JPH06300764A - Carrier for bonding anti-phospholipid antibody, and immunoassay and kit using the carrier - Google Patents

Abstract

PURPOSE:To provide a measuring method for an autoantibody specific to an anti-phospholipid antibody syndrome and a carrier and a kit used in the method. CONSTITUTION:A using carrier for bonding an anti-phospholipid antibody used in an immunodiagnosis for an anti-phospholipid antibody syndrome is a carrier combining phospholipid and moreover processed by refined serum albumin and a surfactant. Therefore, the anti-phospholipid antibody syndrome can be highly accurately diagnosed immunologically. At the same time, a fractioned substance acting to enhance the bonding properties between an antibody specifically detected in the anti-phospholipid antibody syndrome and phospholipid, or protein is used to immonologically diagnose the anti-phospholipid antibody syndrome. Therefore, the correct diagnosis is achieved.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a carrier for binding an antiphospholipid antibody, an immunological assay using the same, a kit used for the assay, and a serum or plasma that can be used in these assays. The present invention relates to a fraction and a protein, particularly to the measurement of autoantibodies specific to antiphospholipid antibody syndrome.

[0002]

2. Description of the Related Art A living body causes an immune response to an exogenous foreign substance such as a pathogenic virus, bacterium, fungus, or parasite that has entered the living body, and acts to eliminate the immune response. This phenomenon is generally classified into two types: humoral immunity in which antibodies are involved, and cell-mediated immunity in which foreign immunocompetent cells are involved to eliminate foreign substances. However, in the pathological condition generally referred to as autoimmune disease, the self and non-self recognition mechanisms do not work normally, and a humoral or cell-mediated immune response occurs to the cells and tissues of the self. Systemic lupus erythematosus (SLE) is a typical clinical example in which a carrier (autoantibody) that reacts with self-components appears, and anti-single-stranded DNA antibody, anti-double-stranded DNA antibody, Appearance of Sm antibody, anti-cardiolipin antibody, etc. is observed. Rheumatoid arthritis (RA)
Rheumatoid factor, Sjogren's syndrome (SJS), anti-SS-A antibody, anti-SS-B antibody and anti-mitochondrial antibody, systemic sclerosis (PSS), anti-scl antibody and anti-single-stranded DNA antibody, Also, mixed connective tissue disease (MCT
Anti-RNP antibodies are found in D). At present, there are many unclear points about the relationship between the appearance of these antibodies and the significance of the onset of the pathological condition, but measuring the antibody is an important means for diagnosing the disease and knowing the change in the pathological condition of the prognosis.

In order to detect these various autoantibodies, immunodiffusion, face-to-face immunoelectrophoresis, hemagglutination, radioimmunoassay, depending on the antigen specificity, biochemical characteristics, and physicochemical properties of the antibody. (RIA method), enzyme immunoassay method (EIA method), and latex agglutination method have been developed. By the way, regarding the above-mentioned assay method for anti-cardiolipin antibody, an immunoprecipitation method was first developed for the diagnosis (mass screening) of syphilis in the latter half of the 1940s. However, in addition to the drawback of low sensitivity in diagnosis using this method, systemic lupus erythematosus (SL)
The appearance of a high frequency of biological false positives in the patient serum of E) has been a problem. However, in recent years, the appearance of high concentrations of anti-cardioliopine antibodies in the blood of young female patients who develop lupus-like syndrome with symptoms of arterial and venous thrombosis, miscarriage, and thrombocytopenia, and anti-cardiolipin antibodies were measured. Therefore, the clinical significance of diagnosing these diseases has been highly evaluated.

In such a situation, a highly sensitive and highly accurate alternative to conventional methods such as non-specific measurement of loop score ground and anti-cardiolipin antibody immunoprecipitation measurement in which biological false positives are frequently observed. Expectations have been placed on the measurement method (Proctor, RR & Rapaport, SL, Am.J.Clin.
Pathol., 36, 212, (1961); Exner, T. et al., Britis
h J. Haematol., 40, 143, (1978); Margolios, A., Medi
cine 40, 145 (1961)). Then, in 1983, an RIA for measuring an anticardiolipin antibody was developed by Harris et al. (Harris, EN et al., Lancet, 1211-1214, (1983)). After that, as a method other than RIA, an EIA method by a non-competitive method (sandwich method) using a labeled antibody labeled with an enzyme such as alkaline phosphatase has been developed. The measurement of cardiolipin antibody by this kind of EIA method is quantitative, simple and suitable for diagnosis and follow-up of patients with SLE and other collagen diseases, and in the field of basic medicine, it is used for the specificity analysis of anticardiolipin antibody. ing. In recent years, among the areas of collagen disease, the view that habitual miscarriage is due to thrombosis (antiphospholipid antibody syndrome) caused by the emergence of antiphospholipid antibodies such as anticardiolipin antibodies has been strengthened. Therefore, there are high expectations in the field of obstetrics and gynecology for the method for measuring anti-cardiolipin antibody.

There are various subclasses of anti-cardiolipin antibody in blood, such as IgG, IgM, and IgA.
The IgM or IgA class often appears in patient serum. In addition, dsDNA (double-stranded DNA antibody), ssDNA (single-stranded DNA), cardiolipin, phosphatidylserine, phosphatidylinositol, etc., which are considered to be specific antigens of each antibody specific to antiphospholipid antibody syndrome, are A similar molecular structure exists in a limited polar site centered around the phosphate ester, and the heterogeneity of these antibodies is also currently under discussion.

[0006]

As described above, SLE,
RIA for measuring anticardiolipin antibody as a measuring system for diagnosing antiphospholipid antibody syndrome such as habitual abortion
The EIA method and the EIA method have been developed at various facilities, but their measurement systems still have the following problems. That is, in the method using the antigen-immobilized carrier, SL
When a patient serum such as E is used for measurement, it may be difficult to quantify an antibody specific to cardiolipin due to nonspecific adsorption unless a suitable blocking agent is used. Fetal bovine serum (FBS) commonly used as a blocking agent
, It is impossible to separately quantify the SLE-derived anti-cardiolipin antibody and the anti-cardiolipin antibody of other foreign factors (such as infectious diseases), and a blocking agent suitable for such purpose has been found. Absent.

[0007]

[Means for Solving the Problems] The present inventors have proposed an antiphospholipid antibody-binding carrier and an antiphospholipid antibody syndrome which selectively trap antiphospholipid antibodies specifically present in antiphospholipid antibody syndrome. Various studies have been conducted in order to invent a highly accurate and highly sensitive assay method for an antiphospholipid antibody that can be put to practical use as a specific diagnostic method for A. As a result, the above-mentioned problems were solved by the following means, and the present invention was completed.

That is, the present invention provides (1) an antiphospholipid antibody-binding carrier, wherein the phospholipid-bonded carrier substance is treated with purified serum albumin and a surfactant, (2) phospholipid (3) A carrier for binding anti-phospholipid antibody, characterized in that the carrier substance bound with phospholipid is treated with purified serum albumin, surfactant and serum, plasma, a fraction thereof or a protein in the fraction, The lipid-bound antiphospholipid antibody-binding carrier is brought into contact with a test solution to form an immune complex between the antibody specifically present in the antiphospholipid antibody syndrome and the phospholipid bound to the carrier, In the method for detecting an antibody, the above-mentioned carrier (1) or (2) is used as a carrier for binding an antiphospholipid antibody, which is an immunological assay method,

(4) First antigen-antibody for contacting an antiphospholipid antibody-binding carrier with a test solution to form an immune complex between the antiphospholipid antibody in the test solution and the phospholipid on the carrier A reaction (hereinafter sometimes referred to as a primary reaction) step, and reacting the immune complex with a labeled anti-immunoglobulin antibody to form a sandwich immune complex composed of the immune complex and the labeled anti-immunoglobulin antibody. A second antigen-antibody reaction (hereinafter also referred to as a secondary reaction) step, and a separation step of separating the phase containing the sandwich immunocomplex from the phase containing a substance not bound to the carrier. And a detection step of detecting a labeling substance (marker) in any phase, wherein the carrier of (1) or (2) above is used as a carrier for binding an antiphospholipid antibody, and at least a first antigen Anti-antibody Sera from the process to the test liquid and the same type or analogue of an animal to the reaction solution, plasma, immunoassay, which comprises carrying out the addition of a fraction thereof or a protein that fraction thereof in,

(5) A first antigen antibody which forms an immune complex between the antiphospholipid antibody-binding carrier and the test solution by contacting the test solution with the antiphospholipid antibody in the test solution and the phospholipid on the carrier A reaction step, a second antigen-antibody reaction step of reacting the immune complex with a labeled anti-immunoglobulin antibody to form a sandwich-shaped immune complex comprising the immune complex and the labeled anti-immunoglobulin antibody, and In a method comprising a separation step of separating a phase containing the sandwich immunocomplex and a phase containing a substance not bound to a carrier, and a detection step of detecting a labeled substance (marker) in either phase, The phospholipid antibody-binding carrier is a phospholipid-bonded carrier substance treated with a surface-active carrier agent, and at least the first antigen-antibody reaction step is performed in the reaction solution in the same or similar manner as the test solution. Sera from the object, plasma, immunoassay, which comprises carrying out the addition of protein fraction thereof or fraction thereof in their,

(6) A kit for use in the immunological assay method according to (4), which comprises at least the following constituent reagents; (A) a carrier for binding an antiphospholipid antibody according to (1) or (2) above, B) a labeled anti-immunoglobulin antibody, (C) a serum or plasma derived from an animal of the same species or akin to the test liquid, a fraction thereof, or a sample diluent to which a protein in the fraction is added, (7) at least A kit for use in the immunological assay method according to (4), which comprises the following components; (A ') a carrier for antiphospholipid antibody binding, in which a carrier substance having phospholipid bound thereto is treated with a surfactant; (B) a labeled anti-immunoglobulin antibody, (C) a serum or plasma derived from an animal of the same species or akin to the test liquid, a fraction thereof or a sample diluent to which a protein in the fraction is added,

(8) A fraction obtained by gel filtration of serum or plasma, which has an action of enhancing the binding property between an antiphospholipid antibody specifically present in an antiphospholipid antibody syndrome and a phospholipid (9) A fraction obtainable from serum or plasma, which has an action of enhancing the binding property between an antiphospholipid antibody specifically present in an antiphospholipid antibody syndrome and a phospholipid Have,
Fractions having the following physicochemical properties: Nondialyzed fractions are included when dialyzed using a cellulose membrane having a molecular weight cut-off of 6,000 to 8,000; the fractions are separated by gel filtration or SDS-polyacrylamide gel electrophoresis. When it is imaged, it is confirmed in the vicinity of serum albumin or slightly on the low molecular side; it does not bind to protein A; elutes in the vicinity of the IgG fraction in chromatography with a weakly basic anion exchanger containing a diethylaminoethyl group; and Contained in the fraction salted out with 30 to 60% saturated ammonium sulfate,

(10) A protein obtainable from serum or plasma, which has an action of enhancing the binding property between an antibody specifically present in an antiphospholipid antibody syndrome and a phospholipid, and has the following physicochemical properties: Proteins with properties: The molecular weight measured by SDS polyacrylamide electrophoresis is about 50,000 ± 2,000 and the isoelectric point is about 6.
60 ± 0.4; and having a binding property to a phospholipid antibody. (11) In isolating and purifying the protein according to (10) above from serum of a healthy subject, it is represented by the formula [I] described later The method for producing a protein according to (10) above, which comprises a step of purifying using a liposome composed of a phospholipid, an affinity adsorbent having the phospholipid bound thereto, or a liposome having the phospholipid as one constituent Law,

(12) Immunization between an antibody specifically present in the antiphospholipid antibody syndrome by contacting a test liquid with a carrier for binding an antiphospholipid antibody to which a phospholipid is bound, and a phospholipid bound to the antibody In the method of forming a complex to detect the antibody, the fraction of the above (8) or (9) or the above (1) is added to a reaction solution when the immune complex is formed.
0) The immunological assay characterized by allowing the protein described above to be present, (13) The antiphospholipid antibody syndrome-derived carrier is obtained by bringing a test solution into contact with a carrier for binding an antiphospholipid antibody bound to phospholipids. A method for differentially detecting an antiphospholipid antibody and an antiphospholipid antibody derived from an infectious disease, which comprises contacting a test solution with an antiphospholipid antibody-binding carrier having a phospholipid bound thereto in a reaction solution. A method in which a high concentration of serum or plasma is present, or the fraction of (8) or (9) above or the protein of (10) above is present in the reaction solution and brought into contact,
(14) a method for separately detecting an antiphospholipid antibody derived from an antiphospholipid antibody syndrome and an antiphospholipid antibody derived from an infectious disease by carrying out both of the methods of contacting these without making them exist, Immunological assay for measuring an antibody specifically present in an antiphospholipid antibody syndrome, which comprises plasma, the fraction according to (8) or (9) or the protein according to (10) as one of constituent reagents The gist is the kit used for the assay method.

