) Mix with 2.5 volumes of 0.8M sucrose in solution
You. The tissue was placed in a Teflon-glass Potter homogenizer for 100-
200 rpm, 5 reciprocations, then homogenization in a Dounce homogenizer, 6 reciprocations
Genize (B. Mechler,Methods in Enzymolo gy 152: 241-248 (1987)). Then the homogen
The nate is centrifuged at 12,000 × g at 4 ° C. for 15 minutes to sediment the nuclei. 2m
1 of the supernatant₁₅₀Mix with 6 ml of 2.5M sucrose in M and mix this mixture
TK in 38ml polyallomer tube₁₅₀Weight over 4 ml of 2.5 M sucrose in M
The polysomes are isolated by layering. Two additional TKs₁₅₀M solution
Are sequentially layered on the extracted fraction (the first layer of 13 ml of 2.05 M sucrose and
Followed by a second layer of 6 ml 1.3M sucrose). The gradient is 90,000 × g,
The polysome is isolated by centrifugation at 4 ° C. for 5 hours. The fraction is then
Remove from the 1.3M sucrose / 2.05M sucrose interface with a reconeated Pasteur pipette.
And an equal volume of TE (10 mM Tris-HCl, pH 7.4, 1 mM EDTA
Dilute during An equal volume of 90 ° C. SDS buffer (1% SDS, 200 mM N
aCl, 20 mM Tris-HCl, pH 7.4) and add the solution to a boiling water bath.
Incubate for 2 minutes in Next, the protein was converted to proteinase K (50 m
g / ml) at 37 ° C. for 15 minutes. With an equal volume of phenol / chloroform
Extraction, then 0.1 volume 2M sodium acetate (pH 5.2) and 2 volumes
Purification of the mRNA by precipitation with 100% ethanol at −20 ° C. overnight
I do. The precipitated RNA was centrifuged at 12,000 × g at 4 ° C. for 10 minutes.
Collect more. The RNA is dried and resuspended in TE (pH 7.4) or distilled water
You. The resuspended RNA was then used for slot blot or dot blot hybridization.
Use in a dilation assay to confirm the presence of BS106 mRNA.
(See Example 6).

The amounts of nucleic acids and proteins will depend on the manufacturing method used. To maximize the efficiency of target molecule isolation, each sample may require different preparation techniques. These preparation techniques are within the skill of those in the art.

[0196]Example 4: Ribonuclease protection assay  A. A labeled complementary RNA (cRNA) hybridization probe and Synthesis of unlabeled sense strand  BS106 fragment adjacent to opposing SP6 and T7 polymerase promoters
PINCY containing the gene cDNA sequence insert (clone 1662885)
Plasmid is transferred to Qiagen Plasmid Purification kit.
(Qiagen, Chatsworth, CA). Incidentally
10 μg of the plasmid is digested with 10 U of DdeI restriction enzyme at 37 ° C. for 1 hour
did. The cleaved plasmid was used in a QIAprep kit (Qiagen, C
hatsworth, CA) and Riboprobe®.
) In vitro Transcription System (Promega C
corporation, Madison, WI) as described in the supplier's instructions.
6.3 μM (α³²P) UTP (800 Ci / mmol)
(Amersham Life Sciences, Inc. Arlington)
n Heights, IL) labeled with the SP6 promoter.
The plasmid was used for synthesis from the plasmid. To obtain the sense strand,
10 μg of the purified plasmid was digested with 10 U of XbaI and 10 U of NotI.
Cleaved with enzyme, purified and 0.25 μg of plasmid was excised from T7
Transferred from motor. Both sense and antisense transcribed strands are
Isolated by ram chromatography. Unlabeled sense transcribed strand
Quantified by UV absorption.

[0197]B. Hybridization of labeled probe to target  The frozen tissue is pulverized under liquid nitrogen to a powder, and
l lysis buffer (DirectProtect)^TMLysate RNase
Protection Kit (Ambion, Inc., Austin, TX)
Available as a component). Further dissolve using a tissue homogenizer.
Solved. Also, use mouse liver lysate for use as a positive control.
A dilution series of a known amount of the sense strand was prepared. Finally, 45 μl of solubilized tissue or
Represents the diluted sense strand at 1 × 10 5 in 5 μl lysis buffer.⁵cpm radioactive marker
Sense probe (1.5 × 10⁹cpm / μg). Hybridization
The reaction was allowed to proceed overnight at 37 ° C.

[0198]C. RNase digestion  DirectProtect^TMAccording to the protocol, RNase A and R
Hybridize to the probe using a solution of Nase T1 at 37 ° C. for 30 minutes.
Unreacted RNA is removed from the reaction and then removed in the presence of sarcosyl sodium.
RNase was removed by proteinase K digestion. Then an equal volume of isoprop
The addition of phenol allows the hybridized fragment, which is protected from digestion, to be
The precipitate was left at -70 ° C for 3 hours. The precipitate was washed at 12,000 × g for 20 minutes.
Collected by centrifugation between.

[0199]D. Fragment analysis  The precipitate was washed with denaturing gel loading dye (80% formamide, 10 mM E
DTA (pH 8.0), 1 mg / ml xylene cyanol, 1 mg / ml bromo
Phenol blue), heat denatured, 6% polyacrylamide TBE, 8
Electrophoresis was performed in a denaturing M urea gel. STORM^TMStorage phosphor (stora)
Ge phosphor autoradiography system (Molecular D)
ynamics, Sunnyvale, CA).
And analyzed. The peak area obtained from the test sample is
Quantification of the band of the protected fragment by comparison to that from the dilution
Mutograms (expressed in units of fg) were performed (see section B above). Also
Assaying the concentration of DNA in the lysate,
Was evaluated. The results were compared to BS106 RNA molecules / cells and
The results are shown as evaluation scores (Table 1). SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:
No. 4, SEQ ID NO: 5, and fragments or complements thereof.
A high level of expression of the mRNA corresponding to is indicative of the presence of BS106 mRNA,
Suggests a diagnosis of a disease or condition of breast tissue such as breast cancer.

[0200]

[Table 1]

[0201]Example 5: Northern blotting  Agarose gel electrophoresis and nucleic acid hybridization
Northern blots were used to identify specific sized RNA species in the combined population.
Using. Briefly, 40 mM morphinopropanesulfonic acid (MOPS
) (PH 7.0), 10 mM sodium acetate, 1 mM EDTA, 2.2 M
5aldehyde in a solution containing 50% v / v formamide
Incubate μg of total RNA (see Example 3) at 65 ° C. for 15 minutes
did. The denatured RNA was added to 2 liters of loading buffer (50% glycerol,
1 mM EDTA, 0.4% bromophenol blue, 0.4% xylene cyano
), 40 mM MOPS (pH 7.0), 10 mM sodium acetate
1.0% denaturing solution containing 1 mM EDTA and 2.2 M formaldehyde.
Loaded into a garose gel. The gel is electrophoresed at 60 V for 1.5 hours,
Rinse in RNase-free water. Run the gel in RNase-free water
Stained with 0.5 g / ml ethidium bromide, illuminated with UV light,
A band was visualized. Downward alkaline capillary transfer method (dow
nward alkaline capillary transfer me
thod) (Chomczynski,Anal. Biochem. 201:
134-139, 1992) to remove the RNA from the gel using a nylon membrane.
(Brightstar-Plus, Ambion, Inc., Austin,
TX) for 1.5 hours. Rinse the filter with 1 × SSC
, Stratalinker (Stratagene, Inc., La Jol.
la, CA) in an autocrosslinking mode.
RNA was cross-linked to the filter and dried for 15 minutes. Then, the membrane is
Pre-heated 20 ml prehybridization solution (5 × SSC, 50%
Contains formamide, 5 × Denhardt's solution, 100 g / ml denatured salmon sperm DNA)
Place the hybridization tube in a hybridization tube at 42 ° C.
Incubated for at least 3 hours. The blot is prehybridized
Random Primer DNA Labeling Sy
stem (Life Technologies, Inc., Gaithers)
burg, MD) according to the manufacturer's instructions using the BS106 insert (clone 1).
662885 digested with XbaI and NotI)
As a result, a random primed probe labeled with 32 P was generated. The plow
Boil half of the tube for 10 minutes, quench on ice and add to the hybridization tube.
I got it. Hybridization was performed at 42 ° C. for at least 12 hours. The high
Discard the hybridization solution and filter the filter with 30 ml of 3 × SSC, 0.1
Wash (twice) at 42 ° C. for 15 minutes in% SDS, then 0.3 × SSC, 0.1
Washed in 60% of SDS for 15 minutes (twice). Saran wrap the filter
Wrap in Kodak XAR-Omat film for 24-96 hours.
The film was developed and analyzed. Agarose gel and pairs stained with ethidium bromide
The corresponding Northern blots (breast tissue and front, shown in FIGS. 3A and 3B, respectively).
Hybridizes to RNA from prostate tissue and from normal breast and breast cancer tissue
Figure 10 shows the results of RNA quality analysis using
3A and 3B. The position (in kb) based on RNA size is indicated on the left side of each panel.
Shown in As shown in FIG. 3A, the BS106 probe was detected in 3 out of 3 normal breast tissues.
To an RNA band of about 0.7 kb in one and three out of three prostate cancer tissues
Rediscovered three normal prostate tissues or prostate or breast cancer cell lines
No RNA band was detected. FIG. 3B shows that the BS106 probe is normal.
In six breast RNA samples and in two out of six breast cancer RNA samples
It shows that it hybridized to RNA of about 0.7 Kb.

[0202]Example 6: dot blot / slot blot  Dot and slot blot assays are designed for specific nucleic acids in complex nucleic acid mixtures.
It is a quick way to assess the presence of a sequence. To perform such an assay
For up to 50 μg of RNA, add 50 μl of 50% formamide, 7% formal
Dehyde, mixed in 1 × SSC, incubated at 68 ° C. for 15 minutes, then
Cool on top. Then add 100 μl of 20 × SSC to the RNA mixture and prepare
On a manifold device with a filled nitrocellulose or nylon membrane
Under vacuum. Soak the membrane in water, 20 × SSC for 1 hour,
Place on two sheets of Whatman # 3 filter paper pre-wetted with 20 × SSC
Load a slot blot or dot blot in a vacuum manifold device.
To The slot blot was prepared and labeled as described in Example 4 above.
Analyze with the selected probe. SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,
Corresponds to a sequence selected from the group consisting of SEQ ID NO: 5 and fragments or complements thereof
The detection of mRNA that acts as an indicator of the presence of BS106,
Suggests a diagnosis of the disease or condition.

