JP2000279174A - Monoclonal antibody against human LDL receptor and method for determining familial hyperlipidemia using the same - Google Patents

Abstract

(57) [Summary] [PROBLEMS] To provide an excellent means for judging familial hypercholesterolemia. SOLUTION: In human lipoprotein receptor,
From the region of the amino acid sequence specific only to the human LDL receptor, a peptide selected as a peptide consisting of a sufficient number of amino acids that can be recognized to be at least specific to only the human LDL receptor as an antigenic determinant,
A monoclonal antibody that inhibits binding between human LDL and human LDL receptor; and a hybridoma that produces the monoclonal antibody. The human LDL utilizes an antigen-antibody reaction between the monoclonal antibody and a human LDL receptor in a sample. A method for detecting an abnormality in the binding ability between a receptor and human LDL, and a method for determining hyperlipidemia based on the detection method, which determines the presence or absence of familial hyperlipidemia in a sample donor. It has been found that the above-mentioned problem can be solved by performing.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a monoclonal antibody and an invention in the technical field relating to a detection method for detecting information relating to a disease in a specimen using the same. More specifically, the present invention is an invention in the above technical field for human LDL receptor.

[0002]

BACKGROUND OF THE INVENTION Abnormal metabolism of serum lipids leads to abnormalities in the blood levels of serum lipids and, consequently, to certain pathological symptoms. In particular, arteriosclerosis and ischemic heart disease are cited as diseases caused by abnormal metabolism of serum lipids. In addition, abnormalities in serum lipid metabolism causing these diseases present various clinical symptoms, and the diagnosis thereof is also various.

[0003] Among serum lipid metabolism disorders, familial hypercholesterolemia is a typical autosomal chromosome characterized by hypercholesterolemia and early-onset coronary artery disease caused by qualitative and quantitative abnormalities of LDL receptor. It is a dominantly inherited disease. In addition, familial combined hyperlipidemia is characterized by elevated blood cholesterol and triglyceride, and, like familial hypercholesterolemia, causes coronary artery disease and arteriosclerosis, and impairs familial lipid metabolism. It is a disease.

[0004] Among these dyslipidemias, familial hypercholesterolemia is known to cause coronary artery disease at a high frequency, and it is characteristic that it occurs in a young age. Homozygous familial hypercholesterolemia, in which both the inherited alleles are abnormal, is about
It is rare, one in one million, and the serum cholesterol level is extremely high (generally, serum cholesterol level, about 1000 mg / dl), and the discrimination is easy.

[0005] In contrast, heterozygous familial hypercholesterolemia, in which one of the alleles is inherited from an abnormal gene, is a hereditary disease that is frequently observed in daily medical care. It is estimated that approximately 250,000 heterozygous familial hypercholesterolemia patients exist in Japan. This heterozygous familial hypercholesterolemia, unlike other types of hyperlipidemia, has a clear cause and is originally identifiable hyperlipidemia. It is known that it is difficult to discriminate from other hyperlipidemia especially at a young age, as described later.

[0006] In general, treatment of familial hypercholesterolemia is diagnosed at an early stage and a corresponding drug, for example,
HMG-CoA reductase inhibitors, fibrate drugs,
It is preferable to start the treatment as early as possible using an antioxidant or the like in order to prevent the development of coronary atherosclerosis that progresses with age and the development of arteriosclerosis.

[0007]

It is undeniable that the above-mentioned difficulty in diagnosis of heterozygous familial hypercholesterolemia at a young age hinders proper management of this familial disease. . In addition, knowing the genetic constitution from a young age makes it possible to control hypercholesterolemia by paying attention to eating habits and moderate exercise in the sense of preventing coronary artery disease.

[0008] Indices of familial hypercholesterolemia currently include, in addition to hypercholesterolemia, the presence or absence of tendon xanthoma, premature coronary artery disease, etc., a family history and the like. Except for family history and hypercholesterolemia, it is a symptom of hypercholesterolemia that has already progressed considerably. In particular, it cannot be denied that these indexes alone are insufficient as diagnostic indices for heterozygous familial hypercholesterolemia, in which the rise in serum cholesterol at young age is hardly remarkable.

At present, as a means for judging a new familial hypercholesterolemia, in addition to the above judgment criteria,
By culturing tissues or cells collected from biomaterials, the cells express the LDL receptor, measure the binding, uptake, and catabolism of LDL labeled with radioisotopes and compare them with those of cells from healthy subjects. The determination is made by measuring LDL receptor activity. In addition, P using genomic DNA obtained from a patient
It is determined by directly determining the presence or absence of an LDL receptor mutation by the CR method.

However, since the former uses radioisotopes, it is impossible to apply it, for example, it requires special facilities, and the procedure is complicated and requires skill.
In the latter, since about 350 or more types of LDL receptor gene mutations have been discovered so far, it is difficult to determine each of them by a molecular biological technique such as PCR. Difficult in terms of time and expense.

An object of the present invention is to provide a means for determining familial hypercholesterolemia which does not have the above-mentioned disadvantages.

[0012]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors have developed familial hypercholesterolemia in which blood LDL is specifically increased (in a broad sense: hyperlipidemia belonging to type II). The main cause of LDL and LD are LDL receptors expressed on cell membranes such as hepatocytes and peripheral cells in order to take up LDL or abnormalities of apoprotein B-100 in LDL which is a receptor ligand.
Focusing on the fact that it is not possible to bind to the L receptor, intensive studies were conducted. As a result, the present invention provides a monoclonal antibody capable of directly and specifically relating to the binding between the LDL receptor and LDL, and further provides a method for specifying the binding property between the LDL receptor and LDL using the monoclonal antibody. As a result, it has been found that this problem can be solved.

That is, the present inventor has recognized that, in the present application, the human lipoprotein receptor can be recognized from the amino acid sequence region specific only to the human LDL receptor to be at least specific only to the human LDL receptor. A peptide selected as a peptide consisting of a sufficient number of amino acids is used as an antigenic determinant, and human LDL
(Hereinafter referred to as "monoclonal antibody of the present invention") and a hybridoma producing the same (hereinafter referred to as "hybridoma of the present invention"), comprising the monoclonal antibody of the present invention and human LDL in a sample. Human LDL receptor and human LD utilizing antigen-antibody reaction with receptor
A method for detecting an abnormality in the ability to bind to L (hereinafter referred to as the detection method of the present invention); and furthermore, a human LDL receptor abnormally detected by the detection method of the present invention and the monoclonal antibody of the present invention, or human LDL. A method for determining hyperlipidemia, which determines the presence or absence of familial hyperlipidemia of a sample donor due to abnormal binding between a receptor and human LDL (hereinafter referred to as the method for determining hyperlipidemia of the present invention) I will provide a.

[0014]

Embodiments of the present invention will be described below. A. The monoclonal antibody of the present invention or the hybridoma of the present invention: As described above, the monoclonal antibody of the present invention is specific to at least the human LDL receptor from the amino acid sequence region specific to the human LDL receptor in the human lipoprotein receptor. A monoclonal antibody which uses a peptide selected as a peptide comprising a sufficient number of amino acids that can be recognized as an antigenic determinant and inhibits binding between human LDL and a human LDL receptor.

That is, the monoclonal antibody of the present invention comprises
Not only a monoclonal antibody against the human LDL receptor, but also a monoclonal antibody having at least the property of inhibiting the binding between human LDL and the human LDL receptor.

As a precondition for obtaining such a monoclonal antibody, it is necessary to select and use an appropriate antigenic determinant. In the present invention, a monoclonal antibody having desired properties can be obtained by using, as an antigenic determinant, a peptide selected from an amino acid sequence region specific to only a human LDL receptor in various human lipoprotein receptors. Reached.

The "region of the amino acid sequence specific only to the human LDL receptor" selected by the present inventors means the region represented by SEQ ID NO: 1. From such region, at least the human LDL receptor An amino acid sequence sufficient to be recognized as being specific only to the antigen can be selected as a desired antigenic determinant.

The criteria for selecting a peptide that satisfies the condition that "at least it can be recognized that it is specific only to the human LDL receptor" are determined based on the three-dimensional structure of the peptide portion of the human LDL receptor as well as the specific sequence. There are a wide variety of locations and electrical characteristics. Therefore, the amino acid sequence of the peptide to be the antigenic determinant is to be determined individually and specifically, and is not particularly limited. If it is intentionally expressed as the number of amino acids constituting the peptide, 5 to 30 amino acids, preferably 17
If a peptide composed of about amino acids is selected, it is generally specific only to the human LDL receptor. Therefore, the number of amino acids of the selected peptide is preferably 5 to 30 amino acids, preferably about 17 amino acids.