The present invention will be specifically described below. 1. Phospholipid The phospholipid in the present invention may have an electron-donating functional group in the vicinity of its phosphodiester bond,
In particular, the general formula [I]

[Chemical 1] [In the formula, R ¹ and R ² are the same or different, and an acyl group having an alkyl group or an alkenyl group in the carbon chain,
Represents an alkyl group or an alkenyl group, R ³ represents a hydrogen atom or — (CH ₂ ) _n —CHR ⁴ —R ⁵ , and R ⁴ represents a hydrogen atom, a hydroxyl group, a carboxyl group, a formyl group, a mercapto group or a halogen atom. , R ⁵ is an amino group, a hydroxyalkyl group or the general formula [II]

[Chemical 2] [In the formula, R ¹ and R ² have the same meanings as described above. ] Or R ⁴ and R ⁵ represent a sugar or sugar alcohol residue, and n represents an integer of 0 to 3. ] The glycerophospholipid represented by this is preferable. This glycerophospholipid has a negative charge, and specific examples of such glycerophospholipid include cardiolipin, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. Cardiolipin is particularly preferable. Cardiolipin is usually prepared from the heart of mammals such as cows and microorganisms such as Escherichia coli, but those obtained from the hearts of mammals are particularly preferable. These are not limited to their origin, fatty acid composition, degree of purification, etc., and may be in salt form or in free form, and the solvent to be dissolved upon binding to the carrier substance is not limited, either. Is preferably reagent grade or higher.

2. Carrier Material The carrier material used in the present invention is not particularly limited as long as it can bind phospholipids and does not inhibit the immune reaction (antigen-antibody reaction) in the measurement, but it is not limited to BF separation (in the immune reaction). A carrier substance (solid phase) insoluble in the reaction solution is preferable in view of the fact that the separation of the labeled body forming the immune complex and the free labeled body can be easily performed. Examples of the material of the carrier substance insoluble in the reaction solution include polyvinyl chloride, polystyrene, styrene-divinylbenzene copolymer, styrene-maleic anhydride copolymer, nylon, polyvinyl alcohol, polyacrylamide, polyacrylonitrile, polypropylene, and poly. Synthetic organic polymer compounds such as methylene methacrylate, dextran derivatives (Sephadex, etc.),
Polysaccharides such as agarose gel (sepharose, biogel, etc.), cellulose (paper disc, filter paper, etc.), inorganic polymer compounds such as glass, silica gel, silicone, etc. are mentioned. These are amino groups, aminoalkyl groups, carboxyl groups, It may be one in which a functional group such as an acyl group or a hydroxyl group is introduced. The material of the carrier substance is preferably one having a low protein binding ability, and examples of such a material include untreated polystyrene and polyvinyl chloride. The shape of the carrier substance insoluble in the reaction solution is flat (microtiter plate, disk, etc.), particulate (beads, etc.), tubular (test tube, etc.), fibrous, film-like,
Examples include fine particles (latex particles and the like), capsules, endoplasmic reticulums, liposomes (multilayer or monolayer lipid membranes), etc., and a carrier having an appropriate shape can be selected according to the measurement method.

3. Carrier for binding antiphospholipid antibody The carrier for binding antiphospholipid antibody in the present invention is the above phospholipid bound to the above carrier substance. The binding method of the phospholipid and the carrier substance is a physical adsorption method, an ionic binding method,
A publicly known method such as a covalent bond method and an inclusion method (for example, refer to “Immobilized Enzyme” (edited by Ichiro Chibata, March 20, 1975, published by Kodansha Co., Ltd.)) can be adopted. Above all,
The physical adsorption method is preferably simple. The binding between the phospholipid and the carrier substance may be performed directly, or may be performed between the two substances via another substance. In order to bind the phospholipid to the carrier substance by the physical adsorption method, usually, an organic solvent solution of phospholipid (organic solvent capable of dissolving the phospholipid such as methanol, ethanol, chloroform) is contacted with the carrier substance for a certain time, A method of distilling off the organic solvent can be used. The solvent can be distilled off by a known method such as reduced pressure drying or ventilation drying. Further, the solvent is distilled off from the organic solvent solution of the phospholipid so that the phospholipid is once adsorbed on the surface of a substance other than the carrier substance, and the phospholipid is absorbed into a buffer solution (for example, sodium-based, potassium-based, or sodium-potassium-based). Buffer solution) and the carrier substance for a certain period of time (for example, several tens of minutes to several hours) under temperature conditions (for example, 0 to 50 ° C.) that do not inhibit the reaction. ), And then the solution is removed by a method such as suction, decantation, or centrifugation, and if necessary, the phospholipid can be bound to the carrier substance by a method of drying (vacuum drying, ventilation drying, etc.). .

The carrier for binding antiphospholipid antibody used in the present invention is a carrier treated only with a surfactant, a carrier treated with a combination of purified serum albumin and a surfactant, or a purified serum. It is preferable that albumin, a surfactant and serum, plasma, a fraction thereof or a protein in the fraction (which may be hereinafter referred to as a treatment substance) are used in combination. In addition, in the presence of a high concentration of serum or plasma in the reaction solution, or in the presence of a fraction or protein obtained from the serum or plasma described below according to the present invention, the binding to the binding carrier for antiphospholipid antibody is performed. When carrying out an antigen-antibody reaction to form an immune complex between the above-mentioned phospholipid and the anti-phospholipid antibody in the test liquid, the above-mentioned carrier for binding anti-phospholipid antibody of the present invention does not necessarily have to be used. Here, "treatment" refers to an operation in which these treatment substances act on an antiphospholipid antibody-binding carrier so that they can be physically or chemically bound. Specifically, such treatment involves binding the phospholipids to a carrier material and then sequentially treating each of the solutions containing the treatment materials (in no particular order) or containing two or three of these materials. Can be performed by contacting the solution with a carrier substance for a certain period of time (for example, several tens of minutes to several hours) under temperature conditions (for example, 0 to 50 ° C.) that do not inhibit the reaction solution. In carrying out such a treatment, the solvent that dissolves these treatment substances is not particularly limited, but a buffer solution (such as sodium-based, potassium-based or sodium-potassium phosphate buffered saline) is usually used. used.
Further, saccharides (oligosaccharides such as sucrose, polysaccharides such as dextrin, cyclodextrin, dextran, monosaccharides, etc.) may be coexistent in the treatment liquid when performing such treatment.

By using the antiphospholipid antibody-binding carrier that has been subjected to the above-mentioned treatment, the antiphospholipid antibody-binding carrier in the first antigen-antibody reaction (first reaction) step is derived from an infectious disease. It can prevent the antibody from binding. Other effects of such treatment include prevention of non-specific adsorption (prevention of binding of anti-phospholipid antibody and labeled antibody to the non-phospholipid-binding portion on the carrier substance), phosphorus on the carrier substance. Examples thereof include enhancement of binding ability of antiphospholipid antibody specifically present in antiphospholipid antibody syndrome to lipid, stabilization of phospholipid on carrier substance, and the like. Purified serum albumin used in the present invention, cow, horse, dog,
Cats, goats, sheep, pigs, birds, rabbits, rats, mice, those purified by known methods from the serum of animals such as humans,
Fraction V having a higher degree of purification than Fraction V (by Cohn's low-salt ethanol fractionation method, heat shock method, salting-out, etc.), and adapted to the object of the present invention. That is, specifically, for example, fraction V has a degree of purification equivalent to that purified by activated carbon treatment, chromatography (ion exchange chromatography, etc.), crystallization, or fraction V purified, Those purified by other methods, particularly those having a reduced lipid content, are preferred.

Serum, plasma, a fraction thereof, or a protein in the fraction (hereinafter sometimes referred to as "the blood component of the present invention") is produced from animal blood by a known method. Serum or plasma itself, a fraction obtained by fractionating them, or a protein obtained by further purifying the fraction. The origin is preferably an animal of the same species as the test liquid to be measured, but may be derived from a related animal. Such a blood component of the present invention is essentially the same as that added during the primary reaction described below. Conventionally, phospholipid-bonded carriers have been treated with fetal bovine serum, newborn bovine serum, bovine serum albumin, surfactants, etc. It was not possible to prevent the binding of the anti-phospholipid antibody derived from the infectious disease. In addition, conventionally used bovine serum albumin was unpurified such as Fraction V by the Cohn low temperature ethanol fractionation method.

The surfactants used in the present invention include nonionic, amphoteric, anionic and cationic surfactants, with nonionic surfactants being particularly preferred. Specific examples of the nonionic surfactant include polyoxyethylene glycol sorbitan alkyl esters (for example, Tween-based surfactant) and acyl sorbitans (for example, Span-based surfactant,
Examples thereof include Arlacel-based surfactants, polyoxyethylene glycol alkylphenyl ethers (eg, Triton-based surfactants), sucrose fatty acid esters, and the like. The carrier for binding antiphospholipid antibody of the present invention is not limited to immunological measurement,
The antiphospholipid antibody can be used for various purposes for selective adsorption removal or separation purification. For example, it can be used as a selective adsorbent for antiphospholipid antibodies in plasmapheresis (see, for example, JP-A-1-68273). Also,
It can also be used as an adsorbent for affinity chromatography for separating and purifying antiphospholipid antibodies. In the case of carrying out the reaction by allowing the blood component of the present invention to exist in the reaction solution in the primary reaction described below, instead of using the carrier treated with these treating substances, a conventionally known carrier for phospholipid antibody binding is used. Can also be used.

4. Antiphospholipid antibody syndrome The immunological measurement using the carrier for phospholipid antibody binding of the present invention can be used for the purpose of diagnosing antiphospholipid antibody syndrome. Here, the antiphospholipid antibody syndrome refers to a disease in which an antiphospholipid antibody appears in the body fluid of the patient among autoimmune diseases (systemic lupus erythematosus (SLE), recurrent miscarriage, etc.). Such diseases are distinguished from tuberculous meningitis, syphilis, and other infectious diseases in which antiphospholipid antibodies appear in the body fluid. The immunological assay method of the present invention is characterized in that an antibody specific to the antiphospholipid antibody syndrome can be discriminated and measured from the antiphospholipid antibody derived from such an infectious disease.

5. Immunological Assay Method The immunological assay method of the present invention is intended for the measurement of antiphospholipid antibody, and the above-mentioned carrier for binding phospholipid antibody and / or the blood of the present invention in the reaction solution during the primary reaction is used. If the method is based on an antigen-antibody reaction by carrying out a reaction in the presence of components, its operating method, labeling substance (marker), labeled substance, carrier, B
The type of F separation method or the like does not matter. A method suitable for the purpose of the present invention can be appropriately selected from known methods known as immunological assay methods. The following methods are known as known immunological assay methods. The competitive reaction method and the non-competitive reaction method (immunometric assay) are known as the classification according to the reaction mode of the antigen-antibody reaction, and the non-competitive reaction method is preferable in the present invention.

As the classification by the detection method, an unlabeled method (nepherometry etc.) for directly detecting the result of the antigen-antibody reaction and a labeling method for detecting by using some kind of marker are known. Alternatively, the method of Considering the measurement sensitivity and the like, the labeling method is particularly preferable. The labeling substance (marker) used in the labeling method will be described later. 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.
As the classification according to the reaction phase, there are known a liquid phase method in which all reactions are carried out in a liquid phase and a solid phase method in which a partner of an immune reaction is immobilized to carry out the reaction. When using a liquid-soluble substance is equivalent to the liquid phase method,
The case of using a substance insoluble in the reaction solution corresponds to the solid phase method. The relationship between the known immunological measurement and the present invention has been described above, and the general method is described in detail in the following documents.

