JPH0635975B2 - Carrier for binding antiphospholipid antibodies, immunoassay method and kit using the same - Google Patents

Description

[Detailed Description of the Invention] Technical Field The present invention relates to a carrier for binding antiphospholipid antibodies, an immunological assay using the carrier, a kit for use in the assay, and fractions and proteins obtained from serum or plasma that can be used in these assays, and in particular to the measurement of autoantibodies specific to antiphospholipid syndrome.

BACKGROUND ART When foreign substances such as pathogenic viruses, bacteria, fungi, and parasites invade the body, the body mounts an immune response and works to eliminate them. This phenomenon is generally broadly divided into two types: humoral immunity, which involves antibodies, and cellular immunity, which directly involves immune cells and eliminates foreign substances. However,
In the pathological conditions collectively known as autoimmune diseases, the self/non-self recognition mechanism does not function normally, resulting in humoral or cellular immune responses against the body's own cells and tissues. A typical clinical example in which antibodies that react to self components (autoantibodies) appear is systemic lupus erythematosus (SLE), in which anti-single-stranded DNA antibodies, anti-double-stranded DNA antibodies, and
Anti-Sm antibodies, anti-cardiolipin antibodies, etc. are observed. In addition, in rheumatoid arthritis (RA), rheumatoid factor
Sjögren's syndrome (SJS) - Anti-SS-A antibody, anti-S
S-B antibodies and antimitochondrial antibodies are found in systemic sclerosis (PSS), anti-scl antibodies and anti-single-stranded DNA antibodies, and anti-RNP antibodies are found in mixed connective tissue disease (MCTD). At present, much is unknown about the relationship between the appearance of these antibodies and the significance of the onset of the disease, but measuring antibodies is an important means of diagnosing the disease and understanding changes in the pathology after prognosis.

To detect these various autoantibodies, various methods are available, such as immunodiffusion, face-to-face immunoelectrophoresis, hemagglutination, radioimmunoassay (RIA), and enzyme immunoassay (EI), depending on the antigen specificity, biochemical characteristics, and physicochemical properties of the antibodies.
A method and latex agglutination method have been developed.

Regarding the measurement of anticardiolipin antibodies, the immunoprecipitation method was first developed in the late 1940s for the diagnosis of syphilis (mass screening). However, this method has the drawback of low sensitivity, and a high frequency of biological false positives has been observed in the serum of patients with systemic lupus erythematosus (SLE). However, in recent years, the use of immunoprecipitation has become more common in patients with arterial and venous thrombosis,
High concentrations of anticardiolipin antibodies have been observed in the blood of young female patients who develop lupus-like syndromes accompanied by symptoms of miscarriage and thrombocytopenia, and the clinical significance of diagnosing these diseases by measuring anticardiolipin antibodies has come to be highly regarded.

In this situation, there has been a growing expectation for a highly sensitive and accurate measurement method to replace conventional methods such as the nonspecific measurement of loop score grand and the anticardiolipin antibody immunoprecipitation method, which frequently produce biological false positives (Proc
tor, RR & Rapaport, SL, Am. J. Clin. Pathal., 36 ,212,
(1961); Exner, T. et al., British J. Haematol., 40 , 143,
(1987); Margolios, A., Medicine 40 , 145 (1961)). Then, in 1983, Harris, E. et al., Lance
t, 1211-1214, (1983)) developed an RIA for measuring anticardiolipin antibodies. After that, methods other than RIA were developed, such as the non-competitive method (sandwich method) using labeled antibodies with enzymes such as alkaline phosphatase.
The measurement of cardiolipin antibodies by this type of EIA method is quantitative, simple, and
It is suitable for diagnosing and monitoring the progress of patients with collagen diseases such as E.
In the field of basic medicine, it is used to analyze the specificity of anticardiolipin antibodies.In recent years, in the field of collagen diseases, there has been a growing view that habitual miscarriage is caused by thrombosis (antiphospholipid syndrome) due to the appearance of antiphospholipid antibodies such as anticardiolipin antibodies, and so there are high hopes for measuring anticardiolipin antibodies in the field of obstetrics and gynecology as well.

The subclasses of anticardiolipin antibodies in the blood are IgG, I
There are various types of IgA, such as IgG and IgM, but IgG or IgM classes appear relatively frequently in the serum of SLE patients.

In addition, dsDNA (double-stranded DNA antibody), which is thought to be the specific antigen of each antibody specific to antiphospholipid syndrome,
sDNA (single-stranded DNA), cardiolipin, phosphatidylserine, phosphatidylinositol, etc.
They have similar molecular structures in the limited polar regions centered on their phosphate esters, and the similarity between these antibodies is currently a subject of debate.

As mentioned above, RIA and EIA methods for measuring anticardiolipin antibodies have been developed at various facilities as diagnostic systems for antiphospholipid antibody syndromes such as SLE and habitual abortion. However, these systems still have the following problems:

Specifically, when measuring serum from patients with SLE or other conditions, methods using a solid-phase antigen carrier can make it difficult to quantify cardiolipin-specific antibodies due to nonspecific adsorption unless an appropriate blocking agent is used. When fetal bovine serum (FBS), which is commonly used as a blocking agent, is used, it is impossible to differentially quantify anticardiolipin antibodies derived from SLE and anticardiolipin antibodies due to other exogenous factors (e.g., infectious diseases), and no blocking agent suitable for such a purpose has yet been found.

Disclosure of the Invention The present inventors have conducted extensive research to develop an antiphospholipid antibody-binding carrier that selectively captures antiphospholipid antibodies specifically present in antiphospholipid syndrome, and a highly accurate and sensitive method for measuring antiphospholipid antibodies that can be put to practical use as a specific diagnostic method for antiphospholipid syndrome. As a result, they have solved the above-mentioned problems by the following means and completed the present invention.

That is, the present invention provides: (1) a carrier for binding antiphospholipid antibodies, characterized in that the carrier material having bound phospholipids is treated with purified serum albumin and a surfactant; (2) a carrier for binding antiphospholipid antibodies, characterized in that the carrier material having bound phospholipids is treated with purified serum albumin, a surfactant, and serum, plasma, a fraction thereof, or a protein in the fraction thereof; and (3) an immunological assay method in which a phospholipid-bound carrier for binding antiphospholipid antibodies is contacted with a test liquid to form an immune complex between an antibody that is specifically present in antiphospholipid antibody syndrome and the phospholipid bound to the carrier, thereby detecting the antibody, characterized in that the carrier described above in (1) or (2) is used as the carrier for binding antiphospholipid antibodies.

