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CN101042402A - Autoantibody detection for cancer diagnostics - Google Patents

Autoantibody detection for cancer diagnostics Download PDF

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CN101042402A
CN101042402A CNA2006100717486A CN200610071748A CN101042402A CN 101042402 A CN101042402 A CN 101042402A CN A2006100717486 A CNA2006100717486 A CN A2006100717486A CN 200610071748 A CN200610071748 A CN 200610071748A CN 101042402 A CN101042402 A CN 101042402A
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ecpka
autoantibody
ecpka autoantibody
antigen
immunoassay
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尹S·调正
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BIOGEMAX Ltd
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Abstract

The invention discloses a composition and method for detecting anti-ECPKA autoantibody in biological sample, and the application of the composition and method in cancer diagnostics of human and non-human mammal.

Description

The autoantibody that is used for cancer diagnosis detects
Invention field
The present invention relates to be used for detecting the composition and the method for anti--ECPKA autoantibody at biological sample, and the application of these compositions and method cancer diagnosis in people and non-human mammal.
Background of invention
Be released into blood with the synthetic tumor marker of malignant cell and with it.These marks also can generate replying of invading by host tissue, or generate as the result of the metabolic alterations of tumor inducing.The level of tumor marker in blood or tissue fluid be for diagnosis, examination and monitoring tumour progression or to disappear be helpful.Desirable tumor marker allows simple blood testing to detect cancer, and its level is relevant with the stage of tumour progression.Yet, owing to lack susceptibility and specificity, in general health crowd or people at highest risk, do not set up single mark as yet so far.The use of tumor marker in cancer diagnosis is described in (Sluss, P.M et al well.[2004]“ESTABLISHMENT?OF?A?CENTRAL?LABORATORY?SERUM?TUMORMARKER?SERVICE?ON?A?CONSOLIDATED?IMMUNODIAGNOSTICPLATFORM：DEVELOPMENT?OF?PRACTICE?STANDARS，SERVICEIMPROVEMENTS，AND?OPERATIONAL?EFFICIENCY”Clin?LeadershManag?Rev.18[1]25-31；Gion，M.[2000]“SERUM?TUMOUR?MARKERS：FROM?QUALITY?CONTROL?To?TOTAL?QUALITY?MANAGEMENT，”Breast?9[6]：306-11；Wiesner，A[2004]“DETECTION?OF?TUMORMARKERS?WITH?PROTEINCHIP TECHNOLOGY，Curr?PharmBiotechnol.Feb；5[1]：45-67；Crawford，NP.et?al.[2003]“TUMOR?MARKERSAND?COLORECTAL?CANCER：UTILITY?IN?MANACEMENT，”J?SurgOncol.84[4]：239-48；Agnantis，N.J.et?al.[2003]“TUMOR?MARKERS.ANUPDATE?APPROACH?FOR?THEIR?PROGNOSTIC?SIGNIFICANCE.PARTI.IN?VIVO，”17[6]609-18；Riley，R.D.et?al.[2004]“A?SYSTEMATICREVIEW?OF?MOLECULAR?AND?BIOLOGICAL?TUMOR?MARKERS?INNEUROBLASTOMA，”Clin?Cancer?Res.10[1?Pt?1]：4-12；Given，M.et?al.[2000]“THE?PREDICTIVE?OF?TUMOUR?MARKERS?CA?15-3，TPS?ANDCEA?IN?BREAST?CANCER?RECURRENCE，”Breast.9[5]：277-80)。
At present available cancer markers is measured cancer antigen.For example prostate cancer can be diagnosed (Gretzer by measuring prostate specific antigen (PSA) cancer markers, M.B.et al.[2003] " PSAMARKERS IN PROSTATE CANCER DETECTION, " Urol Clin North Am.30[4]: 677-86).Have been found that carcinomebryonic antigen (CEA) is marked at and has diagnostic uses in the assessing colorectal cancer.(Crawford，NP.et?al.[2003]“TUMOR?MARKERS?ANDCOLORECTAL?CANCER：UTILITY?IN?MANAGEMENT.J.Surg?Oncol.84[4]：239-48)。Cancer antigen CA15-3, (Cheung has been associated with breast cancer, K.L.et al.[2003] " OBJECTIVE MEASUREMENT OF REMISSION ANDPROGRES SION IN METASTATIC BREAST CANCER BY THE USE OFSERUM TUMOUR MARKERS, " Minerva Chir.Jun; 58[3]: 297-303).Cancer antigen CA 19-9 has been used for diagnosing human primary gastrointestinal cancers (Grotowski, M.[2002] and " ANTIGENS[CEA AND CA 19-9] IN DIGNOS IS AND PRGNOSIS COLORECTALCANCER, " Pol Merkuriusz Lek.12[67]: 77-80; Trompetas, V.et al.[2002] " GIANT BENIGN TRUE CYST OF THE SPLEEN WITH HIGH SERUMLEVERL OF CA19-9, " Eur J Gastroenterol Hepatol.14[1]: 85-8).Cancer antigen CA125 be used to diagnosis of ovarian cancer (Anderiesz, C.et al.[2003] " SCREEING FOROVARIAN CANCER, " Med J Aust 178[12]: 655-6).
Most entity tumors show the chromosome instability that is caused by the distortion of the chromosome separation in the fission process.Several kinase enzymes participate in keeping normal chromosome separation and cell cycle regulation progress.CAMP deopendent protein kinase (PKA) is a kind of such kinases, its as if the many aspects of cellular signal transduction, metabolism and propagation have functional effect (Matyakhina L et al.[2002] " PROTEIN KINASE A AND CHROMOSOMAL STABILITY, " Ann NYAcad Sci.968:139-47; Tortora, G.et al.[2002] " PROTEIN KINASE A ASTARGET FOR NOVEL INTEGRATED STRATEGIES OF CANCERTHERAPY, ' Ann NY Acad Sci.968:139-47).
Mammalian cell has two types cAMP deopendent protein kinase (PKA) kind (Krebs, E.G.et al.[1979] " PHOSPHORYLATION-DEPHOSPHORYLATION OF ENZYMES, " Annu Rev Biochem.48:923-39).These protein kinases are designated as type i (PKA-I) and Type II (PKA-II); It is distinguished by different adjusting subunits (R subunit) RI and RII, and its share a common catalytic subunit (C subunit) (Beebe.S.J.et al.[1986] " CYCLIC NUCLEOTIDE-DEPENDENTPROTEIN KINASES, " In:The Enzymes:Control by Phosphorylation; E.G.Krebs et al.[Eds] Academic Press:Orlando and London.pp.43-111).