Hiroshi Irie, edited by "Radioimmunoassay" (Kodansha Co., Ltd., published May 1, 1979) Eiji Ishikawa, et al., "Enzyme Immunoassay" (2nd edition) (Co., Ltd.)
Medical Institute, published December 15, 1982) Clinical pathology Extra edition special issue No. 53 "immunoassay for clinical examination-technology and application-" (Clinical Pathology Press,
Published in 1983) "Biotechnology Encyclopedia" (CMC, Inc.)
Issued October 9, 1986) "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 Immun
oassay)) "Methods in ENZYMOLOGY" Vol.92 "(Immunochemical techniques (Part E: Monoclonal Ant
ibodies and General Immunoassay Methods)) [published by Academic Press, Inc.] The immunological assay method of the present invention will be specifically described below.

5.1 Test Liquid A test liquid whose presence or content thereof is to be measured by the immunological assay method of the present invention (hereinafter also referred to as the present method) Means blood, serum, ascites, lymph, synovial fluid or fractionated components obtained therefrom, or other biologically derived liquid components. Further, the test liquid may be diluted with a diluting liquid so as to have an appropriate antibody titer. As such a diluting solution, sodium-based,
Potassium or sodium-potassium phosphate buffer saline (PBS), Good's buffer (eg, N-
2-hydroxyethylpyrazine-N′-2-ethanesulfonic acid (HEPES), N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES),
3- (N-morpholino) propanesulfonic acid (MPO
Buffer solutions such as S)), buffer solutions such as glycine buffer solution and veronal buffer solution are preferred.

5.2 First Antigen-Antibody Reaction (Primary Reaction) Step In the present invention, the “first antigen-antibody reaction step” means that an antiphospholipid antibody-binding carrier is contacted with a test solution to be tested. The step of forming an immune complex between an antiphospholipid antibody in a liquid and a phospholipid on a carrier. The method of the present invention uses a carrier for binding an antiphospholipid antibody that has been treated with the treatment substance, and / or serum derived from an animal of the same species or akin to the test fluid in the reaction solution in this step. Alternatively, it is characterized in that the reaction is carried out in the presence of plasma, or in the presence of the fraction thereof or the protein in the purified fraction thereof. By adopting such a method, it is possible to prevent the binding of the antiphospholipid antibody derived from the infectious disease to the carrier for binding the antiphospholipid antibody, and the antibody that is specifically present in the antiphospholipid antibody syndrome specifically to the carrier. Can be combined.

In this step, a standard fluid containing a body fluid of an antiphospholipid antibody syndrome patient or an antiphospholipid antibody of a known concentration is used as a test liquid and a carrier for binding the antiphospholipid antibody for a predetermined time (for example, several tens of minutes to several tens). It can be carried out by contacting under a temperature condition (for example, 0 to 50 ° C.) that does not inhibit the reaction. In the case of the primary reaction, the above-mentioned purified serum albumin can be added to carry out the primary reaction. The purified serum albumin used at this time is preferably used at a concentration of 5% or less, particularly 1% or less. Usually, after the completion of the primary reaction step, the carrier for binding anti-phospholipid antibody which has formed an immune complex with the anti-phospholipid antibody and the test liquid are separated by an appropriate separating means. The applicable separation means depends on the type of carrier substance used, but when a carrier substance insoluble in the reaction solution is used, it can be separated by a known method such as suction, decantation, filtration or centrifugation. After separation, the carrier for binding the antiphospholipid antibody may be washed with a buffer or the like. The primary reaction step and the secondary reaction step described below may be performed at the same time, or after the primary reaction step, the test solution may be continuously performed without separating the test solution.

5.3 Fractions and proteins "Serum and plasma derived from an animal of the same species as the test liquid" used in the primary reaction step are the same species as the animal for which the antiphospholipid antibody in the body fluid is to be measured. Any animal may be used as long as it is produced from the blood of the animal (including relatives) by the known method. For example, when human body fluid is used as the measurement target, human serum or human plasma is used, and particularly serum or plasma of a healthy person is suitable. Further, the fraction is, for example, a known separation / purification method for human serum or human plasma (gel filtration (molecular sieve chromatography); ion exchange chromatography using DEAE-cellulose, etc .; protein A sepharose, pepperin sepharose, cardiolipin). -Affinity chromatography with polyacrylamide etc .; affinity adsorption with liposomes containing phospholipids such as cardiolipin; membrane separation by dialysis, ultrafiltration etc .; electrophoresis; solvent extraction etc.) It is a fraction showing the effect of increasing the binding property between the antibody specifically present in the antiphospholipid antibody syndrome and the phospholipid bound to the carrier when added to the reaction solution in the first reaction step.

As the above-mentioned serum or plasma fraction, for example, human serum or plasma is subjected to gel filtration using, for example, an Ultragel ACA-34 (manufactured by LKB) column to obtain the serum of a patient with antiphospholipid antibody syndrome. Examples thereof include those obtained by collecting a fraction showing an action of enhancing the binding property between an antibody specifically present therein and a phospholipid.
Alternatively, a fractionated product having the physicochemical properties shown below can be mentioned. It is included in the non-dialyzed fraction when dialyzed using a cellulose membrane having a molecular weight cut-off of 6,000 to 8,000. When fractionated by gel filtration or SDS-polyacrylamide gel electrophoresis, it is confirmed in the vicinity of serum albumin or slightly on the low molecular side. Does not bind to protein A. By chromatography with a weakly basic anion exchanger containing a diethylaminoethyl group, it is eluted near the IgG fraction. For example, in chromatography on a DEAE-cellulose column, when eluted with 0.014M phosphate buffer (pH 7.4), the fraction that passes through is eluted with a slight delay in the vicinity of the IgG fraction. It is contained in the fraction salted out with 30 to 60% saturated ammonium sulfate.

The concentration of the following proteins in the above-mentioned serum, plasma or its fraction or its fraction in the above-mentioned antigen-antibody reaction solution is high enough to achieve the effects of the present invention as described above. There is no particular limitation as long as it exists. Serum or plasma, for example, serum or plasma prepared by a conventional method from blood is diluted about 200-fold or more, particularly about 40-fold or more (fraction, in the case of the following proteins, serum Alternatively, it is preferable that the reaction solution be present at a high concentration (equal to or higher than the concentration corresponding to the above-mentioned dilution of plasma).
The presence of a high concentration of serum, plasma or a fraction thereof or a protein in the fraction during the reaction may affect the binding property between an antibody specifically present in serum of a patient with antiphospholipid antibody syndrome and phospholipid. On the other hand, it has the effect of suppressing the reaction between antiphospholipid antibody and phospholipid present in the serum of patients with infectious diseases such as tuberculous meningitis and syphilis.

The protein which is the active ingredient of the above fraction can be isolated and purified as a homogeneous protein as follows. First, serum or plasma of a healthy person, for example, DE
It is subjected to ion exchange column chromatography using AE-cellulose or the like, and the fraction containing the active ingredient is collected. The active fraction is collected using as an index the action of enhancing the binding property between the phospholipid and the antibody specifically present in the serum of the patient with the antiphospholipid antibody syndrome. Then, the obtained active fraction is subjected to protein A-Sepharose column chromatography to remove human IgG contained in the active fraction. Furthermore, an anti-human IgG antibody is used, for example, Sepharose CL-4B.
Affinity column chromatography using a column prepared by binding to a resin (Pharmacia) is performed to remove human IgG that does not adsorb protein A.
Incidentally, each active fraction obtained after subjecting to each of the above column chromatography is dialyzed by a conventional method, if necessary,
Further, it may be appropriately concentrated by salting out with an ultrafilter or saturated ammonium sulfate.

Then, the purified fraction from which human IgG has been removed is brought into contact with the liposome to adsorb the active ingredient to the liposome to purify the active ingredient. The liposome used here is a liposome composed of a phospholipid represented by the formula [I] such as cardiolipin, phosphatidylserine, phosphatidylinositol, or phosphatidic acid, or a liposome having the phospholipid as one component. The liposomes can be prepared by a conventional method [Immunological Experiment Operation Method IX, pages 2989-2994, issued December 4, 1980, published by the Japanese Society of Immunology]. For example, a cardiolipin liposome can be prepared by placing an alcohol solution of cardiolipin in a pear-shaped flask, drying to dryness under reduced pressure, adding an appropriate buffer thereto, and vigorously stirring with a vortex mixer. The fraction containing the active ingredient was added to the buffer solution containing the liposome to adsorb the active ingredient to the liposome, and then the liposome suspension on which the active ingredient was adsorbed was subjected to centrifugation to purify the activity purified from the obtained supernatant. The components are recovered. The active ingredient may be further purified by subjecting it to chromatography such as HPLC.

The purified active ingredient thus obtained exhibits the following physicochemical properties. It has a molecular weight of about 50,000 ± 2,000 and an isoelectric point of about 6.60 ± 0.4 as measured by SDS polyacrylamide gel electrophoresis, and has binding properties to phospholipids. The active ingredient exhibits specific binding properties to phospholipids such as cardiolipin, which has a negative charge. Further, an antibody specifically present in the antiphospholipid antibody syndrome, for example, an anticardiolipin antibody in a patient with autoimmune disease and cardiolipin bound to a carrier react with each other depending on the active ingredient. It has the effect of suppressing the reactivity of antiphospholipid antibodies derived from infectious diseases such as tuberculous peritonitis and syphilis with phospholipids.

From these properties, when the above-mentioned active ingredient is added to the reaction system containing the cardiolipin bound to the carrier and the antibody specifically present in the antiphospholipid antibody syndrome, first, the active ingredient and the carrier are added. It is considered that the bound cardiolipin reacts with each other to form a complex to which an antibody specific to the antiphospholipid antibody syndrome binds. Also,
Since it has the above properties and,
Using this active ingredient, the antiphospholipid antibody derived from the antiphospholipid antibody syndrome and the antiphospholipid antibody derived from the infectious disease can be separately detected. It should be noted that even if a high concentration of serum or plasma or a fraction thereof is present in the reaction solution, it can be similarly fractionated and detected. By adding the above-mentioned active ingredient protein obtained in the present invention during the above-mentioned first antigen-antibody reaction, it is possible to selectively detect the antibody specifically present in the antiphospholipid antibody syndrome. Becomes

5.4 Second Antigen-Antibody Reaction (Secondary Reaction) Step In the present invention, the “second antigen-antibody reaction step” means the immune complex formed in the first reaction step and the labeled anti-immunoglobulin antibody. And reacting with each other to form a sandwich immune complex composed of the immune complex and a labeled anti-immunoglogrin antibody. The labeled anti-immunoglobulin antibody used in this step is an anti-immunoglobulin antibody labeled with an appropriate marker corresponding to the detection method in the detection step described later. The anti-immunoglobulin antibody used here is not particularly limited in its origin and production method as long as it is an antibody or a fragment thereof that can bind to the anti-phospholipid antibody to be measured.

Since the subclasses of antiphospholipid antibodies in body fluids are usually IgG, IgM, IgA, etc., antiimmunoglobulin antibodies are anti-IgG antibodies, anti-IgM antibodies, anti-Ig antibodies.
It can be appropriately selected from A antibody and the like according to the purpose. Also,
It goes without saying that when the antiphospholipid antibody to be measured is of human origin, an anti-human immunoglobulin antibody is used. Such an anti-immunoglobulin antibody is prepared by administering an immunoglobulin derived from an animal of the same species as the animal producing the anti-phospholipid antibody to be measured to another animal for immunization, and separating and purifying from the serum of the animal. Methods, or known methods such as antibody-producing cells of the animal (spleen cells, lymph node cells, peripheral blood lymphocytes, etc.) such as hybridoma method, transformation method (by EB virus, etc.) (for example, Eur. J. Immunol. , 6, 511 (1976)) and allowed to grow semi-permanently.
It can be performed by a method of separating and purifying from a culture grown in vitro (in vitro) or in vivo (in vivo). These anti-immunoglobulin antibodies are commercially available, and such antibodies readily available to those skilled in the art can be used in the method of the present invention.

