WO1992019755A1

WO1992019755A1 - MONOCLONAL ANTIBODY AGAINST HUMAN β2-GLYCOPROTEIN I AND USE THEREOF

Info

Publication number: WO1992019755A1
Application number: PCT/JP1992/000528
Authority: WO
Inventors: Takao Koike; Eiji Matsuura; Hisato Nagae
Original assignee: Yamasa Shoyu Kabushiki Kaisha
Priority date: 1991-04-24
Filing date: 1992-04-23
Publication date: 1992-11-12
Also published as: AU1673292A

Abstract

The invention makes it possible to obtain a monoclonal antibody which specifically reacts with human β2-glycoprotein I and to determine specifically human β2-glycoprotein I in a specimen by using the antibody.

Description

Monoclonal antibody against human / S2-glycoprotein I and its application

The present invention relates to a monoclonal antibody specifically reacting with human; 82-glycoprotein I, and use thereof.

Background art

For the measurement of anti-cardiolipin antibodies, the RI method by Norris et al. (Lancet iii: 1211, 1983) and the ELISA method by Koike et al. (Clin, Exp. Immunol., 56: 193) , 1984) have been reported.

However, these conventional methods suffer from problems such as the inability to quantitatively measure anti-cardiolipin antibodies and the inability to separately measure antibodies from infectious diseases and antibodies from anti-phospholipid antibody syndrome. has, was one cry than those that can always be satisfied ₀

Recently, Matsuura et al. Reported that anti-cardiolipin antibodies from the anti-phospholipid antibody syndrome do not bind directly to cardiolipin, but rather to cardiolipin and / 32-glycoprotein I (also known as apolipolipin). It was found to show binding to a complex with popprotein H). Then, a method for measuring anti-cardiolipin antibodies using this principle was developed (Lancet, 336: 177, 1,990, clinical immunity, 22 (Suppl. 15): 170, 1990). This method for measuring anti-cardiolipin antibodies is an extremely excellent method for overcoming the problems of the conventional method described above, and is expected to be widely used in clinical settings in the future. 5 Anti-cardiolipin antibodies derived from anti-cardiolipin antibody syndrome were found to have binding properties to the complex of cardiolipin and S2-glycoprotein I as described above. However, its clinical significance remains unclear.

10 Clarifying the clinical significance of the binding properties of anti-cardiolipin antibodies will lead to elucidation of the pathogenesis of anti-cardiolipin antibody syndrome, and is considered an important issue for the future. Have been.

The first step in solving this challenge is to use anti-Cardio

It is important to accurately measure the / 32-glycoprotein I, one of the components of the antigen to which the 15-lipin antibody binds, but no method has been reported to achieve this purpose. In other words, although egret antisera (polyclonal antibody) against yS2-glycoprotein I is already known,

9 / (Publisher: DIA GN OSTICAS TA GO), it is unknown whether this antiserum specifically reacts with human 2-glycoprotein I, and There has been no report that the human ^ 2-glycoprotein I was specifically measured by application.

J Disclosure of the Invention Therefore, an object of the present invention is to provide a monoclonal antibody capable of specifically measuring human / 3-glycoprotein I, and another object is to provide a monoclonal antibody capable of specifically measuring human / 3-2 glycoprotein I. An object of the present invention is to provide a method and a kit for specifically measuring human / 3-glycoprotein I using a monoclonal antibody.

The present inventors have conducted studies to achieve the above object, and as a result, have been able to efficiently obtain a monoclonal antibody that meets the above object, and to use this monoclonal antibody. The present inventors have found that human / 32-glycoprotein I can be specifically measured, and completed the present invention.

That is, the present invention relates to a monoclonal antibody that specifically reacts with human / 5-glycoprotein I (hereinafter, may be referred to as “monoclonal antibody of the present invention”). Is concerned.

The present invention also provides a kit for measuring human / 32-glycoprotein I containing the monoclonal antibody of the present invention as one of its constituent reagents (hereinafter referred to as “the present invention”). (Kit of the invention).)

Furthermore, the present invention provides a method for measuring human 2-glycoprotein I, which comprises using the monoclonal antibody of the present invention as a measuring reagent (hereinafter referred to as “the measuring method of the present invention”). ).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the reactivity of the monoclonal antibody CoF15. FIG. 2 shows the reactivity of the monoclonal antibody CoF16.

Figure 3 shows the cross-reactivity of the monoclonal antibody CoF5 against various human apolipoproteins.

FIG. 4 shows the cross-reactivity of the monoclonal antibody CoF15 with various human-derived apolipoproteins.

Figure 5 shows the cross-reactivity of the monoclonal antibody CoF16 against various human apolipoproteins from humans.

FIG. 6 shows the species specificity of the monoclonal antibody CoF15. (-) In the figure means no serum added o

FIG. 7 shows the species specificity of the monoclonal antibody CoF16. (1) in the figure means no serum added. FIG. 8 shows a standard curve in the competitive ELISA method described later.