Traditionally, the enzymatic activity of protein kinase by follow the trail of from (γ- 32P) ATP be delivered to the radiophosphorus acid groups on the residue of suitable albumen or peptide substrates and measure (referring to for example Witt, J.J.et al.[1975] Anal Biochem.66:253-8; Casnellie.J.E.[1991] Methods Enzymol.200:115-20; U.S. Patent number 6,498,005).The PKA enzymatic determination is existing to be described (Cohen, C.B.et al.[1999] " A MICROCHIP-BASED ENZYME ASSAY FOR PROTEINKINASE A, " Anal Chem.[1999] 273:39-97; Cho, Y.S.et al.[2000] " EXTRACELLULAR PROTEIN KINASE A AS A CANCERBIOMARKER:ITS EXPRESSION BY TUMOR CELLS AND REVERSALBY A MYRISTATE-LACKING C ALPMAAND RII BETASUBUNITOVEREXPRESSION, " Proc Natl Acad Sci USA.97[2]: 835-40).
By Biochemical Research and gene clone, identified the R subunit of four isotypes, RI α, RI β, RII α, with RII β (Amieux, P.S.et al.[2002] " THE ESSENTIAL ROLE OF RIALPHA IN THE MAINTENANCE OF REGULATED PKA ACTIVITY, " Ann NY Acad Sci.968:75-95; McKnight, G.S.et al.[1988] " ANALYSIS OFcAMP-DEPENDENT PROTEIN KINASE SYSTEM USING MOLECULARGENETIC APFROACHES, " Recent Prog Honn Res.44:307-35; Levy, F.O.etal.[1998] " MOLECULAR CLONING; COMPLEMENTARYDEOXYRIBONUCLEIC ACID STRUCTURE AND PREDICTEDFULL-LENGTH AMINO ACID SEQUENCE OF THEHORMONE-INDUCIBLE REGULATORY SUBUNIT OF 3 ', 5 '-CYCLICADENOSINE MONOPHOSPHATE-DEPENDENT PROTEIN KINASEFROM HUMAN TESTIS " Mol Endocrinol 2:1364-73).
Importantly, the ratio of PKA-I and PKA-II can be at cell development, marked change (Lohmann takes place in differentiation and the conversion process, S.M.et al.[1984] " REGULATION OF THECELLULAR AND SUB CELLULAR CONCENTRATIONS ANDDISTRIBUTION OF CYCLIC NUCLEOTIDE-DEPENDENT PROTEINKINASES; " In:Advances in CYclic Nucleotide and Protein PhosphorylationResearch, P. ' Greengard et al.[Eds] Raven Press:New York.pp 63-117; Cho-Chung, Y.S.[1990] and " ROLE OF CYCLIC AMP RECEPTOR PROTEINSIN GROWTH; DIFFERENTIATION, AND SUPPRESSION OFMALIGNANCY:NEW APPROACHES TO THERAPY, " Cancer Res.50:7093-100; Cho-Chung, Y.S.[2003] " cAMP SIGNALING IN CANCERGENESIS AND TREATMENT " Cancer Treat Res.115:123-43).
The cAMP signal transduction path has been suggested in lymph sample malignant tumour as treatment target (Lerner, A.et al.[2000] " THE cAMP SIGNALING PATHWAY As ATHERAPEUTIC TARGET IN LYMPHOID MALIGNANCIES, " LeukLymphoma.37[1-2]: 39-51; Cho-Chung, Y.S.et al.[1995] " cAMP-DEPENDENT PROTEIN KINASE; ROLE IN NORMAL ANDMALIGNANT GROWTH, " Crit Rev Oncol Hematol.21[1-3]: 33-61; Cho-Chung, Y.S.et al.[1993] " THE REGULATORY SUBUNIT OFcAMP-DEPENDENT PROTEIN KINASE AS A TARGET FORCHEMOTHERAPY OF CANCER AND OTHER CELLULARDYSFUNCTIONAL-RELATED DISEASES, " Pharmacol Ther.60[2]: 265-88).In by the cell after chemical substance or viral carcinogen and Ki-ras proto-oncogene or the transforming growth factor conversion, with (Cho-Chung when carrying out the cell growth stimulation with granulocyte-macrophage colony stimutaing factor or Buddhist ripple ester, Y.S.[1990] " ROLE OF CYCLIC AMPRECEPTOR PROTEINS IN GROWTH; DIFFERENTIATION.ANDSUPPRESSION OF MALIGNANCY:NEW APPROACHES TOTHERAPY, " Cancer Res.50:7093-100; Cho-Chung, Y.S.et al.[2002] " DISSECTING THE CIRCUITRY OF PROTEIN KINASE A AND cAMPSIGNALING IN CANCER GENESIS:ANTISENS E.MICROARRAY; GENEOVEREXPRESSION; AND TRANSCRIPTION FACTOR DECOY; " Ann NYAcad Sci.968:22-36), than normal homologue, increase the RI α/PKA-I that expresses and appeared at human carcinoma cell line and primary tumo(u)r (Cho-Chung, Y.S.[1990] " ROLE OF CYCLICAMP RECEPTOR PROTEINS IN GROWTH; DIFFERENTIATION.ANDSUPPRESSION OF MALIGNANCY:NEW APPROACHES TOTHERAPY, " Cancer Res.50:7093-100; Miller, W.R.et al.[1993] " TYPESOF CYCLIC AMP BINDING PROTEINS IN HUMAN BREASTCANCERS, " Eur J Cancer.29A:989-91).On the contrary, the reduction of RI α/PKA-I expression is associated the RI α subunit of PKA in the human carcinoma cell line of described antisense oligonucleotides target wide spectrum in nude mice and the people's tumor growth with the growth inhibited that the cAMP analog and the antisense oligonucleotides of site selectivity are induced.(Cho-Chung，Y.S.et?al.[1989]“SITE-SELECTIVE?CYCLIC?AMPANALOGS?AS?NEW?BIOLOGICAL?TooLs?IN?GROWTH?CONTROL，DIFFERENTIATION?AND?PROTO-ONCOGENE?REGULATION，”CancerInv.7：161-77；Cho-Chung，Y.S.et?al.[1999]“ANTISENSE?DNA--TARGETING?PROTEIN?KINASE?A-RIα?SUBUNIT：A?NOVELAPPROACH?TO?CANCER?TREATMENT，”Front?Biosci?4：D898-D907)。
Before proved the polytype cancer cell PKA is secreted in the conditioned medium (Cho, Y.S.et al.[2000] " EXTRACELLULAR PROTEIN KINASE A As A CANCERBIOMARKER:ITS EXPRESSION BY TUMOR CELLS AND REVERSALBY A MYRISTATE-LACKING C ALPHAAnd RII BETASUBUNITOVEREXPRESSION, " Proc Natl Acad Sci USA.97[2]: 835-40).There is (" PKAC α ") in this extracellular protein kinases A (ECPKA) with active, free catalytic subunit (C subunit) form, and its activity is by the special inhibition of PKA Profilin PKI.The C α of PKA or RI α subunit gene crossing in expression vector expressed (it raises PKA-I in the cell) and raised the ECPKA expression significantly.Contrast therewith, cross the expressing of RII β subunit-it is eliminated PKA-I, raises PKA-II, and recover the phenotype-downward modulation ECPKA of conversion.The sudden change that prevents myristylation in C α gene allows PKA rise and blocking-up ECPKA increase in the cell, and hint ECPKA expresses the NH that needs C α 2-end myristyl.Compare with normal serum, in cancer patient's serum, ECPKA expresses and is significantly raised.(Cho，Y.S.et?al.[2000]“EXTRACELLULAR?PROTEIN?KINASE?A?As?ACANCER?BIOMARKER：ITS?EXPRESSION?BY?TUMOR?CELLS?ANDREVERSAL?BY?A?MYRISTATE-LACKING?C ALPHA?AND?RII BETASUBUNIT?OVEREXPRESSION，”Proc?Natl?Acad?Sci?USA.97[2]835-40)。