Other methods and buffers that can be used in the methods described in Examples 5 and 6, but not specifically described herein, are known in the art and can be referred to by reference. J., incorporated herein by reference. (Sambrook et al., Supra).

[0204]Example 7: In situ hybridization  This method involves detecting cells using a detectable nucleic acid hybridization probe.
It is useful for directly detecting a specific target nucleic acid sequence therein.

Tissue is prepared using a cross-linking fixative, such as paraformaldehyde, glutaraldehyde, to maximize retention of cellular RNA. L. Angerer et al., Me thods in Cell Biol. 35: 37-71 (1991). Briefly, the tissue was treated with 50 mM sodium phosphate (pH 7).
. 5) Leave in 5% or more of 1% glutaraldehyde in 4 ° C. for 30 minutes.
The solution is mixed with a fresh glutaraldehyde solution (1% glutaraldehyde, 50 mM
Replace in sodium phosphate (pH 7.5) and fix for an additional 30 minutes. The fixation solution should have an osmolarity of about 0.375% NaCl. The tissue is washed once in isotonic NaCl to remove the phosphate.

The immobilized tissue is then embedded in paraffin as follows. 50% (2
The tissue is dehydrated with a series of ethanol concentrations of 70% (twice), 85%, 90%, and then 100% (twice) for 15 minutes each. Next, the tissue is immersed in xylene (two changes) at room temperature for 20 minutes each. The tissue is then immersed in a 1: 1 mixture of xylene and paraffin (two changes) at 60 ° C. for 20 minutes each, and then in paraffin (three final changes) for 15 minutes each.

Next, the tissue was cut into 5 μm sections using a standard microtome,
Place on slides that have been pre-treated with a tissue adhesive such as 3-aminopropyltriethoxysilane.

The paraffin was removed from the tissue by soaking twice in xylene for 10 minutes,
Rehydrate in a range of ethanol concentrations of 99% (twice), 95%, 85%, 70%, 50%, 30% and then in distilled water (twice). The sections were made with 0.2 M HCl
For 10 minutes and increase permeability with 2 μg / ml proteinase K at 37 ° C. for 15 minutes.

Labeled riboprobes transcribed from the BS106 gene plasmid (see Example 4) were hybridized to the prepared tissue sections and 56 ° C. in 3 × standard saline extract and 50% formamide. Incubate overnight. 2x
Wash in standard citrate saline and 50% formamide, then 100 μg /
Excess probe is removed by digestion with ml RNase A for 30 minutes at 37 ° C. The fluorescent probe is visualized by illumination with ultraviolet (UV) light under a microscope. Fluorescence in the cytoplasm indicates BS106 mRNA. Alternatively,
The sections can be visualized by autoradiography.

[0210]Example 8: Reverse transcription PCR  A. One-step RT-PCR assay  Detecting the target sequence by reverse transcription PCR using methods known in the art
To do so, target-specific primers are designed. One-step RT-PCR consists of RT and
And PCR in a single reaction mixture. The method comprises the steps of:
mM (N, N-bis [2-hydroxyethyl] glycine) (pH 8.15), 8
1.7 mM KOAc, 33.33 mM KOH, 0.01 mg / ml bovine serum
Albumin, 0.1 mM ethylenediaminetetraacetic acid, 0.02 mg / ml NaN ₃ , 8% w / v glycerol, 150 μM of each dNTP, 0.25 μM of each
Immer, 5U rTth polymerase, 3.25 mM Mn (OAc)₂and
200 μl reaction mix containing 5 μl target RNA (see Example 3)
Perform in compound. RNA and the rTth polymerase enzyme are Mn (OAc) ₂ Is unstable in the presence of Mn (OAc)₂Immediately before the addition of the target
Should be added. For those skilled in the art, cDNA synthesis and thermal cycling
It is possible to easily determine the optimal conditions for The reaction is performed using Perkin
-Incubate in Elmer Thermal Cycler 480.
Those skilled in the art will be able to determine the optimal conditions for cDNA synthesis and thermal cycling.
It can be easily determined. Conditions that are considered useful include 60 ° C.
CDNA synthesis for 15-45 minutes at 70 ° C and 1 minute at 94 ° C, 55-70 ° C
For 1 minute and 2 minutes at 72 ° C. for 30-45 cycles. In addition,
RT-PCR involves Taq polymerase and reverse transcriptase (eg, MMLV or
AMV RT enzyme).

[0211]B. Traditional RT-PCR  The traditional two-step RT-PCR reaction is described in Q. Hu et al.Virology 1
81: 721-726 (1991). Briefly explain
And 0.5 μg of the extracted mRNA (see Example 3)
R II buffer (Perkin-Elmer), 5 mM MgCl₂1 mM
dNTP, 20 U RNasin, 2.5 μM random hexamer and 50 U
 2 containing MMLV (Moloney murine leukemia virus) reverse transcriptase (RT)
Reverse transcription was performed in 0 μl of the reaction mixture. Reverse transcription, PE-480 thermal cycler
Incubate at room temperature for 10 minutes, at 42 ° C for 60 minutes, and then at 95 ° C for 5 minutes.
To inactivate the RT. PCR was performed with 10 mM Tris-HCl
(PH 8.3), 50 mM KCl, 2 mM MgCl₂, 200 μM dNT
P, 0.5 μM of each sense and antisense primer (SEQ ID NO: 1 respectively)
2 and SEQ ID NO: 13) and 50 μl containing 2.5 U of Taq polymerase.
Performed using 2 μl of the cDNA reaction in a final PCR reaction volume of 1 μl. The
The reaction was carried out in the MJ Research Model PTC-200 as follows.
40 cycles of amplification (94 ° C for 20 seconds, 58 ° C for 3 seconds).
0 sec, 30 sec at 72 ° C.), final extension (10 min at 72 ° C.) and immersion at 4 ° C.
Pickles.

[0212]C. Analysis of PCR fragments  Correct products are stained with SYBR® Green nucleic acid gel stain (Molecu
(Lar Probes, Eugene, OR).
Confirmed by the decision of the company. Gels were diluted 1: 10,000 SY in IX TBE.
Stained with BR Green I for 45 minutes. The gel is then placed in 1 × TBE for 30 minutes
And was imaged using a STORM imaging system (FIG. 4).
A and 4B). FIG. 4A shows normal (lanes 1-5) and cancerous (lanes 1-5).
DN at position 201 bases derived from RNA of both breast tissues in lanes 6-10)
A band is shown. This band is the BS106 mRNA specific RT-PCR product
Is shown. As shown in FIG. 4B, lung (lanes 1-5) or colon (lanes 6-10)
The product was not detected when RNA isolated from the tissue of (2) was used.
SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and their
Detection of a product containing a sequence selected from the group consisting of fragments or complement
 Indicate the presence of mRNA and suggest a diagnosis of disease or condition of breast tissue such as breast cancer
You.

[0213]Example 9: OH-PCR  A. Probe selection and labeling  Detection of the target sequence by oligonucleotide hybridization PCR
To do this, target-specific primers and probes are designed. June 1992
International Publication WO 92/10505 published July 25 and July 9, 1992
WO 92/11388 has oligos at the 5 'and 3' ends, respectively.
Disclosed are methods for labeling nucleotides. Label oligonucleotides
According to one known method for preparing a labeled-phosphoramidite reagent,
It is used to add the label to the oligonucleotide during its synthesis. An example
For example, N. T. Thong et al.Tet. Letters 29 (46): 5
905-5908 (1988) or J.A. S. Cohen et al., Rice being published
No. 07 / 246,688 (NTIS ORDER No. PAT-
APPL-7-246, 688) (1989). Preferably, the
The lobe is labeled at its 3 'end to participate in PCR and produce unwanted extension.
Prevent the formation of things. In one-step OH-PCR, the probe uses the T _M T at least 15 ° C lower_MShould have. Well-known in the art
Using standard phosphoramidite chemistry and / or post-synthesis labeling techniques
Specific for the primers and probes, with or without detectable labels
Use as a binding member.

[0214]B. One-step oligo hybridization PCR  50 mM (N, N-bis [2-hydroxyethyl] glycine) (pH 8.15)
), 81.7 mM KOAc, 33.33 mM KOH, 0.01 mg / ml
Ciserum albumin, 0.1 mM ethylenediaminetetraacetic acid, 0.02 mg / ml
NaN₃, 8% w / v glycerol, 150 μM of each dNTP, 0.25 μM
3.75 nM probe, 5 U rTth polymerase, 3.
25 mM Mn (OAc)₂And 5 μl of the target blood equivalent (see Example 3)
OH-PCR is performed in a 200 μl reaction containing RNA and
And the rTth polymerase enzyme is Mn (OAc)₂Unstable in the presence of
Therefore, the Mn (OAc)₂Should be added just before the addition of the target. The reaction
Ink in Perkin-Elmer Thermal Cycler 480
Lab. Those skilled in the art will appreciate the advantages of cDNA synthesis and thermal cycling.
It is possible to easily determine the optimal conditions for this. Conditions that are considered useful
Include cDNA synthesis (60 ° C, 30 minutes), 30-45 cycles of amplification (94 ° C).
Oligo-hybridization (9 seconds at 55-70 ° C for 60 seconds).
7 ° C. for 5 minutes, 15 ° C. for 5 minutes, 15 ° C. immersion). Correct reaction product
Is a probe internally hybridized with at least one of the strands of the PCR product.
And

[0215]C. Analysis of OH-PCR products  The amplification reaction product was analyzed by LCx (registered trademark) analysis system (Abbott Laborat).
ories, Abbott Park, IL). Simple
Explained below, antibody-labeled microparticles on the PCR product chain or the high
At the captureable site on the hybridization probe, capture the correct reaction product,
Detectable antibody conjugate to a detectable site on the probe or on the PCR strand
The complex is detected by the binding of PCR hybridized to the internal probe
Only complexes containing the strand are detectable. Therefore, the detection of this complex
, Indicating the presence of BS106 mRNA and diagnosing breast diseases or conditions such as breast cancer
Suggests.

There are a number of other detection modalities that one of skill in the art can use and / or modify to detect the presence of amplified or unamplified BS106-derived nucleic acid sequences, including those in the ligase chain reaction ( LCR, Abbott Laboratori
es, Abbott Park, IL); Qβ replicase (Gene-Tra
k ^TM , Naperville, Illinois), branch chain (b
ranked chain reaction (Chiron, Emeryville, C
A) and a strand displacement assay (Be
cton Dickinson, Research Triangle Pa
rk, NC), but is not limited thereto.