BEST MODE FOR CARRYING OUT THE INVENTION

1. Monoclonal antibody of the present invention

(1) Characteristics

The monoclonal antibody of the present invention typically has the following characteristics, and the use of such a monoclonal antibody only makes it possible to obtain human / 32-glycoprotein in a sample for the first time. I It has become possible to specifically measure

① Reactivity

When the reactivity of the antibody is examined by the ELISA method described in the Examples below, it reacts with human 2-glycoside tin I depending on the antibody concentration.

②Specificity

When the specificity of the antibody was examined by the immunoblotting method described in the Examples below, it was found that the antibody reacted only with human; 52-glycoprotein I, and other protein derived from human. Does not substantially react with any antigenic substance.

③ crossing

It does not substantially react with human various apolipoproteins, or even if it does, it has no effect on the measurement of human / 3-glycoprotein I.

④Species specificity

It does not substantially react with δ2-glycoprotein I derived from other animals and is specific to / 32-glycoprotein I derived from human.

(2) Manufacturing method

The method for producing the monoclonal antibody of the present invention is not particularly limited, and it can be produced by appropriately applying a known method.

The immunogen used is human / 3-glycoprotein I, which is described, for example, by the method of McNeill et al. (Pro Natl. Acad. ScL. USA 87: 4120, 1). 990) Can be prepared from fresh water. The degree of purification of the immunogen is not particularly limited.

In addition, the amino acid sequence and base sequence of human / 5-glycoprotein I are known, and peptides corresponding to a part or all of this known amino acid sequence can be obtained. It may be prepared chemically by a known method and used as an immunogen. If the synthesized peptide has poor antigenicity, it may be combined with a polymer carrier commonly used for the production of antibodies for hapten antigens such as serum albumin and keyhole lysate. It is preferable to use the body as an immunogen. Furthermore, human / 52-glycoprotein I may be prepared by a DNA recombination technique using a known nucleotide sequence, and this may be used as an immunogen.

The animals to which the immunogen is to be administered include: horses, horses, pigs, sheep, goats, rats, mice, guinea pigs, dogs, dogs, bushes, rabbits, rabbits, pigeons, nits The mouse, rat, guinea pig, egret, goat, sheep, etc. are particularly convenient for use.

The administration of the immunogen to such animals may be performed in a conventional manner, for example, complete Freund's adjuvant, incomplete Freund's adjuvant, myocardial adjuvant, or water. A suspension of various adjuvants such as aluminum oxide adjuvant and B. pertussis adjuvant and the above-mentioned immunogen may be prepared and administered to the above animals intravenously, intraperitoneally, subcutaneously or intradermally. . Dosage should be 0.1 to 10 mgZ per mouse or rat, or 0.1 to 10 mgZ if a guinea pig is used. About 01 to lmg animals are preferred.

After the initial administration, booster immunization against human / S2-daricoprotein I is performed in the animal body by boosting about 1 to 5 times as above every 1 to 4 weeks. Induces antibody production. Next, antibody-producing cells such as spleen cells, lymph node cells, and peripheral blood lymphocytes from the animals that have acquired immunity are obtained by a conventional method.

As the myeloma cells to be fused with the antibody-producing cells, cell lines derived from various animals such as mice, rats, and humans and commonly available to those skilled in the art are used. As a cell line to be used, a cell line which has drug resistance, cannot survive in a selection medium in an unfused state, and can survive only in a state fused to an antibody-producing cell is preferable. Usually, an 8-azaguanine resistant strain is used, and this cell line is deficient in hypoxanthine-guanine phosphophorase (Hypoxantine guanine p osphoribosyl transferase). Hypoxanthine 'cannot grow on AMINO-Pterin-Thymidine (HAT) medium, and it is a so-called non-secretory cell line that does not secrete immunoglobulin. I like it. '

A specific example of a myeloma cell line is P3X63Ag8 (ATCCTIB-9) (Nature, 256, 495- 497 (1975 ", P 3 x 63 Ag 8 U. 1 (P3U 1) (ATCCCRL-1597) (Current Topics in Microbiology and Immunology, 81, 1-7 (1978)), P 3 x63Ag8.653 (ATCCCRL-1580) (J. Immunology.123.1548-1550 (1979)), P2 / NSI-Ag4-1 (ATCCTIB-18) (European J. Immunology, 6, 511-519 (1976)). Sp 2/0 -Ag14 (ATCCCRL-1581) Nature, 276, 269—270 (1978)) E. Kazuma cell line, 210.RC Y.A 1.2,3 (Υ3-Ag 1.2.3) (ATCCCRL-1631) (Nature, 277, 131-133 ( Ratmyeloma cell strains such as U-266-AR1 (Pro atl. Acad. Sci. USA, 77, 5429 (1980)), GM150 (Nature, 288). , 488 (1980)) and KR-4 (Pro Natl. Acad. Sci. USA, 79, 6651 (1982)).