The development of monoclonal antibody has caused having identified many tumor associated antigens in the serum of malignant tumor patient and tissue.The protein product of proto-oncogene and tumor suppressor gene can be detected out in the fluid of extracellular and it is used as the potential mark of carcinogenesis in the body.In these growth factors some are encoded by proto-oncogene.For example, higher levels of p21-ras albumen is by the ras proto-oncogene coding of finding in blood samples of patients.To the circulating antibody of p53 tumor suppressor protein at breast cancer and patients with lung cancer and suffer from the lymphadenomatous children's of B-the serum and be found.(Winter，S.F.et?al.[1992]“DEVELOPMENT?OF?ANTIB?ODIES?AGAINST?p53?IN?LUNG?CANCERPATENTS?APPEARS?TO?BE?DEPENDENT?ON?THE?TYPE?OF?p53MUTATION.”Cancer?Res.52：4168-74；Lubin，R..et?al.[1993]“ANALYSISOf?p53?ANTIBODIES?IN?PATIENTS?WITH?VARIOUS?CANCERS?DEFINEB-CELL?EPITOPES?OF?HUMAN?p53：DISTRIBUTION?ON?PRIMARYSTRUCTURE?AND?EXPOSURE?ON?PROTEIN?SURFACE?Cancer?Res.53：5872-6；Crawford，L.V.et?al.[1982]“DETECTION?OF?ANTIBODIESAGAINST?THE?CELLULAR?PROTEIN?p53?IN?SERA?FROM?PATIENTSWITH?BREAST?CANCER.”Int.J.Cancer.30：403-8)。Antibody to proto-oncogene such as c-myc and c-myb also in the serum of suffering from colorectum tumour and tumor of breast patient, be found (Sorokine.L, K.et al.[1991] " PRESENCE OF CIRCULATINGANTI-C-MYB ONCOGENE PRODUCT ANTIBODIES IN HUMAN SERA. " Int.J.Cancer.47:665-9; Ben-Mahrez, K,, et.al.[1988] and " DETECTION OFCIRCULATING ANTIBODIES AGAINST C-MYC PROTEIN IN CANCERPATIENT SERA. " Br J Cancer.57:529-34; Ben-Mahrez, K., et al.[1990] " CIRCULATING ANTIBODIES AGAINST C-MYC ONCOGENEPRODUCT IN SERA OF COLORECTAL CANCER PATIENTS. " Int JCancer, 46:35-8).
Except the above-mentioned new mark of mentioning, other protein, hormone and enzyme were used as mark 30 years.Wherein famous have carcinomebryonic antigen (CEA), alpha fetoprotein (AFP) (AFP), a human chorionic gonadotrophin (HCG), and prostate acid phosphatase (PAP).But, the most specificitys that lack of these marks.These levels also increase under benign conditions and in pregnant process.All these marks are all determined method based on antigen; These marks lack specificity and susceptibility.Therefore be badly in need of finding novel biomarker and change it over to routine clinical using.The present invention is exactly in order to solve such needs.
Summary of the invention
The present invention relates to the enzyme immunoassay (EIA) of the IgG antibody of a kind of extremely sensitive measurement extracellular protein kinases A (ECPKA).Serum to the people of the patient that suffers from all kinds cancer from 295 and 100 no cancers is tested.The frequency (92%) that found that anti--ECPKA IgG antibody among the cancer patient will not have cancered people (14%) apparently higher than those.At anti--ECPKA IgG antibody of being measured by EIA and significantly not related between the measured ECPKA antigen by the PKA enzymatic determination, and anti-ECPKA IgG antibody-EIA method has obtained higher susceptibility and specificity than ECPKA enzymatic determination.These results prove the method that autoantibody is analyzed, rather than traditional antigenic analysis, and the process useful that is used for cancer diagnosis is provided.
In detail, the invention provides a kind of anti--existence of ECPKA autoantibody or immunoassay of concentration of measuring in mammiferous biological sample, wherein immunoassay comprises the following step:
A) biological sample is contacted with the antigen that is specific to anti--ECPKA autoantibody, described contact be enough to allow to resist-the ECPKA autoantibody is in case be present under the situation in the sample, combine and forms with antigen under the condition of antigen-resist-ECPKA autoantibody compound to carry out;
B) antigen-anti--ECPKA autoantibody compound that forms contact with anti--ECPKA autoantibody binding molecule, described contact be enough to allow to resist-ECPKA autoantibody binding molecule is attached on anti--ECPKA autoantibody of antigen-anti--ECPKA autoantibody compound of formation and under the condition of the compound of formation extension and carries out; With
C) existence or the concentration of the compound by the extension that determine to form are determined to resist-existence or the concentration of ECPKA autoantibody in described biological sample.
The invention still further relates to the embodiment of above-mentioned immunoassay, the antigen that wherein is specific to anti--ECPKA autoantibody is extracellular PKA albumen.
The invention still further relates to the embodiment of above-mentioned immunoassay, wherein anti--the ECPKA autoantibody is the antibody of mammal, they are different with mammiferous antibody and be specific to antibody by the mammal generation.
The invention still further relates to the embodiment of above-mentioned immunoassay, wherein mammal is that people and anti--the ECPKA autoantibody is the human IgG antibody.The invention still further relates to the embodiment of above-mentioned immunoassay, wherein anti--ECPKA autoantibody binding molecule can be detected ground mark (particularly using chemistry or enzyme labeling).
The invention still further relates to the embodiment of above-mentioned immunoassay, wherein in step (a), the antigen that is specific to anti--ECPKA autoantibody is fixed on the solid support before coming in contact with biological sample.
The invention still further relates to the embodiment of above-mentioned immunoassay, wherein in step (a), the antigen that is specific to anti--ECPKA autoantibody is fixed on the solid support after coming in contact with biological sample.