[0217]Example 10: Production of synthetic peptide  Represented by SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively
The synthetic peptide is identified as the predicted amino acid in the open reading frame of BS106.
Manufactured based on the sequence (SEQ ID NO: 16) (see Example 1). All
Peptides were prepared using Fmoc chemistry, standard cycling and in situ HBTU.
Activation using Symphony Peptide Synthesizer (
Rainin Instrument Co, Emeryville Cali
(available from Fornia). The conditions for cleavage and deprotection are as follows:
Cage: 2.5 ml volume of cleavage reagent (77.5% v / v trifluoro
Acetic acid, 15% v / v ethanedithiol, 2.5% v / v water, 5% v / v thiol
Oanisol, 1-2% w / v phenol) to the resin and add at room temperature for 2-4 hours
Stirred. The filtrate is then removed and the peptide is cleaved with cold diethyl ether.
Precipitated from reagent. Then, each peptide was filtered, and water / acetonitrile / 0.1
Purified by reverse phase preparative HPLC using a% TFA gradient and lyophilized. Mass of the product
Confirmed by analysis (data not shown).

The purified peptide was then mixed with an adjuvant and injected into rabbits (Example 1).
4). Alternatively, the purified peptide can be conjugated to keyhole limpet hemocyanin with glutaraldehyde, mixed with an adjuvant, and injected into rabbits (see Example 14).

[0219]Example 11a: Expression of a protein in a cell line using plasmid 577  A. Construction of BS106 expression plasmid  U.S. Application No. 08 / filed June 7, 1995, which is hereby incorporated by reference.
No. 478,073 describes a plasmid 577 which is capable of generating secreted antigens in permanent cell lines.
Built for working. This plasmid contains the following DNA segments:
: (A) of pBR322 containing bacterial β-lactamase and DNA origin of replication
2.3 kb fragment, (b) HSV-1 thymidine kinase promoter and poly
1.8 Kb directs expression of neomycin resistance gene under control of A-addition signal
Under the control of (c) SV-40 promoter and poly-A addition signal
A 1.9 Kb cassette that directs the expression of the dihydrofolate reductase gene in
(D) Simian virus 40 T-Ag promoter and transcription enhancer,
Hepatitis B virus surface antigen (HBsAg) enhancer I followed by poly
A fragment of the herpes simplex virus 1 (HSV-1) genome
Under control, a rabbit isolated fused to the modified hepatitis C virus (HCV) E2 protein
A 3.5 Kb cassette that directs expression of the immunoglobulin heavy chain signal sequence, (e)
The remaining region of the simian virus 40 genome late region that does not function in this plasmid
0.7 Kb fragment. All segments of the vector are designated as a standard known to those skilled in molecular biology.
Combined in a standard manner.

[0220] The plasmid for expression of the selectable BS106 protein is plasmid 5
No. 77 comprises a BS106 polynucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and fragments or complements thereof. Construct by substituting as follows. Digestion of plasmid 577 with XbaI releases the hepatitis C virus E2 gene fragment. The resulting plasmid backbone allows for the insertion of a BS106 cDNA insert downstream of the rabbit immunoglobulin heavy chain signal sequence, which directs the expressed protein into the cell's secretory pathway. The BS106
cDNA fragments are obtained by PCR using standard methods. Encoded within the sense PCR primer sequence is an XbaI site, immediately followed by the amino acid sequence Ser-, which enhances signal protease processing, efficient secretion and stability of the end product in the culture fluid. Asn-Glu-Leu ("SNE
L ") followed by a sequence of 12 nucleotides. The primer is
Immediately after the nucleotide sequence, it contains nucleotides complementary to the template sequence encoding the amino acids of the BS106 gene. The antisense primer comprises the following 8
The sequence encoding the amino acid is incorporated immediately before the stop codon: Asp-Tyr-L
ys-Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 21). Within this sequence, a recognition site is incorporated to facilitate analysis and purification of the BS106 protein product. Anti-FLAG M2 (Eastman Kodak, Co., New
It is possible to use the recognition sites (designated "FLAG") and other comparable sequences and their corresponding antibodies that are recognized by commercially available monoclonal antibodies, which are referred to as Haven, CT). is there. For example, Perkin-Elm
PCR is performed using GeneAmp® reagent from er-Cetus according to the supplier's instructions. PCR primers are used at a final concentration of 0.5 μM. PCR was performed for 35 cycles in a 100 μl reaction on the BS106 plasmid template (94 ° C. for 30 seconds, 55 ° C. for 30 seconds, 72 ° C. for 90 seconds)
This is followed by a 10 minute extension cycle at 72 ° C.

[0221]B. Tigers in Chinese hamster ovary cells deficient in dihydrofolate reductase Infection  The plasmid was transferred to CHO / dhfr cells (DXB-111, Uriacio et al.).
,PNAS 77: 4451-4466 (1980)).
You. These cells are T. C. C. , 12301 Parklawn Dr
No. CRL 9096 from ive, Rockville, MD 20852
It is available as Transfection was performed by P. L. Felgner et al.
,PNAS 84: 7413-7414 (1987)
This is performed using a sosome-mediated method. In particular, CHO / dhfr cells were transformed with 10% fetal bovine
The cells were cultured in Ham F-12 medium supplemented with serum and L-glutamine (1 mM).
5-8 × 10 in the flask⁵Freshly seed at the density of cells / flask. Troff
The cells are grown to 60-80% confluency for injection. 2
0 micrograms (20 μg) of plasmid DNA was added to 1.5 ml of Opti-M
In addition to the EMI medium, 100 μl of Lipofectin Reagent (G
ibco-BRL; Grand Island, NY)
 Add to another 1.5 ml portion of medium I. Mix those two solutions,
Incubate for 20 minutes at room temperature. After removing the medium from the cells, the cells are
Rinse three times with ml of Opti-MEM I medium. Then, the Opti-MEM
 Overlay the I-Lipofectin-plasmid DNA solution on the cells. The
The cells were incubated at 37 ° C. for 3 hours, then the Opti-MEM I-Li
The pofectin-DNA solution is replaced with medium for an additional 24 hours before selection.

[0222]C. Selection and amplification  One day after transfection, cells were passaged 1: 3 and dhfr / G418
Incubate with selective medium (hereinafter referred to as "F-12 minus medium G")
I do. The selection medium contains L-glutamine and contains hypoxanthine, thymidine and guanidine.
Ham F-12 containing no lysine (JRH Biosciences, Le.
Nexa, Kansas) and 300 μg / ml G418 (Gibco-
BRL; Grand Island, NY). The volume to surface area ratio of the medium
5ml / 25cm²To maintain. After about two weeks, the cells were transferred in F-12 minus medium G.
DHFR / G418 cells are grown to allow for passage and continuous maintenance.

Each amplification of the transfected BS106 cDNA sequence is performed by sequential selection of DHFR ⁺ , G418 ⁺ cells with methotrexate (R.
Schimke, Cell 37: 705-713 (1984)). Until resistant colonies emerge, 150 nM methotrexate (MTX) (Sigma)
, St. The cells are incubated for about 2 weeks with F-12 minus medium G containing (Louis, MO). In addition, gene amplification was performed with 5 μM MTX for 15 minutes.
Performed by selection of 0 nM compatible cells.

[0224]D. Production of antigen  F-12 minus medium G supplemented with 5 μM MTX was transferred to a confluent monolayer.
5% CO on top₂Overlay at 37 ° C. for 12-24 hours. Remove the growth medium and remove the cells
With Dulbecco's phosphate buffered saline (PBS) (calcium and magnesium)
3 times with Gibco-BRL: Grand Island, NY
To remove any remaining media / serum that may be present. The cells were then purchased as VAS custom (c
phenol medium containing L-glutamine together with HEPES
-Free VAS-free formulation, JRH Bioscience; Lenexa
Available from KS, product number 52-08678P) with 5% CO₂Medium 37 ° C
And incubate for 1 hour. The cells were then prepared in 5 ml / T flasks.
Cover with VAS. After 7 days of incubation, the medium is removed and maintained,
It is then frozen with harvest 2, 3 and 4 until purification. Of three 7-day harvests
To this end, the monolayer is covered with VAS.

[0225]E. FIG. Analysis of expression of breast tissue gene BS106 antigen  Aliquots of VAS supernatant from cells expressing the BS106 protein construct
, Standard methods and reagents known in the art (Laemmli discontinuous gel)
By SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using
Or analyze by mass spectrometry.

[0226]F. Purification  Anti-FLAG M2 monochrome covalently attached to agarose via hydrazine linkage
Affinity matrix containing null antibody (Eastman Kodak C
o. , New Haven, CT)
Purify BS106 protein containing FLAG sequence by luffy
. Prior to affinity purification, pooled VAS media harvest from roller bottles
Of the protein in Sephadex G-25 (Pharmacia Bio
tech Inc. , Uppsala, Sweden) column.
0 mM Tris-HCl (pH 7.5), 150 mM NaCl buffer
Replace. The protein in this buffer was used as an anti-FLAG M2 antibody affinity
Apply to ram. The column was washed with 50 mM Tris-HCl (pH 7.5), 15 mM.
Unbound protein is eluted by washing with 0 mM NaCl buffer.
Excess in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl
The bound protein is eluted using the FLAG peptide. That excess FLAG
Peptide was purified from the purified BS106 protein by gel electrophoresis or HPLC.
Can be removed.

Although this example uses plasmid 577, other comparable expression systems (eg,
For example, it is known to those of skill in the art that CMV) can be used in the present invention with appropriate modifications to the reagents and / or techniques and is within the skill of those in the art.

The largest cloned insert containing the coding region for the BS106 gene was identified as (i
Eukaryotic expression vectors which may contain, for example, the cytomegalovirus (CMV) promoter and / or a protein fusible sequence (which aids in protein expression and detection), or (ii) superoxide dismutase (SOD) and CMP-KDO synthase Subcloned into a bacterial expression vector containing (CKS) or other protein fusion gene (for expression of the protein sequence). Methods and vectors useful for the production of polypeptides containing the fusion sequence of SOD are described in E, published October 1, 1986, which is hereby incorporated by reference.
PO 0196056, which contains the fusion sequence of CKS,
EPO Publication 03, published September 13, 1989, which is incorporated herein by reference.
No. 31961. Proteins purified in this manner include, but are not limited to, animal immunity studies, solid phase immunoassays, etc.
It can be used in various technologies.