For cell fusion, select myeloma cells that match the antibody-producing cells.

Cell fusion, Eagle's minimum essential medium (ME M), da几Beck co-modified Eagle's medium (DMEM), RPMI - 1 6 4 0 medium of any animal cell culture medium 1 0 ^6-1 0 ³ For efficient fusion, mix Zml myeloma cells and antibody-producing cells at a mixing ratio of I: 4 to 10 and contact the cells for 1 to 10 minutes at 37. Can . In order to promote cell fusion, polyethylen glycol (PEG) with an average molecular weight of 1,000 to 6,000 and polyvinyl alcohol (PEG) are used to promote cell fusion. And fusion promoters such as Sendai virus can be used. In addition, antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device using electric pulses.

As a means for selecting a desired hybridoma from cells after the cell fusion treatment, a method utilizing selective growth of cells in a selective medium can be used. For example, cell suspension 1 5% © Shi calf serum (FCS) containing RPMI - 1 6 4 0 medium, etc. By appropriately diluted, microphones LOP rate on the 1 0 ³ to 1 0 ⁶ Add a selection medium (for example, HAT medium) to each well until the wells are about the same volume, and then exchange the selection medium appropriately before culturing. When 8–azaguanine-resistant strains were used as myeloma cells and HAT medium was used as the selection medium, unfused myeloma cells died by about day 10 of culture, and were normal. Since antibody-producing cells, which are cells, cannot grow in vitro (in vitro) for a long period of time, the cells that grow on the 10th to 14th day of cultivation are defined as Neubridomas. Obtainable.

The search for hybridomas producing monoclonal antibodies recognizing human / S2-glycoprotein I was performed by enzyme-linked immunosorbent assay (EIA, ELISA) and radioimmunoassay. This can be done by using an RIA, for example. For example, 96-well E with human / 32-glycoprotein I adsorbed A culture supernatant containing a monoclonal antibody is added to the microplate for LISA to react with human ^ 2-glycoprotein I, and then the enzyme-labeled anti-immunoglobulin antibody is added to the bound specific antibody. After the reaction, or after reaction with a biotin-labeled anti-immune globulin antibody, an avidin D—enzyme receptor is reacted. Then, in each case, an enzyme substrate is added to each well to develop color. Reacts specifically with human jS2-glycoprotein I by selecting the culture supernatant that develops color only with the wells on which human 32-glycoprotein I is immobilized. You can search for hybridomas that produce antibodies.

Further, as the human 2-glycoprotein I used in the above-mentioned screening, those having a high degree of purification are preferred, and specifically, those having a degree of purification of 90% or more are preferably used. As a result, it is possible to efficiently screen a hybridoma that produces a monoclonal antibody that specifically reacts with human ^ 2-glycoprotein I.

Cloning of the hybridoma can be performed by a limiting dilution method, a soft agar method, a fibringel method, a fluorescence excitation cell sorter method, or the like.

As a method for producing a monoclonal antibody obtained in this manner, a normal cell culture method, an ascites formation method, or the like may be employed.

In the cell culture method, the hybridomas are RPMI-164 medium containing 10 to 15% FCS, serum-free medium, etc. The antibody can be obtained from the culture supernatant by culturing in an animal cell culture medium in a usual manner.

The method of recovery from ascites involves the use of minerals such as pristane (2,6,10,14—tetramethyl pentyldecane) in animals of high predominance and tumor tissue compatibility. After the oil is intraperitoneally administered, for example, in the case of a mouse, about 100 ^{s of} hybridomas are intraperitoneally administered. Hybridomas form ascites tumors around 10 to 18 days and produce high concentrations of antibodies in serum and ascites.

If antibody purification is required, ammonium sulfate precipitation, ion exchange chromatography using an anion exchanger such as DEAE cellulose, protein A-Seph. Known methods, such as affinity chromatography and molecular sieve chromatography, using appropriate colors, etc., are appropriately selected and combined. Can be purified. 2. Measurement method of the present invention

The measurement method of the present invention is characterized by using the above-described monoclonal antibody of the present invention as a reagent, and is not limited by the measurement principle, conditions, and the like.

As a classification according to the reaction mode, a competitive reaction method and a non-competitive reaction method (immunometric assay) are known, and either method can be employed in the present invention.

Classification according to the detection method includes a non-labeling method that directly detects the results of the antigen-antibody reaction (such as nephrometry) and a labeling method that detects using a marker. Is known However, in the present invention, any method may be used.

Heterogeneous methods that require BF separation and homogenous methods that do not require BF separation are known, and any of these methods may be applied to the present invention.