The invention still further relates to the embodiment of above-mentioned immunoassay, immunoassay wherein is the immune chromatograph immunoassay, wherein:
In step (a), biological sample is placed with first porous carrier and contacts, the antigen that first porous carrier contains is revocable, be specific to the mark of anti--ECPKA autoantibody;
In step (b), the antigen of formation-anti--ECPKA autoantibody compound is placed with second porous carrier and contacts, and second porous carrier is communicated with first porous carrier, and contains fixing anti--ECPKA autoantibody binding molecule; With
In step (c), the existence of the antigen by detecting the mark be specific to anti--ECPKA autoantibody in second porous carrier is determined to resist-existence or the concentration of ECPKA autoantibody in biological sample.
The invention provides a kind of immunology compound, its contain be attached to being specific to of anti--ECPKA autoantibody anti--antigen of ECPKA autoantibody, wherein anti--ECPKA autoantibody also additionally is attached on anti--ECPKA autoantibody binding molecule.
The invention still further relates to the embodiment of above-mentioned immunology compound, the antigen that wherein is specific to anti--ECPKA autoantibody is extracellular PKA albumen.
The invention still further relates to the embodiment of above-mentioned immunology compound, wherein anti--ECPKA autoantibody binding molecule can be detected ground mark (particularly using chemistry or enzyme labeling).
The invention still further relates to the embodiment of above-mentioned immunology compound, wherein anti--ECPKA autoantibody binding molecule is the immunology molecule.The invention still further relates to the embodiment of above-mentioned immunology compound, wherein anti--the ECPKA autoantibody is people's autoantibody, and anti--ECPKA autoantibody binding molecule is the human IgG antibody.
The invention still further relates to a kind of kit, it is used for measuring existence or the concentration that mammiferous biological sample is measured anti--ECPKA autoantibody, wherein kit contains and comprises the shell that multilayer filters the hollow of system, with first and second porous carrier, wherein second porous carrier communicates with first porous carrier, and first porous carrier communicate with multilayer filtration system, its part can arrive from shell, wherein:
That first porous carrier contains is revocable, the PKA C α or the C α fragment of mark; With
The antibody that second porous carrier contains is fixing, unlabelled, be attached to human IgG.
The invention still further relates to the embodiment of mentioned reagent box, wherein the PKA C α of mark is ECPKA (particularly using chemistry or enzyme labeling).
The invention still further relates to the embodiment of mentioned reagent box, wherein kit detects the people anti--ECPKA autoantibody, and be inhuman mammiferous antibody in conjunction with the antibody of human IgG.
Description of drawings
Fig. 1 has shown the numerical value of the use preferred immunoassays form of the present invention to obtaining among normal individual and the cancer patient.Patient's frequency and average titer (frequency=92%, average titer 2.2) are all apparently higher than those (frequency=14%, average titers 0.6) of normal control.It is positive that numerical value surpasses 1.0 (on the dotted lines).
Fig. 2 has shown receptacle operating characteristic (ROC) curve of anti-ECPKA autoantibody ELISA (Fig. 1).At joint, the cutoff that ELISA measures is 1.0 titres, and susceptibility and specificity are respectively 90% and 89%.
Fig. 3 has shown the western blot analysis result to anti-ECPKA autoantibody in cancer patient's serum.The swimming lane that contains M (mw mark) and C α (1 μ g) with Coomassie blue stain; The anti-C Alpha antibodies of band 1 usefulness Santa Cruz Ab trace (Santa Cruz Biotechnology, Santa Cruz, CA); Band 2-7 cancer patient's serum trace (10,000 times of dilutions); Band 8-12 normal human serum trace (10,000 times of dilutions); Band 1-12 contains the PKA C α (each 1 μ g) of purifying.
Fig. 4 has shown the numerical value that obtains as the contrast of the susceptibility of immune detection form of the present invention and resolving ability by with the ECPKA enzymatic determination.The figure illustrates from numerical value (cancer patient, frequency=83%, the mean value=130mU/ml of normal individual and cancer patient's acquisition; The normal individual contrast, frequency=21%, mean value=60mU/ml), its demonstration lacks susceptibility and specificity.Numerical value greater than 75mU/ml (on the dotted line) is positive.
Fig. 5 has shown from the anti--ECPKA antibody titer of cancer patient and healthy people's serum and the result of the correlative study between the ECPKA enzymatic activity.0.003 cancer patient's coefficient and healthy people's coefficient of 0.001 meaningless statistically.
Fig. 6 shown the HCT-15Z tumor extract as the source of cancer antigen, from the titre of the anticancer antigen-antibody of cancer patient and healthy people's serum.Numerical value is positive greater than 1.5 (dotted lines).
Fig. 7 has shown the titre of the antigen-antibody that observes in the serum from the cancer patient who suffers from dissimilar cancers (lung, kidney, pancreas, ovary, colon, liver, stomach, bladder, and cervical carcinoma, melanoma, sarcoma, and liomyoma).
The description of preferred implementation
The present invention relates to be used for detect the composition and the method for anti--ECPKA autoantibody, and these compositions and method are in people and non-human mammal (Canidae particularly, cat family at biological sample, Bovidae, sheep section (ovine), Suidae and equine mammal) in application in the cancer diagnosis.
The PKA of extracellular form (ECPKA) from cancer cell, secrete (Cho, Y.S et al.[2000] " EXTRACELLULAR PROTEIN KINASE A As A CANCERBIOMARKER:ITS EXPRES SION BY TUMOR CELLS AND REVERSALBy A MYRISTATE-LACKING C ALPHAAND RII BETASUBUNITOVEREXPRESCTON, " Proc Natl Acad Sci USA.97[2]: 835-40).Motivation of the present invention part is from such understanding, and the ECPKA secretion excites the formation of serum autoantibody, and the existence of this autoantibody and/or concentration can be used as the mark of cancer diagnosis and prediction.As described herein, the invention provides in the biological sample of suspecting or be proved the cancer patient, measure anti--ECPKA autoantibody (particularly anti-ECPKA IgG antibody) concentration and/the super-sensitive immunoassay that exists.This immunoassay based on autoantibody provides the routine diagnosis program that is used to detect all kinds cancer cell.
The present invention relates to combining of antigen and antibody.As used herein, " epi-position " be identified and specific bond in 2 or 3 dimension zones of the antigen of antibody.As used herein, if it can special the mutually combining of immunology, then we can say antigen and antibody " special " mutually, perhaps " identification ", perhaps mutual " combination " mutually.
The mode determination of any wide range of types all can use according to method of the present invention.These patterns can be heterogeneous or homogeneities, and are continuous or simultaneously, emulative or noncompetitive.U.S. Patent number 5,563,036; 5,627,, 080; 5,633,141; 5,679,525; 5,691,147; 5,698,411; 5,747,352; 5,811,526; 5,851,778; With 5,976,822 for example understand several different test patterns and application.Such test can be turned to the concentration or the amount of quantitative-ECPKA autoantibody anti-to measure by pattern, and perhaps it can be turned to existing of anti-to measure qualitatively-ECPKA autoantibody by pattern or lack.
