JPH05249118A - Classifying method for anti-phospholipid antibody and kit used therefor - Google Patents

Abstract

PURPOSE:To classify anti-phospholipid antibodies based on the bond patterns of the antibodies by making the antibodies to react to lipids having two or more kinds of negative charges under the presence of 2-glycoprotein I. CONSTITUTION:After, for example, a 2-glycoprotein I solution is dropped onto each well of a plate in which two or more kinds of lipids having negative charges are coupled and the solution is made to react with the lipids, an antibody contained in a liquid to be checked is made to react to the complex of the lipids and 2-glycoprotein I by adding a sample to be checked to the wells. Then, after washing the wells, enzyme-labeled anti-immunity globulin is made to react to the complex and to separate a solid phase from a liquid phase. The anti-phospholipid antibodies are classified by adding a substrate, such as hydrogen peroxide to the solid or liquid phase, measuring ferment activity contained in the phase, and plotting coupling patterns against each kind of lipid of the antibodies from measured values.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for classifying antiphospholipid antibodies and a kit used therefor.

[0002]

2. Description of the Related Art The appearance of anti-cardiolipin antibody, which is a kind of anti-phospholipid antibody, is recognized in both anti-phospholipid antibody syndromes such as systemic lupus erythematosus (SLE) and infectious diseases such as syphilis. Conventionally, the above-mentioned two kinds of anti-cardiolipin antibodies having different origins are the same,
All of them were considered to specifically recognize cardiolipin. However, recently, by Matsuura et al., The anti-cardiolipin antibody derived from the infectious disease recognizes only cardiolipin, and the anti-cardiolipin antibody derived from the antiphospholipid antibody syndrome is identified as cardiolipin and β2-glycoprotein I (alias:
It was revealed for the first time that it recognizes a complex with apolipoprotein H or cofactor (La
ncet, 336: 177,1990, Clinical Immunity, 22 (Suppl.15): 170,199
0, WO91 / 06006).

[0003]

A study by Matsuura et al. Revealed a qualitative difference between an antibody specific to an infectious disease and an antibody specific to an antiphospholipid antibody syndrome. Diagnosis is possible for the first time. However, the clinical significance of antiphospholipid antibodies is still unclear, and clarifying the binding specificity of antibodies specific to antiphospholipid antibody syndromes is particularly important for elucidating the pathogenic mechanism of antiphospholipid antibody syndromes. Can lead to
It is considered to be an important issue for the future. Therefore, the present invention is
It is an object of the present invention to provide a method capable of clarifying the binding specificity of antiphospholipid antibody specific to antiphospholipid antibody syndrome and classifying the antiphospholipid antibody.

[0004]

[Means for Solving the Problems] As a result of repeated studies to achieve the above object, the present inventors found that all antibodies specific to the antiphospholipid antibody syndrome do not have uniform binding specificity. , And found that there is diversity in binding specificity. That is, cardiolipin and β
It was also found that there is an antibody that more strongly recognizes the complex of β2-glycoprotein I with other negatively charged phospholipids than the complex with 2-glycoprotein I,
This knowledge has been developed to complete the present invention.

Accordingly, the present invention provides a method of reacting an antiphospholipid antibody with two or more kinds of negatively charged lipids in the presence of β2-glycoprotein I, and classifying the antiphospholipid antibody based on its binding pattern (see The method of the invention). Further, the present invention is for carrying out the method of the present invention, which comprises as a constituent reagent a solid-phase reagent obtained by coating two or more kinds of negatively charged lipids at different locations on a single carrier. It's about the kit. further,
The present invention relates to a kit for carrying out the method of the present invention, which contains, as constituent reagents, two or more types of solid phase reagents obtained by coating two or more types of negatively charged lipids on different carriers, respectively. It is a thing.