As the anti-immunoglobulin antibody, an antibody molecule may be used as it is, or an antibody fragment (F (ab ′) ₂ , Fab ′ etc.) which is decomposed by a proteolytic enzyme (eg papain, pepsin etc.) or the like. May be used as. The labeling substance (marker) that labels the anti-immunoglobulin antibody is not particularly limited as long as it corresponds to the detection step. For example, radioisotopes ( ¹²⁵ I, ¹³¹ I,
³ H, ¹⁴ C, etc.), enzymes (peroxidase, β-galactosidase, alkaline phosphatase, glucose oxidase, glutamate oxidase, glucose-
6-phosphate dehydrogenase, lysozyme, glucoamylase, acetylcholinesterase, mylenate dehydrogenase, etc.), other in vivo ligand / receptor (biotin, avidin, streptavidin, etc.), coenzyme / prosthetic group (FAD, FMN, ATP, etc.) ), Fluorescent substance (fluorescein isothiocyanate (F
ITC), europium, phycoerythrin, etc.), luminescent substance (luminol derivative, etc.), electron spin resonance (E
SR) marker substance (piperidine-1-N-oxyl compound, pyrrolidine-1-N-oxyl compound, etc.),
It can be labeled with a known marker such as a liposome (containing a labeling substance such as an enzyme, a fluorescent substance, and a luminescent substance) (see the above-mentioned documents,).

The method of labeling the anti-immunoglobulin antibody with these markers can also be performed by known techniques. For example, when a radioisotope is used as a marker, the chloramine T method, an enzymatic method using lactoperoxidase, Bolton-Hunt
er) method or the like (see the above-mentioned document). Further, for example, when labeling with an enzyme as a marker, a maleimide cross-linking method (for example, succimidyl 6-maleimide hexanoate (EMCS) is used), a glutaraldehyde cross-linking method, a periodate cross-linking method (Nakane method), an isocyanate. Crosslinking method (for example, isocyanates (toluene diisocyanate, etc.), isothiocyanates are used) or benzoquinone crosslinking method can be used (see the above-mentioned document). When this secondary reaction step is performed separately from the primary reaction step, a solution containing an antiphospholipid antibody-binding carrier to which the immune complex obtained in the primary reaction step is bound and a labeled anti-immunoglobulin antibody (buffer) A solution such as a liquid) for a certain period of time (for example, several tens of minutes to several hours) under temperature conditions (for example, 0
The reaction can be carried out by contacting at -50 ° C. When the reaction is carried out simultaneously with the primary reaction step or without separating the reaction solution, the labeled anti-immunoglobulin antibody may be allowed to exist in the reaction solution when the primary reaction is carried out or after the reaction is completed. Good.

5.5 BF Separation Step The BF separation step of the method of the present invention is a separation step for separating the phase containing the sandwich immune complex formed in the secondary reaction step from the phase containing the substance not bound to the carrier. It is a process. This step is not necessary when the method of the present invention is carried out by the homogeneous method, but it is an essential step when carried out by the heterogeneous method. This step can be carried out by a known separation means such as suction, decantation, filtration or centrifugation when a carrier substance insoluble in the reaction solution is used. The carrier may be washed with a buffer (PBS or the like) after or simultaneously with the above separating operation.

5.6 Detection Step In the case of the heterogeneous method, the “detection step” of the method of the present invention means the labeling substance (marker) of the labeled anti-immunoglobulin antibody contained in the sandwich immune complex separated in the BF separation step. ) Is detected. This detection may be quantitative or qualitative. The detection step may be carried out by a known method as long as it corresponds to the marker used (see the above literatures). For example, when a radioisotope is used as a marker, detection can be performed by a scintillation counter, and when an enzyme is used as a marker, detection can be performed by a known method used for measuring the activity of the enzyme used.

The measurement of enzyme activity when peroxidase is used as a marker will be described below. The peroxidase activity can be measured by various methods that detect the transfer of electrons when the substrate hydrogen peroxide is decomposed into water. That is, for example, chromogen
Known methods such as measurement of a change in absorbance due to oxidation of, and measurement of a change in redox potential with an electrode can be used. In particular, the method of using a chromogen is common. For example, tetraalkylkilbendidine (3,3 ', 5,
5'-tetramethylbenzidine (TMBZ) etc., o
Known substances such as -phenylenediamine (OPD), 2,2'-azinodi (3-ethyl) benzothiazolinone-6-sulfonic acid (ABTS), dianisidine, dicarboxidine, and diaminobenzidine can be used ( For example, Tokushusho Sho 62-502653 (WO86 / 04
610)).

A measurement using a peroxidase chromogen is carried out by a solution containing hydrogen peroxide and chromogen in either phase separated by BF separation, preferably a buffer (citrate buffer). Solution, tartrate buffer, PBS, etc.) for a certain period of time (for example, several minutes to several hours),
It can be carried out by reacting at room temperature (for example, at room temperature), stopping the reaction with an enzyme reaction stop solution (for example, sulfuric acid), and then measuring the absorbance (in the case of TMBZ, the absorbance at 450 nm). The quantification of the anti-phospholipid antibody in the test liquid includes a known concentration of the anti-phospholipid antibody, for example, serum derived from an animal of the same species or akin to the test liquid, plasma its fraction or its fraction. Standard curve measured using a standard reagent to which the protein in the product was added (showing the relationship between absorbance and antibody concentration (or antibody titer))
And the measured absorbance can be compared.

5.7 Differential Detection The presence of a high concentration of serum or plasma, a fraction thereof, or a protein which is an active ingredient in the fraction in the reaction solution causes the presence of antiphospholipid antibody syndrome-derived antiphospholipid. The differential detection of the antibody and the antiphospholipid antibody derived from the infectious disease can be performed as follows. That is, the above-mentioned antigen-antibody reaction (1
In the second reaction step, both a system in which the primary reaction is carried out in the presence of a high concentration of serum or plasma, a fraction thereof, or a protein, and a system in which the primary reaction is carried out in the absence thereof are carried out. . And when the reactivity of the antiphospholipid antibody and the phospholipid immobilized on the carrier is dependently enhanced by the presence of these blood components of the present invention, the antiphospholipid antibody derived from the antiphospholipid antibody syndrome Can be judged. When the reactivity is dependently reduced by the presence of these blood components of the present invention, it can be determined that the antiphospholipid antibody is derived from an infectious disease.

6. Kit 6.1 Kit (6) The immunological assay kit (6) used in the method of the present invention of the above (4) is composed of at least the constituent reagents of the above (A) to (C). The “antiphospholipid antibody-binding carrier” of the constituent reagent (A) is treated by using two kinds of purified serum albumin and a surfactant in combination among those described in “3. Antiphospholipid antibody-binding carrier” above. Or a purified serum albumin, a surfactant, and serum derived from an animal of the same species or an analogue as the test solution, plasma, a fraction thereof, or a protein in the fraction was used in combination. Stuff used. The “labeled anti-immunoblobulin antibody” of the constituent reagent (B) is the same as the “5.3 Second antigen-antibody reaction (2
Next reaction) step ”is used. Constituent reagent (C) "Sample dilution liquid to which serum, plasma or a fraction thereof derived from an animal of the same species as the test liquid is added" is for diluting the test liquid or standard reagent to be measured as necessary. The blood component of the present invention is added for the same reason as above.

In addition to the above-mentioned constituent reagents, the kit (6) of the present invention may be accompanied by a standard reagent or a reagent for detecting a labeling substance, if necessary. Examples of the standard reagent include standard reagents containing a known concentration of antiphospholipid antibody and optionally containing the blood component of the present invention. This is an antiphospholipid antibody that is usually isolated from a patient with antiphospholipid antibody syndrome, and is serially diluted as necessary, and is described in "5.2 First antigen-antibody reaction (primary reaction) step". For this reason, the blood component of the present invention is added. As a reagent for detecting a labeling substance, for example, when the kit is a kit for an enzyme immunoassay, a reagent for measuring an enzyme activity as needed, that is, an enzyme substrate and an indicator of its reaction. It is preferable to attach (for example, the chromogen of the above-mentioned “5.5 detection step”). In addition, an enzyme reaction stop solution (for example, a solution containing sulfuric acid) may be attached.

6.2 Kit (7) The kit for immunological assay (7) used in the method of the present invention of the above (5) is composed of at least the constituent reagents of the above (A ′) to (C). If necessary, standard reagents and reagents for detecting labeling substances can be attached as in the case of the kit (6). The constituent reagent (A ′) is the antiphospholipid antibody-binding carrier described in “3. Antiphospholipid antibody-binding carrier” that has been treated only with a surfactant.
The other constituent reagents are the same as those in 6.1 above.

6.3 Kit (14) The kit used for the immunological assay of (12) above is
As one of the constituent reagents, a high concentration of serum or plasma, or the above-mentioned fraction or the protein in the fraction is contained. The other constituent reagent may be any conventionally used carrier for binding anti-phospholipid antibody, of course, the carrier for binding anti-phospholipid antibody of the present invention described above. Further, as the other constituent reagents, the reagents usually used can be used as they are.

[0049]

EXAMPLES Hereinafter, the present invention will be described more specifically by showing Reference Examples and Examples. Reference example A general operation method by the conventional method and an example of measurement by the method are shown below. A 50 μg / ml ethanol solution of cardiolipin (manufactured by Sigma) derived from bovine heart (50 μl / well) was placed in each well of a 96-well microtiter plate (polystyrene; manufactured by Titer Tech), and ethanol in the well was dried under reduced pressure. Dried by. After drying, phosphate buffered saline containing 10% fetal bovine serum (P
BS) (hereinafter abbreviated as PBS-FBS) (pH 7.
4) was added at 250 μl / well, and blocking was performed at room temperature for 1 hour.
V) Tween 20 (Tween 20, trade name, manufactured by Kishida Chemical Co., Ltd.)-Containing PBS (hereinafter, referred to as PBS-Tween)
Abbreviated as n. ) Washed 3 times. Next, P of the test liquid (serum)
Add 100 μl each of the appropriate dilutions of BS-FBS to the wells and allow them to react at room temperature for 1 hour.
Wash 5 times with 250 μl. 100 μl of horseradish peroxidase-labeled anti-human IgG antibody was added to each well, reacted at room temperature for 1 hour, and washed 5 times with PBS-Tween. 100 μl of substrate solution (0.3 mM 3,3 ′, 5,5′-tetramethylbenzidine (TMBZ) solution containing 0.003% hydrogen peroxide solution) was added, and the mixture was allowed to stand at room temperature for 15 minutes.
After reacting separately, 100 μl of a reaction stop solution (2N sulfuric acid) was added to the reaction solution, and the absorbance (450 nm) was measured. The results are shown in Fig. 1 (A). The enzyme-labeled antibody used was horseradish peroxidase and a mouse monoclonal anti-human IgG antibody (G-02, I by the periodate crosslinking method).
gG class; Yamasa Shoyu Co., Ltd. combined. The test liquid As serum used here is patient serum of typical antiphospholipid antibody syndrome (habitual abortion in SLE) (hereinafter referred to as As serum), and the test liquid Ya serum is completely antiphospholipid. It is a serum of a patient with tuberculous meningitis without signs of antibody syndrome (hereinafter referred to as Ya serum). In the conventional method using fetal bovine serum (FBS) in the primary reaction, as shown in FIG. 1 (A), the antibody in Ya serum (hereinafter referred to as Ya antibody) and the antibody in As serum (hereinafter referred to as As antibody).
It was impossible to separately quantify.

Example 1 Detection of Antiphospholipid Antibody Syndrome-Specific Antibody by Addition of Serum from Healthy Subjects (1) Preparation of Carrier for Binding Antiphospholipid Antibody The carrier for binding antiphospholipid antibody was prepared using the following two methods. Created. [Method (I)] A 50 μg / ml ethanol solution of 50 μg / ml cardiolipin derived from bovine heart (manufactured by Sigma) was placed in each well of a 96-well microtiter plate (polystyrene; manufactured by Titer Tech Co., Ltd.), and ethanol in the wells was added. Was dried by vacuum drying. Then, PBS (pH 7.) containing 1% (W / V) purified bovine (bovine) serum albumin (manufactured by Sigma; No. 7511, Fatty acid free; hereinafter abbreviated as pBSA).
4) (Hereinafter, abbreviated as PBS-pBSA.) 250 μl was added and treated at 4 ° C. for 1 hour, and PBS containing 0.05% (V / V) Tween 20 (PBS-Tween) 250
It was washed 3 times with μl to obtain the carrier for binding antiphospholipid antibody of the present invention.