Heterogeneous immunoassay typically relates to the solid phase material that uses reaction product to combine with it, but also is suitable for relating to combine (that is the solution phase immunoassays) of revocable antigen and antibody.By solid phase being removed (for example by clean) from reaction mixture, reaction product is from excess sample, and test agent and is separated in other material.A kind of solid-phase immunoassay type that can use according to the present invention is the sandwich immunoassay method.In sandwich assay, the analyte in the sample is many more, and the labelled amount that exists on the solid phase is many more.Common preferred such mode determination, especially for the analyte of naked eyes detection low concentration, this is because appear at easier being detected of mark of solid phase.
According to the preferred embodiments of the invention, specifically with the antigen of anti-ECPKA autoantibody reaction be incorporated into (that is: fixing) on the solid support and be used to test the biological sample that anti--ECPKA IgG antibody exists and contact incubation.As will being understood, can be with antigen with biological sample with the unbound state incubation, and it is attached on the solid support subsequently and (can fixes).Preferably holder is handled up hill and dale (for example by clean or the like) subsequently but may have the non-ECPKA IgG antibody that is not attached to conjugated antigen to remove basically.The result of Chu Liing forms immune complex between antigen and anti-ECPKA IgG antibody like this.
Preferred second antibody of adding detectable mark subsequently and holder is placed at is enough to make second antibody to be attached to incubation under the condition of any anti--ECPKA IgG antibody that may exist.Preferably holder is handled up hill and dale (for example by cleaning or the like) subsequently to remove any second antibody that does not have combination basically.If there is anti--ECPKA IgG antibody in specimen, two kinds of antibody will form immune complex (that is: second antibody/anti--ECPKA IgG antibody/antigen is sandwich) with analyte so.In such test, be measured to the existence that the second antibody that is attached to holder shows anti--ECPKA IgG antibody in the tested fluid.The sandwich assay pattern is described in the Schuurs et al. U.S. Patent number 3,791,932 and 4,016,043, and in the Pankratz et al. U.S. Patent number 5,876,935.Second antibody can be the native immunoglobulin separated from inhuman Primate (a for example anti-human IgG murine antibody, anti-human IgG goat-anti body, or the like), perhaps can be through reorganization or synthetic the generation.It can be complete immunoglobulin (Ig), perhaps immunoglobulin fragment (Fab for example, F[Ab] 2, etc.).If desired, can use other binding molecule (can in conjunction with anti--ECPKA autoantibody) to coordinate or replace this class second antibody.For example, anti--ECPKA autoantibody can be biotinylated and can replace second antibody with the avidin or the streptavidin of mark.
In order to eliminate unconjugated separating step and to reduce chemical bond and measure required time and equipment, also can optionally use the homogeneity mode determination.In such test, can remain in conjunction with right a kind of component and to be fixed; But, need not be not in conjunction with separating the existence of measuring in conjunction with the second right component.The example of the optical means of homogeneity is Syva, and (it operates by measuring fluorescent quenching Inc. for Sunnyvale, EMIT method CA); (Marburg, laser Germany) are than turbid latex particle agglutination, and it is operated by the variation of measuring light scattering for Behringwerke; Mitsubishi ChemicalIndustries (Tokyo, LPIA latex particle agglutination Japan); Abbott Laboratories (Abbott Park, TDX FD method IL); Cis Bio Intemational (Paris, fluorescence energy transfer method France).Any of these determination method can be changed to be applicable to purpose of the present invention.
Combination of the present invention is measured and can be configured to competitive assay.In competitive assay, exist in the specimen anti--ECPKA IgG antibody is many more, the labelled amount that exists on the solid phase is few more.
Being similar to the mode of sandwich assay, anti--ECPKA IgG antibody that competitive assay can be by providing quantitative mark also determines whether tested fluid contains the anti--ECPKA IgG antibody that will combine holder with the labelled antibody competition and carries out.In such competitive assay, the labelled antibody amount that is captured is inversely proportional to the amount of analyte that is present in the specimen.Smith (U.S. Patent number 4,401,764) has described alternative competitive assay pattern, it use to mix can bound analyte or labelled analyte in conjunction with compound, but analyte wherein and labelled analyte can not combine with compound simultaneously.Clagett (U.S. Patent number 4,746,631) immunoassay of using reaction chamber has been described, in reaction chamber, analyte/part/mark conjugate is replaced from reaction surface under the situation that the specimen analyte exists, and substituted analyte/part/mark conjugate is fixed on second reaction site.Conjugate comprises biotin, bovine serum albumin(BSA) and as the synthetic peptide of the part component of conjugate, and enzyme, chemiluminescent material, enzyme inhibitor and as the radioactive nucleus thuja acid of the marker components of conjugate.Li (U.S. Patent number 4,661,444) has described the competitive immunometric assay method of the conjugate that uses anti-idiotype and second antibody, and it is specific to detectable mark, wherein detectable reply with sample in the existence of analyte to be inverse ratio relevant.Allen (European Patent Application No. 177,191) combination of having described the conjugate that relates to ligand analogs and second reagent such as fluorescein is measured, wherein conjugate and analyte (part) competition is attached on the binding partners that is specific to part of mark, and wherein the conjugate of resulting mark is separated from reaction mixture by the solid phase of carrying the second reagent binding partners subsequently.This in conjunction with mode determination with the use of competitive combination technology and oppositely the sandwich assay configuration combine; That is: by conjugate is attached to solid phase, before conjugate and mixture separation, conjugate is attached to the bonded block of mark.But this measurement result is determined to be in traditional competitive assay, and the amount that wherein is attached to analyte in the amount of mark of solid phase and the specimen is inversely proportional to.Chieregatt et al. (the GB patent No. 2,084,317) has described similar mode determination, and its use is specific to the indirect labelling binding partners of analyte.Mochida et al. (U.S. Patent number 4,185,084) has also described the conjugate that uses two antigens, and itself and the competition of antigenic analysis thing are attached on the fixing antibody and are labeled subsequently.This method also causes detecting the mark on solid phase, and wherein the amount of analyte is inversely proportional in the amount of mark and the specimen.Sadeh et al. (U.S. Patent number 4,243,749) has described similar enzyme immunoassay (EIA), and wherein the haptens conjugate combines the antibody that is fixed on the solid phase with the analyte competition.The variant of any of these determination method all is applicable to according to the present invention and uses.