The present invention will be described in detail below. (1) Method of the present invention In the method of the present invention, an antiphospholipid antibody is reacted with two or more kinds of negatively charged lipids in the presence of β2-glycoprotein I, and the antiphospholipid antibody is classified based on its binding pattern. To do. The antibodies targeted by the present invention are all antibodies that specifically appear in the serum of patients with antiphospholipid antibody syndrome such as SLE and habitual abortion. Therefore, in the present specification, an antibody that specifically appears in the antiphospholipid antibody syndrome is referred to as "antiphospholipid antibody", and all of these antibodies are the subject of the present invention.

The lipid used in the method of the present invention is not particularly limited, and may be any one as long as it has a negative charge. Examples of such lipids include phospholipids and glycolipids. Specific examples of phospholipids include glycerophospholipids such as cardiolipin, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. Further, specific examples of glycolipids include monogalactosyl diglyceride, digalactosyl diglyceride, phosphatidyl glyceroglycolipid,
A glyceroglycolipid such as glycerophosphoglyceroglycolipid can be exemplified. The type and quantity of lipid are not particularly limited. In the method of the present invention, at least two types of lipids may be used, and the type and number of lipids may be appropriately selected according to the range to be tested and used in the method of the present invention.

Such a lipid is bound to a water-insoluble carrier and used in the method of the present invention. The carrier for binding the lipid is not particularly limited as long as it is usually used for binding the lipid. Examples thereof include synthetic resins such as polyvinyl chloride, polystyrene, styrene-divinylbenzene copolymer, styrene-maleic anhydride copolymer, nylon, polyacrylamide, polyacrylonitrile, polypropylene and polymethylene methacrylate. The shape of the carrier material may be flat (microtiter plate, disc, etc.), particulate (beads, etc.), tubular (test tube, etc.), fibrous, membranous,
Any of fine particles (latex particles, etc.) can be used, and flat particles are particularly preferable.

The binding between the lipid and the above-mentioned carrier is a method or condition generally used as a method for immobilizing lipid (Biochemistry Experiment Course 11; Enzyme Immunoassay, 1989).
Tokyo Kagaku Doujinshi, etc.) can be selected as appropriate. For example, any method such as a physical adsorption method, an ionic binding method, and a covalent binding method can be used. In particular, the physical adsorption method is preferable because the binding operation is simple. Binding of a lipid to a carrier by a physical adsorption method is carried out by, for example, contacting a carrier of a solution of an organic solvent for lipid (eg, one capable of dissolving lipids such as methanol, ethanol, chloroform) with a carrier for a certain period of time, and distilling the solvent It can be performed by a method.

Β2-glycoprotein I coexisting in the reaction
Is not particularly limited as long as it is derived from a mammal, and for example, those derived from human, bovine, pig, etc. can be used. Further, β2-glycoprotein I with the sugar chain removed can also be used in the method of the present invention. The β2-glycoprotein I can be prepared by a known method, for example, the method of McNeill et al. (Proc. Natl. Acad.
Sci. USA 87: 4120, 1990). Further, the amino acid sequence and the base sequence of β2-glycoprotein I have already been clarified, and those prepared by ordinary peptide synthesis means or DNA recombination technique can be used in the present invention. The degree of purification of β2-glycoprotein I is not particularly limited, and the concentration of β2-glycoprotein I in the reaction solution is 10 to 100 μl / ml,
It may be added to the reaction solution preferably at 20 to 70 μl / ml. Also, β2-glycoprotein I may be bound to a carrier together with a lipid.

The reaction principle between the lipid and the antiphospholipid antibody is not limited as long as it is a method capable of assaying the binding specificity of the antibody present in the test solution. For example,
The competitive reaction method and the non-competitive reaction method are known as the classification according to the reaction mode, and either method can be adopted in the present invention. Further, as the classification by the detection method, there are known a non-labeling method for directly detecting the result of the antigen-antibody reaction (nepherometry etc.) and a labeling method for detecting by using some kind of marker. May be Furthermore, a heterogeneous method that requires BF separation and a homogeneous method that does not require BF separation are known, and any method may be applied to the present invention. That is, a method suitable for the purpose of the measuring method of the present invention may be appropriately selected from these known general methods.