(4) A first antigen-antibody reaction (hereinafter sometimes referred to as the primary reaction) step in which the test liquid is contacted with the carrier for binding antiphospholipid antibodies to form an immune complex between the antiphospholipid antibody in the test liquid and the phospholipid on the carrier, and a second antigen-antibody reaction (hereinafter sometimes referred to as the secondary reaction) step in which the immune complex is reacted with a labeled anti-immunoglobulin antibody to form a sandwich-type immune complex consisting of the immune complex and the labeled anti-immunoglobulin antibody.
a separation step of separating a phase containing the sandwich-shaped immune complex from a phase containing a substance not bound to the carrier, and a detection step of detecting a labeling substance (marker) in either phase, wherein the carrier (1) or (2) above is used as the carrier for binding the antiphospholipid antibody, and at least the first antigen-antibody reaction step is carried out by adding to the reaction solution serum, plasma, fractions thereof or proteins in fractions thereof derived from the same species as or related to the test solution, (5) An immunoassay comprising a first antigen-antibody reaction step of contacting a test solution with an antiphospholipid antibody-binding carrier to form an immune complex between the antiphospholipid antibody in the test solution 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 consisting of the immune complex and the labeled anti-immunoglobulin antibody, a separation step of separating a phase containing the sandwich immune complex from a phase containing a substance not bound to the carrier, and a detection step of detecting a labeled substance (marker) in either phase, wherein the antiphospholipid antibody-binding carrier is a carrier material having phospholipids bound thereto that has been treated with a surfactant, and at least the first antigen-antibody reaction step is carried out by adding serum, plasma, fractions thereof, or proteins in fractions thereof derived from the same species as or related to the test solution to the reaction solution; (6) A kit for use in the immunoassay of (4) above, comprising at least the following constituent reagents: (A) the antiphospholipid antibody-binding carrier of (1) or (2) above, (B) a labeled anti-immunoglobulin antibody, (C) a specimen dilution solution to which serum, plasma, a fraction thereof or a protein in the fraction derived from an animal of the same species as or related to the test liquid has been added, (7) a kit for use in the immunological assay of (4) above, which comprises at least the following constituent reagents: (A') a carrier for binding antiphospholipid antibodies, in which a carrier substance to which phospholipids have been bound has been treated with a surfactant, (B) a labeled anti-immunoglobulin antibody, (C) a specimen dilution solution to which serum, plasma, a fraction thereof or a protein in the fraction derived from an animal of the same species as the test liquid has been added, (8) a fraction obtainable by gel filtration of serum or plasma, which has the effect of increasing the binding affinity between antiphospholipid antibodies specifically present in antiphospholipid syndrome and phospholipids, (9) a fraction obtainable from serum or plasma, which has the effect of increasing the binding affinity between antiphospholipid antibodies specifically present in antiphospholipid syndrome and phospholipids,
and a fraction having the following physicochemical properties: when dialyzed using a cellulose membrane with a molecular weight cutoff of 6,000 to 8,000, it is contained in the non-dialyzed fraction; when fractionated by gel filtration or SDS-polyacrylamide gel electrophoresis, it is found near serum albumin or slightly on the lower molecular weight side; it does not bind to protein A; it is eluted near the IgG fraction in chromatography using a weakly basic anion exchanger containing diethylaminoethyl groups; and it is contained in the fraction salted out with 30 to 60% saturated ammonium sulfate (10) A protein that can be obtained from serum or plasma, which has the effect of increasing the binding affinity between antibodies that are specifically present in antiphospholipid syndrome and phospholipids, and which has the following physicochemical properties: a molecular weight of approximately 50,000 ± 2,000 and an isoelectric point of approximately 6.
(11) A method for producing the protein described in (10) above, which comprises, when isolating and purifying the protein described in (10) above from the serum of a healthy person, a purification step using a liposome consisting of a phospholipid represented by the formula [1] described below, an affinity adsorbent to which the phospholipid is bound, or a liposome having the phospholipid as one of its constituent components.

(12) A method for detecting an antibody that is specifically present in antiphospholipid syndrome by contacting a phospholipid-bound carrier for binding an antiphospholipid antibody with a test liquid to form an immune complex between the antibody and the phospholipid bound to the carrier, characterized in that, when forming the immune complex, a high concentration of serum or plasma is present in the reaction liquid, or the fraction described in (8) or (9) above or the protein described in (10) above is present in the reaction liquid. (13) A method for differentially detecting antiphospholipid antibodies derived from antiphospholipid syndrome and antiphospholipid antibodies derived from an infectious disease by contacting a phospholipid-bound carrier for binding an antiphospholipid antibody with a test liquid, characterized in that, when contacting a phospholipid-bound carrier for binding an antiphospholipid antibody with a test liquid, a high concentration of serum or plasma is present in the reaction liquid, or the fraction described in (8) or (9) above or the protein described in (10) above is present in the reaction liquid.
0) and in the absence of the protein,
A method for differentially detecting antiphospholipid antibodies derived from antiphospholipid syndrome and antiphospholipid antibodies derived from an infectious disease, more specifically, a method for differentially detecting or differentially quantifying antiphospholipid antibodies derived from antiphospholipid syndrome and anticardiolipin antibodies derived from an infectious disease by contacting a test liquid with a carrier for binding anticardiolipin antibodies to which cardiolipin has been bound, wherein when contacting the carrier for binding anticardiolipin antibodies with the test liquid, both a method in which the carrier is contacted in the presence of an anticardiolipin cofactor and a method in which the carrier is contacted in the absence of an anticardiolipin cofactor are carried out, thereby differentially detecting or differentially quantifying antiphospholipid antibodies derived from antiphospholipid syndrome and anticardiolipin antibodies derived from an infectious disease; (14) A kit for use in an immunological assay for measuring antibodies specifically present in antiphospholipid syndrome, comprising high-concentration serum or plasma, the fraction described in (8) or (9), or the protein described in (10) as one of the constituent reagents; and (15) a kit for carrying out a method for differentially detecting or differentially quantifying anticardiolipin antibodies derived from antiphospholipid syndrome and anticardiolipin antibodies derived from an infectious disease, comprising anticardiolipin cofactor and cardiolipin as constituent reagents.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1 (A), (B), and (C) show the results of measuring absorbance (4500 nm) as an index of antiphospholipid antibody titer in As serum derived from antiphospholipid syndrome and Ya serum derived from an infectious disease, respectively, in a reference example using a conventional method, a comparative experimental example showing the effect of the present invention, and an example of the method of the present invention.

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

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

FIG. 4 shows the gel filtration pattern of serum from a healthy person and the As measured by adding the fractions and carrying out the primary reaction.
Absorbance as an indicator of antibody titer of serum and Ya serum (4
50 nm).

Figure 5 shows the relationship between the gel filtration pattern of healthy human serum by HPLC and the absorbance (450 nm) as an index of the antibody titer of As serum measured by adding the fraction and carrying out the primary reaction.

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

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

Figure 8 shows the absorbance (450 nm) of the antibody titer of the serum of healthy subjects fractionated with ammonium sulfate.
This shows the relationship between

FIG. 9 shows the antibody titers (units: unit/m;
1 shows the results of measuring the temperature (hereinafter referred to as U).

FIG. 10 shows the results of determining the correlation between the antibody titers (U) of the sera of autoimmune disease patients 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 determined for the serum of an SLE patient.

FIG. 12 shows the results of measuring absorbance (450 nm) as an index of antibody titer for the sera of patients with autoimmune diseases and infectious diseases (tuberculous meningitis, syphilis) by the method of the present invention (shaded) and the control method (open).

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

FIG. 14 shows the DEAE-cellulose (DEAE) activity of serum from healthy individuals.
The figure shows the fractionation pattern of ion exchange chromatography using the 1-52 column.

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

Figure 16 shows the fractionation pattern when the fraction containing anticardiolipin cofactor activity obtained by the chromatography shown in Figure 15 was subjected to affinity column chromatography using an anti-human IgG antibody-Sepharose CL-4B column.