In all these mode determinations, at least a composition of preferred marker determination reagent, perhaps sending or extinguish and detect by light in other cases.This component can be a second antibody, and is anti--ECPKA IgG antibody, perhaps is attached to the antigen of anti--ECPKA IgG antibody, and this depends on employed immunoassays pattern.Radioactive isotope in conjunction with test pattern (for example: radioimmunoassay method of testing or the like) use radioactive isotope as this mark; In the presence of fluorescence or fluorescence part, send measured signal (see Lucas et al.[U.S. Patent number 5,698,411] and Landrumet al.[U.S. Patent number 5,976,822]) by light.Enzyme uses enzyme to serve as a mark in conjunction with mode determination (for example ELISA or the like); In the presence of chromophore or fluorescence part, by the measured signal that sends of color or light.Other mark, spin labeling for example, as the material of colored particles, latex particle, colloidal metal such as selenium and gold, and dye granule (is seen U.S. Patent number 4,313,734; 4,373,932 and 5,501,985) also can be used.The preferred enzyme (particularly alkaline phosphatase, beta galactosidase, horseradish peroxidase, perhaps urase) that uses is as detectable label (that is: enzyme immunoassay (EIA) or EIA).
The existence of enzyme labeling can detect by the chromophoric substrate (comprise send or absorb fluorescence, UV, those of visible light or the like) that use responds to enzyme labeling catalysis.Preferred, can use chemical labeling (for example collaurum, latex beads mark or the like).Can be by a plurality of detectors of use, multipass filter (multipass filter), grating, perhaps the different fluorescent material of spectrum is realized marker detection (seeing United States Patent (USP) 5,759,781) etc.Especially preferably use peroxidase as enzyme labeling, particularly with chromophoric substrate 3,3 ', 5,5 '-tetramethyl benzidine (TMB) is collaborative mutually.Coming in the process of labelled antibody as enzyme with peroxidase, can use periodate technology (Nakane, P.K.et al.[1974] " PEROXIDASE-LABELED ANTIBODY.A NEW METEOD OFCONJUGATION; " J Histochem Cytochem.22:1084-90) or reported method, gametophyte is wherein rolled into a ball reagent with Heterobifunctional and is connected (Ishikawa, E.et al.[1983] " ENZYME-LABLELING OF ANTIBODIES AND THEIR FRAGMENTSFOR ENZYME IMMUNOAS SAY AND IMMUNOHISTOCHEMICALSTAINING, " J.Immunoassay.4[3]: 209-327).
In immunoassays of the present invention, can use any of multiple holder.The suitable material of solid support is a for example polystyrene of synthetic material, Polyvinylchloride, polyamide, perhaps other synthetic polymer, natural polymer is cellulose for example, and the natural polymer of deriving such as cellulose acetate or NC Nitroncellulose and glass, particularly glass fibre.The form of holder can be spherical, shaft-like, the fixed or microtiter plate of tubulose and micrometering.Schistose texture is paper slip for example, and platelet and film are suitable for too.For aqueous solution, carrier surface can be permeable or impermeable.
Although the description of front relates to the mensuration that anti--ECPKA autoantibody exists in the biological sample, described biological sample is a fluid (serum for example, blood, urine, saliva, pancreatic juice, cerebrospinal fluid, seminal fluid etc.), should be appreciated that still any fluidity biological sample (for example organizing or the biopsy extract extract of ight soil, phlegm or the like) can similarly use in determination method of the present invention.Most preferably, detected biological sample is a serum.
Ideally, be used for the material that the present invention measures and be applicable to the preparation kit.Thereby such kit can contain the carrier arrangement of being accepted with closing form by compartmentation, one or more case bottle, pipe etc.; Each case contains the element of a separation of using in method.For example, case can contain the suitable antigen (for example ECPKA (PKA C α or C α fragment)) that is attached to solid support or the extract of one or more dissimilar cancer cell and tumour).The second antibody of the mark that second container can contain is soluble, can detect is preferably with the form of freeze-drying, perhaps in solution.In addition, kit can also contain one or more container, and each container contains the ECPKA (PKA C α or C α fragment) or anti--ECPKA (the PKA C α) autoantibody of (different) scheduled volume.Container is planted in these backs can be used to the preparation standard curve, and curve can insert resulting result from contain the sample of unknown quantity at the autoantibody of ECPKA.
When using kit, the whole of the required work of user are exactly the sample of premeasuring is joined in the container, but described sample suspection contains can be measured the autoantibody at ECPKA of unknown quantity, the second antibody of the detectable mark of the holder conjugated antigen of the premeasuring that in first container, exists and the premeasuring that in second container, exists.Behind the incubation reasonable time, immune composition forms and separates from the supernatant fluid, by radiocounting, add zymolyte, and color development, perhaps detect immune complex or supernatant fluid by incorporating chemical labeling (for example collaurum, latex beads or the like) into.
Anti--ECPKA the autoantibody that The present invention be more particularly directed to use the immune chromatograph mode determination to detect.In preferred immune chromatograph mode determination, use two kinds of contacts but the different porous carrier in space.That first kind of such carrier will contain will be unfixed, the PKA C α of mark or C α fragment (ECPKA[C α] or protease digestion thing or C α), and second kind of such carrier will contain fixing still unlabelled antibody, this antibody (for example combines with IgG, wherein the people anti--when the ECPKA autoantibody was determined, unlabelled antibody can be anti-human IgG antibody).
Preferably, device will contain the shell of hollow, it is by manufacturings such as for example plastic materials, wherein the inside of first carrier and shell is not by can directly communicating (for example by stretch out from it or not exclusively covered by device) from the multilayer filter system that device arrives, like this, serum, blood plasma, perhaps the whole blood specimen can be applied directly to the filter system and infiltrate through first porous carrier from it.In such device, the infiltration that contains the fluid of anti--ECPKA autoantibody will cause that the PKA C α or the C α fragment of revocable mark of first carrier are attached on the antibody of migration, and infiltrates through second carrier subsequently.Because second carrier contains the fixing antibody in conjunction with human IgG, any PKA C α or C α fragment that enters the mark of second carrier will be hunted down wherein.
Contain the PKA C α of the mark in the carrier of fixing unmarked antibody or the detection of C α fragment and show that therefore anti--ECPKA autoantibody is existed by in the sample of estimating.The amount that is combined in mark PKA C α in second porous carrier or C α fragment by mensuration can be so that this mensuration becomes quantitative mensuration.
Described prevailingly after the present invention, can be by further understanding the present invention with reference to the following examples, these embodiment are that the mode that illustrates by way of example provides, unless otherwise noted, it can not be considered to limit the present invention.
Embodiment
The contrast that anti--ECPKA autoantibody immune detection and PKA enzyme detect in the diagnosis of embodiment 1 human cancer
Estimate
Materials and methods
The experimenter: blood serum sample is obtained from 295 people patient and 100 not cancered philtrums with dissimilar cancer cells.