[Method (II)] A 500 μg / ml ethanol solution of bovine heart-derived cardiolipin (manufactured by Sigma) was placed in a glass test tube, dried under reduced pressure, and then PBS.
Was added to suspend cardiolipin. 50 μl / well of this cardiolipin suspension was placed in each well of a 96-well microtiter plate (polystyrene; manufactured by Titer Tech Co., Ltd.) and reacted at 4 ° C. for 1 hour to remove the suspension by suction. Thereafter, the same procedure as in method (I) was performed to obtain the antiphospholipid antibody-binding carrier of the present invention. (2) Measurement of antiphospholipid antibody To each of the same patient sera (As serum and Ya serum) as used in Reference Example, 5% of healthy human serum was added, and PB was added.
Diluted with S-pBSA. Each of these dilutions is 100
Add 1 μl each to the cardiolipin-bound titer plate prepared by method (I) and method (II), and add 1 at 4 ° C.
The reaction was carried out for a time, and the plate was washed 5 times with 250 μl of PBS-Tween. For the subsequent reaction, the absorbance was measured by performing the same operation as in Reference Example (the method of the present invention). Regarding the plate prepared by the method (I), pBSA was added in the primary reaction and the reaction was performed in the same manner as above, and the absorbance was measured (comparative experiment). The results obtained by each method are shown as a dilution curve. FIG. 1 (B) shows the result of the comparative experiment, FIG. 1 (C) shows the result of the method of the present invention performed on the plate prepared by the method (I), and FIG. 2 shows the result obtained by the method (II). The results of the method of the present invention performed on the plates are shown below.

As is apparent from FIG. 1 (B), the cardiolipin-bonded plate according to the present invention was used, and the reference example 1 was used.
By replacing the fetal bovine serum (FBS) used in the next reaction system with a commercially available purified bovine serum albumin (pBSA) (comparative experiment), the binding property of the anti-cardiolipin antibody (Ya antibody) in the Ya serum was significantly (20). %). Samples with high dilution (ie,
Loss of binding activity was observed with a 200-fold or more diluted solution). By adding healthy human serum (about 5%) to the basic measurement system of the comparative experiment in which pBSA was added in the first reaction, only the As antibody titer was the same as that of the conventional method using fetal bovine serum (FBS) (reference example). It recovered to a certain degree and completely disappeared with respect to the Ya antibody titer. When the antibody titer of As serum was measured by changing the concentration of healthy human serum to be added in the primary reaction and performing the reaction in the same manner as above, it was found that the dilution was 1
Sufficient absorbance was obtained when healthy human serum was added to a high concentration of / 40 or more (Fig. 3). The measured samples are 24 units (24U) and 12 units (12U).
As serum (by the unit of Harris et al.).

Example 2 Detection of Antiphospholipid Antibody Syndrome-Specific Antibodies by Addition of Serum Fractions from Healthy Subjects Serum from healthy subjects was fractionated by gel filtration, and each fraction was separated into 1 fraction.
It was added to the reaction solution of the next reaction. The cardiolipin-bound plate was prepared by the method (I) of Example 1, and the absorbance as an index of antibody titer was obtained by the same procedure as in Example 1 except that the serum of a healthy person was replaced by the fraction obtained by gel filtration. (450
nm) was measured. The fraction of the healthy human serum is PBS (pH
Ultrogel ACA previously equilibrated in 7.4)
3 m with 34 (LKB) column (2.0 x 55 cm)
It was performed by gel filtration of 1 healthy human serum and collecting 2 ml of each. FIG. 4 shows the relationship between the gel filtration pattern by Ultrogel ACA-34 and the absorbance (450 nm) of As serum and Ya serum as an index of the antibody titer obtained by the above method. As a result, 3 in FIG.
A component having the effect of increasing the binding property of As antibody and eliminating the binding of Ya antibody in the fraction of the second peak appearing in the absorption peak at 280 nm of the book (hereinafter, also referred to as the present active ingredient). Was confirmed to exist. Serum albumin was also contained in this fraction, but it was confirmed that it was not the effect of serum albumin since the activity (effect) was not observed in purified human serum albumin itself.

Further, the serum of a healthy person was subjected to H using G2000SW (manufactured by Tosoh Corporation) column (0.75 × 30 cm).
When fractionated by gel filtration by the PLC method, a fraction containing this active ingredient (activity peak) appeared near the serum albumin fraction and was slightly eluted on the low molecular side (FIG. 5).
Next, the serum of a healthy person was treated with 1 M Tris-HCl buffer (pH 9.
After dialysis with 0), it was passed through a Protein A-Sepharose (Pharmacia) column and passed through (non-IgG fraction) and a fraction (IgG fraction) eluted with 0.1 M citrate buffer (pH 3.0). Min) was prepared. FIG. 6 shows the elution pattern. Also, after dialyzing each fraction with PBS, the 1/20 serum concentration equivalent amount of As serum (24 U) was used.
And the absorbance (45%) as an index of the antibody titer in the same manner as in Example 1 using the plate prepared by the method (I).
0 nm) was measured. The results are shown in Table 1.

[0055]

[Table 1] Table 1 ────────────────────────── Absorbance (450 nm) ────────────── ───────────── Non-IgG fraction 1.154 (0.022) IgG fraction 0.806 (0.676) ─────────────── ─────────────

The numerical values in the table represent the absorbance at 450 nm. In addition, the number in parentheses indicates the result of measurement on a sample containing no As serum (control test). As shown in Table 1, this active ingredient is present in the non-IgG fraction. Furthermore, when healthy human serum was passed through DEAE-cellulose (DE-52 manufactured by Whatman), this active ingredient appeared in the flow-through fraction eluted with 0.014M phosphate buffer (pH 7.4). (Fig. 7) Further, salting out with ammonium sulfate revealed that the active ingredient was 30-60.
It precipitated at a% saturation concentration (Fig. 8). The effect of increasing the binding of As antibody is that the above-mentioned components and immobilized cardiolipin
It is considered that the effect of eliminating the binding of the Ya antibody by the interaction with the micelle is due to the factor reacting with the antibody and being absorbed in the liquid phase.

Example 3 Detection of Antiphospholipid Antibody Syndrome-Specific Antibodies by Surfactant-Treated Antigen-Binding Carrier Basically, the method (I) and method (I) of Example 1 (1) were used.
The effect of the method of the present invention was investigated using an antiphospholipid antibody-binding carrier treated with Tween 20 alone according to I). That is, bovine heart cardiolipin was bound to each well of a 96-well microtiter plate according to method (I). 0.05% PBS-
After washing with Tween, serum of a healthy person was added to the reaction solution of the primary reaction (Experiment I-1) or reaction was performed without addition (Experiment I-2) in the same manner as in Example 1, and thereafter, as in Example 1. The same operation was performed to measure the absorbance (450 nm) as an index of antibody titer. Also, wells to which cardiolipin was bound by the same method were washed with PBS containing no Tween 20, and human serum of healthy human was added to the reaction solution of the first reaction (Comparison I-1) or without addition ( Comparative I-2) The reaction was carried out, and thereafter the same operation as in Example 1 was carried out to measure the absorbance (450 nm) as an index of the antibody titer. For the washing after the primary reaction step and after the secondary reaction step, those corresponding to the above washing solutions were used in each experimental section.

96 wells of bovine heart cardiolipin were prepared in the same manner as in the above method (II) except that dry cardiolipin was suspended in a test tube with PBS (pH 6.0) containing 0.01% BSA. Bound to each well of a microtiter plate. Add 0.05% pP to this well
It was washed with BS-Tween and reacted in the same manner as in Example 1 for the secondary reaction II-2). Thereafter, the same operation as in Example 1 was performed,
Absorbance (450 nm) was measured as an index of antibody titer. In addition, wells to which cardiolipin was bound by the same method were washed with pBS without Tween 20,
Normal person serum was added to the reaction solution of the primary reaction (Comparison II-
The reaction was performed 1) or without addition (Comparison II-2), and thereafter the same operation as in Example 1 was carried out to measure the absorbance (450 nm) as an index of the antibody titer. For the washing after the first reaction step and after the second reaction step, PBS without Tween 20 was used in each experimental section. Also,
In each of the above experiments, As serum and Y were used as test liquids.
a Serum was used. In addition, these sera are Harris (H
arris E. N. ) Et al., The anti-phospholipid antibody titer proposed at the 3rd Kingston Antiphospholipid Antibody Symposium (KAPS) was diluted so as to be constant. The results of these experiments are shown in Table 2. The numerical values in the table represent the absorbance at 450 nm. The values in parentheses are the values measured using a carrier to which cardiolipin is not bound (control test).

[0059]

[Table 2] Table 2 ──────────────────────────── Experimental group As serum (24U) Ya serum (24U) ─── ───────────────────────── Experiment I-1 0.982 0.032 (0.011) (0.012) Experiment I-20 0.066 0.054 (0.010) (0.012) Comparison I-1 1.280 1.109 (1.124) (1.082) Comparison I-2 1.260 1.102 (1.102) (1.164) Experiment II-1 0.998 0.053 (0.022) (0.050) Experiment II-2 0.084 0.097 (0.076) (0.045) Comparison II-1 1 .125 1.100 (1.025) (1.028) Comparison II-2 1.147 1.163 (1.101) (1.1 3) ────────────────────────────

As shown in Table 2, the washing treatment with Tween 20 caused antigenic expression of cardiolipin,
The binding property of As antibody to immobilized calciopine is enhanced and the adsorption of immunoglobulin to a non-specific plate is suppressed. In the carrier for binding antiphospholipid antibody prepared by the method (I), it is considered that physically adsorbed cardiolipin is lipid-transferred to the micelle of Tween 20, and formation of "surfactant-phospholipid / micelle" is antigenicity of cardiolipin. It is essential for expressing a three-dimensional structure having the above-mentioned structure on the plate surface, and the micelle of Tween 20 has an action of suppressing non-specific adsorption of immunoglobulin. Immobilization of “surfactant-phospholipid / micelle” can be achieved by not only the method (I) but also the method (II). In this method (II), micelles of cardiolipin were first formed in a buffer solution, and then incubated in a plate to adsorb cardiolipin to the plate surface through a lipid bilayer membrane of micelles, followed by washing with Tween 20. Is the method of doing.

Example 4 Effect of maintaining cardiolipin antigenic expression by treatment with pBSA When a cardiolipin-bound plate prepared by the same method as the above method (I) was used and this plate was treated with pBSA (invention), In the case of treatment (comparative example), the absorbance (450 nm) as an index of anti-cardiolipin antibody antibody titer was measured by changing the addition concentration of Tween 20 for treating the plate. This result is the third
Shown in the table. The treatment with the surfactant was performed using PBS (pH 7.4) containing Tween 20 at each concentration. The measured sample is 24 units (24
U) and 12 units (12 U) (according to the unit of Harris et al.) As serum.

[0062]

[Table 3] ──────────────────────────────── Tween concentration The present invention Comparative Example (%) 24U 12U 24U 12U ─ ─────────────────────────────── 0 0.621 0.492 0.082 0.079 0.005 1.383 0.962 1.023 0.823 0.05 1.399 0.983 1.079 1.854 0.5 1.401 0.981 1.106 0.877 ──────────── ─────────────────────

As is clear from Table 3, tween 2
The effect of 0 antigenic expression was demonstrated with or without treatment with pBSA. However, the cardiolipin antigenic expression was promoted by the presence or absence of treatment with 1% pBSA for 1 hour at 4 ° C. That is, in order to form a physiological three-dimensional conformation having antigenicity after the dry treatment of the ethanol solution of cardiolipin by the method (I), not only the tween treatment but also the BSA treatment occurs, and both When used in combination, the antigenic expression is promoted, the stability is enhanced, and the so-called "blocking promoting effect" of non-specific adsorption of the antibody is also obtained.

Example 5 Effect of Various Surfactants As serum (2
Absorbance (450 nm) as an index of antibody titer of 4 U) was measured. The results are shown in Table 4. A 0.05% solution was used as each surfactant.

[0065]

[Table 4] Table 4 ─────────────────────────── Surfactant Absorbance (450 nm) ────────── ────────────────── Tween 20 1.136 Tween 60 1.025 Tween 80 1.119 ───────────────── ──────────

As is clear from Table 4, the tween
The same effect as Tween 20 was obtained even with a surfactant having a hydrophobic group length different from 20. Example 6 Detection of Antiphospholipid Syndrome-Specific Antibody by Carrier Treated with Human Serum, pBSA and Surfactant Instead of treatment with PBS containing 1% pBSA in the method of Example 1 (I) The cardiolipin-conjugated plate was treated with PBS containing 5% normal human serum and 1% (W / V) pBSA in the same manner as in Example 1 (I) except that the antiphospholipid antibody-conjugated product of the invention was bound. A carrier for use was obtained. In addition to the above carrier (invention) and the carrier (control) obtained by the method (I) of Example 1, 1% PBS-BSA containing no healthy human serum was used in the reaction solution of the primary reaction. In the same manner as in Example 1, the absorbance (450 nm) as an index of the antibody titer of As serum was measured. The results are shown in Table 5.