FIG. 17 shows the anticardiolipin cofactor in SDS.
-The results obtained by polyacrylamide gel electrophoresis are shown.

FIG. 18 shows the separation pattern of the anticardiolipin cofactor purified by liposomes when it was subjected to HPLC, and the peak indicated by the arrow corresponds to the anticardiolipin cofactor.

FIG. 19 shows the anticardiolipin antibody obtained by affinity column chromatography using the anti-human IgG antibody-Sepharose CL-4B column shown in FIG.
1 shows the results obtained by subjecting fractions containing cofactor activity to ISODALT electrophoresis.

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

FIG. 21 is a graph showing the binding specificity of anticardiolipin cofactors to phospholipids.

FIG. 22 is a graph showing the anticardiolipin cofactor dependency of anticardiolipin antibody (As antibody).

FIG. 23 is a graph showing the species specificity of anticardiolipin cofactors in the anticardiolipin antibody reaction system.

FIG. 24 shows the fractionation pattern of serum from healthy subjects by DEAE-cellulose column chromatography.

FIG. 25 shows the fractionation pattern of serum from healthy subjects by heparin Sepharose column chromatography.

FIG. 26 shows the fractionation pattern of serum from healthy subjects by cardiolipin-polyacrylamide gel column chromatography.

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

Best Mode for Carrying Out the Invention The present invention will now be described in detail.

1. Phospholipids The phospholipids in the present invention may be any phospholipids as long as they have an electron-donating functional group proximal to the phosphodiester bond.
In particular, the general formula [I] [wherein R ¹ and R ² are the same or different and represent an acyl group having an alkyl group or an alkenyl group in the carbon chain;
^R3 represents a hydrogen molecule or -( _CH2 )n- ^CHR4 - ^R5 , ^R4 represents a hydrogen atom, a hydroxyl group, a carboxyl group, a formyl group, a mercapto group or a halogen atom, ^R5 represents an amino group, a hydroxyalkyl group or a group of the general formula [wherein ^R1 and ^R2 are as defined above], or ^R4 and ^R5 together represent a sugar or sugar alcohol residue, and n represents an integer of 0 to 3.] is preferred. This glycerophospholipid has a negative charge, and specific examples of such glycerophospholipids include cardiolipin, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid.

Cardiolipin is particularly preferred. Cardiolipin is usually prepared from mammalian hearts such as bovine hearts or microorganisms such as Escherichia coli, and cardiolipin obtained from mammalian hearts is particularly preferred.

These are not limited in terms of origin, fatty acid composition, degree of purification, etc., and may be in the salt form or the free form. There are also no limitations on the solvent in which they should be dissolved when bound to the carrier substance, but the degree of purification is preferably at least reagent grade.

2. Carrier Substance The carrier substance 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) during measurement. However, in order to facilitate BF separation (the separation of a labeled substance that has formed an immune complex in an immune reaction from a free labeled substance), a carrier substance (solid phase) that is insoluble in the reaction solution is preferred.

Examples of materials for the carrier substance insoluble in the reaction solution include synthetic organic polymers such as polyvinyl chloride, polystyrene, styrene-divinylbenzene copolymer, styrene-maleic anhydride copolymer, nylon, polyvinyl alcohol, polyacrylamide, polyacrylonitrile, polypropylene, and polymethylene methacrylate; polysaccharides such as dextran derivatives (e.g., Sephadex), agarose gels (e.g., Sepharose, Biogel), and cellulose (e.g., paper disks, filter paper); and inorganic polymers such as glass, silica gel, and silicone, which may contain functional groups such as amino groups, aminoalkyl groups, carboxyl groups, acyl groups, and hydroxyl groups. Preferably, the carrier substance has low protein binding capacity, and examples of such materials include untreated polystyrene and polyvinyl chloride.

Examples of the shape of the carrier substance insoluble in the reaction solution include flat plates (microtiter plates, disks, etc.), particles (beads, etc.), tubular shapes (test tubes, etc.), fibers, membranes, microparticles (latex particles, etc.), capsules, endoplasmic reticulum, and liposomes (multilayer or single-layer lipid membranes), and a carrier of an appropriate shape can be selected depending on the measurement method.

3. Carrier for binding antiphospholipid antibodies The carrier for binding antiphospholipid antibodies in the present invention is obtained by binding the above-mentioned phospholipid to the above-mentioned carrier substance. The binding method of the phospholipid to the carrier substance can be a physical adsorption method, an ionic bonding method, or a hydroxylase method.
Known methods such as covalent bonding and entrapment methods (see, for example, "Immobilized Enzymes" (edited by Ichiro Senbata, published by Kodansha on March 20, 1975)) can be used.
The physical adsorption method is preferable because it is simple. The phospholipid and the carrier substance may be bound directly to each other, or may be bound to each other via another substance.

To bind phospholipids to a carrier substance by physical adsorption, a method can usually be used in which a solution of phospholipids in an organic solvent (such as methanol, ethanol, or chloroform, which can dissolve phospholipids) is brought into contact with the carrier substance for a certain period of time, and the organic solvent is then evaporated from the solution. The solvent can be evaporated by known methods such as vacuum drying or forced air drying. Alternatively, the phospholipids can be adsorbed onto the surface of a substance other than the carrier substance by evaporating the solvent from the organic solvent solution of phospholipids, and then suspended in a buffer solution (such as a sodium-based, potassium-based, or sodium-potassium-based phosphate buffered saline solution), and then the suspension is brought into contact with the carrier substance for a certain period of time (such as several tens of minutes to several hours) under a temperature condition that does not inhibit the reaction (such as 0 to 50°C), and the solution is then removed by aspiration, decantation, centrifugation, or other methods, and further dried (such as vacuum drying or forced air drying) as necessary, thereby binding the phospholipids to the carrier substance.

The antiphospholipid antibody binding carrier used in the present invention is preferably one treated with a surfactant alone, one treated with a combination of purified serum albumin and a surfactant, or one treated with a combination of three substances: purified serum albumin, a surfactant, and serum, plasma, a fraction thereof, or a protein contained in the fraction (hereinafter, these substances may be referred to as "treatment substances"). When a high concentration of serum or plasma is present in the reaction solution, or a fraction or protein obtained from serum or plasma as described below in the present invention is present, and an antigen-antibody reaction is carried out to form an immune complex between the phospholipid bound to the antiphospholipid antibody binding carrier and the antiphospholipid antibody in the test solution, the antiphospholipid antibody binding carrier of the present invention described above does not necessarily need to be used. Here, "treatment" refers to a procedure in which these treatment substances act on the antiphospholipid antibody binding carrier, resulting in physical or chemical binding. Specifically,
In this treatment, after binding the phospholipid to the carrier substance, solutions containing the treatment substances are sequentially added (in any order), or a solution containing two or three of these substances is added to the carrier substance for a certain period of time (for example, several tens of minutes to several hours) under a temperature condition that does not inhibit the reaction (for example, 0 to 5°C).
The treatment can be carried out by contacting the substance with the solution at a temperature of 0°C (0°C). When carrying out such treatment, there are no particular limitations on the solvent used to dissolve the substance, but typically a buffer solution (such as sodium-based, potassium-based, or sodium-potassium-based phosphate buffered saline) is used. When carrying out such treatment, sugars (such as oligosaccharides such as sucrose, polysaccharides such as detoxrin, cyclodextrin, and dextran, and monosaccharides) may be present in the treatment solution.