Enzyme linked immunosorbent assay (ELISA) (ELISA): measure anti--ECPKA IgG autoantibody in cancer patient and the healthy individual serum by solid phase ELISA determination method.In order to carry out such mensuration, with (50 μ g/ml concentration are used PBS) antigen coated round bottom Polyvinylchloride of purification of recombinant human PKA C α microwell plate of the dilution of 100 μ l (Thermolab System, Helsinki, Finland).With these plates incubation 1 hour at room temperature, then use cleaning buffer solution (20mM Hepes, 0.9%NaCl, the pure albumin of 30mM sucrose-0.1% ox blood [BSA], pH 7.0) clean once, each hole is with the Blockace (Serotec of 100 μ l, http://www.serotec.com) aqueous solution (4g/400ml) was at room temperature sealed 2 hours, then use sodium citrate washing lotion (50mM sodium citrate, 0.15M NaCl, 0.1% polyoxyethylene sorbitan mono laurate fat tween  20, pH 5.0-5.2) plate is cleaned 2 times.With diluted sample damping fluid (PBS pH 7.4,0.25%BSA[be fatty acids level VI not], 0.05% polysorbas20) with serum samples diluted to 25,000 times, the sample of 100 μ l dilution is joined in each hole, 37 ℃ of following incubations are 1 hour subsequently.After cleaning 3 times, with anti-human IgG-HRP antibody-enzyme conjugate (the Jackson ImmunoResearchLaboratories of 20,000 times of PBS dilutions of 100 μ l with the sodium citrate washing lotion, West Grove, PA) (1%BSA) join in the hole, then, with plate incubation 1 hour at room temperature.After with sodium citrate washing lotion clean plate 5 times, add the tmb substrate of 100 μ l.With the 0.45M sulphate reagent cessation reaction of 100 μ l, then on ELISA reader (BioRad[Hercules, CA] micropiate reader benchmark), be recorded in the absorption of 450nm.By simultaneously suppressing to test the specificity that confirms ELISA at blood serum sample, wherein blood serum sample at room temperature with final concentration be 1mg/ml above-mentioned antigen incubation 1 hour.The purifying of PKA C α: will from OT1529-C α plasmid (Cho, Y.S.et al.[2000] " EXTRACELLULAR PROTEIN KINASE A As A CANCER BIOMAKER:ITS EXPRESSION By TUMOR CELLS AND REVERSAL BY AMYRISTATE-LACKING C ALPHAAND RII BETASUBUNITOVEREXPRESSION; " Proc Natl Acad Sci USA.97[2] 835-40) recombined human PKAC α (1.1kb) inject (infuse) thus pQE31 DNA produces pQE-C α (Hong, S.H.SeoulNational University, Korea S does not deliver).PQE-C α plasmid is at expression in escherichia coli and realized the purifying (Paragon, Baltimore MD) of natural PKA C α albumen.PKA measures: the method for the measurement of PKA enzymatic activity by Rohlff C. (1993) etc. (" 8-Cl-cAMPINDUCES TRUNCATION AND DOWN-REGULATION OF THE RIALPHA SUBUNIT AND UP-REGULATION OF THE RII BETA SUBUNITOF CAMP-DEPENDENT PROTEIN KINASE LEADING TO TYPE IIHOLOENZYME-DEPENDENT GROWTH INHIBITION ANDDIFFERENTIATION OF HL-60 LEUKEMIA CELLS; " J Biol Chem, 268:5774-82).In order to measure serum ECPKA activity, will measure (cumulative volume 50 μ l) under 37 ℃ reaction mixture (contain 50mM Tris HCI[pH 7.5], 1mMDTT, 10mM MgCl 2, 5 μ M kemptide (Kemptide) [the synthetic substrates of a kind of PKA; GIBCO/BRL], 1.2 μ M[γ- 32P] ATP[25Ci/mmol; ICN], have or do not have 5 μ M cAMP and 5 μ M PKI) and 10 μ l serum in carried out 20 minutes.Behind incubation, the reaction mixture point is coated onto cellulose phosphate plate (disk) (GLBCO/BRL) goes up and with 0.5% phosphoric acid cleaning three times.Filter is air-dry and count with liquid scintillation counter.The PKA activity of a unit is defined in 37 ℃ the standard test system in one minute time 1pmol 32P from (γ- 32P) ATP transfers to the amount of the enzyme that reclaims albumen.LDH is active to be measured by commercial kit (Sigma).
The result
Use above-mentioned ELISA to measure the serum of people patient and normal control individuality, anti--ECPKA autoantibody titre is expressed as the ratio to the average absorption degree of normal control serum arbitrarily.Than the anti--ECPKA autoantibody titre of normal control, it is positive that the ratio greater than 1.0 is considered to.
Mensuration is repeatably, in its flow process between (within-run) and flow process the CV of (between-run) be respectively＜8.8% and＜9.0%.Fig. 1 has shown the numerical value of anti--ECPKA autoantibody in cancer patient's serum.Patient's frequency and average titer (frequency=92%, average titer 2.2) all are significantly higher than normal control (frequency=14%, average titer 0.60).
Receiver operating characteristic (ROC) curve (Fig. 2) has diagrammatized the sensitivity and the specificity of the ELISA mensuration of calculating at different cutoffs.At joint, the cutoff of anti--ECPKA autoantibody titre is 1.0 (Fig. 1), and susceptibility and specificity that ELISA measures are respectively 90% and 89%.
Western blotting identifies in cancer patient's serum anti--ECPKA autoantibody (Fig. 3).The patients serum of Xuan Zeing has shown the immune cross-reactivity (Fig. 3, band 2-7) to purifying PKA C α albumen (40kDa) at random, yet does not observe this immune cross-reactivity (Fig. 3, band 8-12) to C α albumen in normal serum.
Frequency and the average value of ECPKA enzymatic determination (it measures antigen concentration) between cancer patient (n=66) and normal control (n=66) obtained significant overlapping (patient, frequency=83%, mean values=130mU/ml; Normal control, frequency=21%; Mean values=60mU/ml), show to lack susceptibility and specificity (Fig. 4).
By the individuality that ELISA obtains anti--ECPKA autoantibody titre with by relatively seeing Fig. 5 between the ECPKA of PKA enzymatic determination measurement.There is not association between anti--ECPKA IgG antibody titer that obtains by ELISA and the ECPKA titre (measurement of ECPKA antigen) measured by enzymatic determination.
Embodiment 2 is about the conclusion of anti--ECPKA autoantibody immunoassays and the purposes in human cancer thereof
The present invention confirms to point to existence and the cancer height correlation of autoantibody in serum of ECPKA.For anti--ELISA that the ECPKA autoantibody is developed is extremely sensitive and special, it has shown very high (average titer 2.2 in the cancer patient, frequency 92%) anti--ECPKA autoantibody titre, and in the normal individual contrast, show low or negative titre and frequency (14%).By the frequency of ELISA detection and by the relatively demonstration between the frequency of enzymatic determination detection, compare with the enzymatic determination of measuring antigen active, the ELISA that detects the ECPKA autoantibody is extremely sensitive and special.