As the classification according to the reaction phase, the liquid phase method in which the entire reaction is carried out in the liquid phase and the solid phase method in which the reaction of the immune reaction is performed by immobilizing the partner on the solid phase are known. Can also be adopted.

A method suitable for the purpose of the measurement method of the present invention may be appropriately selected from these known general methods.

The details of the general method are described in detail, for example, in the following literature.

(1) edited by Hiroshi Irie, “Radio Munno Atsusei” (Kodansha Co., Ltd., published May 1, 1980)

(2) Eiji Ishikawa et al. “Enzyme immunoassay” (2nd edition) (Medical Publishing Co., Ltd., published on Feb. 15, 1982)

③Clinical Pathology Special Issue No. 53 Special Issue on “Imno-Two Technology for Clinical Examination and Its Application I” (Clinical Pathology Publishing Association, published in 1983)

φ “Biotechnology Encyclopedia” (CMC, Inc., issued on October 9, 1998)

⑤ rMethods in ENZYM0 and OGY Vol.70j (Immunochemical techniques (Part A))

⑥ `` Methods in ENZYM0L0GY Vol.73j (Immunochemical techniques (Part B) ⑦ “Methods in ENZY 0L0GY Vol.74” (Immunochemical techniques (Part C))

⑧ “Methods in ENZYM0L0GY Vol.84” (Immunochemical techniques (Part D: Selected Immunoassay))

⑨ “Methods in ENZYM0L0GY Vol.92” (Immunochemical techniques (Part E .-Monoclonal Antibodies and General Immunoassay Methods))

[⑤-⑨ is published by Aki Derick Press Co.]

In addition, the mode of use of the monoclonal antibody of the present invention used in the assay of the present invention may be appropriately derived according to the assay used. Specific examples include a labeled antibody and a solid-phased antibody.

The antibody to be used may be the antibody itself, but it is preferable to use an active fragment of the antibody from the viewpoint of preventing nonspecific adsorption. The active fragment of an antibody can be any fragment that retains the characteristics of the antibody (for example, F (ab ') 2Fab', Fab, etc.). It may be. Preparation of these active fragments can be carried out by applying a known method such as a method of subjecting the purified antibody to limited degradation using a protease such as papine, pepsin, or trypsin. (See, for example, "Research Methods for Immunochemical Chemistry (Seismic Chemistry Laboratory Course 5) J, edited by The Biochemical Society of Japan, p. 89 (1989))."

Labeling agents that bind to antibodies include radioisotopes (for example, ³² P, ³ H, ¹⁴ C, etc.), enzymes (for example, Lactosidase, peroxidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, power tarase, glucosoxidase, lactate oxidase, alcoholoxidase, monoamido Oxidases), coenzyme / prosthetic groups (eg, FAD, FMN, ATP, piotin, heme), fluorescein derivatives (eg, fluorescein isothiocyanate, fluorescein thiofluva) Mil), rhodamin derivatives (eg, tetramethyl rhodamin B isothiocyanate), umbelliferone, and 1-anilino-1 8 Fluorescent dyes such as naphthalenesulfonate, luminol derivatives (for example, luminol, isolminol, N— (6 Hexyl) one N - etc. Echirui Sonore real Roh Ichiru), etc. Ru can and child to use. The binding between the antibody or its active fragment and the labeling agent was described in a written book [eg, “Sequence Chemistry Experimental Course 5 Immunobiochemical Research Method” Tokyo Chemical Co., Ltd., (1996) Issue) Pages 102 to 112] may be appropriately selected from publicly known methods described above.

As a material of the carrier substance for immobilizing the antibody, for example, poly (vinyl chloride), polystyrene, styrene-vinyl divinylbenzene copolymer, styrene-monovinyl maleate copolymer are used. Polymer, Nylon, Polyvinyl alcohol, Polyacrylamide, Polyacrylonitrile, Polypropylene, Polymethylenemethacrylate Synthetic organic polymer compounds such as Polysaccharides such as kisstran derivatives (such as Sephadex), agarose gels (such as Sepharose and biogel), cellulose (such as paper discs and filter paper), glass, silica gel, Examples thereof include inorganic polymer compounds such as silicones, which may have a functional group such as an amino group, an aminoalkyl group, a carboxyl group, an acyl group, or a hydroxyl group introduced therein. . The material of the carrier substance is preferably a substance having a low protein-binding ability, and examples of such a material include untreated polystyrene and polyvinyl chloride.

The carrier material can be in the form of a plate (microtiter plate, disk, etc.), particulate (beads, etc.), tubular (test tube, etc.), fibrous, film-like, particulate (lambda). Examples thereof include particles such as particles, capsules, and endoplasmic reticulum, and a carrier having an appropriate shape can be selected according to the measurement method. In addition, ribosomes (multilayer or monolayer lipid membrane) can also be used as a carrier substance for immobilizing antibodies.