By using the antiphospholipid antibody-binding carrier that has undergone the above-described treatment, it is possible to prevent antiphospholipid antibodies derived from infectious diseases from binding to the antiphospholipid antibody-binding carrier in the first antigen-antibody reaction (primary reaction) step. Other effects of such treatment include the prevention of nonspecific adsorption (prevention of binding of antiphospholipid antibodies and labeled antibodies to non-phospholipid-binding portions on the carrier material), enhancement of the binding ability of antiphospholipid antibodies that are specifically present in antiphospholipid syndrome to phospholipids on the carrier material, and stabilization of phospholipids on the carrier material.

The purified serum albumin used in the present invention is a purified serum albumin from bovine, equine,
The term "fraction V" refers to a substance that has been purified from the serum of animals such as dogs, cats, goats, sheep, pigs, chickens, rabbits, rats, mice, and humans by a known method, has a higher degree of purity than fraction V (obtained by low-temperature corn ethanol fractionation, heat shock, salting out, etc.), and is suitable for the purposes of the present invention. Specifically, for example, fraction V has been purified by activated carbon treatment, chromatography (e.g., ion exchange chromatography), or crystallization, or fraction V has been purified by other methods to a degree equivalent to that of fraction V, and is particularly preferred when the lipid content has been reduced.

Furthermore, serum, plasma, fractions thereof, or proteins in such fractions (hereinafter, these may be referred to as "blood components of the present invention") refer to serum or plasma itself produced from animal blood by known methods, fractions obtained by fractionating these, or proteins obtained by further purifying such fractions. The origin of these is preferably the same species of animal as the test liquid to be measured, but may also be derived from related animals. Such blood components of the present invention are essentially the same as those added during the primary reaction described below.

In the past, phospholipid-bound carriers have been treated with fetal bovine serum, newborn bovine serum, bovine serum albumin, surfactants, etc., but these conventional methods have not been able to prevent the binding of antiphospholipid antibodies derived from infectious diseases to the carriers for binding antiphospholipid antibodies. Furthermore, the bovine serum albumin used in the past has been unpurified, such as Fraction V obtained by low-temperature ethanol fractionation of corn.

The surfactants used in the present invention include nonionic, amphoteric,
Although anionic and cationic surfactants are included, nonionic surfactants are particularly preferred. Specific examples of nonionic surfactants include polyoxyethylene glycol sorbitan alkyl esters (e.g., Tween surfactants), acyl sorbitans (e.g., Span surfactants, Arlacel surfactants, etc.), and the like.
lacel-based surfactants, etc.), polyoxyethylene glycol alkylphenyl ethers (for example, Triton-based surfactants, etc.), sucrose fatty acid esters, etc. can be exemplified.

The antiphospholipid antibody-binding carrier of the present invention can be used not only for immunological measurements but also for various applications such as selective adsorption and removal or separation and purification of antiphospholipid antibodies. For example, it can be used as a selective adsorbent for antiphospholipid antibodies in plasma exchange therapy (see, for example, Japanese Patent Application Laid-Open No. 1-682).
73) It can also be used as an adsorbent for affinity chromatography to separate and purify antiphospholipid antibodies.

Furthermore, when the blood component of the present invention is present in the reaction solution in the primary reaction described below, a conventionally known carrier for binding phospholipid antibodies can also be used instead of using a carrier treated with these treating substances.

4. Antiphospholipid Antibody Syndrome Immunological assays using the phospholipid antibody-binding carrier of the present invention can be used for the purpose of diagnosing antiphospholipid antibody syndrome. Here, antiphospholipid antibody syndrome refers to an autoimmune disease (such as systemic lupus erythematosus (SLE) or recurrent miscarriage) in which antiphospholipid antibodies appear in the body fluids of the patient. Such diseases are distinguished from infectious diseases in which antiphospholipid antibodies appear in the body fluids, such as tuberculous meningitis and syphilis.

The immunological assay method of the present invention is characterized by its ability to distinguish and measure antibodies specific to antiphospholipid syndrome from antiphospholipid antibodies derived from such infectious diseases.

5. Immunoassay The immunoassay of the present invention is intended to measure antiphospholipid antibodies, and is carried out by causing the above-mentioned phospholipid antibody-binding carrier and/or the blood component of the present invention to be present in the reaction solution during the primary reaction. If the method is based on an antigen-antibody reaction, the operation method, labeling substance (marker), substance to be labeled, carrier, B, etc.
The type of F separation method is not important, and a method suitable for the purpose of the present invention can be appropriately selected from known methods known as immunological assays.

The following methods are known as known immunological assay methods.

Antigen-antibody reactions are classified by reaction type into competitive reaction methods and non-competitive reaction methods (immunometric assays), but in the present invention, the non-competitive reaction method is preferred.

Known detection methods include non-labeling methods (such as nephelometry) that directly detect the results of antigen-antibody reactions, and labeled methods that use some kind of marker for detection. Either method may be used in the present invention. Considering measurement sensitivity, labeled methods are particularly preferred. The labeling substances (markers) used in the labeled methods will be described later.

There are known heterogeneous methods that require BF separation and homogeneous methods that do not require BF separation, and either method may be applied to the present invention.

As a classification based on the reaction phase, there are known two methods: a liquid phase method in which the entire reaction is carried out in a liquid phase, and a solid phase method in which the partner of the immune reaction is immobilized and the reaction is carried out. In the present invention, the liquid phase method is one in which a substance soluble in the reaction solution is used as the carrier substance.
The case where a substance insoluble in the reaction solution is used corresponds to the solid phase method.

The relationship between known immunological assays and the present invention has been explained above, but general methods are described in detail in the following documents.

"Radioimmunoassay" edited by Hiroshi Irie (published by Kodansha Co., Ltd. on May 1, 1979) "Enzyme Immunoassay Method" edited by Eiji Ishikawa et al. (2nd edition) (published by Kodansha Co., Ltd.)
Igaku Shoin, published December 15, 1982) Clinical Pathology Clinical Special Issue No. 53 "Immunoassay for Clinical Testing - Technology and Applications -" (Clinical Pathology Publishing Association, published in 1983) "Biotechnology Encyclopedia" (CMC Co., Ltd.,
Published 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
The immunoassay method of the present invention will now be described in detail.

5.1 Test Fluid The test fluid to be measured for the presence or content of antiphospholipid antibodies by the immunological assay method of the present invention (hereinafter sometimes referred to as the method of the present invention) is a body fluid of an animal, including a human. Here, body fluid refers to blood, serum, ascites, lymph, synovial fluid, fractions obtained from these, or other liquid components derived from a living organism.

Alternatively, the test solution may be diluted with a diluent to an appropriate antibody titer. Examples of such diluents include sodium-, potassium-, or sodium-potassium-based phosphate buffered saline (PBS), Good's buffer (e.g., N-2-hydroxyethylpiperazine-N'-2-
Ethanesulfonic acid (HEPES), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (T
Preferred are buffers such as glycine-buffered saline, veronal-buffered saline, and buffers containing buffering agents such as 3-(N-morpholino)propanesulfonic acid (MOPS), glycine-buffered saline, and veronal-buffered saline.