The amino acid sequence of the peptide selected from SEQ ID NO: 1 and contained in the amino acid sequence constituting the original human LDL receptor may be changed, if necessary, within a range not to lose the desired antigenic determinant properties. It can be modified and used as an antigenic determinant.

Specifically, as a peptide selected from the region of the amino acid sequence represented by SEQ ID NO: 1, for example, the 7th to 25th amino acids of the amino acid sequence represented by SEQ ID NO: 1
~ 37th, 47 ~ 60th, 68 ~ 80th, 83 ~
94th, 96th to 107th, 107th to 119th, 1st
46th-158th, 159-176th, 195-20
7th, 210-221st, 222-233th, 2
34th to 246th, 256th to 275th and 275-2
A peptide consisting of an amino acid sequence selected from the group consisting of the amino acid sequence selected from the group consisting of the 87th region is selected. Among these peptides, 159-1
It is preferable to select a peptide consisting of the 76th amino acid, based on the number of amino acids constituting the peptide. The peptide that can be selected as described above can be easily synthesized by a generally known peptide synthesis method, for example, a solid phase method or a liquid phase method. Of course, it is also possible to synthesize using a peptide synthesizer.

In addition, particularly when the number of amino acids in the selected peptide is large and is not suitable for peptide synthesis, other methods such as isolation and purification from isolated and purified human LDL receptor-expressing cells and the like can be used. A method of obtaining a desired peptide by subjecting a human LDL receptor protein to an enzymatic treatment or the like, a method of obtaining a peptide having the amino acid sequence from a recombinant obtained by incorporating a gene encoding the desired amino acid sequence, and the like. It is also possible to use. In addition, the amino acid sequence of the peptide obtained by such other methods can be modified as necessary, as long as the desired properties as the antigenic determinant are not lost.

A hapten can be bound to the above-obtained peptide in order to improve the immunoreactivity of the monoclonal antibody.
As this hapten, it is possible to arbitrarily select a substance that can be normally used as a hapten, for example,
Umbrella hemocyanin (KLH), chicken egg albumin (OVA), bovine serum albumin (BSA) and the like can be selected as haptens.

A cross-linking agent can be used to bind the above-mentioned peptide to the hapten. Such crosslinkers are
It can be appropriately selected according to the type of hapten to be selected. For example, when umbrella hemocyanin is selected as the hapten as in the examples described below, m-maleimide benzyl-N-hydroxysuccinimide ester (MBS) or the like can be used. In addition, necessary processing can be performed on the peptide according to the type of the crosslinking agent to be selected. For example, when the above-mentioned MBS is used as a cross-linking agent, it is necessary to add a cysteine residue having a sulfide bond required for a binding reaction to either of both ends of the above-mentioned peptide. The cysteine residue in the selected peptide is preferably protected by substituting it with another amino acid or attaching a protecting group.

In order to produce the monoclonal antibody of the present invention, animals immunized with the immunizing antigen obtained as described above are not particularly limited, and mice, rats, and the like can be widely used. It is desirable to select in consideration of compatibility with myeloma cells used for cell fusion.

Immunization can be performed by a general method, for example, by administering the above-mentioned immunizing antigen to an animal to be immunized intravenously, intradermally, subcutaneously, intraperitoneally or the like. Than,
Specifically, the above-described immunizing antigen is optionally used in combination with a usual adjuvant, and administered to an animal to be immunized several times about every two weeks by the above-described method to give an immunity for producing the monoclonal antibody of the present invention. Cells, such as spleen cells after immunization, can be obtained.

When a monoclonal antibody is produced, the myeloma cell as the other parent cell to be fused with the immune cell is already known, for example, SP2 / 0-Ag.
14, P3-NS1-1-Ag4-1, MPC11-4
5,6. TG1.7 (all derived from mouse); 210. R
CY. Ag 1.2.3 (from rat); SKO-00
7, GM15006TG-A12 (all derived from humans) and the like can be used.

Cell fusion between the above-mentioned immune cells and the myeloma cells is generally carried out by a known method, for example, the method of Kohler, G. and Milstein, C., Nature , 256 , 495.
(1975)).

More specifically, the cell fusion is carried out by using a generally known fusion promoter such as polyethylene glycol (PE).
G) or in the presence of Sendai virus (HVJ) or the like, a hybridoma is prepared in a usual culture medium to which an auxiliary agent such as dimethyl sulfoxide is added as necessary to improve the fusion efficiency.

The desired hybridoma can be isolated by culturing it in a usual selection medium, for example, a HAT (hypoxanthine, aminopterin and thymidine) medium. That is, hybridomas can be separated by culturing them in this selection medium for a time sufficient to kill cells other than the target hybridomas. The hybridoma thus obtained can be subjected to a search for a target monoclonal antibody and a single cloning by a usual limiting dilution method.

The target monoclonal antibody producing strain can be searched by, for example, ELISA, plaque, spot,
It can be performed according to a general search method such as an agglutination reaction method, an octalony method, and an RIA method.

The hybridoma of the present invention that produces the monoclonal antibody of the present invention that recognizes the human LDL receptor protein thus obtained can be subcultured in a usual medium, and is further stored in liquid nitrogen for a long time. You can also.

The monoclonal antibody of the present invention can be collected from the hybridoma of the present invention by culturing the hybridoma according to a conventional method to obtain a culture supernatant, or by administering the hybridoma to an animal that is compatible with the hybridoma. A method of growing the cells and obtaining the ascites can be used.

[0033] In vitro, the immune cells were replaced with human LDL.
The monoclonal antibody of the present invention is cultured in the presence of the receptor protein or a part thereof, and after a certain period of time, using the above-described cell fusion means, to prepare a hybridoma between the immune cells and myeloma cells, and to screen for an antibody-producing hybridoma. (Reading, CL, J.Immun
ol. Meth. , 53, 261 (1982); Pardue, RL, et al., J. Cell.
Biol., 96 , 1149 (1983)).

The monoclonal antibody obtained as described above can be further purified by usual means such as salting out, gel filtration, affinity chromatography and the like.

The thus obtained monoclonal antibody of the present invention is an antibody having specific reactivity to the human LDL receptor protein. The expression "having specific reactivity with human LDL receptor protein" means that the monoclonal antibody of the present invention shows cross-reactivity with human LDL receptor protein but other human lipoprotein receptors such as VLDL receptor protein. Etc. means that they show no cross-reactivity. At the same time, when the monoclonal antibody of the present invention is a lipoprotein (human LD) containing apoB protein or apoE protein,
Lipoprotein in which L receptor protein is a ligand)
And LDL receptor binding can be inhibited. Furthermore, it also means that the binding between the monoclonal antibody of the present invention and the LDL receptor can be inhibited by a synthetic peptide containing a binding region of apoB protein or apoE protein as a ligand.

As described later, the monoclonal antibody of the present invention having such properties can be used not only clinically but also as a reagent for separating and purifying the LDL receptor protein itself.

The specific contents of the monoclonal antibody of the present invention will be described later in Examples. The thus obtained monoclonal antibody of the present invention can be labeled with a labeling substance, if necessary, and used in a measurement method or the like according to the present invention described later.

Such a labeling substance can be used alone or by reacting the labeling substance with another substance.
It is a labeling substance that provides a detectable signal, and specifically, for example, horseradish peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase , Fluorescent substances such as penicillinase, catalase, apoglucose oxidase, urease, luciferase or acetylcholinesterase, fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelates, dansyl chloride or tetramethylrhodamine isothiocyanate, ¹²⁵ I, ¹⁴ C, ³
Radioisotopes such as H; chemical substances such as biotin, avidin and digokesigenin; and chemiluminescent substances.

As the labeling method of the monoclonal antibody of the present invention with these labeling substances, a known labeling method can be appropriately used depending on the type of the labeling substance to be selected.

Further, the monoclonal antibody of the present invention (including a labeled antibody) may be immobilized on an insoluble carrier and used as an immobilized monoclonal antibody in a measurement method and the like according to the present invention described later.

In general, when a monoclonal antibody is subjected to various labeling treatments, the characteristics of the monoclonal antibody may be lost. It is characterized by not losing its ability to bind to the LDL receptor.

The monoclonal antibody of the present invention can be used by immobilizing it on an insoluble carrier, if necessary. As such an insoluble carrier, various insoluble carriers already used as an insoluble carrier for an antibody can be used. Specifically, for example, a plate represented by a microplate, a test tube, a tube, a bead, a ball,
Filter, membrane, or cellulosic carrier,
Insoluble carriers used in affinity chromatography, such as an agarose-based carrier, a polyacrylamide-based carrier, a dextran-based carrier, a polystyrene-based carrier, a polyvinyl alcohol-based carrier, a polyamino acid-based carrier, and a porous silica-based carrier.