[0229]Example 11b: In a cell line using pcDNA3.1 / Myc-His Protein expression  A. Construction of BS106 expression plasmid  Plasmid pcDNA3.1 / Myc-His (Cat. # V855-20)
, Invitrogen, Carlsbad. CA) has so far
Has been constructed for expression of secreted antigens by mammalian cell lines. Tampa expressed
The cytoplasmic insert is fused to a myc-his peptide tag. The myc-hi
The s tag (SEQ ID NO: 22) can be used for anti-myc or anti-his affinity column
Or c useful for purification of expressed fusion proteins using metalloprotein binding columns
-Includes myc oncoprotein epitope and polyhistidine sequence.

A plasmid for expression of the secretable BS106 protein was clone 1662.
885-derived BS106 polynucleotide sequence with pcDNA3.1 / Myc-H
It was constructed by insertion into an is vector. Prior to constructing the BS106 expression plasmid, the BS106 cDNA sequence was first converted to pCR®-Blu
cloned into the nt vector. The BS106 cDNA fragment was obtained from Stra
PCR was performed using Stratagene® reagent available from Tagene according to the supplier's instructions. PCR primer is 0
. Use at a final concentration of 5 μM. 5U pfu polymerase (Stratage
ne, La Jolla, Calif.) for 30 cycles (at 94 ° C. for 1 minute) in a 50 μl reaction on BS106 plasmid template (Example 2).
(65 ° C. for 1.5 minutes, 72 ° C. for 3 minutes) followed by an extension cycle at 72 ° C. for 8 minutes. The sense PCR primer sequence (SEQ ID NO: 14)
Contains the same nucleotides as the pINCY vector immediately upstream of the S106 gene insert. The antisense primer (SEQ ID NO: 15) incorporates a 5 'NotI restriction sequence and a sequence complementary to the 3' end of the BS106 cDNA insert immediately upstream of the 3 'most in-frame stop codon. Five microliters (5 μl) of the resulting blunt-ended PCR product was combined with 25 ng of linearized pCR®-Bl.
unt vector (Invitrogen, Carlsbad, CA) to block the lethal ccdB gene of the vector. Using the One Shot ^™ Transformation Kit (Invitrogen, Carlsbad, Calif.) According to the supplier's instructions, the resulting ligated vector was transferred to TOP10 E. coli (E. coli).
(Invitrogen, Carlsbad, CA). The transformed cells were plated on an LB-Kan (50 μg / ml kanamycin) selection plate for 37 hours.
Grow at ℃. Only cells containing the plasmid with the blocked ccdB gene grew after transformation (Grant, SGN, PNAS 87:
4645-4649 (1990)). Transformed colonies were picked and grown at 37 ° C. in 3 ml LB-Kan broth. QIAprep (registered trademark) (Q
iagen Inc. Plasmid DNA was isolated using the (Santa Clarita, CA) method according to the manufacturer's instructions. The DNA was digested with EcoRI and NotI restriction enzymes to release the BS106 insert. The fragment
% Seakem® LE Agarose (FMC, Rockland, ME
) /0.5 μg / ml ethidium bromide / TE gel, visualized by UV illumination, QIAquick ^™ (Qiagen Inc, Santa Cl.)
arita, CA) method according to the supplier's instructions.

The pcDNA3.1 / Myc-His plasmid DNA was replaced with the plasmid DNA.
Were linearized by digestion at the EcoRI and NotI sites present in the polylinker region. The purified BS106 fragment described above was ligated to the resulting plasmid DNA backbone, downstream of the CMV promoter, and DH5alpha ^™ cells (GibcoBRL Gaithersburg, MD) according to the supplier's instructions.
). Briefly, 10n containing the BS106 insert fragment.
g of pcDNA3.1 / Myc-His was added to 50 μl of competent DH5alpha cells and the contents were mixed gently. The mixture was incubated on ice for 30 minutes, heated at 37 ° C. for 20 seconds and left on ice for another 2 minutes. 0.95
Once ml of LB medium was added, the mixture was incubated at 37 ° C. for 1 hour with shaking at 225 rpm. Then, the transformed cells were subjected to 100 mm LB / Amp
(50 μg / ml ampicillin) plated on plate and grown at 37 ° C. Colonies were picked and grown in 3 ml LB / ampicillin broth.
Plasmid DNA was purified using the QIAprep kit. The presence of the insert was confirmed by restriction enzyme digestion and gel analysis (J. Sambrook et al., Supra).

[0232]B. Transfection of human fetal kidney 293 cells  Reforming the BS106 expression plasmid described in Section A above into DH5alpha cells
And plated on LB / ampicillin agar, 10 ml LB / a
Grow in ampicillin broth (as described above). The plasmid was transformed with QIAf
ilter ™ Maxi kit (Qiagen, Chatsworth, CA)
) And purified using HEK293 cells (FL Graham et al.,J. Gen . Vir . 36: 59-72 (1977)). these
Cells of A. T. C. C. , 12301 Parklawn Drive, R
ockville, MD 20852, accession number CRL 1573
It is possible. P. Hawley-Nelson et al.Focus 15:73 (
1993) using the cationic lipofectamine-mediated method described in US Pat.
Was performed. In particular, HEK293 cells were transformed with 10% fetal bovine serum (FB
S), cultured in 10 ml DMEM medium supplemented with L-glutamine (2 mM)
8 × 10 in a 12 × 100 mm culture plate⁶New at cell / plate density
Sown. 70-80% confluency for transfection
The cells are grown at 37 ° C. until they are. 8 micrograms (8 μg) of plasmid
DNA was added to 800 μl of Opti-MEM I® medium (Gibco-
BRL; Grand Island, NY) and 48-96 μl of Lipo
featuremine^TM Reagent (Gibco-BRL; Grand)
Island, NY) with another 800 μl portion of Opti-MEM I medium
Minutes. Mix the two solutions and incubate at room temperature for 15-30 minutes.
I did it. After removing the medium from the cells, the cells were added to 10 ml of serum-free DMEM medium.
Was washed once. Then, the Opti-MEM I-Lipofectamine
-Dilute the plasmid DNA solution in 6.4 ml of serum-free DMEM and place on the cells
Overlaid. The cells were incubated at 37 ° C. for 5 hours, and then 20% FBS was added.
Add an additional 8 ml of DMEM containing. 18-24 hours later, the old medium
And the cells are overlaid with 5 ml of fresh DMEM containing 10% FBS.
Was. At 72 hours after transfection, the supernatant and cell extracts were
Analyzed for 6 gene activities.

[0233]C. Analysis of expression of breast tissue gene BS106 antigen  The culture supernatant was transferred to a cryotube and stored on ice.
. HEK293 cells were washed twice with 10 ml cold Dulbecco PBS, 1
. 5 ml of CAT lysis buffer (Boehringer Mannhein, Ind.)
harvested by adding and dissolving dianapolis, IN) followed by room temperature
For 30 minutes. Lysate 1.7 ml polypropylene micro
Transferred to a centrifuge tube and centrifuged at 1000 × g for 10 minutes. Transfer the supernatant to a new cryotube
And stored on ice. An aliquot of the supernatant from the cells and the BS106 protein
A lysate of cells expressing the cytoplasmic construct is used in the presence of the BS106 recombinant protein.
We analyzed for the presence. S using standard methods and reagents known in the art
The DS was analyzed by DS polyacrylamide gel electrophoresis (SDS-PAGE).
(See J. Sambrook et al., Supra). In particular, sump
Desired with Tricine buffer (Novex, San Diego, CA)
Adjusted to a protein concentration of 2 × Tricine sample buffer (No.
vex, San Diego, CA) at 100 ° C in a thermal cycler.
For 5 minutes. Samples were then precast Novex 10-2
Apply to 0% Tricine gel (Novex, San Diego, CA)
And electrophoresed. After electrophoresis, samples were removed from the gel using Novex Tris-Glu.
Electricity to Novex nitrocellulose membrane in lysine transfer buffer
Electrophoretically transferred. The BS106 protein band was ligated with anti-myc
Pitope monoclonal antibodies (Invitrogen Carlsbad, CA
) Was visualized by Western blotting using a dilution of 1: 5000 and W
ester Lights Plus chemiluminescence detection reagent (Tropix, Be
dford, MA). The cell lysate is an anti-myc monoclonal
The following two intense bands were stained specifically with the antibody: a light of approximately 20 kD.
A clear band, and a wider band of about 120-200 kD. The supernatant was
A single 30-45 kD single peptide showing differences in post-translational modifications between the 106 intracellular and secretory forms.
I gave one dark broad band. Alternatively, anti-BS106 polyclonal serum (
(See Example 14) to detect the recombinant BS106 protein.
Or analyzing the expressed BS106 recombinant protein by mass spectrometry.
(See Example 12).

[0234]D. Purification  Contains nickel-filled agarose resin that specifically binds to polyhistidine residues
Xpress® affinity chromatography system (Invit
(gengen. Carlsbad, Calif.) using the myc-his sequence.
Purification of the BS106 recombinant protein is performed. Prepared as above,
Supernatants from 10 × 100 mm plates were pooled and passed through the nickel-packed column.
You. The column was washed with 50 mM Tris-HCl (pH 7.5) / 150 mM Na.
The unbound protein was eluted by washing with a Cl buffer, resulting in myc-h
Only the is fusion protein remains. Then an excess of imidazole or histidine
The bound BS106 recombinant protein is purified using a column or low pH buffer.
Eluted from Alternatively, hydrazine or other linkage to agarose resin
Affinity comprising bound anti-myc or anti-histidine monoclonal antibody
-Column with myc-his sequence and excess myc peptide or
Alternatively, the recombinant protein can be purified by elution with histidine or histidine.
Noh.

The purified recombinant protein was then purified using an N-hydroxysuccinimide-activated Sepharose column (Pharmacia Biotech, Piscataawa).
y, NJ) can be covalently crosslinked according to the supplier's instructions. These columns containing the covalently linked BS106 recombinant protein can then be used to purify anti-BS106 antibodies from rabbit or mouse serum (see Examples 13 and 14).

[0236]E. FIG. Coating of microtiter plate with BS106 expressed protein G  Dilute the supernatant from the 100 mm plate in an appropriate volume of PBS. One
Then, 100 μl of the obtained mixture was added to Reaci-Bind^TMMetal chelate
Microtiter plates (Pierce, Rockford, IL)
And incubated at room temperature with shaking, then with 0.05% T
Wash three times with 200 μl each of PBS containing ween® 20
You. Then, using the prepared microtiter plate, the anti-BS10
Polyclonal antisera can be screened for the presence of 6 antibodies
(See Example 17).