5.2 First antigen-antibody reaction (primary reaction) step In the present invention, the "first antigen-antibody reaction step" refers to a step in which a test liquid is contacted with a carrier for binding antiphospholipid antibodies to form an immune complex between the antiphospholipid antibodies in the test liquid and the phospholipids on the carrier.

The method of the present invention is characterized by using a carrier for binding antiphospholipid antibodies that has been treated with the above-mentioned treatment substance, and/or by carrying out the reaction in this step in the presence of serum or plasma derived from the same or similar animal species as the test solution, or a fraction thereof, or a protein in a purified fraction thereof, in the reaction solution. By employing such a method, it is possible to prevent antiphospholipid antibodies derived from infectious diseases from binding to the carrier for binding antiphospholipid antibodies, and to enable antibodies specifically present in antiphospholipid syndrome to bind specifically to the carrier.

In this step, the test solution is a body fluid of a patient with antiphospholipid syndrome or a standard reagent containing a known concentration of antiphospholipid antibodies, and the test solution is incubated with the carrier for binding antiphospholipid antibodies for a certain period of time (for example, several tens of minutes to several hours) under a temperature condition that does not inhibit the reaction (for example, 0
The contact can be carried out by contacting the mixture at a temperature of 100°C to 50°C.

The primary reaction can be carried out by adding the purified serum albumin described above. The purified serum albumin used in this case is preferably used at a concentration of 5% or less, particularly 1% or less.

Usually, after the first reaction step is completed, the reaction mixture is separated by an appropriate separation means.
The antiphospholipid antibody-binding carrier that has formed an immune complex with the antiphospholipid antibody is separated from the test solution. The separation method that can be applied depends on the type of carrier material used. When a liquid-insoluble carrier material is used, separation can be performed by known methods such as aspiration, decantation, filtration, and centrifugation.
After separation, the carrier for binding antiphospholipid antibodies may be washed with a buffer solution or the like.

The first reaction step and the second reaction step described below may be carried out simultaneously, or the first reaction step may be followed by the second reaction step without separating the test liquid.

5.3 Fractions and Proteins The "serum or plasma derived from an animal of the same species as the test fluid" used in the primary reaction step may be prepared by a known method from the blood of an animal of the same species (including related species) as the animal whose bodily fluid antiphospholipid antibodies are to be measured. For example, when measuring human bodily fluids, human serum or human plasma is used, with serum or plasma from healthy individuals being particularly preferred. Furthermore, the fraction is, for example, human serum or human plasma fractionated by a known separation and purification method (gel filtration (molecular sieve chromatography); ion exchange chromatography using DEAE-cellulose or the like; affinity chromatography using protein A Sepharose, heparin Sepharose, cardiolipin-polyacrylamide or the like; affinity adsorption using liposomes containing phospholipids such as cardiolipin; membrane separation by dialysis, ultrafiltration or the like; electrophoresis; solvent extraction, etc.), and is a fraction that, when added to the reaction solution in the primary reaction step of the method of the present invention, exhibits the effect of enhancing the binding between antibodies specific to antiphospholipid syndrome and phospholipids bound to a carrier.

As the serum or plasma fraction, for example, human serum or plasma may be used, for example, Ultrogel ACA-3
Examples of such a substance include those obtained by gel filtration using a 4 (LKB) column and collecting fractions that exhibit the effect of enhancing the binding between phospholipids and antibodies specifically present in the serum of patients with antiphospholipid syndrome.

Alternatively, fractions having the following physicochemical properties may be mentioned.

When dialyzed using a cellulose membrane with a molecular weight cutoff of 6,000 to 8,000, it is contained in the non-dialyzed fraction.

When fractionated by gel filtration or SDS-polyacrylamide gel electrophoresis, it is found to be in the vicinity of serum albumin or slightly lower in molecular weight.

Does not bind to Protein A.

In chromatography using a weakly basic anion exchanger containing diethylaminoethyl groups, it is eluted near the IgG fraction. For example, in chromatography using a DEAE-cellulose column, when eluted with 0.014 M phosphate buffer (pH 7.4), it is eluted in the fraction that passes through.
It is eluted near or slightly later than fraction G.

It is contained in the fraction salted out with 30 to 60% saturated ammonium sulfate.

The concentrations of the above-mentioned serum, plasma, fractions thereof, or the proteins described below in the fractions in the antigen-antibody reaction solution are not particularly limited, as long as they are high enough to achieve the effects of the present invention as described above. It is preferable to have serum or plasma present in the reaction solution at a high concentration of at least about 200-fold diluted serum or plasma prepared from blood by a conventional method, particularly at least about 40-fold diluted serum or plasma (in the case of fractions and the proteins described below, at a concentration equivalent to the above dilution of serum or plasma).

The presence of high concentrations of serum, plasma, fractions thereof, or proteins in such fractions during the reaction has the effect of increasing the binding affinity of antibodies specifically present in the serum of patients with antiphospholipid syndrome with phospholipids, and on the other hand, has the effect of suppressing the reaction between antiphospholipid antibodies present in the serum of patients with infectious diseases such as tuberculous meningitis and syphilis and phospholipids.

The protein, which is the active component of the above fraction, can be isolated and purified as a homogeneous protein as follows.

First, serum or plasma from a healthy person is purified by, for example, DEAE-
The mixture is subjected to ion exchange column chromatography using cellulose or the like, and fractions containing the active ingredient are collected. The active fractions are collected based on their ability to enhance the binding between phospholipids and antibodies specifically present in the serum of patients with antiphospholipid syndrome.

The resulting active fraction is then subjected to Protein A-Sepharose column chromatography to remove human IgG contained in the active fraction, and then further subjected to affinity column chromatography using a column prepared by binding anti-human IgG antibody to, for example, Sepharose CL-4B resin (Pharmacia) to remove human IgG that does not adsorb to Protein A.

Each active fraction obtained after each of the above column chromatography steps may be dialyzed by a conventional method, if necessary.
Alternatively, the concentrate may be appropriately concentrated using an ultrafilter or by salting out with saturated ammonium sulfate.

Next, the purified fraction from which human IgG has been removed is contacted with liposomes to adsorb the active ingredient to the liposomes, thereby purifying the active ingredient. The liposomes used here are liposomes made of phospholipids represented by formula [I], such as cardiolipin, phosphatidylserine, phosphatidylinositol, and phosphatidic acid, or liposomes containing the phospholipid as one of their components. Liposomes can be prepared by a conventional method [Immunological Experimental Procedures IX, No. 2989-299
For example, an alcohol solution of cardiolipin can be placed in a pear-shaped flask, evaporated to dryness under reduced pressure, and an appropriate buffer solution added thereto, followed by vigorous stirring in a vortex mixer to prepare cardiolipin liposomes.

The fraction containing the active ingredient is added to the buffer solution containing the liposomes to adsorb the active ingredient to the liposomes, and the liposome suspension with the adsorbed active ingredient is then centrifuged to recover the purified active ingredient from the resulting supernatant. The active ingredient may be further purified by 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 a binding affinity to phospholipids.