In the method for immobilizing the monoclonal antibody of the present invention on these insoluble carriers, the method for immobilizing the antibody already established on various insoluble carriers is appropriately selected according to the type of the insoluble carrier to be selected. be able to.

B. The detection method of the present invention:
As described above, a method for detecting an abnormality in the binding ability between a human LDL receptor and human LDL, utilizing an antigen-antibody reaction between the monoclonal antibody of the present invention and a human LDL receptor in a sample. When the binding ability between human LDL receptor and human LDL is abnormal, the antigen-antibody reaction that should be originally observed is not observed between the monoclonal antibody of the present invention and the human LDL receptor. The detection method of the present invention detects this directly or indirectly.

Examples of the measurement method of the present invention utilizing such an antigen-antibody reaction include enzyme immunoassay, radioimmunoassay, analysis by flow cytometry, western blotting, and the like. Considering the simplicity of handling and the measurement sensitivity, as well as the specificity of the present measurement method, it is desirable to select a method based on analysis by flow cytometry.

The feature of the method by the analysis by flow cytometry is that, unlike the observation by a fluorescence microscope or the like (fluorescent antibody method) for detecting the cell surface antigen or the ELISA method by the enzyme antibody method, the fluorescence intensity of each cell is determined. Measured and measured fluorescence intensity can be expressed as a two-dimensional or three-dimensional point by optical parameters such as cell size and cell type.Moreover, high sensitivity measurement is possible and many The ability to measure cells. Further, the fact that the positive rate of the cells to which the fluorescently labeled antibody is bound can be easily obtained is the reason why the analysis by flow cytometry is preferable.

As described above, the "abnormal binding ability between human LDL receptor and human LDL" to be detected by the detection method of the present invention is usually a phenomenon caused by genetic factors. In a group of patients having abnormal LDL receptor abnormally, the expression of LDL receptor protein in cells expressing LDL receptor, for example, leukocytes, hepatocytes, skin fibroblasts, etc., is about half that of normal subjects, or not at all. It is clear that it is missing. In the detection method of the present invention, it is extremely preferable that the protein expressed on the surface of the above-described LDL receptor-expressing cell obtained from a patient suspected of having abnormal LDL receptor can be quantitatively measured. In this regard, detection with a flow cytometer is useful in that labeled antibodies bound to individual cells can be quantified.

Further, it is possible to determine the presence or content of familial hyperlipidemia of the sample donor based on the abnormality in the binding ability between the detected human LDL receptor and human LDL as described above ( The method of the present invention for determining hyperlipidemia).

That is, in the method of the present invention for determining hyperlipidemia, either one of the parameters indicating the abnormal binding ability between the detected human LDL receptor and human LDL and the parameter of a normal human is used. However, when it is about １ ／ of the parameter of a normal person, it is determined that the sample provider is a patient with heterozygous familial hypercholesterolemia. In addition, the parameter indicating the above abnormality shows an extreme value with respect to the parameter of a normal person, and the monoclonal antibody of the present invention and the L
When the antigen-antibody reaction with the DL receptor is hardly observed (including the case where the antigen-antibody reaction is not observed at all), it is determined that the sample provider is a patient with homozygous familial hypercholesterolemia. Is done.

As described above, according to the method for determining hyperlipidemia of the present invention, the presence or the content of familial hyperlipidemia of the sample provider can be determined. The specimen used in the method for determining hyperlipidemia of the present invention (including the detection method of the present invention) can originally express the LDL receptor.
The sample is not particularly limited as long as it contains the above-mentioned LDL-expressing cells. For example, any sample containing the above-mentioned leukocytes, hepatocytes, and skin fibroblasts is acceptable. However, considering the availability of the sample, it is preferable to select a blood sample containing leukocytes, particularly a blood sample containing leukocytes derived from peripheral blood as a main component.

In the present invention, a kit for determining familial hyperlipidemia containing the monoclonal antibody of the present invention for use in the method of determining hyperlipidemia of the present invention (including the detection method of the present invention). [Hereinafter referred to as the kit for determining hyperlipidemia of the present invention (kit for measurement of the present invention)].

In the kit for determining hyperlipidemia of the present invention,
Elements for providing the monoclonal antibody of the present invention for detection of LDL receptor protein abnormality in a sample, such as the above-described labeled monoclonal antibody of the present invention or immobilized monoclonal antibody of the present invention, may be included.

The measurement kit of the present invention may also be a kit having a structure that can be easily determined and detected by flow cytometry in accordance with the above-described method for determining hyperlipidemia (detection method of the present invention). preferable.

[0054]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to Examples, but the Examples should not be construed as limiting the technical scope of the present invention. 1. Construction of LDL Receptor Activity Measurement System In the present invention, establishing an accurate LDL receptor expression protein measurement system is indispensable for appropriately evaluating experimental results.

In this example, a measurement system constructed according to the method of Ranganathan et al. (J Lab Clin Med, 125: 479-86, 1995) was used as a measurement system for LDL receptor expression protein. In Examples, the expression level of LDL receptor protein was quantified by this measuring system unless otherwise specified.

Hereinafter, the contents of this measuring system will be described in detail.
Will be described.Establishment of LDL receptor expressing cells 10 ml of whole blood ACD collected from peripheral blood
Layered on a board L (Muto Chemical), using a Kubota centrifuge 8900
And centrifuged at 1000 rpm for 40 minutes. After centrifugation, intermediate
The lymphocyte layer of the layer was collected with a pipette. Collected lymph
The spheres were washed twice with 10 ml of RPMI medium (manufactured by Gibco).
Was. After washing, 1% BSA (bovine serum albumin: Sigma)
Cells) in an RPMI medium containing⁶Pieces/
Adjust to a concentration of 25 ml, 25 cm^TwoIn this culture flask,
6 ml of the mixture, and at 37 ° C., 5% CO 2 _TwoIncubator
And cultured for 3 days. After culturing, remove the cells by centrifugation
Into a tube (Falcon) and run at 1500 rpm for 10 minutes.
I thought. After centrifugation, the cells were further washed with R containing 1% BSA.
After washing twice with PMI medium, LDL receptor derived from peripheral blood
-Expressing cells were obtained as monocytes.

<Detection of protein expressing LDL receptor>
The LDL receptor protein-expressing cells obtained as described above are prepared into a cell suspension of 1 × 10 ⁶ cells / ml with ice-cooled PBS containing 1% BSA (phosphate buffer, pH 7.4). did. Further, the cell suspension (100 μl
) Was collected in a microfuge tube (manufactured by Eppendorf). Add 10 μg / ml of diluted L
A fluorescently labeled antibody against the DL receptor (such as the monoclonal antibody of the present invention) was added and reacted at 4 ° C. for 30 minutes. After completion of the reaction, unreacted antibody was removed by ice-cooling 1%
After washing three times with PBS containing BSA, the cells were suspended in 500 μl of PBS containing 1% BSA.

[0058] Flow flow detection of the antibody bound to the LDL receptors expressed in the detection cell membrane surface at cytometer cytometer FACScan (Becton
Dickinson) and measuring the fluorescence intensity of the antibody bound to the cells. That is, the cells reacted with the antibodies as described above were subjected to laser power 15 W and excitation wavelength 4 using a flow cytometer.
It was measured under the conditions of 88 nm, a fluorescence wavelength of 530 nm, and a PMT voltage of 500 mV, and the forward scattered light (FSC) and side scattered light (SS
Gating lymphocyte and monocyte cell regions from C), FITC phosphor (FL1), PE phosphor (FL2)
Was measured for fluorescence intensity. The fluorescence intensity of the cells can be obtained by measuring the fluorescence intensity of 10,000 cells. The measured fluorescence intensity of the cells was analyzed using the analysis software CELLQUEST of the connected computer.