The details of the general method are described in the following documents, for example. Irie Hiroshi "Sequel Radioimmunoassay" Kodansha Co., Ltd., published on May 1, 1979 Eiji Ishikawa et al. "Enzyme Immunoassay (2nd Edition)", Medical Shoin Co., Ltd., published December 15, 1982 Clinical Pathology Extra edition special issue No. 53 “Immunoassay for clinical examination-Technology and application-”, Clinical Pathology Press, 1
Published in 983 "Biotechnology Encyclopedia" CMC Co., Ltd., 1
Published October 9, 986 "Methods in ENZYMOLOGY Vol.70" (Immunochemical
techniques (Part A)) "Methods in ENZYMOLOGY Vol.73" (Immunochemical
techniques (Part B)) "Methods in ENZYMOLOGY Vol.74" (Immunochemical
techniques (Part C)) "Methods in ENZYMOLOGY Vol.84" (Immunochemical
techniques (Part D: Selected Immunoassay)) "Methods in ENZYMOLOGY Vol.92" (Immunochemical
techniques (Part E: Monoclonal Antibodies and Gene
ral Immunoassay Methods)) (is published by Academic Press)

[0013] For example, the method of the present invention will be described in more detail by taking as an example the ELISA method using a microtiter plate to which a lipid is bound and using a solid-phase reagent. First, β2- in each well of the plate to which various lipids are bound
After the glycoprotein I solution is added and reacted, a test sample (for example, blood, serum, etc.) is added and the antibody in the test solution is reacted with the lipid-β2-glycoprotein I complex. .. Then, after washing the wells, an enzyme-labeled anti-immunoglobulin antibody (eg, peroxidase-labeled anti-I
gG antibody, etc.) to separate the solid phase from the liquid phase. A substrate (for example, hydrogen peroxide and tetramethylbenzidine in the case of peroxidase) is added to the solid phase or the liquid phase, and the enzyme activity contained in either phase is measured. Finally, the binding pattern of the antibody to various lipids is plotted from the obtained measured values (absorbance, etc.), and the antiphospholipid antibody present in the test liquid can be classified based on this.

(2) Kit of the present invention As a kit used for carrying out the method of the present invention, for example, two or more kinds of negatively charged lipids are coated on different locations on a single carrier. One containing the obtained solid phase reagent as a constituent reagent (Kit A), or two or more kinds of solid phase reagents obtained by coating two or more kinds of negatively charged lipids on different carriers as constituent reagents The thing (Kit B) contained can be illustrated. Regarding the combination of the constituent reagents of such a kit and the preparation of the constituent reagents, nothing special is required. The composition of the reagent may be determined according to the measurement method used, and each reagent may be prepared by a conventional method. For example,
When the above ELISA method is applied to kit A and kit B, each kit is composed of the following reagents. Kit A: Solid phase reagent obtained by coating two or more kinds of negatively charged lipids at different locations on one microtiter plate, enzyme-labeled anti-immunoglobulin antibody substrate solution Kit B; two or more kinds of negatively charged Two or more kinds of solid-phase reagents obtained by coating lipids having different amounts on separate microtiter plates, enzyme-labeled anti-immunoglobulin antibody, substrate solution

[0015]

INDUSTRIAL APPLICABILITY The method of the present invention is a useful method for classifying antiphospholipid antibodies, which were conventionally considered to have uniform binding specificity, based on their binding specificity. In addition, the kit of the present invention is essential for carrying out the method of the present invention.