Although this example uses pcDNA3.1 / Myc-His, other comparable expression systems can be used in the present invention with appropriate modifications to the reagents and / or techniques. Is known to those skilled in the art and is within the skill of those skilled in the art. The largest cloned insert containing the coding region of the BS106 gene is identified as (i) a plasmid that may contain, for example, a cytomegalovirus (CMV) promoter and / or a protein fusible sequence (to aid protein expression and detection). A nuclear expression vector, or (ii) superoxide dismutase (SOD)
And CMP-KDO synthase (CKS) or other protein fusion genes (
(For expression of the protein sequence) into a bacterial expression vector. Methods and vectors useful for the production of polypeptides containing the fusion sequence of SOD are described in European Patent Application EP 0 196 056 published October 1, 1986, which is hereby incorporated by reference, Vectors containing the fusion sequence are described in European Patent Application EP 0 331 961, published September 13, 1989, which is hereby incorporated by reference. The purified protein can be used in various techniques, including animal immunity studies, solid phase immunoassays, and the like.

[0238]Example 12: Chemical analysis of breast tissue proteins  A. Analysis of tryptic peptide fragments by MS  Serum from patients with breast disease such as breast cancer, from patients without breast disease
Serum, extracts of breast tissue or cells from patients with breast diseases such as breast cancer, milk
Extracts of breast tissue or cells from patients without atrial disease, and other non-
Extract tissue or cells from the disease or diseased organ using standard methods.
Run on a acrylamide gel and stain with Coomassie Blue. The unknown poly
Sections of gels suspected of containing peptides were cut out, reduced in gels,
And trypsin digestion (P. Jeno et al.,Anal. Bio. 224
: 451-455 (1995) and J.C. Rosenfeld et al.,Anal. B io . 203: 173 (1992)). The gel slice was treated with 100 mM NH₄HCO ₃ And wash with acetonitrile. The shrunken gel piece was digested with digestion buffer (50 mM
 NH₄HCO₃, 5 mM CaCl₂And 12.5 μg / ml trypsin)
Swell for 45 minutes at 4 ° C. The supernatant is aspirated and trypsin free 5-
Replace with 10 μl digestion buffer and incubate at 37 ° C. overnight. Peptide
Extract with 3 changes of 5% formic acid and acetonitrile and evaporate to dryness.
The peptide was trapped at about 0.4 μg at the tip of a stretched gas chromatography capillary.
1 μl of POROS R2 adsorbent (Perseptive Biosystem)
s, Framingham, Massachusetts).
Dissolve in 1% 5% formic acid and adsorb it by passing it through the capillary. Adsorbed pep
The tide is washed with water and eluted with 5% formic acid in 60% methanol. Nano Electrosp
The eluate was analyzed using an API III mass spectrometer (Pe
rkin-Elmer Sciex, Thornhill, Ontario, C
(M. Wilm et al.,Int. J. M ass Spectrom. Ion Process 136: 167-180
(1994) and M.E. Wilm et al.Anal. Chem. 66: 1-8 (1
994)). The mass of the trypsin peptide was determined by the mass spectrum obtained from the first quadrupole.
Measured from the Furthermore, the mass corresponding to the expected peptide is assigned to the MS / MS mode.
Analysis of the peptide to obtain the amino acid sequence of the peptide.

[0239]B. Analysis of peptide fragments by LC / MS  Also, polypeptides deduced from mRNA sequences found in proliferative disease tissues
Is detected by liquid chromatography / tandem mass spectrometry (LC / MS / MS
) (D. Hess et al., METHODS. A Compa).
Nion to Methods in Enzymology 6: 227-
238 (1994)). Denature serum sample or tumor extract from the patient with SDS
And reduced with dithiothreitol (1.5 mg / ml) at 90 ° C for 30 minutes.
Alkylation with iodoacetamide (4 mg / ml) at 25 ° C. for 15 minutes.
After acrylamide electrophoresis, the polypeptide is electrophoresed on a cationic membrane.
Troblot and stain with Coomassie Blue. After staining, wash the membrane
Then, the section thought to contain the unknown polypeptide is cut and cut into small pieces.
Place the membrane in a 500 μl microcentrifuge tube and add 10-20 μl of protein.
Digestion buffer (0.1 M NaCl, 10% acetonitrile, 2 mM Ca
Cl₂And 100 mM Tris-HCl containing 5 μg / ml trypsin
, PH 8.2) (Sigma, St. Louis, MO). 1 at 37 ° C
After 5 hours, 3 μl of saturated urea and 1 μl of 100 μg / ml trypsin were added.
And further incubated at 37 ° C. for 5 hours. Add 3 μl of the digestion mixture to 10%
Acidify with trifluoroacetic acid, centrifuge and separate the supernatant from the membrane. The supernatant
Was injected directly onto a microbore reverse-phase HPLC column with 0.05% trifluoroacetic acid.
Elution with a linear gradient of acetonitrile in. The eluate is adjusted for the volume of the substance
Electrospray mass spectrometry after passing through a flow splitter if necessary
Send to total. Data are analyzed according to the method described in Example 12, Section A.

[0240]Example 13: Gene Immunization Protocol  A. In vitro antigen expression  Gene immunization is performed by using a suitable expression vector.
Protein expression by direct expression of the antigen in vivo after inoculation
Eliminates the need for steps. In addition, the production of antigen by this method is
Rudding and glycosylation may be possible. Because the protein is
This is because it is produced in dairy tissue. The method includes the use of a CMV promoter.
Insertion of the gene sequence into a plasmid, growth and purification of the plasmid, etc.
In addition, the plasmid DNA is injected into the muscle tissue of the animal. Preferred animals
Include mice and rabbits. For example, H. Davis et al.Human Molecular Genetics 2: 1847-1851 (1993)
Please refer to). After one or two booster immunizations, the animals were bled.
The spleen of the animal to collect ascites fluid or to produce hybridomas.
Can be gathered.

[0241]B. Plasmid preparation and purification  Digestion with EcoRI and NotI restriction enzymes resulted in B described in Example 11b.
The BS106 cDNA insert was released from the S106 vector. Digested
Plasmid fragment was digested with 1% Seakem KE agarose / 0.5 μg / ml bromide
Electrophoresed on ethidium / TE gel and the bands were visualized by UV illumination.
The insert is excised from the gel and purified using the QIAquick method described above.
You. The fragment was digested with EcoRI / NotI pcDNA3.1 vector (
(Invitrogen, Carlsbad, CA).
5 alpha cells (as described above). Use QIAprep column
The plasmid DNA was purified from the bacterial lysate. All of these
Techniques are well known to those skilled in molecular biology.

[0242]C. Immunization protocol  Anesthetized animals are treated with PBS or other DNA uptake enhancer (Cardiotox).
in, 25% sucrose) from 0.1 to 100 μg of the purified plasmid.
Immunize intramuscularly. For example, H. Davis et al.Human Gene Th erapy 4: 733-740 (1993) and P.E. W. Wolff et al.B iotechniques 11: 474-485 (1991).
. One or two booster injections are given at monthly intervals.

[0243]D. Testing and use of antisera  Animals are bled and the resulting serum is purified using techniques known in the art (eg, wafers).
(Stan blot method or EIA technique).
Test for antibodies using peptides. Then, the anti-blood produced by this method
The presence of antigen in the patient's tissue or cell extract or in the patient's serum is
ELISA or Western blotting (for example, as described in Examples 15-18)
).

[0244]Example 14: Production of antibodies to BS106  A. Production of polyclonal antiserum  A sequence derived from the deduced amino acid sequence of BS106 consensus sequence (SEQ ID NO: 4)
Antiserum against BS106 by injecting rabbits with peptide with
Is prepared. The peptides (SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and the sequence
The synthesis of number 20) is described in Example 10.

[0245] 1. Peptide binding Maleimide-activated keyhole limpet hemocyanin (KLH, Imject
Commercially available as ®, Pierce Chemical Co.
mpany, Rockford, IL). Imject® contains about 250 moles of reactive maleimide groups per mole of hemocyanin. The activated KLH is dissolved at a concentration of about 7.7 mg / ml in phosphate buffered saline (PBS, pH 8.4). The peptide is linked via a cysteine present in the peptide sequence or to a cysteine previously added to the synthetic peptide to provide a point of attachment. The peptide was treated with dimethyl sulfoxide (DMSO, Sigma Chemical Compa).
ny, St. Louis, MO) and react with the activated KLH at a molar ratio of about 1.5 moles of peptide per mole of reactive maleimide bound to the KLH. Methods for conjugation of peptides are described below. It is known to those skilled in the art that the amount, time and conditions in such methods can be varied to optimize peptide binding.

The coupling reaction described below was performed using 3 mg containing about 0.77 μmol of reactive maleimide groups.
KLH peptide conjugate ("binding peptide"). This amount of peptide conjugate is usually appropriate for one initial injection and four booster injections to produce a polyclonal antiserum in the rabbit. Briefly, peptides are dissolved in DMSO at a concentration of 1.16 μmol / 100 μl DMSO. 100 μl of the DMSO solution was added to 380 μl of the activated KLH solution prepared as described above, and 20 μl of PBS (pH 8.4) was added to make the volume 500 μl.
to l. The reaction is incubated overnight at room temperature with stirring. The degree of reaction is determined by measuring the amount of unreacted thiol in the reaction mixture. The difference between the starting and final thiol concentrations is considered the concentration of the peptide bound to the activated KLH. The amount of residual thiol was determined using the Ellman reagent (5,5'-dithiobis (2-nitrobenzoic acid), Pierce Chemical Company,
Rockford, IL). 35 mg of cysteine HCl (
(Pierce Chemical Company, Rockford, IL)
Was dissolved in 10 ml of PBS (pH 7.2) and the cysteine standard was diluted to 0, 0.1, 0.5, 2, 5, and 2 by diluting the stock solution to the desired concentration.
Prepare at a concentration of 0 mM. Photometric determination of thiol concentration is performed by placing 200 μl of PBS (pH 8.4) in each well of an Immulon 2® microwell plate (Dynex Technologies, Chantilly, VA). Next, 10 μl of standard or reaction mixture is added to each well. Finally, Ell at a concentration of 1 mg / ml in PBS (pH 8.4)
Add 20 μl of man reagent to each well. The wells are incubated at room temperature for 10 minutes, and the absorbance of all wells is measured using a microplate reader (eg, BioRad).
(Model 3550, BioRad, Richmond, CA) at 415.
Read in nm. A standard curve is created using the absorbance of the standard, and the thiol concentration of the reaction mixture is determined from the standard curve. A decrease in free thiol concentration indicates a successful binding reaction. Also, the calculation of free thiol in the peptide solution before adding maleimide-activated KLH and at the completion of the reaction allows for the determination of the substitution ratio of moles of peptide / moles of KLH for each peptide x KLH bond. . In all cases, about 250 per KLH molecule for each of the peptide conjugates
The reaction will be completed so that the peptide is obtained. Any unreacted peptides are removed by dialysis against PBS (pH 7.2) at room temperature for 6 hours. The conjugate is stored at 2-8 ° C for immediate use, otherwise at -20 ° C or lower.