The average fluorescence intensity calculated from the fluorescence intensity of the cells obtained by the flow cytometer was determined as the amount of LDL receptor protein expressed in the cells. 2. Preparation of hybridoma of the present invention Preparation of KLH-binding peptide 16 mg of KLH (keyhole limpet hemocyanin: Pierce) was added to 1 ml of 10 mM sodium phosphate buffer (p
H7.2), and after the dissolution, centrifugation was performed at 16,000 rpm for 5 minutes, and the supernatant was transferred to a test tube. Then
MBS (m-maleimidbenzoyl-N-hydrosuccin) dissolved in dimethylformamide to a concentration of 0.3 g / ml
100 μl of imede ester (Pierce) was added to the test tube and stirred at room temperature for 30 minutes. This MBA solution was transferred to a 1.5 ml tube (manufactured by Eppendorf) and centrifuged at 4 ° C. and 16,000 rpm for 5 minutes. The supernatant thus obtained was previously packed in a gel filtration column (inner diameter 1 cm × 30 cm: manufactured by Bio-Rad) at 4 ° C., and 50 mM sodium phosphate buffer (pH 6.0) was added.
And equilibrated on a column of Sephadex G25 (Pharmacia), and equilibrated with 50 mM sodium phosphate buffer (p
H6.0) and eluted in 1 ml portions. KLH-MB
For fractions, 1 μl of each fraction was spotted on a PVDF membrane, and then the PVDF membrane was washed with a methanol-acetic acid solution (50/5 v).
/ v). Further, the PVDF membrane is coated with 0.1% CB.
It was immersed in methanol containing B for about 10 seconds, and then decolorized using the methanol-acetic acid solution described above. The fraction stained blue in this way is designated as KLH-MB by K
The LB-MB fractions were pooled, aliquoted into 0.5 ml aliquots into 1.5 ml tubes and stored at -80 ° C.

Binding of Synthetic Peptide and KLH-MB 1 mg of the synthetic peptide represented by SEQ ID NO: 2 synthesized by a peptide synthesizer was dissolved in distilled water (0.5 ml) to a concentration of 2 mg / ml. And the KLH-
Added to MB and 0.25 ml of 0.2 M disodium phosphate solution. The reaction solution was blown with argon gas, and the reaction solution tube replaced with argon gas was shaken at room temperature for 3 hours. The reaction solution thus prepared was used as a KLH-binding synthetic peptide. The reaction solution was divided into 10 equal parts in 1.5 ml tubes, stored at -20 ° C, dissolved at the time of use, and used for the following immunization.

Preparation of Hybridoma The above KLH binding peptide (LDL receptor amino acid 1)
58-178) were mixed with an equal volume of Freund's complete adjuvant, and 0.2 ml (containing 25 μg of the binding peptide) of a 6-week-old Balb / c mouse (female, Nippon SLC)
Was immunized into the abdominal cavity. Two weeks later, the mixture was similarly mixed and immunized using Freund's incomplete adjuvant, and thereafter, immunization was performed once every two weeks for a total of six times.

3 × 10 4 spleen cells of the mouse 3 days after the final immunization
^Eight cells were used as mouse myeloma cells (SP2 / 0-AG14).
(8 × 10 ⁷ cells)), and cell fusion was carried out using a HEPES solution containing 50% polyethylene glycol (average molecular weight 1500: manufactured by Boehringer Mannheim).
The number of cells on the plate was adjusted with 10% FCS-containing RPMI medium to which HAT reagent (manufactured by Sigma) was added so that the number of cells per well was 5 × 10 ⁵ cells, and selective culture was performed for 7 days. However, hybridoma growth was observed in almost all wells.

Screening by ELISA Method The presence or absence of a monoclonal antibody against the human LDL receptor in the culture supernatant of each hybridoma was determined by an ELISA method using a peptide having the amino acid sequence represented by SEQ ID NO: 2 used as an immunizing antigen as an antigen. Screened.

That is, first, a peptide having the amino acid sequence represented by SEQ ID NO: 2 was added to each well by 250 ng.
It was immobilized on a 96-well microplate (referred to as immobilized peptide plate). Then, 1% skim milk (D
200 μl of a PBS solution containing 0.1% NaN ₃ and 0.1% NaN ₃ were added to each well to block the portion of the peptide that was not immobilized. Plates are 0.1% Tween
After washing four times with a PBS solution containing n20, the culture supernatant of the hybridoma (0.1% Tw) was added to each well of the plate.
50 μl each of which was added at room temperature.
The reaction was carried out for 1 hour (primary reaction).

Next, the plate was placed on a 0.1% Tween.
After washing four times with a PBS solution containing No. 20, a peroxidase-labeled rabbit anti-mouse IgG antibody (manufactured by Zymed Laboratories) diluted 4000 times with the same PBS solution was added.
The reaction was performed at room temperature for 1 hour (secondary reaction). Plate P
After washing four times with a BS solution, 100 μl of a 0.4 mg / ml OPD solution (manufactured by Sigma) containing 0.009% hydrogen peroxide was added to each well, and reacted in a greenhouse for 30 minutes (substrate reaction). After the reaction, add 25 μl of 6N sulfuric acid to each well.
In addition, the reaction was stopped. Then, the absorbance at a wavelength of 492 nm was measured with a microplate reader (manufactured by Lab System). As a result, a hybridoma culture supernatant having an absorbance of 1.0 or more was defined as a specific monoclonal antibody positive.

Screening by Western Blotting The supernatant of the hybridoma culture that showed a positive result in the above screening method was further subjected to Western blotting to a recombinant cell line expressing human LDL receptor [by a conventional method.
A cell line in which the human LDL receptor cDNA is incorporated into an expression vector and introduced into CHO (chinese hamster ovary) cells: hereinafter also referred to as human LDL receptor CHO cells: Kingsley DM and Krieger M, Proc Natl Acad
Sci USA, 81: 5454-5458, 1984]. The reactivity of the membrane fraction protein with the human LDL receptor protein was further confirmed.

That is, the recombinant CHO cells expressing human LDL receptor were prepared by adding 1% Triton X-100 and 2 mM
The cells solubilized in a PBS solution containing MSF at 4 ° C. for 30 minutes were centrifuged at 15000 rpm for 30 minutes, and the supernatant was used as a human LDL receptor membrane fraction.

The human LDL receptor membrane fraction obtained as described above was electrophoresed on a 7.5% acrylamide gel, and after electrophoresis, the protein was transferred to a PVDF membrane (Millipore). After the transfer, the PVDF membrane was made of PB containing 5% skim milk (manufactured by Difco) and 0.1% NaN _3.
After blocking with an S solution, 100 μl of each culture supernatant (containing 0.1% Tween 20) was added to each lane using a slot blotter (manufactured by Sunplate Tech), and the mixture was reacted at room temperature for 2 hours. After the reaction, the membrane is
After washing three times with a PBS solution containing 20% Tween 20, a 4000-fold diluted peroxidase-labeled rabbit anti-mouse IgG antibody (manufactured by Zymed) was added, and reacted at room temperature for 1 hour. After the reaction, the membrane was washed three times with a PBS solution containing 0.1% Tween 20, then a chemiluminescence reagent (manufactured by NEN Life Science Products) was added, and the film was exposed to a film (manufactured by Amersham) to give a specific A band was detected.

As described above, the HT reagent (manufactured by Sigma) and 10
In a 96-well microplate, RPMI1640 medium containing 10% FCS supplemented with 0.1% Hybridoma Cloning Factor (manufactured by IGEN) was added in an amount of 0.1% per well.
The operation of seeding five cells was repeated three times, and the hybridoma was cloned to obtain the monoclonal antibody-producing hybridoma of the present invention.

[0070] Of these hybridomas, 2
The hybridoma of the strain has been deposited with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology [mouse hybridom.
aLL8H6 (Deposit No .: FERM P-1724
0), mouse hybridoma LL12D1
0 (deposit number: FERM P-17239)]. 3. Preparation of Monoclonal Antibodies of the Present Invention Monoclonal antibodies purified in large quantities from Balb / c mice (female, 8 weeks old, 10 mice, Japanese SLC
Was injected intraperitoneally one week before the injection of hybridoma cells into Bristol (Wako Pure Chemical Industries, Ltd., 0.5 ml / mouse). The hybridomas obtained as described above [LL8H6, LL12D10 (5 × 10 ⁶ to 1 each)
× 10 ⁷ /0.5 ml / mouse)] were introduced into mice by intraperitoneal injection. Ten days after the introduction, the mice were laparotomized under anesthesia, and monoclonal antibodies (LL8H6, LL12D10) were each prepared in large amounts from the ascites fluid collected by a conventional method.

Determination of Isotype The isotype of the monoclonal antibody was determined using a mouse monoclonal antibody isotype determination kit (Sang Stat Medic).
cal Corporation) according to the protocol of the experiment attached to the kit, and the isotype of the monoclonal antibody (LL8H6, LL12D10) was determined.