[0016]

EXAMPLES The present invention will be described in detail below with reference to examples. Example Cardiolipin (CL; bovine heart, Sigma), phosphatidylserine (PS; bovine brain, Avanti), phosphatidylinositol (PI; bovine liver, Avanti), dioleoylphosphatidylglycerol (DO)
PG; Avanti), dioleoylphosphatidic acid (DOPA; Avanti), and dioleoylphosphatidylcholine (DOPC; Avanti) each ethanol solution (50 μg / ml) 50 μl / well 96
Wells were added to each well of a microtiter plate (polystyrene; manufactured by Lab Systems), and all wells were dried under reduced pressure. After drying, 200 μl / well of 1% bovine purified albumin (without bovine β2-glycoprotein I) -containing phosphate buffered saline (PBS) (hereinafter abbreviated as PBS-pBSA) (pH 7.4) was added, After leaving at room temperature for 1 hour, 0.05% (V / V) T was applied to all wells.
PBS containing ween 20 (manufactured by Kishida Chemical Co., Ltd.)
Abbreviated as PBS-Tween. ) Washed 3 times with 250 μl.

Then, each well was diluted with purified human or bovine β2-glycoprotein I (PBS-pBSA to 3 wells).
50 μl / well) (prepared to 0 μ / ml) (PBS-pBSA 50 μl / well was added to the control group), left still for 10 minutes, and then further PBS-pBS
50 μl of a test serum solution diluted 100 times with A was added to the wells and reacted at room temperature for 30 minutes. PBS-Twe
After washing three times with 200 μl of en, 100 μl of horseradish peroxidase-labeled anti-human IgG antibody was added to each well and reacted at room temperature for 1 hour, and then PBS-Tw was added.
Washed 3 times with 200 μl of een. Substrate solution (0.0
0.3 mM containing 3% hydrogen peroxide 3, 3 ', 5,
5'-tetramethylbenzidine (TMBZ) solution) 10
After adding 0 μl and reacting at room temperature for 10 minutes, 100 μl of a reaction stop solution (2N sulfuric acid) was added to the reaction solution, and the absorbance (45
0 nm) was measured.

The results are shown in FIGS. 1 and 2. Fig. 1 shows the results of measurement when human β2-glycoprotein I was added, and Fig. 2 shows bovine β2-glycoprotein I.
It shows the result when the measurement was performed by adding. Considering the tendency of antibody binding specificity, it was revealed that sera 1 to 4 can be classified into two groups, group A (sera 1 and 2) and group B (serum 3 and 4). By using different phospholipid / β2-glycoprotein I complexes as described above, the antiphospholipid antibody derived from the antiphospholipid antibody syndrome can be classified based on its binding specificity.

[0019]

[Brief description of drawings]

FIG. 1 shows the presence of human β2-glycoprotein I,
It shows the binding properties of antiphospholipid antibodies to various phospholipids.

FIG. 1 is a graph showing coexistence of bovine β2-glycoprotein I,
It shows the binding properties of antiphospholipid antibodies to various phospholipids.

Claims (7)

[Claims] 1. A method of classifying an antiphospholipid antibody based on its binding pattern by reacting an antiphospholipid antibody with two or more negatively charged lipids in the presence of β2-glycoprotein I. 2. Use of two or more kinds of phospholipids and / or glycolipids as negatively charged lipids,
The method of claim 1. 3. A glycerophospholipid and / or a glyceroglycolipid as a lipid having a negative charge is 2
The method according to claim 1, wherein one or more kinds are used. 4. The method according to claim 1, which comprises as a constituent reagent a solid-phase reagent obtained by coating two or more kinds of negatively charged lipids at different locations on a single carrier. Kit. 5. The method according to claim 1, which comprises, as constituent reagents, two or more kinds of solid phase reagents obtained by coating two or more kinds of negatively charged lipids on different carriers, respectively. Kit. 6. Two or more kinds of phospholipids and / or glycolipids are used as the negatively charged lipid.
The kit according to claim 4 or 5. 7. A glycerophospholipid and / or a glyceroglycolipid as a lipid having a negative charge is 2
The kit according to claim 4 or 5, which is used in one or more kinds.