The active ingredient exhibits specific binding affinity to negatively charged phospholipids, such as cardiolipin, and antibodies specifically present in antiphospholipid syndrome, such as anticardiolipin antibodies in patients with autoimmune diseases, react with carrier-bound cardiolipin in a manner dependent on the active ingredient.

It has the effect of suppressing the reactivity of phospholipids with antiphospholipid antibodies derived from infectious diseases such as tuberculous meningitis and syphilis.

Based on these properties, when the above-mentioned active ingredient is added to a reaction system containing cardiolipin bound to a carrier and an antibody specific to antiphospholipid syndrome, it is thought that the active ingredient first reacts with the cardiolipin bound to the carrier to form a complex, to which the antibody specific to antiphospholipid syndrome binds.

Furthermore, because of the above-mentioned properties (1) and (2), the active ingredient can be used to differentially detect antiphospholipid antibodies derived from antiphospholipid syndrome and antiphospholipid antibodies derived from infectious diseases, as will be described later. Furthermore, differential detection can be achieved in the same way even when high concentrations of serum or plasma, or fractions thereof, are present in the reaction solution.

In this specification, proteins having the above physicochemical properties (1) to (4) are referred to as anticardiolipin cofactors.

By adding the above-mentioned active ingredient protein (anticardiolipin cofactor) obtained by the present invention during the first antigen-antibody reaction, it becomes possible to selectively detect antibodies that are specifically present in antiphospholipid syndrome.

5.4 Second Antigen-Antibody Reaction (Secondary Reaction) Step In the present invention, the "second antigen-antibody reaction step" refers to a step in which the immune complex formed in the first reaction step is reacted with a labeled anti-immunoglobulin antibody to form a sandwich-shaped immune complex consisting of the immune complex and the labeled anti-immunoglobulin antibody.

The labeled anti-immunoglobulin antibody used in this step is
The anti-immunoglobulin antibody is 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 terms of origin or production method, as long as it is an antibody or a fragment thereof that can bind to the antiphospholipid antibody to be measured.

Generally, the subclasses of antiphospholipid antibodies in body fluids are IgG,
Since the anti-immunoglobulin antibody is IgM, IgA, etc., the anti-immunoglobulin antibody can be appropriately selected depending on the purpose from anti-IgG antibody, anti-IgM antibody, anti-IgA antibody, etc. Furthermore, when the antiphospholipid antibody to be measured is of human origin, it goes without saying that an anti-human immunoglobulin antibody is used.

Such anti-immunoglobulin antibodies can be prepared by immunizing an animal with immunoglobulins derived from the same species as the animal producing the antiphospholipid antibody to be measured, and then isolating and purifying the antibodies from the serum of that animal, or by transforming antibody-producing cells (spleen cells, lymph node cells, peripheral blood lymphocytes, etc.) of that animal using a known method (e.g., hybridoma method, transformation method (using EB virus, etc.))
Eur. J. Immunol., 6 , 511 (1976) and other similar publications) to allow the cells to proliferate semi-permanently, and then these cells are grown in vitro (in a test tube) or
These antibodies can be isolated and purified from cultures grown in vivo (in a living organism). These anti-immunoglobulin antibodies are commercially available, and such antibodies that are readily available to those skilled in the art can be used in the method of the present invention.

The anti-immunoglobulin antibody may be used as it is, or may be decomposed with a protease (e.g., papain, pepsin, etc.) to produce an antibody fragment (F(a
b') ₂ , Fab', etc.).

The labeling substance (marker) used to label the anti-immunoglobulin antibody is not particularly limited as long as it is compatible with the detection step. For example, radioisotopes ( ¹²³ 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, maleate dehydrogenase, etc.), other in vivo ligands and receptors (biotin, avidin, streptavidin, etc.), coenzymes and prosthetic groups (FAD, FMN, ATP, etc.), fluorescent substances (fluorescein isothiocyanate (FITC),
They can be labeled with known markers such as europium, phycoerythrin, etc.), luminescent substances (luminol derivatives, etc.), marker substances for electron spin resonance (ESR) (piperidine-1-N-oxyl compounds, pyrrolidine-1-N-oxyl compounds, etc.), liposomes (containing labeling substances such as enzymes, fluorescent substances, luminescent substances, etc.) (see the aforementioned literature, etc.).

Labeling of anti-immunoglobulin antibodies with these markers can also be carried out by known techniques. For example, when using radioisotopes as markers, the chloramine T method, the enzyme method using lactoperoxidase, the Bolton-Hunter method, etc. can be used (see the above-mentioned literature). In addition, when using enzymes as markers, the labeling can be carried out by the maleimide crosslinking method (e.g., using succinimide 6-maleimidohexanoate (EMCS)), the glutaraldehyde crosslinking method, the periodate crosslinking method (Nakane method), the isocyanate crosslinking method (e.g., using isocyanates (e.g., toluene diisocyanate), isothiocyanates), or the benzoquinone crosslinking method (see the above-mentioned literature).

When this second reaction step is carried out separately from the first reaction step,
The reaction can be carried out by contacting the antiphospholipid antibody binding carrier to which the immune complex obtained in the primary reaction step is bound with a solution containing labeled anti-immunoglobulin antibody (a solution such as a buffer solution) for a certain period of time (e.g., several tens of minutes to several hours) under temperature conditions that do not inhibit the reaction (e.g., 0 to 50°C).

When the reaction is carried out simultaneously with the primary reaction step or successively without separating the reaction solution, the labeled anti-immunoglobulin antibody may be present in the reaction solution when the primary reaction is carried out or after completion of the reaction.

5.5 BF Separation Step The BF separation step of the method of the present invention is a separation step for separating a phase containing the sandwich-shaped immune complex formed in the secondary reaction step from a phase containing substances not bound to the carrier.

This step is not necessary when the method of the present invention is carried out by a homogeneous method, but it is an essential step when the method is carried out by a heterogeneous method.

When a carrier substance insoluble in the reaction solution is used, this step can be carried out by known separation means such as suction, decantation, filtration, or centrifugation.

After or at the same time as the above separation procedure, a buffer solution (e.g., PBS)
The carrier may be washed with

5.6 Detection step The "detection step" of the method of the present invention refers to the step of detecting the labeled anti-immunoglobulin antibody contained in the sandwich immune complex separated in the BF separation step in the case of the heterogeneous method.
Alternatively, it refers to a step of detecting a labeling substance (marker) of the labeled anti-immunoglobulin antibody that has not formed the complex.
This detection may be quantitative or qualitative.

The detection step may be carried out by any known method as long as it corresponds to the marker used (see the above-mentioned documents 1 to 4). For example, when a radioisotope is used as the marker, detection can be carried out by a scintillation counter, and when an enzyme is used as the marker, detection can be carried out by a known method used to measure the activity of the enzyme used.

The following describes the measurement of enzyme activity when peroxidase is used as a marker.

Peroxidase activity can be measured by various methods that detect the transfer of electrons when hydrogen peroxide, a substrate, is decomposed into water.
Known methods can be used, such as measuring the change in absorbance due to the oxidation of Benzidine (gen) or measuring the change in redox potential using an electrode. In particular, methods using a chromogen are common. Examples of chromogens include tetraalkylbenzidine (3,3',5,5'-tetramethylbenzidine (TM),
BZ), o-phenylenediamine (OPD),
Known substances such as 2,2'-azinodi(3-ethyl)benzothiazolinone-6-sulfonic acid (ABTS), dianisidine, dicarboxydine, and diaminobenzidine can be used (for example, Japanese Patent Publication No. 62-50265).
3 (see WO86/04610).