[0247] 2. Animal immunization Female white New Zealand rabbits (weighing 2 kg or more) were used to raise polyclonal antisera. One animal was immunized per peptide (SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively). One week prior to the first immunization, 5-10 ml of blood was obtained from the animal as a non-immunized pre-bleed sample.

Using each peptide of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, 0.5 ml of the peptide at a concentration of 2 mg / ml and 0.5 ml of complete Freund's adjuvant (CFA) ( The primary immunogen was prepared by emulsification in PBS (pH 7.2) with Difco, Detroit, MI). The immunogen was injected into the animal at several sites by the subcutaneous, intraperitoneal and / or intramuscular route of administration. Four weeks after the first immunization, a booster immunization was administered. The immunogen used for the booster immunization was the same peptide 0 used in the primary immunogen except that it was diluted to 1 mg / ml in 0.5 ml incomplete Freund's adjuvant (IFA) (Difco, Detroit, MI). It was prepared by emulsifying 0.5 ml. Again, the booster administration is performed at several sites,
Subcutaneous, intraperitoneal and intramuscular injection modes were used. Two weeks after the booster immunization, the animals were bled (5 ml) and the sera tested for immunoreactivity to the peptides described below. The booster and blood collection procedure is repeated at 4 week intervals until appropriate titers are obtained. The titer or concentration of the antiserum is measured by the microtiter EIA described in Example 17 below. The apparent affinity value [K _d (app)] is also determined for some of the antisera (see Example 17). 1:
Antibody titers of 500 or more are considered suitable for further use and study.

[0249]B. Production of monoclonal antibody molecules  1. Immunization Protocol Unbound or Bound Peptide for Production of Monoclonal Antibodies in Mice
10 minutes of the amount used for the production of polyclonal antiserum in rabbits
Immunize mice using the immunogen prepared as described above, except that
. Therefore, the primary immunogen is 100 μg in 0.1 ml CFA emulsion.
Immunization used for booster immunization
The stock consists of 50 μg unbound or bound peptide in 0.1 ml IFA.
Using standard techniques, prepare hybridomas for monoclonal antibody production,
Screen. The method used to produce the monoclonal antibody was Kohler et al.
And Milstein,Nature 256: 494 (1975)
J. as a review. G. FIG. R. Hurrel,Monoclonal Hybridoma Antibodies: Techniques and Applications , CRC Press, Inc, Boca Rato
n, FL (1982), according to procedures known in the art. K
Methods for producing monoclonal antibodies based on the method of Ohler and Milstein
Another method for this is described in L. et al., Incorporated herein by reference. T. Mimms et al.V irology 176: 604-619 (1990).

The immunization regime (per mouse) consists of a primary immunization with an additional booster immunization. The primary immunogen used for the primary immunization consists of 100 μg of unbound or bound peptide in 50 μl of PBS (pH 7.2) pre-emulsified in 50 μl of CFA. Booster immunizations performed approximately two and four weeks after the primary immunization consist of 50 μl of PBS (pH 7.2) emulsified with 50 μl of IFA.
) Consists of 50 μg of unbound or bound peptide. A total of 100 μl of this immunogen is inoculated intraperitoneally and subcutaneously into each mouse. About 4 weeks after the third immunization, individual mice are screened for an immune response by the microtiter plate enzyme immunoassay (EIA) described in Example 17. About 15 of the third immunization
After a week, mice are inoculated intravenously, spleen or intraperitoneally with 50 μg of unbound or bound peptide in PBS (pH 7.2).

Three days after this intravenous boost (boost), splenocytes were purified by the polyethylene glycol (PEG) method, for example, using Sp2 / 0-Ag14 myeloma cells (Mils
tein Laboratories, England). The fusion was prepared using Iscove's Modified D containing 10% fetal calf serum (FCS) and 1% hypoxanthine, aminopterin and thymidine (HAT).
Culture in ulbecco's Medium (IMDM). The bulk culture is screened by microtiter plate EIA according to the protocol of Example 17. The peptide used as the immunogen is reactive with other peptides (
That is, clones that are non-reactive to the BS106 peptide not used as an immunogen) are selected for final expansion. The clones thus selected are expanded, aliquoted and frozen in IMDM containing 10% FCS and 10% dimethyl sulfoxide.

[0252] 2. Production of Ascites Fluid Containing Monoclonal Antibodies Frozen hybridoma cells prepared as described above are thawed and placed in a growth culture. Viable hybridoma cells are inoculated intraperitoneally into Prisane-treated mice. Ascites fluid is removed from the mice, pooled, filtered through a 0.2μ filter, and subjected to immunoglobulin class G (IgG) analysis to determine the volume of Protein A column required for the purification.

[0253] 3. Purification of Monoclonal Antibody from Ascites Fluid Briefly, filtered and thawed ascites fluid was mixed with an equal volume of Protein A Sepharose binding buffer (1.5 M glycine, 3.0 M NaCl, pH 8.9). Refilter with a 2μ filter. The capacity of the protein A column is determined from the amount of IgG present in the ascites fluid. The eluate was then washed with PBS (2-8 ° C. overnight).
Dialyze against pH 7.2). The dialyzed monoclonal antibody is sterile filtered,
Dispense into aliquots. The immunoreactivity of the purified monoclonal antibody is confirmed by measuring its ability to specifically bind to the peptide used as the immunogen in the EIA microtiter plate assay of Example 17. The specificity of the purified monoclonal antibody is confirmed by determining that it does not bind to an irrelevant peptide (eg, a peptide of BS106 not used as an immunogen). The purified anti-BS106 monoclonal prepared and characterized in this way is left at 2-8 ° C for short-term storage and at -80 ° C for long-term storage.

[0254] 4. Further Characterization of Monoclonal Antibodies Isotypes and subtypes of monoclonal antibodies produced as described above can be obtained from commercially available kits (Amersham. Inc., Arlington).
on Heights, available from IL). Stability testing also involves storing aliquots of the monoclonal antibody continuously at 2-8 ° C. and assaying optical density (OD) over the course of a period of time.
It can be performed on the monoclonal antibody.

[0255]C. Use of recombinant proteins as immunogens  Recombinant protein produced as described herein may be prepared using reagents known to those of skill in the art.
And with concomitant changes to the technology, polyclonal and monoclonal antibody
Use as an immunogen in production is included within the scope of the present invention.

[0256]Example 15: Purification of serum antibodies that specifically bind to BS106 peptide  The immune serum obtained as described in the above Examples 13 and / or 14
Immobilized synthetic peptide prepared as described in Example 10 or as described in Example 11.
Affinity purification is performed using the recombinant protein prepared in the cage. Diluted
The crude antiserum was applied to a protein A column (Affi-Gel protein A).
, Bio-Rad, Hercules, CA).
A clear IgG fraction is obtained. Buffer (binding buffer supplied by the manufacturer)
Elution removes substantially all proteins except immunoglobulins.
Elution with 0.1 M buffered glycine (pH 3) allows albumin and other serum
An immunoglobulin preparation substantially free of proteins is obtained.

To obtain a preparation having a higher fraction of specific antigen binding antibodies, immunoaffinity chromatography is performed. The peptide used to produce the antiserum is immobilized on a chromatography resin, and a specific antibody against the epitope is adsorbed to the resin. After washing off unbound components, the specific antibody is eluted with 0.1 M glycine buffer (pH 2.3). Antibody fraction was added to 1.0M Tr
Immediately neutralize with is buffer (pH 8.0) to maintain immunoreactivity. The chromatography resin chosen will depend on the reactive groups present in the peptide. When the peptide has an amino group, Affi-Gel 10 or Affi-Gel 10
-Use a resin such as Gel 15 (Bio-Rad, Hercules, CA). If it is desired to bind via a carboxy group on the peptide, Affe
-Gel 102 (Bio-Rad, Hercules, CA) can be used. When the peptide has a free sulfhydryl group, Affi-G
el 501 (Bio-Rad, Hercules, CA), SulkfoLi
Organic mercury resins such as nk ^™ (Pierce, Rockford, IL) can be used. The amount of the peptide immobilized on the resin was determined by Nano Orange
Ge ^™ (Molecular Probes, Eugene, OR).

Alternatively, the spleen can be harvested and used for the production of hybridomas for producing monoclonal antibodies according to the usual methods known in the art.

[0259]Example 16: Western blotting of tissue samples  0.1 M Tris-HCl (pH 7.5), 15% (w / v) glycerol
0.2 mM EDTA, 1.0 mM 1,4-dithiothreitol, 10 μg
/ Ml leupeptin and 1.0 mM phenylmethylsulfonyl fluoride (K
ain et al.Biotechniques, 17: 982 (1994))
A protein extract is prepared by homogenizing the sample. Homozygous
After homogenization, the homogenate is centrifuged at 4 ° C. for 5 minutes to separate the supernatant from the residue.
You. For protein quantification, 3-10 μl of the supernatant was mixed with 1.5 ml of bicinchoninic acid
(Bicinonicinic acid) reagent (Sigma, St. Louis)
s, MO) and the absorbance obtained at 562 nm is measured.

In SDS-PAGE, a sample is transferred to Tricine Buffer (Nov.
ex, San Diego, CA) and adjust to the desired protein concentration, and an equal volume of 2 × Tricine sample buffer (Novex, San Diego, CA).
And heat at 100 ° C. for 5 minutes in a thermal cycler. The sample was then applied to a precast Novex 10-20% Tricine gel,
Perform electrophoresis. After electrophoresis, samples are transferred from the gel to a nitrocellulose membrane in Novex Tris-glycine transfer buffer. The membrane is then transferred to Western Lights or Western
Probe with a specific anti-peptide antibody using the reagents and methods provided in Lights Plus (Tropix, Bedford, MA) chemiluminescence detection reagents. The developed membrane is connected to Hyperfilm ECL (Amersham, A
(Lingington Heights, IL) to visualize the chemiluminescent band.

Competition experiments are performed as described above, except that the primary antibody (anti-peptide polyclonal antiserum) is preincubated with various concentrations of peptide immunogen for 30 minutes at room temperature before exposure to the nitrocellulose filter. . The Western development is performed as described above.