The binding method between the monoclonal antibody of the present invention and the carrier substance may be a known method such as a physical adsorption method, an ion binding method, a covalent binding method, and an inclusive method (for example, “immobilized enzyme” (Chibatake) (Ichiro Edition, March 20, 1980, published by Kodansha Co., Ltd.)). In particular, the physical adsorption method is preferred because it is simple. Further, the above-mentioned binding may be performed directly, or may be performed via another substance between the two substances.

3. Kit of the present invention The kit of the present invention is characterized in that the monoclonal antibody of the present invention is contained as one of the constituent reagents of the kit. It may be different depending on the law.

In the case of a kit based on the competitive reaction method, for example, the following reagent composition is used.

(1) Anticancer antigen (antibody)

② Labeled antibody (antigen) solution

③ Antigen solution of known concentration

In the case of a kit based on the sandwich method, for example, the following reagent configuration is used.

① Immobilized first antibody

② Second antibody solution

Labeled anti-immunoglobulin antibody solution

抗原 Antigen solution of known concentration

In the above kit, "Antibody J is the monoclonal antibody of the present invention, and" antigen "is human yS2-glycoprotein I, needless to say. Absent. In the case of a kit based on the sandwich method, the "first antibody" and the "second antibody j" recognize different antigenic determinants on human S2-glycoprotein I. This can be easily understood from the measurement principle, and the kit configuration based on the sandwich method is, for example, as follows.

① Immobilized first antibody

Labeled second antibody solution ③ Antigen solution of known concentration

By using the monoclonal antibody of the present invention, it has become possible for the first time to specifically measure human / S2-glycoprotein I. Therefore, the present invention is useful as a tool for elucidating the mechanism of onset of anti-cardiolipin antibody syndrome.

Hereinafter, the present invention will be described specifically with reference to Examples. Example 1 Preparation of mouse monoclonal antibody against human / S2-glycoprotein I (82-GPI) (1) Monoclonal antibody 產 Preparation of live hybridoma

Human / S2-GPI (0.2 mg / m 1) prepared by a known method (Pro Natl. Acad. Sci. USA 87: 4120 (1990)) was dissolved in physiological saline and completely dissolved. BALBZc mice (female, 6-week-old) were intraperitoneally administered 10 g / 00 a1 (ip, mixed with Freund's adjuvant in a 1: 1 ratio to give emalgiones). ) And the first immunization. After the first immunization, immunizations (i.P.) were performed several times every two weeks in the same manner, and the final immunization was performed using a human / 32-GPI saline solution (1 OzgZlO0 / 1 ) Was administered (i.V.) to the tail vein of the mouse. Three days after the final immunization, the spleen of the mouse was removed and washed with Eagle's minimum essential medium (MEM) to prepare a spleen cell suspension. At the same time, the mouse myeloma P3X63Ag8U1 (P3U1) (ATCCCRL-1579) is washed with MEM, and spleen cells and P3U1 are mixed at a ratio of 10: 1. After centrifugation, add 50% poly to the pellet obtained by centrifugation. Cell fusion was performed by gradually adding 1 ml of a MEM solution containing 1000 g of polyethylene glycol (PEG). Further, add the MEM solution to make up to 1 Oml, and centrifuge the pellet, and add P3U1 to the RFMI-164 medium containing 10% fetal calf serum (FCS). to 3 xl 0 ⁴ were suspended at a cell Z 0. 1 ml, aliquoted Kakuu E blur 0. 1 ml min 9 6 c Erumai click furnace data one pre one Bok. The next day, add 0.1 ml of MEMS medium (HAT medium) containing hypoxanthin-thymidine-aminobuterin, and then remove half of the medium every 3 to 4 days with a new HAT medium. Was replaced by

Hybridomas were screened on day 14 after fusion. That is, 96-Elmic was coated with purified human 52-GPI (purity: 999 or more) (10 g / ml) and blocked with 1% serum albumin (BSA). (B) The culture supernatant (501) was added to the evening plate and reacted at room temperature for 1 hour. After washing three times with PBS200Z1, 5001 of a solution of a biotinylated anti-mouse IgG (manufactured by Vector) was added, and the mixture was further reacted at room temperature for 1 hour. After the reaction, the plate was washed three times with PBS, added with a solution of avidin D-peroxidase (manufactured by Vector Inc.) 50 // 1 and reacted at room temperature for 30 minutes. After washing similarly with PBS, the substrate solution (4-aminoantipyrine (0.25 mgZm1)), phenol (0.25 mg / ml), 0.2% Add 4 25 M hydrogen peroxide), react at room temperature, measure the absorbance at 550 nm, and specifically react with human 2-GPI. Reactive antibodies were detected, and specific antibody-producing hybridomas were selected (Table 1).