As a result, the monoclonal antibody LL8H6
Has the isotype of IgG2a and LL12D10 of l
gG2b. Purification of monoclonal antibody The above-mentioned monoclonal antibody (LL8H6, LL12D1
0) Each of the centrifugal supernatants (5 ml each) was mixed with 5 ml of a saturated ammonium sulfate solution, cooled on ice, allowed to stand for 1 hour, and then centrifuged at 15000 rpm at 4 ° C. for 30 minutes. After centrifugation, the precipitate was washed with a binding buffer (3 M sodium chloride / 1.5 M glycine solution, p
H8.9), mixed with 1 ml of Protein A Sepharose (Pharmacia), and allowed to stand at 4 ° C overnight. The next day, protein A Sepharose was washed 6 times with 6 ml of binding buffer, then packed in a column, and eluted with 2 ml of elution buffer (0.1 M citric acid solution, pH 4.0). Tris solution (p
(H10.0) 0.2 ml. The absorbance (wavelength 280 nm) of each fraction was measured, and the eluted fraction of the monoclonal antibody was collected and dialyzed (4 ° C., overnight) with a phosphate buffer (pH 7.4) containing 0.02% NaN ₃ , A purified monoclonal antibody (LL8H6, LL12D10) was obtained.

Characterization of Monoclonal Antibody Reactivity to Human LDL Receptor The purified monoclonal antibody (L
The reactivity of (L8H6, LL12D10) to the human LDL receptor was confirmed by Western blotting using a cell membrane fraction prepared from human LDL receptor recombinant CHO cells, as well as human hepatocyte-derived HepG2 cells.

That is, human LDL receptor recombinant C
The concentration of the HO cell membrane fraction was 8 μg / lane, and the concentration of the HepG2 cell membrane fraction was 40 μg / lane. After boiling for 5 minutes in the presence of 5% mercaptoethanol (manufactured by Bio-Rad), 7.5% SDS polyacrylamide gel ( SDS-PAGE electrophoresis was carried out by using ATTO.
The gel was then transferred to a PVDF membrane. Further, the transfer membrane was blocked with a blocking reagent (PBS containing 5% skim milk, p
H7.4) at 4 ° C. overnight. The transfer membrane was washed five times with PBS containing 0.1% Tween 20 (pH 7.4), and then washed with PBS containing 0.1% Tween 20 (pH 7.4).
In 7.4), each purified monoclonal antibody (LL8H6, LL12D10) adjusted to a concentration of 2 μg / ml was reacted at room temperature for 2 hours.

After the reaction, the membrane was washed 5 times with PBS (pH 7.4) containing 0.1% Tween 20, and the membrane was washed with 0.1% Tween 20.
Secondary antibody (HRP-labeled anti-mouse IgG antibody: Zym) diluted 4000-fold with n20-containing PBS (pH 7.4)
ed) was allowed to react in a greenhouse for 1 hour. After the reaction, the membrane was washed with PBS containing 0.1% Tween 20 (pH 7.4).
5 times with Chemiluminescence Reagent (NEN Life Sci
ence Products) for 1 minute. Next, the chemiluminescence reagent was removed from the film, and after exposing the film to an X-ray film, the film was developed and a reaction band was detected.

As a result, each purified monoclonal antibody (L
The reactivity of (L8H6, LL12D10) with the human LDL receptor recombinant CHO cell membrane fraction and the HepG2 cell membrane fraction was confirmed to specifically recognize a protein of about 160 kDa. In addition, it was confirmed that each of the purified monoclonal antibodies (LL8H6, LL12D10) did not show reactivity with the protein against the CHO cell membrane fraction into which the LDL receptor had not been introduced as control cells (FIG. 1).

Reactivity to other human lipoprotein receptors In addition, other lipoprotein receptors belonging to the LDL receptor family, namely Very Low Density
Cross-reactivity with Lipoprotein (VLDL) receptor and apolipoprotein E receptor 2 (apoER2) was examined.

That is, a cell line obtained by incorporating human VLDL receptor and apoER2 cDNA into an expression vector and introducing them into CHO cells by a conventional method [VLDL receptor CHO cells and apoER2C, respectively.
HO cells: Takahashi S, et al, Proc Matl Acad Sci US
A, 89: 9252-9256, 1992; Kim DH, et al, J Biol Chem, 27
2: 8498-8504, 1997], the cell membrane fraction was prepared as described above, and SD was performed using 7.5% polyacrylamide gel.
S-PAGE electrophoresis was performed, and as described above, the above cross-reactivity was confirmed by Western blotting using a cell membrane fraction.

As a result, each purified monoclonal antibody (L
L8H6, LL12D10) showed no band reacting with the non-introduced CHO cell membrane fraction, VLDL receptor CHO cell membrane fraction, and apoER2CHO cell membrane fraction, which were the controls.
A band was detected in a protein of about 160 kDa only in the cell membrane fraction.
It was shown that only the DL receptor was specifically recognized (FIG. 2).

These facts indicate that human LDL receptor was recognized as a membrane protein of about 160 kDa from a similar experiment using hepatocytes (Beisiegel et al.).
al, J BiolChem, 257: 13150-13156, 1982, Leit
erdorf et al, Proc NatlAcad Sci, 85: 7912-79.
16, 1988), each purified monoclonal antibody (LL8H6,
LL12D10) was shown to be an antibody that specifically reacts with the human LDL receptor.

Labeling of Purified Monoclonal Antibody Labeling of each purified monoclonal antibody is preferably performed by a detection method using an antigen-antibody reaction in living cells as a reaction system. It is preferable to select a simpler reaction system in consideration of the measurement using a flow cytometer, which is a simple embodiment. That is, it is important to minimize the number of reaction steps in the reaction system between the membrane protein present on the cell surface and the monoclonal antibody of the present invention. In view of these conditions, as shown below, the following two methods are mainly considered as the reaction system to be selected.

That is, 1) a method in which a monoclonal antibody is directly labeled with a fluorescent dye or the like, and the labeled antibody is reacted with cells to measure using a flow cytometer (1 step method); A method (two-step method) in which an antibody bound to a cell membrane protein is measured using a flow cytometer by labeling with a compound and then reacting with fluorescently labeled streptavidin is considered.

The above-described one-step method and two-step method are based on a method of measuring an antibody reacted with a combination of a conventional mouse monoclonal antibody, a biotin-labeled goat anti-mouse antibody, and a fluorescence-labeled streptavidin with a flow cytometer (3 Compared to the step method, the operation is simpler, the sensitivity is further improved, and the method is considered to be excellent in reproducibility.

First, a labeling method for a labeled monoclonal antibody used in these reaction systems will be described. Biotin Labeling of Purified Monoclonal Antibody Each of the above purified monoclonal antibodies (LL8H6, LL1
2D10: 1 mg / ml) with 0.1 M NaHCO ₃ (p
H8.2-8.3) solution (4 ° C, overnight). Next, NHS-biotin (60 μl of 1 mg / ml, Pierce) was added, and the mixture was stirred vigorously and reacted at room temperature for 4 hours. Then, the mixture was dialyzed (4 ° C., overnight) with a phosphate buffer to prepare a desired biotin-labeled monoclonal antibody (hereinafter referred to as a biotin-labeled monoclonal antibody) for each monoclonal antibody.

Fluorescent Labeling of Purified Monoclonal Antibody Each of the above purified monoclonal antibodies (LL8H6, LL1
2D10: 2 mg / ml) with 0.1 M NaHCO ₃ (p
H8.2-8.3) solution (4 ° C./overnight). Next, a fluorescent dye FlTC (100 μl of 1 mg / ml, manufactured by Pierce Co.) was gradually added with stirring, and the mixture was stirred.
For 16 hours. Next, the obtained reaction product was subjected to gel filtration using a NAP-5 column (manufactured by Pharmacia) to remove unreacted fluorescent dye. Then
The gel-filtered reaction product was dialyzed (4 ° C.—overnight) with a phosphate buffer to prepare a desired FlTC-labeled monoclonal antibody (hereinafter referred to as FITC-labeled monoclonal antibody) for each monoclonal antibody. 4. Preparation of cells for detection of human LDL receptor by flow cytometer and three-step antigen-antibody reaction method (conventional method) Further, each purified monoclonal antibody (LL8H6, LL
12D10) was examined as to whether detection by a flow cytometer, which is a measurement method that focuses on the present invention, is possible.

That is, the monoclonal antibody of the present invention (L
L8H6, LL12D10) specifically binds to the human LDL receptor expressed on the cell surface and is capable of detecting an antibody bound to the receptor in a flow cytometer. It examined using.