After visualization of the band on the film, the band is directly visualized on the membrane by adding and developing a chromogenic substrate such as 5-bromo-4-chloro-3-indolyl phosphate (BCIP). be able to. This chromogenic solution is 100
mM NaCl, 5 mM MgCl ₂ and 100 mM Tris-HCl (p
0.016% BCIP in the solution containing H9.5). Incubate the filter in the solution at room temperature until the band develops at the desired intensity. Based on mobilities based on pre-stain molecular weight standards (Novex, San Diego, CA) or biotinylated molecular weight standards (Tropix, Bedford, MA)
The molecular weight is measured.

[0263]Example 17: EIA microtiter plate assay  Antibodies obtained from rabbits or mice as described in Example 13 or Example 14.
Serum immunoreactivity was measured by microtiter plate EIA as follows.
Set. Synthetic peptide prepared as described in Example 10 or
Recombinant protein prepared as described was combined with 50 mM carbonate buffer (pH 9.6).
) To a final concentration of 2 μg / ml. Next, 100 μl of the pepti
Solution or protein solution with Immulon2® microtiter
Rate (Dynex Technologies, Chantilly, VA
) In each well. Incubate the plate at room temperature overnight, then
Wash 4 times with deionized water. 12 in phosphate buffered saline (PBS, pH 7.4)
5 μl of a suitable protein blocking agent (eg, Superblock
Registered trademark) (Pierce Chemical Company, Rockfor
d, IL)) to each well, and then immediately discard the solution.
Block the well. Perform this blocking procedure three times. Already listed
Antisera from immunized rabbits or mice prepared as described
Tween-20 (registered trademark) (monolaurate polyoxyethylene ether)
(Sigma Chemical Company, St. Louis, MO)
And 1: 500, 1: 2500, 1: 12,500, 1: 62,500, and 1:
312,500 in 0.05% sodium azide in PBS
Protein blocking agents (eg, 3% Superblock® solution)
Solution) and place in each well of the coated microtiter plate
. The wells are then incubated at room temperature for 3 hours. Each well with deionized water
Wash 4 times. 0.05% Tween-20® and 0.05% sodium azide
3% Superblock in phosphate buffered saline containing thorium (registered)
®) 100 μl alkaline phosphatase binding diluted 1: 2000 in solution
Goat anti-rabbit IgG or goat anti-mouse IgG antiserum (Southern Bi
otech, Birmingham, AB) is added to each well. The well
Incubate for 2 hours at room temperature. Next, each well is washed four times with deionized water.
You. Then 100 μl of paranitrophenyl phosphate substrate (Kirkega)
ard and Perry Laboratories, Gaithersb
urge, MD) is added to each well. Incubate the wells for 30 minutes at room temperature
I do. Read the absorbance at 405 nm of each well. 405 in the test well
Absorbance at nm is increased compared to that obtained from non-immune serum (negative control)
By doing so, a positive reaction is identified. Positive reaction: detectable anti-BS106 antibody
Indicates the presence of

In addition to titer, apparent affinity [K _d (app)] can also be determined for some of the anti-peptide antisera. Using the results of the EIA microtiter plate assay, a similar formula of the Michaelis-Menten equation (V. Van Hey) was used.
Ningen, Methods in Enzymology , Vol. 121
, P. 472 (1986), and X Qui et al., Journal of Immunology , Vol. 156, p. 3350 (1996)):

[0265]

(Equation 1) (Where [Ag-Ab] represents the concentration of the antigen-antibody complex, [Ag-Ab] _max represents the maximum complex concentration, [Ab] represents the antibody concentration, and _Kd represents the dissociation constant). ,
An apparent dissociation constant (K _d ) can be derived. At the time of curve fitting, [Ag-
Ab] is replaced by the background-subtracted value of OD _{405 nm} at the given Ab concentration. K _d and _{[OD 405nm] max ([Ag} -Ab] corresponds to _{ma x)} are both treated as adaptation (Fitted) parameter. The software program Origin can be used for curve fitting.

[0266]Example 18: Coating of solid particles  A. Coating of microparticles with an antibody that specifically binds to BS106 antigen  Affinity purified antibody that specifically binds to BS106 protein (actually
(See Example 15) with a poly having a radius in the range of about 0.1-20 μm.
Fine particles of styrene, carboxylated polystyrene, polymethyl acrylate
Coats on similar particles. Microparticles can be either passive or active
It is possible to coat with. One coating method is EDAC (
1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (Al
drich Chemical Co. , Milwaukee, WI))
Carboxylated latex microparticles specifically for BS106 protein
Coating with the antibody that binds as follows. Briefly explain
And a carboxylated latex that was finally washed with a 0.375% resin solids suspension.
Fine particles (Bangs Laboratories, Carmel, IN or
(Available from Serodyn, Indianapolis, IN) with the appropriate
In a container, 50 mM MES buffer (pH 4.0) and 150 mg / l affinity
Tea-purified anti-BS106 antibody (see Example 14)
Mix in solution for 15 minutes. EDAC binder was brought to a final concentration of 5.5 μg / ml.
Add to the mixture until complete and mix at room temperature for 2.5 hours.

The microparticles are then washed with 8 volumes of Tween 20® / sodium phosphate wash buffer (pH 7.2) by tangential flow filtration using a 0.2 μm Microgon Filtration module. The washed microparticles are stored until needed, in a suitable buffer usually containing a dilute detergent and an irrelevant protein (blocking agent).

[0268]B. 1/4 inch bead coating  Further, an antibody that specifically binds to the BS106 antigen can be obtained by a conventional method known in the art.
(Snitman et al., US Pat. No. 5,273,88, incorporated herein by reference.
No. 2) to coat on the surface of 1/4 inch polystyrene beads.
Can be used in a binding or EIA sandwich assay.₃Buffer solution (pH 8.0)
Clean in sonication for about 15 seconds in water. Then all the fines
The beads are washed with deionized water until is removed. Then put the beads 10m
Soak in antibody solution in M carbonate buffer (pH 8.0-9.5). The antibody solution is high
In the case of an affinity monoclonal antibody, it is as low as 1 μg / ml or
In the case of a polyclonal antibody which has not been purified to about 500 μg / ml,
It can be as dilute as the concentration. Allow the beads to reach at least 12
Coat for an hour and then wash with deionized water. The beads are allowed to air dry or
Alternatively, it can be stored wet (in PBS, pH 7.4). Also,
They can be protein stabilizers (eg, sucrose) or non-specific binding blocks.
Carr (eg irrelevant protein, Carnation skim milk, Superb
lock (registered trademark)).
Bar coating can be applied.

[0269]Example 19: Microparticle enzyme immunoassay (MEIA)  Microparticles (ME) were subjected to standard antigen competition EIA or antibody sandwich EIA.
By using a solid phase such as IA), BS106 antibodies in patient test samples can be
Detect the original. The assay is an IMx® Analyzer (Abobo)
tt Laboratories, Abbott Park, IL)
It can be performed on an analyzer.

[0270]A. Antibody sandwich EIA  Briefly, to form an antigen / antibody complex, the BS106 antigen was
A sample suspected of containing microparticles coated with anti-BS106 antibody
(Prepared as described in Example 17).
The microparticles are then washed and the signal producing compound (ie, alkaline phosphatase)
And antibodies conjugated to enzymes such as horseradish peroxidase
An indicator reagent is added to the antigen / antibody complex or the microparticle and incubated. The
Wash microparticles and react with signal-generating compound to generate measurable signal
Substrates (eg, 4-methylumbelliferyl phosphate (MUP), respectively)
Or OPD / peroxide) to form the conjugated antibody / antigen / antibody complex
Is detected. Increased signal in the test sample compared to the signal generated from the negative control
The signal detects the presence of the BS106 antigen. BS10 in test sample
The presence of the six antigens is indicative of a breast disease or condition such as breast cancer.

[0271]B. Competitive binding assays  In the competitive binding assay, microparticles coated with anti-peptide antibody and labeled
Peptides or proteins that produce a measurable signal when contacted with a peptide
Use Parkin. This assay is based on the IMx® Analyzer (A
(Bobbot Laboratories, Abbott Park, IL)
Can do it. The labeled peptide is suspected of containing the BS106 antigen
Microparticles coated with BS106 antibody in the presence of test sample (Example 1
7) as well as labeled BS106 peptide (or labeled
Protein / binding antibody complex and / or patient's BS106 antigen / binding antibody
Incubate for a time and under conditions sufficient for the formation of the body complex. Test sample
The BS106 antigen in is labeled BS106 peptide for the binding site on the microparticle
(Or BS106 protein). In the assay, the test sump
BS106 antigen in the sample is labeled microparticles coated with a peptide or antibody.
Reduces the binding of Because the antigen in the test sample and the BS106 pepti
Or BS106 protein competes for the antibody binding site
. Reduced signal (compared to control) is due to BS106 antigen in test sample
Indicates the presence of The presence of the BS106 antigen is diagnostic of breast diseases or conditions, such as breast cancer.
Suggest disconnection.

The BS106 polynucleotides provided herein and discussed above are useful as markers for diseases of breast tissue, especially breast cancer. Tests based on the appearance of this marker in test samples such as blood, plasma, serum, etc. provide inexpensive and non-invasive diagnostic information to assist physicians in diagnosing cancer,
Helps select a therapy protocol or monitor the achievement of the selected therapy. This marker may appear in readily available body fluids, such as blood, urine, feces, etc., as an antigen from diseased tissue that can be detected by immunological methods. The marker can be a normal protein of the breast that is elevated in the pathology, changes in the pathology, or appears in inappropriate body fractions.

[Sequence list]

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE FIGURES FIG. 1: Clones 166885 (SEQ ID NO: 1), 89988 (SEQ ID NO: 2), 12
Figure 9 shows the nucleotide alignment of 09814 (SEQ ID NO: 3) and the consensus sequence derived therefrom (SEQ ID NO: 4). G / position 54 of the consensus sequence
The T polymorphism is shown as "K" and the C / T polymorphism at position 312 of the consensus sequence is shown as "Y".

FIG. 2. Overlapping clones 166885 (SEQ ID NO: 1), 89988 (SEQ ID NO: 2)
) And 1209814 (SEQ ID NO: 3) from a nucleotide alignment (co.
ntig map).

FIG. 3A is a scan of an agarose gel stained with ethidium bromide of RNA from various tissues, and the corresponding Northern blot of RNA using a BS106 radiolabeled probe.

FIG. 3B is a scan of another agarose gel stained with ethidium bromide of RNA from normal breast and breast cancer tissue, and the corresponding Northern blot of RNA using a BS106 radiolabeled probe.

FIG. 4A is a scan of a stained agarose gel of BS106-specific primed PCR amplification products.