Table 1 Antigen-specific antibody positive cell proliferation Total number of cells

ゥ Electric web | ¾β2-GPI 120 4 0 0 7 6 8 Each hybridoma selected in this way is further cloned using the limiting dilution method, and the human / S 2-Hydroma strains producing antibodies highly reactive against 2-GPI (Cof5, Cof6, Cof15, Cof16, CofF) 18, COF 19, COF20, COF21, COF22, COF23) were established.

②Preparation and purification of monoclonal antibodies

Three types of established cell lines (C0F5, C0F15, C0F16) were intraperitoneally injected with mouse treated with 0.5 ml of virgin pristine. 3 × 10 ⁶ were administered to the mice, and ascites was collected about 2 weeks later. After diluting by adding an equal volume of PBS in the collected ascites, addition of sulfuric acid A Nmoniu厶, 5 fractions precipitated at 0% saturation A emissions monitor © beam sulfate concentration was recovered by centrifugation _{(precipitated} fraction of this Was re-dissolved in PBS and dialyzed against the same buffer for 2 days, followed by protein A-Sepharose scalar to purify IgG from the crude ammonium sulfate fraction described above. First, a chromatographic study was performed: Protein A—Sepharose (manufactured by Fanoremasia) 20 The ml was filled into a 1.5 × 20 cm glass column, and equilibrated with 1.5 M glycine buffer (pH 8.9) containing 3 mM sodium chloride. Next, the above ammonium sulfate fraction was diluted 2-fold with an equal volume of the same glycine buffer, added to the column, and the non-adsorbed proteins were washed away with a sufficient amount of the same glycine buffer, and the adsorbed IgG was removed. Elution was performed with 1 M citrate buffer (pH 3.0). The IgG fraction thus obtained was immediately dialyzed against PBS for 1 μm to avoid denaturation to obtain the monoclonal antibody of the present invention.

Example 2 Analysis of Properties of Monoclonal Antibody 1 (Search for Class and Type)

96-well microcloth coated with human ^ 2—GPI (5〃gZml) in 50〃1Z ゥ L and blocked with PBS containing 1% ゥ serum albumin (BSA) Add the culture supernatant of the hybridoma or purified monoclonal antibody to the titer plate and use the Mono Ab-1 DEIA kit (Zymed) to determine the class and type of the antibody. Performed a search. The results are as shown in Table 2.

Type fnF18 CnFl k / Lanos type 1 1 k IeGl K 1 '*' * ^u ^ a? Class CoF20 CoF21 COF22 CoF23 class, type I Gi, K IgGi, K IgGi, K IgGi, Example 3 Analysis of Monoclonal Antibody Properties② (Reaction Specificity of Monoclonal Monoclonal Anti-Hit ^ 2-GPI Antibody) Neubridoma (C0F5C0F15C0F) The antigen specificity of the monoclonal antibody produced by 16) (the antibody name is the same as the name of the hybridoma producing it) was confirmed by SDS-polyacrylamide gel electrophoresis. The immunoassay was performed by electrophoresis (SDS-PAGE) and enzyme-linked immunosorbent assay (ELISA). (A) Analysis of specificity by the immunoblotting method

SDS-PAGE was performed according to the method of Lammli (Nature, 227: 680, 1972). As reported by Matsuura et al. (Lancet, 336: 177-178, 1990), human / 32-GPI exhibits a molecular weight of 50 kD under reduced and non-reduced conditions. In the present invention, purified human / S2-GPI and serum from a healthy subject were subjected to electrophoresis, and immunoblotting was performed by the method described below. The separation gel obtained by SDS-PAGE was placed on a nitrocellulose membrane, and electricity was transferred to the nitrocellulose membrane at 60 V for 12 hours. The nitrocellulose membrane thus obtained was cut into strips along the electrophoresis line of the sample, and a portion was stained for protein using an amide black. The other membrane was blocked by immersing it in a 3% gelatin solution at 37 ° C for 1 hour, and then appropriately diluted with PBS. Monoclonal anti-human / 52-GPI antibody (Cof5, C O F 15 and C O F 16) were reacted at room temperature for 1 hour. After sufficient washing with PBS, the cells were reacted with a peroxidase-labeled anti-mouse IgG antibody for 1 hour at room temperature. Further, the nitrose cellulose membrane was washed in the same manner, and a substrate solution (30 mg of color developer (manufactured by Biorad), 10 ml of methanol, 50 ml of PBS, and 50 ml of PBS) And hydrogen peroxide solution (containing 301)), and when the color of the mixture became suitable, the reaction was stopped by washing with water. As a result, all three monoclonal antibodies (Cof5, Cofl5, and Cofl6) showed extremely strong reactivity with human 2-GPI with a molecular weight of 5 OkD. It did not react with any other human-derived serum proteins.