The above human LDL receptor CHO cell
Was cultured in F12 medium containing 5% FCS, and the medium was removed.
Excluding 5 mM EDTA for 5 minutes in the presence of PBS containing EDTA
And the cells were detached from the dish. Then
Transfer the suspended cells to a 15 ml Falcon tube,
Centrifugation was performed at 1500 rpm for 10 minutes. Cells
These were washed twice with 15 ml of PBS containing 1% BSA. Most
Eventually, 1 × 10 cells ⁶1% BSA to a concentration of
This was adjusted with PBS containing the solution to obtain a cell suspension.

Further, 100 μl of this cell suspension was dispensed into a 0.5 ml microtube, and each purified monoclonal antibody (LL8H6, L
L12D10) and reacted at 4 ° C. for 30 minutes.
After the reaction, the cells were washed three times with PBS containing 1% BSA,
Unreacted purified monoclonal antibodies were removed. Then
Biotin-labeled goat anti-mouse IgG antibody (BlOSOURCE INTERNATIONA) diluted 500-fold with PBS containing 1% BSA
(Company L) was added, and reacted at 4 ° C. for 30 minutes.
After the reaction, the cells were washed three times with PBS containing 1% BSA,
Unreacted biotin-labeled goat anti-mouse IgG antibody was removed. Further, a PE-labeled streptavidin solution diluted 160-fold with PBS containing 1% BSA (Life Technolog
was added to the mixture, and the mixture was reacted at 4 ° C. for 30 minutes.
After the reaction, the cells were washed three times with PBS containing 1% BSA,
Unreacted PE-labeled streptavidin solution was removed, and the cells were finally suspended in 500 μl of PBS containing 1% BSA.

Two-Step Method The cell suspension of the human LDL receptor CHO cells prepared as described above was placed in a 0.5 ml microtube and placed in a microtube.
Each biotin-labeled purified monoclonal antibody (LL8H6, LL12D1
0) was added and reacted at 4 ° C. for 30 minutes. After the reaction, the cells were washed three times with 1% BSA-containing PBS to remove unreacted purified monoclonal antibodies. Then, the PE-labeled streptavidin solution diluted and prepared as described above was
μl was added and reacted at 4 ° C. for 30 minutes. After the reaction, the cells were washed three times with PBS containing 1% BSA to remove the unreacted PE-labeled streptavidin solution.
l of PBS containing 1% BSA.

One-Step Method The cell suspension of the human LDL receptor CHO cells prepared as described above was placed in a 0.5 ml microtube at 100 μl.
of each FITC-labeled purified monoclonal antibody (LL8H6, LL12D1
0) was added and reacted at 4 ° C. for 30 minutes. After the reaction, the cells were washed three times with PBS containing 1% BSA, and unreacted F
The ITC-labeled purified monoclonal antibody was removed, and the cells were finally suspended in 500 μl of PBS containing 1% BSA.

Measurement by Flow Cytometer As described above, the cells to which the antibody is bound by reacting with the antibody are measured for the fluorescence intensity of each cell using a flow cytometer, and the number of cells is determined to be 10,000 cells. By measuring the fluorescence intensity of, and performing analysis using the analysis program of CELLQUEST, it becomes possible to express the average fluorescence intensity, that is, the quantification of the target protein expressed in the cells, as the average fluorescence intensity.

As described above, the purified monoclonal antibodies (LL8H6, LL12D10) in the human LDL receptor CHO cells reacted by the three-step method were as follows:
The fluorescence intensity increased in a concentration-dependent manner with respect to the antibody concentration, and it was confirmed that the fluorescence intensity was maximum at the concentrations of 0.1 μg / ml and 1.0 μg / ml, respectively (1st.
See table: Numbers in table represent fluorescence intensity).

Mouse anti-LD recognizing control N-terminal region
L receptor antibody lgG-C7 (Amersham)
Is maximum at a concentration of 1.0 μg / ml, indicating that it is an antibody that recognizes the human LDL receptor with at least comparable sensitivity.

[0094]

Table 1 Table 1 Antibody concentration LL8H6 LL12D10 IgG-C7 (Μg / ml) ０．００ 0.00 94 94 94 0.01 612 312 280 0.10 1072 735 729 1.00 1094 853 891 10.00 1009 999 864 ─────── Similarly, each biotin-labeled purified monoclonal antibody (LL
8H6, LL12D10) human LDL receptor CH
Reactivity in O cells was examined in a two-step method. 3
Similar to the step method, each of the biotin-labeled purified monoclonal antibodies (LL8H6, LL12D10) showed an increase in fluorescence intensity in an antibody concentration-dependent manner, and was recognized to recognize human LDL receptor protein at a concentration of 1.0 μg / ml. (See Table 2: Numbers in table represent fluorescence intensity).

However, the IgG-C7 monoclonal antibody against the known human LDL receptor, which was used as a control, showed an increase in fluorescence intensity in an antibody concentration-dependent manner, but the maximum binding amount was 10.0 μg / ml.
At a concentration of about 1/5 of the above-mentioned biotin-labeled purified monoclonal antibodies (LL8H6, LL12D10).
This suggests that the monoclonal antibody itself is a vulnerable antibody to modification, and that labeling has reduced the binding activity of the antibody.

[0096]

TABLE 2 Biotin-labeled antibody concentration LL8H6 LL12D10 IgG −C7 (μg / ml) ０．００ 0.00 84 84 840 .01 276 195 106 0.10 820 640 165 1.00 1135 1045 207 10.00 1152 1107 214 ─────────────────────────── ───────── Furthermore, each FlTC-labeled purified monoclonal antibody (LL
8H6, LL12D10), human LDL receptor C
Reactivity in HO cells was examined by a one-step method. As a result, similarly to the three-step method, each FlTC-labeled purified monoclonal antibody (LL8H6, LL12D1
0) indicates that an increase in fluorescence intensity is observed in an antibody concentration-dependent manner,
Recognition of human LDL receptor protein was found at a concentration of 10.0 μg / ml (FIG. 3).

However, IgG-C7 did not show an increase in the fluorescence intensity dependent on the antibody concentration, and showed 10.0 μg / ml.
CHO without human LDL receptor
Fluorescence was observed only to the same degree as the fluorescence intensity of the cells. From this, FlTC-labeled IgG-C7 antibody,
It was shown to be unsuitable for the one-step method of detecting LDL receptor.

LDL receptor on peripheral blood mononuclear cells
As the detection above, from ACD collected blood, the mononuclear cells were separated by density centrifugation, by culturing for 3 days in a lipid deficient medium, with the LDL receptor was expressed maximally culture monocytes, Each purified monoclonal antibody, biotin-labeled and FITC-labeled purified monoclonal antibody (LL8
H6, LL12D10).

That is, 10.0 μg / ml of each purified monoclonal antibody, biotin-labeled purified monoclonal antibody and fluorescence-labeled purified monoclonal antibody were reacted with cultured mononuclear cells as described above, and measured using a flow cytometer. The fluorescence intensity of the antibody bound to the cells was measured. In addition, due to the difference in cell size between mononuclear lymphocytes and monocytes, after measuring the fluorescence intensity of the cells, CELLQUES
Analysis was performed using the T analysis program, each cell was identified, and the fluorescence intensity of the cell was calculated.

As a result, each purified monoclonal antibody was
Compared to gG-C7, lymphocytes are about twice in the 3-step method, 2.5 to 3 times in the 2-step method, and monocytes are 1.5 to 2 times in the 3-step method, and 2 to 2 in the 2-step method. It was shown that a high fluorescence intensity of about 0.5 times was obtained (FIG. 4). In both blood cells, 1
In the step method, no reactivity was observed in IgG-C7, but clear reactivity was observed in each purified monoclonal antibody. In particular, the one-step method was shown to be effective for detecting monocyte LDL receptors (FIG. 5).

Study on the Function of LDL Receptor LDL receptor is highly expressed in the body, especially in the liver and adrenal glands. In the liver, LDL in the bloodstream is taken up and secreted into the blood again as VLDL, thereby increasing the blood level. Keeps cholesterol homeostasis. In the adrenal glands,
The LDL receptor takes in LDL in the blood and supplies cholesterol into cells, thereby synthesizing and secreting steroid hormones. LD on the cell membrane
The L receptor not only binds LDL in the bloodstream, but also takes up the bound LDL into cells (internalization), separates the taken-up LDL from the receptor protein (catabolism), and expresses it again on the cell membrane (li). Cycling) function.

Therefore, in discussing the physiological activity of the LDL receptor, it is important to specify the function of the LDL receptor. That is, in the LDL receptor gene abnormality, a relationship between the mutation site and the functional abnormality has been recognized, and it is important to know the function in the diagnosis of familial hypercholesterolemia (Goldst
ein JL, et al, The Metabolic and Molecular bases of
inherited disease, McGraw-Hill, Inc.New York, pp1981-
2030).