[0067]

[Table 5] Table 5 ───────────────────────────── Experimental Zone 24U 12U 6U ────────── ──────────────────── Present invention 1.158 0.811 0.509 Control 0.169 0.095 0.035 ────────── ────────────────────

As is clear from Table 5, when the antiphospholipid antibody-binding carrier was treated with the serum of normal human, pBSA and the surfactant in combination, human serum was not added in the first reaction. The same effect as when adding serum was obtained. Example 7 Measurement of serum of antiphospholipid antibody syndrome patient A serum sample of a healthy subject and an SLE patient were prepared in the same manner as in Example 1 using the carrier for binding antiphospholipid antibody prepared by the method (I) of Example 1. (Production group and abortion group) Anti-cardiolipin antibody titer of serum sample (unit; unit
(U)) was measured. The results are shown in FIG. As shown in FIG. 9, the absorbance (450 nm) as an index of the anti-cardiolipin antibody titer in the serum of SLE patients was significantly higher than that in the sera group of healthy subjects. Furthermore, the antibody titer in the habitual abortion group was significantly higher than that in the production group (group that succeeded in giving birth).

Further, 248 cases of sera from patients with autoimmune disease including sera from SLE patients were subjected to anti-cardiolipin antibody titer determination by the conventional method (method of Reference Example) using fetal bovine serum in the primary reaction and the above-mentioned method of the present invention. The results are shown in FIG. In FIG. 10, the vertical axis represents the antibody titer (unit) according to the method of the present invention, the horizontal axis represents the antibody titer (unit) according to the conventional method, and the broken line represents the cut-off value (average antibody titer in serum of healthy subjects + 3 × standard deviation (1). Unit))). The correlation coefficient between both measurement systems is 0.664, which shows that the method of the present invention allows high-fractionation quantification. That is,
In the method of the present invention, positive-negative sieving is improved. This is because the antibody derived from the infectious disease (that is, the Ya type antibody) becomes negative as described above.
The results obtained from the dilution curve (representing the relationship between serum dilution and anti-cardiolipin antibody titer (unit)) according to the method of the present invention for 4 SLE patient sera and 2 healthy human sera are shown in FIG. As is clear from FIG. 11, the method of the present invention has extremely good dilution linearity, indicating that the antibody titer is highly quantitative.

Example 8 Comparison of Antiphospholipid Antibody Titers in Serum of Patients with Autoimmune Diseases and Infectious Diseases Antibodies prepared by binding various phospholipids to a carrier by the same method as the method (I) of Example 1. Using a carrier for phospholipid antibody binding, the absorbance (45) as an index of antiphospholipid antibody titer in serum of autoimmune disease (SLE) patients and infectious disease (tuberculosis and syphilis) patients was measured in the same manner as in Example 1.
0 nm) was measured. 5% for each test serum
The results of measurement in the presence and absence of serum from healthy subjects are shown in FIG. As serum was 24 U (diluted to 1/200 concentration), and other test serum was 1/10.
It diluted with 0 times concentration and used for the experiment. In the figure, cardiolipin (CL), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylethanolamine (P) are listed in this order from the top.
E) and phosphatidylcholine (PC) are used as immobilized antigens to show reactivity of antiphospholipid antibody in the test serum (CL, PI, PS, PA are the present invention, P
E and PC are controls). The amount of antigen used at this time was 2.5 μg /
50 μl / well. As is clear from FIG. 12, antigens having a negative charge (that is, CL, PI, PS, PA)
In contrast, autoimmune antibodies (antibodies specific to autoimmune diseases) show increased reactivity depending on the addition of serum, whereas infectious antibodies (antibodies derived from infectious diseases) The reactivity decreased in a dependent manner. This has enabled the differential quantification of infectious and autoimmune antiphospholipid antibodies, which were difficult to measure by conventional methods.

Example 9 Effect of Buffer Solution The carrier for antiphospholipid antibody binding prepared by the method (I) of Example 1 was used, and 1% pB was used as a diluent for the test solution.
HEPES buffer containing SA (10 mM HEPES,
The absorbance (450 nm) as an index of the antibody titer of As serum was measured by the same method as in Example 1 except that 150 mM NaCl; pH 7.4) was used. The result is thirteenth
Shown in the figure. As is clear from FIG. 13, when a reaction solution containing the HEPES was used as the reaction solution of the primary reaction (dilution solution of the test solution), a good calibration curve (anti-cardiolipin antibody titer (unit) and absorbance (450 nm)) was obtained. Was obtained and was found to be useful for the measurement of antiphospholipid antibody derived from the antiphospholipid antibody syndrome.

Example 10 Purification of the present active ingredient The present active ingredient (hereinafter referred to as anticardiolipin cofactor) in the healthy human serum fraction of Example 2 was purified as follows. (1) DEAE-Cellulose (DE-5 of healthy human serum)
2) Ion-exchange column chromatography with a column Ion-exchange column chromatography with DEAE-cellulose (DE-52) was performed in order to purify the active fraction from the serum of a healthy person. That is, DEAE-cellulose (DE-52) which was previously activated by a conventional method
Ion exchange resin (manufactured by Whatman) 150 ml 2.5
It was packed in a 60 cm glass column and equilibrated with a 14 mM sodium phosphate buffer (pH 7.4). To this, human serum 10 dialyzed against 3 L of the same buffer solution for 2 days
0 ml was added from the upper part of the column, and elution was performed with 3 L of the same phosphate buffer solution. The eluate was collected in 10 ml fractions using a fraction collector. Wavelength of each fraction 280n
The elution status of the protein was detected by measuring the absorption of m, and the activity of the anti-cardiolipin cofactor was measured by the method (B) described below. In addition, DEAE-
Since cofactor-independent anticardiolipin antibody activity appears by performing cellulose column chromatography, the independent anticardiolipin antibody activity was simultaneously measured by the method (B). Then, a fraction showing cofactor activity was recovered (FIG. 14). The collected active fraction was concentrated to about 100 ml by salting out with an ultrafilter or 80% saturated ammonium sulfate according to a conventional method, and protein A-sepharose column chromatography shown below was performed.

(2) Protein A Sepharose column chromatography of cofactor activity-containing fraction Protein A-Sepharose column chromatography was performed to remove human IgG from the cofactor activity-containing fraction. That is, 20 ml of Protein A-Sepharose (Pharmacia) was previously added to 1.5 × 20 cm.
The column was made of glass and was equilibrated with 1.5 M glycine buffer (pH 8.9) containing 3 mM sodium chloride. To this, a solution prepared by diluting 10 ml of the above-mentioned active fraction with 10 ml of the same glycine buffer was added from the top of the column and eluted with 100 ml of the same glycine buffer. The eluate was collected in 10 ml fractions using a fraction collector. Each of the thus obtained fractions was measured for protein absorption in the same manner as described above, and the absorption peak portion was recovered (Fig. 15). The recovered portion is a 10 mM HEPES buffer solution (pH 7.4) containing 150 mM sodium chloride.
After dialysis against 2 L overnight, 5 ml by ultrafiltration
It was concentrated to the desired concentration and subjected to the following affinity column chromatography. The adsorbed IgG was eluted with 100 mM sodium citrate buffer (pH 3.0).

(3) Anti-human IgG antibody-Sepharose C
Affinity Chromatography with L-4B Column To remove IgG that does not adsorb protein A, affinity column chromatography was performed using a column prepared by binding an anti-human IgG antibody to Sepharose CL-4B column resin (Pharmacia) by a conventional method. I went. That is, a resin prepared in advance was packed in a 2.0 × 5 cm glass column, and a 10 mM HEPES-sodium buffer solution (pH containing 150 mM sodium chloride) was used.
1 ml of the solution obtained in (2) after equilibrating in 7.4)
Was added from the top of the column and eluted with the same HEPES buffer. The eluate was collected in 1 ml fractions with a fraction collector. The protein absorption of each fraction was measured by the same method as described above, and the peak fractions were collected (Fig. 16). The collected fractions were collected together and subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis by a conventional method.
After confirming that gG has been completely removed (first
Figure 7, Fr1N), concentrated to 2 ml by ultrafiltration. The adsorbed IgG was eluted with 0.1 M glycine hydrochloric acid buffer (pH 3.0).

(4) Purification of cofactors by cardiolipin liposomes The purified fraction obtained in (3) from which IgG was completely removed (hereinafter referred to as Fr.1N) was subjected to affinity adsorption on cardiolipin liposomes. The factor was completely purified. That is, 5m of cardiolipin
2 ml of the g / ml ethanol solution was placed in a pear-shaped flask having a volume of 25 ml, and gently dried under vacuum reduced pressure so as to form a film on the wall of the flask. 150 mM to this
10 mM HEPES buffer containing sodium chloride (p
H7.4) 2 ml was added, and the mixture was vigorously stirred for 15 minutes with a vortex mixer to prepare cardiolipin liposomes. The liposome thus prepared was used as the purified cofactor solution Fr. 1N (1.2mg / ml
The same volume was added to the same HEPES buffer solution, and the mixture was allowed to stand at room temperature for 1 hour to allow the liposome to adsorb the cofactor affinity. Next, the liposomes were recovered by centrifugation at 15,000 rpm for 15 minutes at 4 ° C., and the liposomes were washed with the same HEPES buffer solution three times. Each supernatant was collected, and it was confirmed by SDS polyacrylamide slab gel electrophoresis by a conventional method that there was no unadsorbed cofactor. At this time,
When unadsorbed cofactor was present, it was recovered by adsorbing it on the liposome by repeating the same procedure. The liposome thus adsorbed the cofactor was suspended in 100 to 500 μl of the same HEPES buffer, and stored frozen at 20 ° C. By this operation, by SDS polyacrylamide slab gel electrophoresis by a conventional method using the obtained cofactor-adsorbed liposomes,
A protein was obtained that appeared as two bands very adjacent to each other with a molecular weight of about 50,000 (Fig. 17, F-1). Also,
At the same time, dipalmitoylphosphatidylcholine (DPP
C): 2 mM of cardiolipin (80:20, mol%)
The cofactor was purified by the same procedure using a liposome and a 2 mM liposome of DPPC: dipalmitoylphosphatidylethanolamine (DPPE) (80:20, mol%). As a result, liposomes composed of DPPC and cardiolipin showed similar effects to those of cardiolipin alone (Fig. 17, F).
-2), however, the components thought to be cofactors were not adsorbed on the liposomes composed of DPPC and DPPE (first
(Figure 7, F-3).

(5) Separation of cofactor adsorbed on liposomes by HPLC A cofactor purified by affinity adsorption on cardiolipin liposomes was analyzed by HPLC using a reverse phase column.
Separated. That is, the liposome suspension on which the cofactor is adsorbed is centrifuged at 15,000 rpm for 15 minutes,
The liposomes were collected. To this, 1% SDS aqueous solution was added and well stirred to sufficiently dissolve the liposomes. After re-centrifuging under the same conditions to precipitate insoluble matter, the supernatant portion was subjected to a reverse phase column (Waters Micro Bonder Spare C).
-4 columns (3.9 x 15 cm); manufactured by Waters)
HPLC was carried out under the following conditions, while measuring the absorption at 215 nm by the attached absorbance detector. In this way, a purified cofactor was obtained (Fig. 18).

Cardiolipin with cofactor adsorbed
HPLC conditions for cofactor separated from the liposome: a flow rate of 1 ml / min pressure limit 300 kgf / cm ² pressure lower limit value 0 kgf / cm ² flow rate for 60 minutes using liquid feed A solution 0.1% aqueous trifluoroacetic acid solution Solution B Acetonitrile: isopropanol (3:
7, V / V) with 0.07% trifluoroacetic acid added Gradient conditions Solution A (%) Solution B (%) Time 0 minutes after 100 0 60 minutes after 40 60

(6) Molecular weight and isoelectric point of anticardiolipin cofactor In order to investigate the isoelectric point and molecular weight of cofactor, An
ISODALT gel electrophoresis was performed by the method of Derson et al. First, the solution Fr. Electrophoresis was performed for 1N (Fig. 19). Although some spots appeared as shown in Fig. 19, the cofactor adsorbed on the cardiolipin liposome was spot No. 3 shown in Fig. 19. 10 and No. It was found to be 12 from the result of SDS polyacrylamide electrophoresis of the purified cofactor obtained in (5) (Fig. 17, F-1). That is, from FIG. 19, the isoelectric point of the cofactor is 6.75, the molecular weight is 49,600 (No. 10), and the isoelectric point is 6.6.
The molecular weight was 0 and the molecular weight was 50,000 (No. 12). still,
These two components correspond to proteins having the same amino acid sequence but different sugar chains or partially modified amino acid functional groups, and are considered to be subtypes of cofactors.