Chromogenic measurements of peroxidase activity are performed using BF
A solution containing hydrogen peroxide and a chromogen (preferably a solution of a buffer (citrate buffer, tartrate buffer, PBS, etc.)) is added to either of the phases separated by separation, and the mixture is allowed to react for a certain period of time (for example, several minutes to several hours) at room temperature (for example, at room temperature). The reaction is stopped with an oxygen reaction stop solution (for example, sulfuric acid), and then the absorbance (for TMBZ, absorbance at 450 nm) is measured. Quantitation of antiphospholipid antibodies in a test solution can be performed by plotting a standard curve (absorbance vs. antibody concentration (or antibody titer)) measured using a standard reagent containing a known concentration of antiphospholipid antibodies, for example, serum, plasma, fractions thereof, or proteins in fractions thereof (anticardiolipin cofactor) derived from the same or related animal species as the test solution.
This can be done by comparing the measured absorbance with the measured value.

5.1 Differential detection Differential detection of antiphospholipid antibodies derived from antiphospholipid syndrome and antiphospholipid antibodies derived from infectious diseases can be performed as follows by adding a high concentration of serum or plasma, a fraction thereof, or a protein (anticardiolipin cofactor) which is an active component of the fraction in the reaction solution.

That is, in the above-mentioned antigen-antibody reaction (primary reaction) step, there are two systems in which the primary reaction is carried out in the presence of high concentrations of serum or plasma, their fractions, or proteins (anticardiolipin cofactors), and the primary reaction is carried out in the absence of these.
If the presence of these blood components of the present invention (anticardiolipin cofactors) enhances the reactivity of antiphospholipid antibodies with phospholipids immobilized on a carrier, it can be determined that the antiphospholipid antibodies are derived from antiphospholipid syndrome.
If the reactivity is reduced in a dependent manner by the presence of a cofactor, it can be determined that the antibody is an antiphospholipid antibody derived from an infectious disease.

6. Kit 6.1 Kit (6) The immunoassay kit (6) used in the method of the present invention (4) above comprises at least the constituent reagents (A) to (C) described above.

The "antiphospholipid antibody binding carrier" of component reagent (A) is one of those described in "3. Antiphospholipid antibody binding carrier" above, which is treated with a combination of two types of carriers: purified serum albumin and surfactant, or one of those treated with a combination of three types: purified serum albumin, surfactant, and serum, plasma, fractions thereof, or proteins in fractions thereof (anticardiolipin cofactor) derived from the same or similar animal species as the test liquid. The "labeled anti-immunoglobulin antibody" of component reagent (B) is one described in "5.3 Second antigen-antibody reaction (secondary reaction) step" above. The "specimen dilution solution to which serum, plasma, or fractions thereof derived from the same animal species as the test liquid has been added" of component reagent (C).
is used to dilute the test liquid or standard reagent to be measured as needed, and for the same reason as above, the blood component of the present invention (anticardiolipin cofactor) is added.

In addition to the above-mentioned constituent reagents, the kit (6) of the present invention can be provided with a standard reagent or a reagent for detecting a labeled substance, if necessary. An example of a standard reagent is a standard reagent containing a known concentration of antiphospholipid antibody, to which the blood component of the present invention (anticardiolipin cofactor) is added as necessary. This is a standard reagent obtained by serially diluting antiphospholipid antibodies usually isolated from patients with antiphospholipid syndrome as necessary, to which the blood component of the present invention (anticardiolipin cofactor) is added for the reasons explained in "5.2 First antigen-antibody reaction (primary reaction) step" above. An example of a reagent for detecting a labeled substance is
When the kit is a kit for enzyme immunoassay, it is preferable to attach a reagent for measuring the enzyme activity, i.e., the enzyme substrate and an indicator of the reaction (e.g., the chromogen described in "5.5 Detection step" above), if necessary. Also, an enzyme reaction stop solution (e.g., a solution containing sulfuric acid)
may be attached.

6.2 Kit (7) Kit (7) for immunoassay used in the method of the present invention (5) above is composed of at least the constituent reagents (A') to (C) above. If necessary, standard reagents and reagents for detecting labeling substances can be included, as with kit (6).

Constituent reagent (A') is the antiphospholipid antibody binding carrier described in "3. Carrier for binding antiphospholipid antibody" above, but treated with only 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 method described in (12) above is
One of the constituent reagents includes high-concentration serum or plasma, or the above-mentioned fraction, or the (anticardiolipin cofactor) protein in the fraction. The other constituent reagents may be any conventionally used carrier for binding antiphospholipid antibodies, and may, of course, be the above-mentioned carrier for binding antiphospholipid antibodies of the present invention. Furthermore, commonly used reagents may also be used as they are for the other constituent reagents.

[Example]

The present invention will be explained in more detail below with reference to Reference Examples and Examples.

Reference Example A general procedure according to the conventional method and an example of measurement using this method are shown below.

Bovine heart-derived cardiolipin (Sigma) 5
5 μl of 10 μg/ml ethanol solution was placed in each well of a 96-well microtiter plate (polystyrene; manufactured by Titer Tech) and the ethanol in the wells was dried under reduced pressure. After drying, the plate was resuspended in 10% fetal bovine serum-containing phosphate-buffered saline (PBS) (hereinafter referred to as PBS).
250 μl/well of 0.05% (V/V) Tween 20 (abbreviated as BS-FBS) (pH 7.4) was added, and the plate was blocked at room temperature for 1 hour.
(Tween 20, product name, manufactured by Kishida Chemical Co., Ltd.)-containing PBS
(hereinafter abbreviated as PBS-Tween) three times. Next, 100 μl of a test solution (serum) appropriately diluted with PBS-FBS was added to each well, and the wells were incubated at room temperature for 1 hour, followed by washing five times with 250 μl of PBS-Tween.
Horseradish peroxidase-labeled anti-human IgG antibody
100 μl of each solution was added to each well and allowed to react at room temperature for 1 hour.
The plate was washed five times with PBS-Tween.
% hydrogen peroxide solution containing 0.3 mM 3,3',5,5'
-Tetramethylbenzidine (TMBZ) solution) 100 μ
After adding 111 ml of the reaction mixture and reacting at room temperature for 15 minutes,
100 μl of 1N sulfuric acid was added to the reaction solution, and the absorbance (450 nm) was measured.
The results are shown in Figure 1(A). The enzyme-labeled antibody used was a mouse monoclonal anti-human IgG antibody (G-02, IgG class; manufactured by Yamasa Shoyu Co., Ltd.) conjugated with horseradish peroxidase by the periodate crosslinking method. The test serum As used here was the serum of a patient with typical antiphospholipid syndrome (SLE with habitual abortion) (hereinafter referred to as As serum), while the test serum Ya was the serum of a patient with tuberculous meningitis who showed no signs of antiphospholipid syndrome (hereinafter referred to as Ya serum). Conventional methods using fetal bovine serum (FBS) in the primary reaction did not allow for the differential quantification of the antibodies in the Ya serum (hereinafter referred to as Ya antibodies) and the antibodies in the As serum (hereinafter referred to as As antibodies), as shown in Figure 1(A).