From such a viewpoint, it was further examined whether or not the monoclonal antibody of the present invention is a monoclonal antibody which can also specify the function of the LDL receptor. That is, the LDL binding activity of the LDL receptor is 4 ° C., and the uptake (internalization) activity is 25%.
At 37 ° C., the catabolism was examined at 37 ° C. by confirming the reactivity with the monoclonal antibody, respectively.

As described above, the human LDL receptor CH
O cells were incubated at 4 ° C., 25 ° C. and 37 ° C. for 2 hours at 5 μg / ml.
FITC-labeled purified monoclonal antibodies (LL8H
6, LL12D10), the cells were washed as described above, and the fluorescence intensity of the cells was measured with a flow cytometer.

As described above, at each temperature, the FlTC-labeled purified monoclonal antibody (LL8H6, LL12D1
The fluorescence intensity of the human LDL receptor CHO cells reacted with 0) was larger at 25 ° C. than at 4 ° C., and was similar between 25 ° C. and 37 ° C. . More specifically, rather than the fluorescence intensity at 4 ° C, 25 ° C and 37 ° C (internalization)
At 1.5 to 2.0 times. This means that once the FlTC-labeled purified monoclonal antibody (LL8H6, LL12D1
This was considered to be the result of the FlTC-labeled purified monoclonal antibody again binding to the receptor protein expressed on the cell membrane again by so-called recycling by being taken into the cells (FIG. 6).

Further, the fluorescence intensity of these cells was reduced by the presence of a competitor, ie, coexistence of an excess (100 μg / ml) of LDL. This suggested that the above-described reaction of enhancing the fluorescence intensity was a specific reaction due to the incorporation of the FDLC-labeled purified monoclonal antibody into the LDL receptor (FIG. 7).

As described above, it has been found that the function of the LDL receptor can be sufficiently examined by the monoclonal antibody of the present invention. Investigation of Ligand in Competitive Reaction One of the hyperlipidemias showing clinical symptoms similar to familial hypercholesterolemia is apolipoprotein B-100.
Deficiency (Familial defective apolipoprotein B-10)
0: FDB) is known (Kane JP and Havel RJ, T
he Metabolicand Molecular bases of inherited disea
se, McGraw-Hill, Inc. New York, pp1853-1885).

In this disease, mutations occur in the binding region of apolipoprotein B-100, a ligand of the LDL receptor, which is a major constituent and functional protein of LDL, and LDL in the blood binds to the LDL receptor of cells. LDL in blood, like FH,
Hyperlipidemia, which causes a rise in cholesterol and is very prone to develop coronary artery disease as well as arteriosclerosis.

In order to determine the FDB, it is essential to specify the mutation of the corresponding gene. Mutations in this gene are mostly mutations at the 3500th amino acid substitution in Westerners, but in Japanese,
Not yet discovered. FDB in Japanese may be caused by abnormalities in different amino acids, or abnormalities may occur in LDL, a ligand due to other causes. Searching for this gene abnormality in Japanese is difficult because the corresponding gene is as large as about 43 kbp, and it is difficult to analyze the entire gene region because it takes enormous time and cost.

In view of such circumstances, if the apoB abnormality can be known by examining the binding ability of apolipoprotein B-100 (apoB) to the LDL receptor, the above-mentioned determination can be carried out simply and without cost. It is considered useful in Therefore, it was examined whether the monoclonal antibody of the present invention can be used as a material for knowing the binding activity of apoB as a ligand to the LDL receptor. That is, the LDL receptor-expressing cells and the competitor apoB-containing lipoprotein LDL or VLDL are allowed to coexist, and the monoclonal antibody (LL8H6,
LL12D10) was able to inhibit the binding between the LDL receptor and these lipoproteins.

More specifically, the human LDL receptor CHO
Cells were treated with each FlTC-labeled purified monoclonal antibody (LL
8H6, LL12D10) 2 μg / ml was co-existed with LDL, VLDL or HDL obtained by specific gravity centrifugation from plasma and reacted at 4 ° C. for 2 hours. After the reaction, the cells were washed as described above, Was used to measure the fluorescence intensity of the cells.

As a result, the fluorescence intensity of each FITC-labeled purified monoclonal antibody (LL8H6, LL12D10) bound to the human LDL receptor CHO cells was LDL.
And VLDL in a concentration-dependent manner (1-250 μg /
The decrease in fluorescence intensity was observed in the presence of HDL (1-250 μg / ml), but no change was observed in the fluorescence intensity (FIG. 8).

This indicates that the binding of the monoclonal antibody of the present invention to the LDL receptor is inhibited by the apoB-containing lipoprotein LDL or VLDL, but not by the lipoprotein HDL which does not contain apoB. . Further, the inhibition of the binding of the monoclonal antibody of the present invention to the LDL receptor by LDL or VLDL, which is an apoB-containing lipoprotein, was 90% or more at a concentration of 25 μg / ml. Was found to be suitable as a principle for detecting abnormal binding to LDL receptor.

[0114]

According to the present invention, a monoclonal antibody against the human LDL receptor is provided, and the monoclonal antibody provides a means for determining familial hypercholesterolemia.

[0115]