(B) Specificity analysis by ELISA

The basic ELISA procedure for the specificity analysis of monoclonal antibodies is as follows. Purified human / S2-GPI in PBS (5 μgZml) was added to each of the 96-well micromicroplate evening plates, and left at 4 ° C for 1 2. , 1% BSA-containing PBS 200 // 1 in each well It was left at room temperature for 1 hour before blocking. After washing three times with PBS, various monoclonal antibodies

50 β 1 was added and reacted at room temperature for 1 hour. In the competitive inhibition test, an inhibitor was added to this reaction system to the concentration shown in each figure. Thereafter, the plate was washed with PBS in the same manner, and the antibody was labeled with alkaline phosphatase.

1 was added and reacted at room temperature for another hour. After washing, 100 μl of a substrate solution (Ρ-diphenylphosphoric acid) was added, and the mixture was reacted at room temperature for 1 hour. Then, the absorbance at 405 nm was measured.

(1) Reactivity of monoclonal antibody to human S2-GPI

Figures 1 and 2 show the reactivity of the monoclonal antibodies C0F15 and CoF16 with the immobilized human S2-GPI, respectively. That is, as shown in the figure, a plate coated with human / 32-GPI (5 μg / m 1, PBS as a control) and blocked with 1% BSA as shown in the figure. When the antibody was reacted with ~ 20 g Zml, CoFl5 and CofF16 were applied to the human / 52-GPI (10 μg / ml) immobilized plate. Both antibodies bound in a concentration-dependent manner.

② Antigen specificity of monoclonal antibodies: comparison of the inhibitory activities of apolipoproteins from various humans

Figures 3 to 5 show apolipoproteins (apolipoprotein H (ap0H), apolipoprotein A1 (apoAl), and apolipoprotein A derived from various human antibodies. 2 (ap 0 A 2), Apolipoprotein B (apo B), And the results of examining the presence / absence of cross-reactivity to apolipoprotein E (apoE)). The binding of any of the monoclonal antibodies (Cof5, Cof15, Cof16) to immobilized human 52-GPI was 0.4 to 10 gm aml. Addition of lipoprotein H, ie, 2-GPI, inhibits in a concentration-dependent (competitive) manner, but other apolipoproteins (ie, apo A1, apo A2, apo B, and apo E) are added. However, no significant inhibitory effect was observed.

(3) Antigen specificity of monoclonal antibodies: comparison of the inhibitory activities of serum from different animal species

It is known that about 20 ^ 2-GPI, that is, apolipoprotein H, is present in human serum. Therefore, various monoclonal sera (mouse (BALBZc), goat, rabbit, rabbit (fetal serum), and human serum) were used in place of apolipoprotein H, and these monoclonal antibodies were used. (C0F15 and C0F16) It was examined whether it was specific for S2-GPI (apolipoprotein H) derived from humans. As shown in FIGS. 6 and 7, the 10-fold and 50-fold dilutions of human serum significantly inhibited the binding of C0F15 and CoF16 to immobilized human S2-GPI. However, sera from other animal species did not show any inhibitory effects.

In addition, hybridomas other than the above (C0F5, C0F6, C0F18, C0F19, C0F20, C0F21, Similar results were obtained when the same experiment was performed with respect to the monoclonal antibody produced by Cof22 and Cof23).

Example 4 Confirmation of Monoclonal Antibody Specificity by Inhibition Activity Measurement Using Purified Human / 32-GPI, Rat Serum, and Bovine Serum

The specificity of each antibody was confirmed by measuring the inhibitory activity using a human / 82-GPI solid phase plate.

That is, a purified human / 52-GPI antigen solution dissolved in PBS to a concentration of 10 μg nom 1 was applied to 96-well microplates (Falcon) at a volume of 1 μl per 5 μl. Aliquots of 0 to 1 were added, and the mixture was allowed to stand at 4 ° C to bind the antigen to the plate. Remove the residual antigen solution using an aspirator, dispense 2001 PBS per well, and remove the plate using an aspirator. Was washed. The above washing operation was repeated three times. Invert the washed plate and gently tap it several times on a paper towel to remove the liquid remaining in the gel, and then add 3% gelatin-PBS solution to 200 μl per 1 μl. The mixture was dispensed and allowed to stand at 25 ° C for 30 minutes. After standing, the liquid was removed from the gel, and the liquid was thoroughly beaten several times on paper paper to completely remove the remaining liquid.