(B) Method for measuring anti-cardiolipin cofactor activity and cofactor-independent anticardiolipin activity The anticardiolipin cofactor activity and the cofactor-independent anticardiolipin antibody activity contained in the serum of healthy subjects are as follows. It was measured by the method of. Reference example 1
The measurement was performed in almost the same manner as the method shown in. That is, a 96-well microplate coated with cardiolipin,
PBS buffer containing 0.05% Tween (pH 7.
4); the following is referred to as a washing solution), and the operation of washing with 200 μl per well is repeated 3 times to activate the plate. Then, a sample to be measured is dispensed at 50 μl per well. Next, 150 mM sodium chloride and 1% B were previously added to the wells for measuring the cofactor activity.
A diluted 200-fold with a 10 mM HEPES buffer solution (pH 7.4; hereinafter referred to as a dilution buffer solution) containing SA
50 μl of s serum is dispensed into the well for measuring the independent cardiolipin antibody activity, and 50 μl of the dilution buffer is dispensed into each well, and the mixture is allowed to stand at room temperature for 30 minutes. The subsequent operations are the same for both activity measurement methods. That is,
150m after washing both wells 3 times with washing solution
M sodium chloride, 1% BSA and 1 mM EDTA
100 μl of anti-human IgG antibody conjugated with peroxidase derived from horseradish, which was appropriately diluted with 10 mM HEPES buffer (pH 7.4) containing 10 μl, was dispensed into each well and allowed to stand at room temperature for 30 minutes. After washing with the washing solution three more times,
0.3 mM TMBZ and 0.03% oxygen peroxide water (1
00 μl) was added as a substrate solution and reacted for 10 minutes, and then the absorbance at 450 nm was measured by a plate reader.

Test Example The following test was conducted to investigate the properties of the anti-cardiolipin cofactor. (1) Preparation of biotinylated anti-cardiolipin cofactor In accordance with the method of Kumagai and Okumura et al. Of the immunological experiment procedure method (February 20, 1980, published by the Japanese Society of Immunology (Cell Antigen III, 15-59, p2425)). Then, 1 mg of the anti-cardiolipin cofactor purified in (3) of Example 10 was biotinylated to label the anti-cardiolipin cofactor.

(2) Binding of anti-cardiolipin cofactor to cardiolipin 2. In each well of a 96-well microtiter plate.
A cardiolipin / ethanol solution was added at 5 μg / 50 μl / well, dried under reduced pressure, reacted with 1% BSA-containing PBS for 1 hour, and then washed with 0.05% Tween 20-containing PBS (200 μl) three times. On this cardiolipin-immobilized plate, as shown in FIG.
g / ml of biotinylated anti-cardiolipin cofactor (100 μl) was reacted for 30 minutes at room temperature, and after washing three times, avidinylated peroxidase was reacted for 30 minutes at room temperature. After washing three more times, 100 μl of 0.3 mM TMB
After adding Z and 0.003% hydrogen peroxide solution and reacting at room temperature for 10 minutes, the reaction was stopped by adding 100 μl of 2N sulfuric acid. The anti-cardiolipin cofactor bound to the immobilized cardiolipin was quantified by measuring the absorbance of the reaction solution. As shown in FIG. 20, the biotinylated anti-cardiolipin cofactor showed binding properties to immobilized cardiolipin in a concentration-dependent manner.

(3) Binding Specificity of Biotinylated Anticardiolipin Cofactor to Various Phospholipids Various phospholipids (that is, cardiolipin, phosphatidylserine (PS), phosphatidylinositol (PI), di) were prepared in the same manner as in (2) above. 2.5 μg / 50 μl of palmitoylphosphatidic acid (DPPA), dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylethanolamine (DPPE))
The binding specificity of the biotinylated anti-cardiolipin cofactor was examined using an avidinylated peroxidase on a plate immobilized per well. As shown in FIG.
The binding of anti-cardiolipin cofactor was specific for negatively charged lipids (cardiolipin, PS, PI, DPPA).

(4) Dependence of anti-cardiolipin cofactor on the reaction system of anti-cardiolipin antibody An anti-cardiolipin antibody (A which is specifically present in the antiphospholipid antibody syndrome (A) was determined by the same ELISA method as in (2) above.
anti-cardiolipin cofactor (2 μg / well) was added to the reaction system between solid-state cardiolipin and immobilized cardiolipin, and the dependence of anti-cardiolipin cofactor on the reaction between the anti-cardiolipin antibody and immobilized cardioline was investigated. . As shown in FIG. 22, the anti-cardiolipin antibody reacted in a manner dependent on the added antibody cardiolipin cofactor.

(5) Species specificity of anti-cardiolipin cofactors The activity of anticardiolipin cofactors (derived from human and bovine) of different species was evaluated by the same ELISA method as in (2) above. When cofactors are used, both the reactivity of anti-calciolipin antibodies for autoimmune (As serum) and infectious diseases (Ya serum) are increased, but when human-derived cofactors are used, only autoimmune antibodies (As antibodies) react. The activity to enhance the sex was recognized. (Fig. 23).

Example 11 Collection of Fractions from Serum of Healthy Subjects (1) Collection of Fractions by DEAE-Cellulose Column Chromatography With 14 mM sodium phosphate buffer (pH 7.4) (hereinafter, PB7.4) Serum (1 ml) of healthy person dialyzed overnight
Was applied to a DEAE-Sepharose column (10 ml, 1.0φ × 13 cm, Whatman DE-52) preliminarily equilibrated with PB7.4, and 1 ml of the pass-through fraction was collected. Using 25 μl of each fraction, the activity to enhance the reactivity of the anti-cardiolipin antibody that specifically appears in the serum of patients with antiphospholipid antibody syndrome (As) and the immobilized cardiolipin and the serum of syphilis patients (Sy) The activity of suppressing the reactivity between the existing anti-cardiolipin antibody and the immobilized cardiolipin was measured in substantially the same manner as in the method of Reference Example 1 and Example 10 (A). The results are shown in Fig. 24.

(2) Collection of fractions by heparin sepharose column chromatography Heparin-sepharose-equilibrated with 10 mM sodium phosphate buffer containing 50 mM NaCl, pH 7.4.
1 ml serum of a healthy person dialyzed against a column (6 ml, 1.0φ × 8 cm, Pharmacia) against the same buffer solution overnight.
Is adsorbed and the column is thoroughly washed with the same buffer solution.
Elution was performed with a concentration gradient from Cl concentration of 50 mM to 1 M.
Using 15 μl of each fraction, the activity to enhance the reactivity of anti-cardiolipin antibody and immobilized cardiolipin that specifically appears in the serum of patients with antiphospholipid antibody syndrome (As) and the presence in serum of syphilis patients (Sy) The activity of suppressing the reactivity of the anti-calciolipin antibody with the immobilized cardiolipin was measured according to the method of Reference Example 1 and Example (A). The results are shown in Fig. 25.

(3) Collection of fractions by cardiolipin-acrylamide gel column chromatography i) Preparation of cardiolipin-polyacrylamide column Bovine heart cardiolipin (5 μmoles), cholesterol (5 μmoles), dicetyl phosphate (0.5 μmoles) Dry in a pear flask under reduced pressure to form a film, add 500 μl of ethanol, and add 6
The lipid was dissolved by heating to about 0 ° C. Then 5 ml of 15%
Acrylamide, 5% bisacrylamide solution was added, and the mixture was vigorously stirred with a vortex mixer, and further 100 μl of ammonium persulfate (100 mg / ml), and 2 μl
Was added and polymerized. Homogenize the polymerized gel and pack it in a column (1.0φ x 3 cm) 50
10 mM sodium phosphate buffer containing mM NaCl, p
It was thoroughly washed and equilibrated with H7.4.

Ii) Affinity column chromatography 1 ml of healthy human serum dialyzed against 10 mM sodium phosphate buffer containing 50 mM NaCl, pH 7.4 overnight was passed through the column, and then thoroughly washed with the same buffer. 0M NaC
The adsorbable protein was eluted with 10 mM sodium phosphate buffer (pH: 7.4) containing 1-liter. Using 15 μl of each fraction, the activity to enhance the reactivity of anti-cardiolipin antibody that specifically appears in the serum (As) of antiphospholipid antibody syndrome patients and immobilized cardiolipin, and the activity in syphilis patient serum (Sy) The activity of suppressing the reactivity between the anti-cardiolipin antibody and the immobilized cardiolipin was measured according to the method of Reference Example 1 and Example (A). The results are shown in Fig. 26. As described above, the fraction of the present invention can be obtained from the serum of a healthy subject by using DEAE-cellulose column chromatography, heparin sepharose column chromatography, or cardiolipin-acrylamide gel column chromatography.

Example 12 Activity of anti-cardiolipin cofactor The activity to enhance the reactivity of anti-cardiolipin antibody in the serum (As) of patients with antiphospholipid antibody syndrome (As) (As ↑ activity) and the activity in sera of patients with syphilis. The expression of the activity (Sy ↓ activity) of suppressing the reactivity of the anti-cardiolipin antibody against cardiolipin was examined. After washing the cardiolipin-immobilized plate with PBS containing Tween 20,
Each of the serogroups 3 and 4 shown in the table was treated with the cofactor (10 μg / ml) obtained in (3) of Example 10 for 30 minutes, and the control group was treated with the cofactor lysis buffer. . After this treatment, 3 with PBS containing Tween 20
Washed twice, serum As (diluted 1/400, 125 U / m)
1) and Sy (1 / 200-fold diluted) were allowed to react at room temperature for 30 minutes in the presence (2,4 groups) or absence (1,3 groups) of cofactor (10 μg / ml). The following operation is the same as in (A) of Reference Example 1 and Example 10. Sixth
As shown in the table, the cofactor is pretreated as described above to obtain As ↑ activity, is present in the reaction system with the antibody, or is present in both. To obtain Sy ↓ activity, a cofactor may be present during pretreatment, but it must be present in the reaction system with the antibody.

[0090]

[Table 6] Table 6 ──────────────────────────────────── Cofactor Patient serum and cofactor Group Incubation Absorbance Pretreatment (As or Sy) (450 nm) ─────────────────────────────────── ── As 1st group − − 0.102 2 〃 − ＋ 1.051 3 〃 + − 0.826 4 〃 + +1.102 ─────────────────── ───────────────── Sy 1 group − − 1.106 2 〃 − + 0.710 3 〃 + − 1.125 4 〃 ＋ + 0.850 ──── ────────────────────────────────

Example 13 Using a method substantially similar to the method of (A) of Reference Example 1 and Example 10, the serum (A
s) activity for increasing the reactivity of anti-cardioline antibody with cardiolipin (As ↑ activity) and serum of syphilis patients (Sy)
The effect of purified bovine serum albumin on the activity (Sy ↓ activity) of suppressing the reactivity between the anti-cardiolipin antibody present in A. and cardiolipin was examined. When using a highly pure lipid-free bovine serum albumin (BSA) and a purified cofactor (cofactor obtained in (3) of Example 10) in this assay, the Sy ↓ activity was determined by the BSA concentration in the reaction solution. It decreases in a dependent manner (Fig. 27). This is because the Sy ↓ active expression site on the cofactor molecule is purified albumin (BS
Apparently due to being blocked by A), it is therefore desirable to use BSA concentrations in the range 0-5% (particularly 0-1%).

Example 14 Determination of Amino Acid Sequence of Cofactor The cofactor obtained by the method of (3) of Example 10 was further subjected to cardiolipin-acrylamide gel column chromatography used in (3) of Example 11. Purified by co-adsorption and subsequent elution with 1 molar NaCl (which contains the two subtypes identified in Example 10 (6)).
The N-terminal amino acid sequence of was determined. That is, 30 μl (300 pmol) of a sample was adsorbed on a PVDF membrane (polyvinylidene difluoride; manufactured by Millipore; trade name, immobilon), washed with 60% methanol, and then a gas phase protein sequencer (PSQ-1, type) The analysis was performed using Shimadzu Corporation). By the above operation,
A sequence of 5 amino acids was determined from the N-terminal of the cofactor.