In order to check whether autoantibody detection method of the present invention can extend to other cancer antigen (cell exocrine), carries out following experiment: HCT-15 tumour (the multi-drug resistance human colon carcinoma of growth on the nude mice) is used as the cancer antigen source.With tumour at damping fluid #10 (20mM Tris-HClpH7.4,100mMNaCl, 0.5% NaTDC, 5mM MgCl 2, protease inhibitors cocktail combination I[Calbiochem]) middle homogenate (1: 3), 1 part of tumour (weight), 3 portions of damping fluids (volume).With homogenate 10, under the 000rpm centrifugal 10 minutes, and collect supernatant (extract).With extract and albumin A agarose (3: 1) incubation, 4 ℃ of down rotations 1 hour, with preparation with 6, centrifugal 2 minutes of 000rpm.Collect supernatant and 4 ℃ of dialysed overnight in PBS.After the dialysis, extract can be ready for use in ELISA and wrap by microtiter plate.Fig. 6 has shown the titre from anticancer antigen-antibody in cancer patient (n=36) and healthy people (n=25) serum.Numerical value is positive greater than 1.5 (dotted lines).Fig. 7 has shown the titre of the antigen-antibody that observes in the serum from the patient who suffers from dissimilar cancers (lung, kidney, melanoma, pancreas, ovary, colon, liver, stomach, bladder, and cervical carcinoma, and sarcoma, and liomyoma).Patients serum (n=36) and normal serum (n=25) are identical with among Fig. 6 those.
These results prove that the cancer antigen that uses autoantibody detection method of the present invention to detect in the humans and animals is possible.Resulting result's demonstration detects autoantibody with ELISA, rather than detects antigen and other cancer antigen of ECPKA, and the new way of cancer diagnosis in humans and animals is provided.
Publication and patent that all are mentioned in instructions all are incorporated by reference thereto, as each independent publication or patented claim all by distinguishingly and respectively in conjunction with as a reference.
Though invention has been described in conjunction with its specific embodiments, should be appreciated that it can further be revised, and the application is intended to contain and follows principle of the present invention usually, and comprise for example depart from disclosure of the present invention but but under the present invention the field known or the convention scope in and be applicable to the of the present invention various variants of the essential feature of illustrating hereinbefore, purposes, and change.

Claims (21)

1. measure the anti--existence of ECPKA autoantibody or immunoassay of concentration for one kind in mammiferous biological sample, wherein said immunoassay comprises the following step:
(a) described biological sample is contacted with the antigen that is specific to anti--ECPKA autoantibody, described contact be enough to allow to resist-the ECPKA autoantibody is in case be present under the situation in the described sample, combine and forms under the condition of antigen-resist-ECPKA autoantibody compound with described antigen and carry out;
(b) antigen of described formation-anti--ECPKA autoantibody compound contact with anti--ECPKA autoantibody binding molecule, described contact be enough to allow described anti--ECPKA autoantibody binding molecule is attached under the condition of compound of the anti--ECPKA autoantibody of antigen-anti--ECPKA autoantibody compound of described formation and formation extension and carries out; With
(c) existence or the concentration of the compound of the extension by determining described formation are determined described resisting-existence or the concentration of ECPKA autoantibody in described biological sample.
2. the immunoassay of claim 1, the wherein said antigen that is specific to anti--ECPKA autoantibody is extracellular PKA albumen.
3. the immunoassay of claim 1, wherein said anti--the ECPKA autoantibody is the antibody of mammalian species, they are different with described mammiferous antibody and be specific to antibody by described mammal generation.
4. the immunoassay of claim 3, wherein said mammal be the people and also described anti--the ECPKA autoantibody is the human IgG antibody.
5. the immunoassay of claim 1, wherein said anti--ECPKA autoantibody binding molecule can be detected ground mark.
6. the immunoassay of claim 5, wherein said detectable mark is a chemical labeling.
7. the immunoassay of claim 1, wherein in described step (a), the described antigen that is specific to anti--ECPKA autoantibody be fixed on the solid support before described biological sample takes place by described the contact.
8. the immunoassay of claim 1, wherein in described step (a), the described antigen that is specific to anti--ECPKA autoantibody be fixed on the solid support after described biological sample takes place by described the contact.
9. the immunoassay of claim 1, immunoassay wherein is the immune chromatograph immunoassay, wherein:
In described step (a), described biological sample is placed with first porous carrier and contacts, the antigen that described first porous carrier contains is revocable, be specific to the mark of anti--ECPKA autoantibody;
In described step (b), the antigen of described formation-anti--ECPKA autoantibody compound is placed with second porous carrier and contacts, described second porous carrier communicates with described first porous carrier, and contains fixing anti--ECPKA autoantibody binding molecule; With
In described step (c), determine described resisting-existence or the concentration of ECPKA autoantibody in described biological sample in the existence of the antigen of the described mark that is specific to anti--ECPKA autoantibody described in described second porous carrier by detection.
10. immune complex, its contain be attached to being specific to of anti--ECPKA autoantibody anti--antigen of ECPKA autoantibody, wherein said anti--the ECPKA autoantibody also additionally is attached on anti--ECPKA autoantibody binding molecule.
11. the immune complex of claim 10, the wherein said antigen that is specific to anti--ECPKA autoantibody is extracellular PKA albumen.
12. the immune complex of claim 10, wherein said resisting-ECPKA autoantibody binding molecule can be detected ground mark.
13. the immune complex of claim 12, wherein said detectable mark is a chemical labeling.
14. the immune complex of claim 10, wherein said resisting-ECPKA autoantibody binding molecule is an immune molecule.
15. the immune complex of claim 14, wherein said resisting-the ECPKA autoantibody is people's autoantibody, and described resisting-ECPKA autoantibody binding molecule is anti-human IgG antibody.
16. kit, the existence or the concentration of it is used for measuring mammiferous biological sample anti--ECPKA autoantibody, wherein said kit contains and comprises the shell that multilayer filters the hollow of system, with first and second porous carrier, wherein said second porous carrier communicates with described first porous carrier, and described first porous carrier communicates with described multilayer filtration system, and its part can reach from described shell, wherein:
That described first porous carrier contains is revocable, the PKAC α or the C α fragment of mark; With
Described second porous carrier contains fixing, the unlabelled antibody that is attached to human IgG.
17. the kit of claim 16, wherein said PKA C α is ECPKA.
18. the kit of claim 16, the described mark of the PKA C α of wherein said mark is a chemical labeling.
19. the kit of claim 16, wherein said kit detect the people anti--ECPKA autoantibody, and described antibody in conjunction with human IgG is inhuman mammiferous antibody.
Diagnose the method for cancer of non-human mammal 20. utilize the immune complex of any one among the claim 10-15.
Diagnose the method for cancer of non-human mammal 21. utilize the kit of any one among the claim 16-19.
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