[Sequence List] SEQUENCE LISTING <110> BLM Inc. <120> Monoclonal Antibody <130> LDLR <140> <141> <160> 1 <170> PatentIn Ver. 2.0 <210> 1 <211> 839 <212> PRT <213> Hominidae <400> 1 Ala Val Gly Asp Arg Cys Glu Arg Asn Glu Phe Gln Cys Gln Asp Gly 1 5 10 15 Lys Cys Ile Ser Tyr Lys Trp Val Cys Asp Gly Ser Ala Glu Cys Gln 20 25 30 Asp Gly Ser Asp Glu Ser Gln Glu Thr Cys Leu Ser Val Thr Cys Lys 35 40 45 Ser Gly Asp Phe Ser Cys Gly Gly Arg Val Asn Arg Cys Ile Pro Gln 50 55 60 Phe Trp Arg Cys Asp Gly Gln Val Asp Cys Asp Asn Gly Ser Asp Glu 65 70 75 80 Gln Gly Cys Pro Pro Lys Thr Cys Ser Gln Asp Glu Phe Arg Cys His 85 90 95 Asp Gly Lys Cys Ile Ser Arg Gln Phe Val Cys Asp Ser Asp Arg Asp 100 105 110 Cys Leu Asp Gly Ser Asp Glu Ala Ser Cys Pro Val Leu Thr Cys Gly 115 120 125 Pro Ala Ser Phe Gln Cys Asn Ser Ser Thr Cys Ile Pro Gln Leu Trp 130 135 140 Ala Cys Asp Asn Asp Pro Asp Cys Glu Asp Gly Ser Asp Glu Trp Pro 145 150 155 160 Gln Arg Cys Arg Gly Leu Tyr Val Phe Gln Gly Asp Ser Ser Pro Cys 165 170 175 Ser Ala Phe Glu Phe His Cys Leu Ser Gly Glu Cys Ile His Ser Ser 180 185 190 Trp Arg Cys Asp Gly Gly Pro Asp Cys Lys Asp Lys Ser Asp Glu Glu 195 200 205 Asn Cys Ala Val Ala Thr Cys Arg Pro Asp Glu Phe Gln Cys Ser Asp 210 215 220 Gly Asn Cys Ile His Gly Ser Arg Gln Cys Asp Arg Glu Tyr Asp Cys 225 230 235 240 Lys Asp Met Ser Asp Glu Val Gly Cys Val Asn Val Thr Leu Cys Glu 245 250 255 Gly Pro Asn Lys Phe Lys Cys His Ser Gly Glu Cys Ile Thr Leu Asp 260 265 270 Lys Val Cys Asn Met Ala Arg Asp Cys Arg Asp Trp Ser Asp Glu Pro 275 280 285 Ile Lys Glu Cys Gly Thr Asn Glu Cys Leu Asp Asn Asn Gly Gly Cys 290 295 300 Ser His Val Cys Asn Asp Leu Lys Ile Gly Tyr Glu Cys Leu Cys Pro 305 310 315 320 Asp Gly Phe Gln Leu Val Ala Gln Arg Arg Cys Glu Asp Ile Asp Glu 325 330 335 Cys Gln Asp Pro Asp Thr Cys Ser Gln Leu Cys Val Asn Leu Glu Gly 340 345 350 Gly Tyr Lys Cys Gln Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr 355 360 365 365 Lys Ala Cys Lys Ala Val Gly Ser Ile Ala Tyr Leu Phe Phe Thr Asn 370 375 380 Arg His Glu Val Arg Lys Met Thr Leu Asp Arg Ser Glu Tyr Thr Ser 385 390 395 400 400 Leu Ile Pro Asn Leu Arg Asn Val Val Ala Leu Asp Thr Glu Val Ala 405 410 415 Ser Asn Arg Ile Tyr Trp Ser Asp Leu Ser Gln Arg Met Ile Cys Ser 420 425 430 Thr Gln Leu Asp Arg Ala His Gly Val Ser Ser Tyr Asp Thr Val Ile 435 440 445 Ser Arg Asp Ile Gln Ala Pro Asp Gly Leu Ala Val Asp Trp Ile His 450 455 460 Ser Asn Ile Tyr Trp Thr Asp Ser Val Leu Gly Thr Val Ser Val Ala 465 470 475 480 Asp Thr Lys Gly Val Lys Arg Lys Thr Leu Phe Arg Glu Asn Gly Ser 485 490 495 Lys Pro Arg Ala Ile Val Val Asp Pro Val His Gly Phe Met Tyr Trp 500 505 510 Thr Asp Trp Gly Thr Pro Ala Lys Ile Lys Lys Gly Gly Leu Asn Gly 515 520 525 Val Asp Ile Tyr Ser Leu Val Thr Glu Asn Ile Gln Trp Pro Asn Gly 530 535 540 Ile Thr Leu Asp Leu Leu Ser Gly Arg Leu Tyr Trp Val Asp Ser Lys 545 550 555 560 Leu His Ser Ile Ser Ser Ile Asp Val Asn Gly Gly Asn Arg Lys Thr 565 570 575 Ile Leu Glu Asp Glu Lys Arg Leu Ala His Pro Phe Ser Leu Ala Val 580 585 590 Phe Glu Asp Lys Val Phe Trp Thr Asp Ile Ile Asn Glu Ala Ile Phe 595 600 605 Ser Ala Asn Arg Leu Thr Gly Ser Asp Val Asn Leu Leu Ala Glu Asn 610 615 620 Leu Leu Ser Pro Glu Asp Met Val Leu Phe His Asn Leu Thr Gln Pro 625 630 635 640 Arg Gly Val Asn Trp Cys Glu Arg Thr Thr Leu Ser Asn Gly Gly Cys 645 650 655 Gln Tyr Leu Cys Leu Pro Ala Pro Gln Ile Asn Pro His Ser Pro Lys 660 665 670 Phe Thr Cys Ala Cys Pro Asp Gly Met Leu Leu Ala Arg Asp Met Arg 675 680 685 Ser Cys Leu Thr Glu Ala Glu Ala Ala Val Ala Thr Gln Glu Thr Ser 690 695 700 Thr Val Arg Leu Lys Val Ser Ser Thr Ala Val Arg Thr Gln His Thr 705 710 715 720 Thr Thr Arg Pro Val Pro Asp Thr Ser Arg Leu Pro Gly Ala Thr Pro 725 730 735 Gly Leu Thr Thr Val Glu Ile Val Thr Met Ser His Gln Ala Leu Gly 740 745 750 Asp Val Ala Gly Arg Gly Asn Glu Lys Lys Pro Ser Ser Val Arg Ala 755 760 765 Leu Ser Ile Val Leu Pro Ile Val Leu Leu Val Phe Leu Cys Leu Gly 770 775 780 Val Phe Leu Leu Trp Lys Asn Trp Arg Leu Lys Asn Ile Asn Ser Ile 785 790 79 5 800 Asn Phe Asp Asn Pro Val Tyr Gln Lys Thr Thr Glu Asp Glu Val His 805 810 815 Ile Cys His Asn Gln Asp Gly Tyr Ser Tyr Pro Ser Arg Gln Met Val 820 825 830 Ser Leu Glu Asp Asp Val Ala 835 <210 > 2 <211> 18 <212> PRT <213> Hominidae <400> 2 Trp Pro Gln Arg Cys Arg Gly Leu Tyr Val Phe Gln Gly Asp Ser Ser 1 5 10 15 Pro Cys

[Brief description of the drawings]

FIG. 1 is a Western blot diagram for examining the reactivity of the monoclonal antibody of the present invention with human LDL receptor.

FIG. 2 is a Western blot diagram for examining the reactivity of the monoclonal antibody of the present invention with lipoprotein receptors other than human LDL receptor.

FIG. 3 is a drawing showing the results of examining the reactivity of FlTC-labeled monoclonal antibody of the present invention on human LDL receptor CHO cells by a one-step method.

FIG. 4 is a drawing in which the reactivity of the monoclonal antibody of the present invention was examined with a flow cytometer using cultured lymphocytes.

FIG. 5 is a drawing in which the reactivity of the monoclonal antibody of the present invention was examined with a flow cytometer using cultured mononuclear cells.

FIG. 6. LDL binding activity of LDL receptor (4
FIG. 1 is a drawing in which the reactivity of the monoclonal antibody of the present invention with respect to the incorporation activity (25 ° C.), the catabolic activity (37 ° C.), and the uptake activity (25 ° C.) were examined.

FIG. 7 is a drawing in which the function of the LDL receptor was examined using the monoclonal antibody of the present invention.

FIG. 8 is a drawing examining whether the monoclonal antibody of the present invention can be used as a material for knowing the binding activity of apoB as a ligand to an LDL receptor.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/53 C12P 21/08 33/577 C12N 5/00 B // C12P 21/08 15/00 C ( 72) Inventor Makoto Nagano 1361-1, Matoba, Kawagoe-shi, Saitama Pref. Inside the BML Research Institute Co., Ltd. (72) Inventor Toru Egasashi 1361-1, Matoba, Kawagoe-shi, Saitama 1st BML Within the Research Institute (72) Inventor Tokuo Yamamoto 5-18-405 F-term (reference) 4B024 AA11 BA46 CA04 CA05 DA02 EA04 GA03 GA11 GA18 GA23 HA15 4B063 QA01 QA19 QQ03 QQ08 QQ13 QQ14 QQ46 QQ79 QQ96 QS03 QS33 QS36 QX02 4B064 AG27 BH09 CA10 CA19 CA20 CC24 CE04 CE12 DA13 4B065 AA91X AA94Y AB01 AB05 BA02 BA06 CA25 CA46 4H045 AA11 AA30 BA10 BA17 BA40 CA42 DA50 DA76 DA86 EA50 FA34 FA41 F A72 FA74 GA06 GA15 GA26

Claims (9)

[Claims] (1) a human lipoprotein receptor,
A peptide selected from a region having an amino acid sequence specific only to the human LDL receptor as a peptide comprising at least a sufficient number of amino acids that can be recognized to be specific only to the human LDL receptor; A monoclonal antibody that inhibits the binding of to human LDL receptor. 2. The method according to claim 1, wherein the number of amino acids sufficient to be recognized at least as specific to the human LDL receptor is 5 to 5.
The monoclonal antibody according to claim 1, wherein the number is 30. 3. A peptide selected from a region specific only to the human LDL receptor, wherein the amino acid sequence represented by SEQ ID NO: 1 is 7-25, 25-37, 47-60, 68-80, 83-94, 96-107, 107-119, 146-158, 159
176th, 195th to 207th, 210th to 221st, 222th to 233th, 234th to 246th, 256th
A peptide having an amino acid sequence selected from the group consisting of the -275th region and the 275-287th region;
The monoclonal antibody according to claim 1 or 2. 4. The region specific to only the human LDL receptor is the region of the amino acid sequence represented by SEQ ID NO: 2 (the region of the 159th to 176th amino acid sequence in SEQ ID NO: 1). The monoclonal antibody as described. 5. The monoclonal antibody according to any one of claims 1 to 4, wherein the labeling treatment does not cause loss of binding ability to human LDL receptor. 6. A hybridoma producing the monoclonal antibody according to any one of claims 1 to 5. 7. A human LDL utilizing an antigen-antibody reaction between the monoclonal antibody according to any one of claims 1 to 5 and a human LDL receptor in a sample.
A method for detecting an abnormality in the binding ability between a receptor and human LDL. 8. The detection method according to claim 7, wherein the sample is a blood sample containing leukocytes derived from human peripheral blood. 9. The presence or absence or content of familial hyperlipidemia of a sample donor is determined based on an abnormality in the binding ability between human LDL receptor and human LDL detected by the detection method according to claim 7 or 8. Method for determining hyperlipidemia.