[Chemical 3] (X is presumed to be Cys or His)

According to the present invention, an antiphospholipid antibody-binding carrier treated with a surfactant alone, a combination of a surfactant and purified serum albumin, or a combination thereof with a blood component of the present invention is used, and And / or infection with an antiphospholipid antibody derived from an antiphospholipid antibody syndrome, which could not be sufficiently separated by the conventional method, by adding the blood component of the present invention to the reaction solution in the first antigen-antibody reaction (first reaction) We established a method for reproducibly separating anti-phospholipid antibodies derived from the disease. By using the method of the present invention, the diagnosis of antiphospholipid antibody syndrome can be performed extremely accurately. In addition, the blood component of the present invention enhances the binding property between the antibody and the phospholipid specifically present in the antiphospholipid antibody syndrome, and has the action of decreasing the binding property between the antiphospholipid antibody derived from the infectious disease and the phospholipid. Have. Therefore, by using these serum or plasma, a fraction or a protein for an immunological assay of an antibody specifically present in the antiphospholipid antibody syndrome, it is possible to accurately diagnose the antiphospholipid antibody syndrome. In addition, each antiphospholipid antibody in antiphospholipid antibody syndrome and infectious disease can be detected separately.

[Brief description of drawings]

FIG. 1A shows anti-phospholipid antibody titers of As serum derived from antiphospholipid syndrome and Ya serum derived from infectious disease in a reference example by a conventional method, a comparative experimental example showing the effect of the present invention, and an example of the method of the present invention, respectively. Absorbance as an index (450n
It shows the result of measuring m).

FIG. 1B shows the antiphospholipid antibody titers of As serum derived from antiphospholipid syndrome and Ya serum derived from infectious disease in Reference Example by the conventional method, Comparative Experimental Example showing the effect of the present invention, and Example of the method of the present invention, respectively. Absorbance as an index (450n
It shows the result of measuring m).

FIG. 1C shows the antiphospholipid antibody titers of As serum derived from antiphospholipid syndrome and Ya serum derived from infectious disease in Reference Example by the conventional method, Comparative Experimental Example showing the effect of the present invention, and Example of the method of the present invention, respectively. Absorbance as an index (450n
It shows the result of measuring m).

FIG. 2 shows As serum and Ys using a carrier for binding an antiphospholipid antibody prepared by a method different from the example of FIG. 1 (C).
The results of measuring the absorbance (450 nm) as an index of serum antibody titer are shown.

FIG. 3 shows the relationship between the concentration (dilution degree) of the added healthy human serum and the absorbance (450 nm) as an index of antibody titer.

FIG. 4 is a gel filtration pattern of normal human serum and the absorbance (45) as an index of the antibody titer of As serum and Ys serum measured by adding a fraction thereof and performing a primary reaction.
0 nm).

FIG. 5: Gel filtration pattern by normal human serum HPLC method and absorbance (45 as an index of antibody titer of As serum measured by adding a fraction thereof and performing a primary reaction).
0 nm).

FIG. 6 shows a fractionation pattern of serum of a healthy person on a protein A-Sepharose column.

FIG. 7 shows the relationship between the fractionation pattern of normal human serum on a DEAE-cellulose column and the absorbance (450 nm) as an index of the antibody titer of As serum.

FIG. 8: Fractionation of normal human serum with ammonium sulfate and A
It shows the relationship with the absorbance (450 nm) as an index of the antibody titer of s serum.

FIG. 9 shows the antibody titers (unit: unit / m) of serum of healthy subjects and serum of patients with antiphospholipid antibody syndrome by the method of the present invention.
1; hereinafter referred to as U).

FIG. 10 shows the results of obtaining the correlation between the antibody titers (U) of sera from patients with autoimmune diseases measured by the method of the present invention and the conventional method.

FIG. 11 shows the dilution linearity of the method of the present invention for SLE patient sera.

FIG. 12: Absorbance (450 nm) as an index of antibody titer in sera of patients with autoimmune diseases and infectious diseases (tuberculosis, meningitis, syphilis) by the method of the present invention (shaded) and control method (open) ) Shows the result of having measured.

FIG. 13 shows the results of preparing a calibration curve for the antibody titer of As serum using HEPES-containing buffer solution as the primary reaction solution.

FIG. 14: DEAE-cellulose (DE-
52) shows a fractionation pattern of ion exchange chromatography by a column.

FIG. 15 shows a fractionation pattern when the fraction containing the anticardiolipin cofactor activity obtained by the ion exchange chromatography shown in FIG. 14 was subjected to protein A-cellulose column chromatography.

16 shows a fractionation pattern when the fraction containing the anti-cardiolipin cofactor activity obtained by the chromatography shown in FIG. 15 was subjected to affinity column chromatography with an anti-human IgG antibody-Sepharose CL-4B column. It is a thing.

FIG. 17: Anti-cardiolipin cofactor SDS-
It shows the results obtained by subjecting to polyacrylamide gel electrophoresis.

FIG. 18 shows a separation pattern when an anticardiolipin cofactor purified by liposome was subjected to HPLC, and a peak portion indicated by an arrow corresponds to the anticardiolipin cofactor.

FIG. 19 shows the results obtained by subjecting the fraction containing anti-cardiolipin cofactor activity obtained by affinity column chromatography using the anti-human IgG antibody-Sepharose CL-4B column shown in FIG. 16 to ISODALT electrophoresis. It is a thing.

FIG. 20 is a graph showing the concentration-dependent binding of biotinylated anti-cardiolipin cofactor to cardiolipin-immobilized plates.

FIG. 21 is a graph showing the binding specificity of anticardiolipin cofactor for phospholipids.

FIG. 22 is a graph showing the dependence of anti-cardiolipin antibody (As antibody) on anti-cardiolipin cofactor.

FIG. 23 is a graph showing the species specificity of an anti-cardiolipin cofactor in an anti-cardiolipin antibody reaction system.

FIG. 24 shows a fractionation pattern of healthy human serum by DEAE-cellulose column chromatography.

FIG. 25 shows a fractionation pattern of normal human serum by heparin sepharose column chromatography.

FIG. 26 shows a fractionation pattern of normal human serum by cardiolipin-polyacrylamide gel column chromatography.

FIG. 27 shows the effect of purified bovine serum albumin on the immunoassay method of the present invention.

Claims (14)

[Claims] 1. A carrier for binding an antiphospholipid antibody, wherein the carrier substance bound with phospholipid is treated with purified serum albumin and a surfactant. 2. An antiphospholipid characterized in that the carrier substance bound to phospholipid is treated with purified serum albumin, a surfactant and serum, plasma, a fraction thereof or a protein in the fraction. Carrier for antibody binding. 3. The carrier for binding antiphospholipid antibody according to claim 1 or 2, which is used for immunological diagnosis of antiphospholipid antibody syndrome. 4. An immune complex between an antibody specifically present in an antiphospholipid antibody syndrome by contacting a test solution with a carrier for phospholipid-bonded antiphospholipid antibody and a phospholipid bound to the carrier. An immunological assay method, which comprises using the carrier according to claim 1 or 2 as a carrier for binding an antiphospholipid antibody in a method of forming a body to detect the antibody. 5. A first antigen-antibody reaction for bringing an antiphospholipid antibody-binding carrier into contact with a test solution to form an immune complex between the antiphospholipid antibody in the test solution and the phospholipid on the carrier. And a second antigen-antibody reaction step of reacting the immune complex with a labeled anti-immunoglobulin antibody to form a sandwich immune complex comprising the immune complex and the labeled anti-immunoglobulin antibody, and A method comprising a separation step of separating a phase containing a sandwich-like immune complex and a phase containing a substance not bound to a carrier, and a detection step of detecting a labeled substance in either phase Serum derived from an animal of the same species as or similar to the test liquid in at least the first antigen-antibody reaction step, using the carrier according to claim 1 or 2 as a carrier for
An immunological assay method, which is performed by adding plasma, a fraction thereof, or a protein in the fraction. 6. A first antigen-antibody reaction for bringing an antiphospholipid antibody-binding carrier into contact with a test liquid to form an immune complex between the antiphospholipid antibody in the test liquid and the phospholipid on the carrier. A second antigen-antibody reaction step of reacting the immune complex with a labeled anti-immunoglobulin antibody to form a sandwich immune complex comprising the immune complex and the labeled anti-immunoglobulin antibody, and A method comprising a separation step of separating a phase containing a sandwich immune complex and a phase containing a substance not bound to a carrier, and a detection step of detecting a labeled substance in either phase The carrier for phospholipids is a carrier substance treated with a surfactant, and serum or blood derived from an animal of the same species as or similar to the test solution in at least the first antigen-antibody reaction step. An immunological assay method, which is performed by adding plasma, a fraction thereof, or a protein in the fraction. 7. A kit for use in the immunological assay method according to claim 5, which comprises at least the following constituent reagents. (A) A carrier for binding the antiphospholipid antibody according to claim 1 or 2, (B) a labeled anti-immunoglobulin antibody, (C)
A sample diluent containing serum, plasma, a fraction thereof or a protein in the fraction derived from an animal of the same species or akin to the test fluid. 8. A kit for assaying by the immunological assay method according to claim 6, which comprises at least the following constituent reagents. (A ′) A carrier for binding a phospholipid, which is treated with a surfactant, for binding an antiphospholipid antibody, (B) a labeled anti-immunoclobulin antibody, (C) an animal of the same species or relative to a test liquid A sample dilution liquid containing serum, plasma, a fraction thereof or a protein in the fraction derived from the above. 9. A fraction which can be obtained by gel filtration of serum or plasma, the fraction having an action of enhancing the binding property between an antibody specifically present in an antiphospholipid antibody syndrome and a phospholipid. object. 10. A fraction obtainable from serum or plasma, which has an action of enhancing the binding property between an antibody specifically present in antiphospholipid antibody syndrome and phospholipid,
And a fractionated product having the following physicochemical properties: It is included in the non-dialyzed fraction when dialyzed using a cellulose membrane having a molecular weight cut off of 6,000 to 8,000; it is separated by gel filtration or SDS-polyacrylamide gel electrophoresis. When it is drawn, it is confirmed in the vicinity of serum albumin or slightly on the low molecular side. It does not bind to protein A. It is eluted in the vicinity of the IgG fraction by chromatography with a weakly basic anion exchanger containing a diethylaminoethyl group. And a fraction salted out with 30 to 60% saturated ammonium sulfate. 11. A protein obtainable from serum or plasma, which has an action of enhancing the binding property between an antibody specifically present in an antiphospholipid antibody syndrome and a phospholipid,
And a protein having the following physicochemical properties; a molecular weight measured by SDS polyacrylamide electrophoresis of about 50,000 ± 2,000, and an isoelectric point of about 6.
60 ± 0.4; and has binding properties for phospholipids. 12. An immune complex between an antibody specifically present in an antiphospholipid antibody syndrome by contacting a test solution with a carrier for phospholipid-bonded antiphospholipid antibody and a phospholipid bound to the carrier. A method for detecting the antibody by forming a body, wherein when forming the immune complex, a high concentration of serum or plasma in the reaction solution, the fraction according to claim 9 or 10, or An immunological assay characterized by including the protein according to item 11. 13. A specific anti-phospholipid antibody syndrome, which comprises a high concentration of serum or plasma, the fraction of claim 9 or 10 or the protein of claim 11 as one of the constituent reagents. A kit used in an immunological assay method for measuring the antibody present in. 14. An antiphospholipid antibody-binding carrier having phospholipids bound thereto and a test solution are brought into contact with each other to separately detect an antiphospholipid antibody derived from an antiphospholipid antibody syndrome and an antiphospholipid antibody derived from an infectious disease. 11. The method according to claim 9, wherein when the carrier for binding antiphospholipid antibody bound with phospholipid and the test solution are contacted with each other, a high concentration of serum or plasma in the reaction solution, An antiphospholipid antibody derived from an antiphospholipid antibody syndrome and an infectious disease are carried out by carrying out both the method of contacting with the product or the protein of claim 11 and the method of contacting without the presence of these proteins. A method for differentially detecting an anti-phospholipid antibody derived from the same.