FIG. 4B is a scan of a stained agarose gel of BS106-specific primed PCR amplification products.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) A61P 35/00 C07K 16/30 4C085 C07K 14/47 C12N 1/15 4H045 16/30 1/19 C12N 1 / 15 1/21 1/19 C12P 21/02 C 1/21 C12Q 1/68 A 5/10 G01N 33/53 M C12P 21/02 33/574 C12Q 1/68 33/577 G01N 33/53 C12N 15/00 ZNAA 33/574 A61K 37/02 33/577 C12N 5/00 A (72) Inventor Cohen, Maurice United States of America, 60035, Highland Park, Deaffield Road 2026 (72) Inventor Colpitts, Tracy・ El United States of America, Illinois ・ 60073, Round Lake, North Circle Drive ・ 34365 (72) Reedman, Paula N. USA, Illinois 60015, Day Affeld, Camno Court 462 (72) Inventor Gordon, Jiulian U.S.A., Illinois 60044, Lake Bluff, East Sheridan Road 307 (72) Inventor Granados, Edward United States of America, Illinois 60048, Vernon Hills, Montgomery Rain 19 (72) Inventor Hotsujis, Stephen Sea United States of America, Illinois 60089, Batu Fallow Clove, Stonegate Road 169 (72) Inventor Class, Mikle Earl United States of America, Illinois 60048, Libertyville, Mulberry Drive 1606 (72) Inventor Kratokville, Jillon Day United States of America, Wisconsin 53143, Kenoshya, Huifus Avenille 7101 (72) Inventor Donkey Tulap, Liza United States, 60031, Illinois, Gurney, Westfield Drive, 2090 (72) Inventor Ratsell, Jillon Sea, United States, 53142, Wisconsin, 53142, Present Prairie, Sixty Fours Court, 8275 (72) Inventor Strobing, Stephen Day United States, Illinois 60048, Libertyville, Wilshire Drive, 945 F-term (reference) 4B024 AA01 AA12 BA80 CA04 CA07 CA09 CA20 EA04 GA13 GA18 GA27 HA03 HA04 HA13 HA14 4B063 QA01 QA19 QQ08 QQ43 Q QR40 QR42 QR55 QR56 QR62 QR82 QS25 QS33 QS34 QX01 QX02 4B064 AG01 AG27 CA01 CA10 CA19 CA20 CC01 CC24 CE02 CE08 CE12 CE13 DA01 DA05 DA14 4B065 AA90X AA93X AA93Y AB01 AC14 AC15 AC16 BA02 BA05 BA04 ACA14 BD01 A04 BA01 CA53 CA59 NA14 ZB262 ZC512 4C085 AA13 AA14 4H045 AA10 AA11 AA20 AA30 BA10 BA41 CA40 DA75 DA86 EA28 EA51 FA71 FA74 GA01 GA22 GA26

Claims (39)

[Claims] 1. A method for detecting the presence of a target BS106 polynucleotide in a test sample, comprising: (a) contacting the test sample with at least one BS106-specific polynucleotide or its complement; Detecting the presence of the target BS106 polynucleotide in the test sample, wherein the BS106-specific polynucleotide comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3.
, SEQ ID NO: 4, SEQ ID NO: 5 and fragments or complements thereof, having at least 50% identity to a polynucleotide selected from the group consisting of: 2. The method of claim 1, wherein the target BS106 polynucleotide is bound to a solid phase before performing step (a). 3. A method for detecting the mRNA of BS106 in a test sample, comprising: (a) performing reverse transcription with at least one primer to obtain cDNA; and (b) converting the cDNA obtained from step (a) into BS106. The oligonucleotides were amplified using sense and antisense primers to produce BS10
(C) detecting the presence of the BS106 amplicon in the test sample, wherein the BS106 oligonucleotide used in steps (a) and (b) comprises SEQ ID NO: 1; A method characterized by having at least 50% identity to a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and fragments or complements thereof. 4. The method of claim 3, wherein the test sample is reacted with a solid phase before performing one of steps (a), (b) or (c). 5. The method of claim 3, wherein said detecting step comprises using a detectable label capable of producing a measurable signal. 6. A method for detecting a target BS106 polynucleotide in a test sample suspected of containing the target BS106 polynucleotide, comprising: (a) using the test sample with at least one BS1 as a sense primer.
06 oligonucleotide and at least one BS106 oligonucleotide as an antisense primer and amplifying to obtain a first stage reaction product, (b) contacting the first stage reaction product with at least one other BS106 oligonucleotide To obtain a second-stage reaction product (provided that the other BS106 oligonucleotide is 3% of the BS106 oligonucleotide used in the step (a)).
And (c) detecting the second stage reaction product as an indicator of the presence of the target BS106 polynucleotide, comprising the steps of: ) And (b) wherein the BS106 oligonucleotide has at least a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and fragments or complements thereof. A method characterized by having 50% identity. 7. The method of claim 6, wherein the test sample is reacted with a solid phase prior to performing one of steps (a), (b) or (c). 8. The method of claim 6, wherein said detecting step comprises using a detectable label capable of producing a measurable signal. 9. The method of claim 8, wherein said detectable label is reacted with a solid phase. 10. A test kit useful for detecting a target BS106 polynucleotide in a test sample, comprising: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and fragments thereof. A test kit comprising a container containing at least one BS106 polynucleotide having at least 50% identity to a sequence selected from the group consisting of complements. 11. A purified polynucleotide or a fragment thereof derived from the BS106 gene, wherein the polynucleotide has an ability to selectively hybridize to the nucleic acid of the BS106 gene; 2. A purified polynucleotide or a fragment thereof having at least 50% identity to a polynucleotide selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and their complements . 12. The purified polynucleotide according to claim 11, wherein said polynucleotide is produced by a recombinant technique. 13. The method of claim 12, wherein said polynucleotide is produced by synthetic techniques. 14. The purified polynucleotide of claim 11, wherein said polynucleotide comprises a sequence encoding at least one BS106 epitope. 15. A B operably linked to a control sequence compatible with the desired host.
A recombinant expression system comprising a nucleic acid sequence comprising an open reading frame derived from S106, wherein the nucleic acid sequence comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and fragments thereof. Or a recombinant expression system having at least 50% identity to a sequence selected from the group consisting of complements. 16. A cell transfected with the recombinant expression system according to claim 15. 17. SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 1
9. A BS106 polypeptide having at least 50% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 20 and fragments of SEQ ID NOS: 17-20. 18. The polypeptide of claim 1, wherein said polypeptide is produced by recombinant technology.
8. The polypeptide according to 7. 19. The polypeptide of claim 17, wherein said polypeptide is produced by synthetic techniques.
2. The polypeptide according to item 1. 20. An antibody that specifically binds to at least one BS106 epitope, wherein the BS106 epitope comprises SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and fragments thereof. An antibody derived from an amino acid sequence having at least 50% identity to an amino acid sequence selected from the group consisting of: 21. An assay kit for determining the presence of a BS106 antigen or an anti-BS106 antibody in a test sample, the kit comprising: SEQ ID NO: 16, SEQ ID NO: 17
An assay comprising at least one BS106 polypeptide having at least 50% identity to an amino acid sequence selected from the group consisting of: SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and fragments thereof. kit. 22. The assay kit according to claim 21, wherein said polypeptide is bound to a solid phase. 23. An assay kit for determining the presence of a BS106 antigen in a test sample, the kit comprising at least one BS106 epitope.
An assay kit comprising a container containing an antibody that specifically binds to the 06 antigen. 24. The kit according to claim 23, wherein said antibody is bound to a solid phase. 25. A method for producing a polypeptide comprising at least one BS106 epitope, comprising: SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 1.
9, transfected with an expression vector containing a polynucleotide sequence encoding a polypeptide having an amino acid sequence having at least 50% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 20 and fragments thereof. A process comprising incubating a host cell. 26. B in a test sample suspected of containing the BS106 antigen
A method for detecting S106 antigen, comprising: (a) at least one epitope of BS106 antigen selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and fragments thereof An antibody or a fragment thereof that specifically binds to the test sample,
Contacting for a time and under conditions sufficient to form an antibody / antigen complex, and (b) detecting the presence of the complex as an indicator of the presence of the BS106 antigen. 27. The method of claim 26, wherein said antibody is bound to a solid phase. 28. A method for detecting the presence of a BS106 antigen in a test sample suspected of containing the antibody, comprising: (a) SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, No. 19, an amino acid sequence having at least 50% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 20 and fragments thereof, or at least one derived from a fragment thereof
Contacting a BS106 polypeptide containing two BS106 epitopes with the test sample for a time and under conditions sufficient to allow formation of an antigen / antibody complex; and (b) detecting the complex. The method comprising. 29. The method of claim 28, wherein said BS106 polypeptide is bound to a solid phase. 30. A cell transfected with a nucleic acid sequence encoding at least one BS106 epitope, wherein the nucleic acid sequence comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and A cell selected from the group consisting of fragments or complements thereof. 31. Production of an antibody that specifically binds to the BS106 antigen, comprising administering to an individual an amount of an isolated immunogenic polypeptide or fragment thereof sufficient to elicit an immune response. Wherein the immunogenic polypeptide comprises at least one BS106 epitope and is selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and fragments thereof. A production method characterized by having at least 50% identity to the sequence. 32. A method for producing an antibody that specifically binds to a BS106 antigen, which is selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and fragments thereof. A method comprising administering to a mammal a plasmid comprising a sequence encoding at least one BS106 epitope derived from a polypeptide having an amino acid sequence. 33. A composition comprising a BS106 polynucleotide or a fragment thereof, wherein said polynucleotide comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3.
, SEQ ID NO: 4, SEQ ID NO: 5 and fragments or complements thereof, having at least 50% identity to a polynucleotide selected from the group consisting thereof. 34. A composition comprising a polypeptide containing at least one BS106 epitope, wherein the polypeptide comprises SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NOs: 17-20
A composition having at least 50% identity to a sequence selected from the group consisting of: 35. The test kit of claim 10, further comprising a container having means useful for collecting said sample, wherein said means is selected from the group consisting of a lancet, absorbent paper, cloth, swab, and cup. 36. The assay kit of claim 21, further comprising a container having means useful for collecting said sample, wherein said means is selected from the group consisting of a lancet, absorbent paper, cloth, swab, and cup. 37. The test kit of claim 23, further comprising a container having means useful for collecting said sample, wherein said means is selected from the group consisting of a lancet, absorbent paper, cloth, swab and cup. 38. A gene encoding BS106 protein comprising an amino acid sequence having at least 50% identity to SEQ ID NO: 16, or a fragment thereof. 39. A gene or a fragment thereof comprising DNA having at least 50% identity to SEQ ID NO: 4 or SEQ ID NO: 5.