Next, purified human ^ 2-GPI solution dissolved in HEPES buffer to a concentration of 5 g nom 1, rat serum diluted 5 times, and serum serum diluted 5 times HEPES buffer (control) was dispensed into the wells at 25 p.1 each. HE Heron anti / 32-GPI antiserum (DIAGNOSTICAS TAGO), mouse anti- ^ 2-GPI antiserum (Yamasa Shoyu) and Monoclonal, which have been appropriately diluted in PES buffer Dispense each of the antibody (C of 6, C of 20, C of 22 and C of 23) solutions at a rate of 25α 当たり per volume, and gently mix with a macroplate mixer. The reaction was allowed to stand for 1 hour at room temperature. After the residual antibody solution was removed with an aspirator, the plate was washed three times according to the above procedure, and the secondary antibody was dispensed at a concentration of 501 per unit. As the secondary antibody, a peroxidase (HR 標識) -labeled goat anti-money IgG antibody was used for the ale to which the antiserum derived from heron was reacted. HRP-labeled goat anti-mouse IgG antibody was appropriately diluted with a 1% BSA-PBS buffer solution and dispensed into the wells reacted with the clonal antibody solution. After standing at room temperature for 1 hour, remove the remaining secondary antibody solution with an aspirator, wash three times according to the above procedure, and then add 1% hydrogen peroxide solution to TMBZ101111 50 n1 Immediately before use, the substrate solution mixed well was dispensed at a rate of 100/1 per 1 ml of the solution, and reacted at room temperature for 10 minutes. 2 N sulfuric acid was dispensed in IOOI per 1 μl, and after the color reaction was stopped, the absorbance at 450 nm was measured with a microplate reader.

The inhibitory activity was expressed as the inhibition rate (), where the color development of the control group was 100, the color development of the wells containing each solution was calculated, and the value was subtracted from the force of 100. . Two

As a result, as shown in Table 3, the binding of the egret antiserum to human δ2-GPI was significantly inhibited in both rat serum and chicken serum. The binding of mouse antiserum was also inhibited by rat serum. In contrast, the monoclonal antibodies of the present invention (C of 6, C0f20, Cof22, and Cof23) can be used in any of rat serum and human serum. Almost no inhibition of binding was observed, and the monoclonal antibody of the present invention did not react with rat ^ 2-GPI and cis-2-GPI, and was specific for human / 32-GPI This was reconfirmed.

Table 3

Antibody blocking rate (%)

Human; S2-GPI rat serum ゥ serum ゥ heron antiserum 58.24.5.4 34.2 mouse antiserum 40.24.20.0.0.0 monoclonal Antibody

C 0 f 6 36 .2 0 .0 0 .0

C 0 f 2 0 5 6 .7 1 .3 0 .9

C o f 2 25 9 .0 2 .3 0 .0 C o f 2 3 6 .6.5 0 .0 0 .0

Example 5 Computing humans by competitive ELISA method; S2-GPI

To quantitate human S2-GPI, first use a diluted solution of purified human 2-GPI as standard, and then use human 52-GP. I Cappa plate and anti-human; standard curve prepared by competitive ELISA using 52-GPI monoclonal antibody L 7 o

That is, the previously prepared human S 2 -GPI solid phase plate was subjected to washing and blocking treatments in the same procedure as in Example 4, and the standard was treated with HEPES buffer. A purified human ^ 2-GPI solution dissolved at a concentration of 0.06 to 64 g / ml, and a HEPES buffer solution as a control group were dispensed at a ratio of 25 1 per well. Inject the antibody solution of the monoclonal antibody (C of 23) diluted in the same manner as in Example 4 for 25 to 1 minute per 1 ml, and gently stir with a macroplate mixer. Then, the reaction was allowed to stand at room temperature for 1 hour. Thereafter, in the same manner as in the procedure described in Example 4, each operation of washing, secondary reaction, washing, dispensing of the substrate solution, stopping color development, and measuring absorbance was performed.

The measured value obtained as described above is calculated as the inhibition ratio expressed by determining the color development ratio of the control group as 100, and then subtracting that value from 100. Was calculated, and a standard curve of human / S2-GPI was created. Results The results are shown in FIG. Figure 8 As is clear from the above, in the competitive ELISA method using the monoclonal antibody of C of 23 described above, the human ^ 2 in the range of about 1 to 300 μg Zml was used. -It is possible to measure GPI.

Industrial applicability

The present invention specifically reacts with human ^ 2-glycoprotein I By using the monoclonal antibody obtained by the method described above, it is possible to accurately measure human / S2-glycoprotein I in a test sample. . Therefore, the monoclonal antibody of the present invention is extremely useful as a tool for elucidating the onset mechanism of, for example, anti-cardiolipin antibody syndrome.

Claims

The scope of the claims

1. A monoclonal antibody that specifically reacts with human 2-glycoprotein I.

2. The monoclonal antibody according to claim 1, having the following characteristics;

① Reactivity

Reacts with human 52-glycoprotein I depending on antibody concentration;

②Specificity

Does not substantially react with other antigenic substances derived from human; reacts only with human; 52-glycoprotein I;

Does not substantially react with various apolipoproteins derived from humans;

④Species specificity

Does not react substantially with S2-glycoprotein I from other animals;

3. A kit for measuring human / 8-glycoprotein I comprising the monoclonal antibody according to claim 1 as one constituent reagent.

4. A method for measuring human ^ 2-glycoprotein I, which comprises using the monoclonal antibody according to claim 1 as a measuring reagent.