Example 1 Detection of Antiphospholipid Syndrome-Specific Antibodies by Addition of Serum from Healthy Individuals (1) Preparation of Carriers for Binding Antiphospholipid Antibodies Carriers for binding antiphospholipid antibodies were prepared using the following two methods.

[Method (I)] Bovine heart-derived cardiolipin (Sigma) 50 μg/
5 μl of the ethanol solution was added to each well of a 96-well microtiter plate (polystyrene; manufactured by Titer-Tek) at a rate of 5 μl/well, and the ethanol in the wells was dried under reduced pressure. Then, 1% (w/v) purified bovine serum albumin (Sigma; No. 8511, Fat
y acid-free (hereinafter abbreviated as pBSA).
PBS (pH 7.4) (hereinafter abbreviated as PBS-pBSA)
250 μl of the solution was added, and the mixture was treated at 4° C. for 1 hour.
V) PBS containing Tween 20 (PBS-Tween)
The carrier for binding the antiphospholipid antibody of the present invention was obtained by washing three times with 250 μl of the solution.

[Method (II)] 500 μL of bovine heart cardiolipin (Sigma)
The 1000 mg/ml ethanol solution was placed in a glass test tube, dried under reduced pressure, and then PBS was added to suspend the cardiolipin. 50 μl of this cardiolipin suspension was added to each well of a 96-well microtiter plate (polystyrene;
The suspension was then removed by suction. The procedure was then the same as in method (I) to obtain a carrier for binding antiphospholipid antibodies of the present invention.

(2) Measurement of antiphospholipid antibodies: 5% of normal serum was added to each of the same patient serum (As serum and Ya serum) used in the Reference Example, and PB
The diluted solution was diluted with S-pBSA.
Each μl was added to the cardiolipin-binding titer plates prepared by method (I) and method (II), reacted at 4°C for 1 hour, and washed five times with 250 μl of PBS-Tween. The subsequent reactions were performed in the same manner as in the Reference Example, and the absorbance was measured (method of the present invention). For the plate prepared by method (I), the absorbance was measured in the same manner as above, except that pBSA was added in the primary reaction (comparative experiment). The results obtained by each method are shown as dilution curves. Figure 1(B) shows the results of the comparative experiment, Figure 1(C) shows the results of the method of the present invention performed on the plate prepared by method (I), and Figure 2 shows the results of method (I).
The results of the method of the present invention performed on the plates obtained in I) are shown below.

As is clear from Figure 1(B), when the cardiolipin-binding plate of the present invention is used, the fetal bovine serum (FBS) used in the primary reaction system of the Reference Example is replaced with commercially available purified bovine serum albumin (pBSA) (comparison experiment).
The binding activity of anticardiolipin antibodies (Ya antibodies) in Ya serum was significantly reduced (to about 20%). The binding activity of As antibodies was also lost in highly diluted samples (i.e., dilutions of 200 times or more).
By further adding serum from a healthy individual (approximately 5%) to the basic measurement system of the comparative experiment added in the next reaction, only the As antibody titer recovered to the same level as in the conventional method (reference example) using fetal bovine serum (FBS), while the Ya antibody titer completely disappeared.

In the primary reaction, the concentration of the serum from healthy individuals was varied, and the antibody titer of the As serum was measured. Sufficient absorbance was obtained when the serum from healthy individuals was added at a high concentration, particularly at a dilution of 1/40 or more (see Table 3).
The measured sample consisted of 24 units (24
U) and 12 units (12U) (according to the units of Harris et al.) of As serum.

Example 2 Detection of Antiphospholipid Syndrome-Specific Antibodies by Addition of Serum Fractions from Healthy Individuals Serum from healthy individuals was fractionated by gel filtration, and each fraction was
The cardiolipin-binding plate used was the one prepared by the method (I) of Example 1, and the absorbance (450n) was measured as an index of antibody titer in the same manner as in Example 1, except that the serum from a healthy person was replaced with a fraction obtained by gel filtration.
The serum fractions of healthy subjects were diluted with PBS (pH 7.
4) Ultrogel ACA-34 pre-equilibrated with
The gel filtration of 3 ml of normal human serum was carried out on a column (2.0 x 55 cm) manufactured by LKB Co., Ltd., and 2 ml of each fraction was collected. Figure 4 shows the relationship between the gel filtration pattern using Ultrogel ACA-34 and the absorbance (450 nm) as an index of antibody titer obtained by the above method for As serum and Ya serum. As a result, As was detected in the fraction of the second peak of the three 280 nm absorption peaks shown in Figure 4.
A component that has the effect of increasing the binding of antibodies and eliminating the binding of Ya antibodies (hereinafter referred to as the active ingredient (anticardiolipin
It was confirmed that the fraction contained serum albumin, but the purified human serum albumin itself did not exhibit these activities (effects), confirming that the effects were not due to serum albumin.

Serum from healthy individuals was analyzed using a G2000SW (Tosoh Corporation).
When fractionated by gel filtration using a column (0.75 x 30 cm) by HPLC, the fraction containing the active ingredient (activity peak) appeared near the serum albumin fraction and was eluted slightly toward the lower molecular weight side (Figure 5).

Next, the serum of a healthy person was diluted with 1M Tris-HCl buffer (pH 9.0)
After dialysis with 1000 ml of ...
Fractions (IgG fractions) eluted with 0.1 M citrate buffer (pH 3.0) were prepared. Figure 6 shows the elution patterns. Each fraction was dialyzed against PBS, and then an amount equivalent to 1/20 of the serum concentration was added to As serum (24 U). The absorbance (450 nm) was measured as an index of antibody titer using the plate prepared in Method (I) in the same manner as in Example 1. The results are shown in Table 1.

The values in the table represent absorbance at 450 nm, and the values in parentheses represent the results of measurements on samples that did not contain As serum (control test).

As shown in Table 1, the active ingredient is present in the non-IgG fraction.

Furthermore, when healthy human serum was passed through DEAE-cellulose (Whatman, DE-52), the active ingredient appeared in the flow-through fraction eluted with 0.014 M phosphate buffer (pH 7.4) (Fig. 7). When salted out with ammonium sulfate, the active ingredient precipitated at 30-60% saturation (Fig. 8).

The effect of enhancing the binding of the As antibody is due to the interaction between the above components and the immobilized cardiolipin micelles.
The effect of eliminating the binding of Ya antibody is thought to be due to the factor reacting with the antibody in the liquid phase and being absorbed.

Example 3: Detection of antiphospholipid syndrome-specific antibodies using surfactant-treated antigen-binding carriers. Basically, the method (I) and method (I) of Example 1 (1) were performed.
The effect of the method of the present invention was examined using a carrier for binding antiphospholipid antibodies treated only with Tween 20 according to I).

Specifically, bovine heart cardiolipin was bound to each well of a 96-well microtiter plate according to method (I). The wells were washed with 0.05% PBS-Tween, and the primary reaction mixture was incubated with either healthy human serum (Experiment I-1) or no serum (Experiment I-2) in the same manner as in Example 1.
2) reaction, and thereafter, the same procedures as in Example 1 were repeated to measure absorbance (450 nm) as an indicator of antibody titer. Furthermore, wells to which cardiolipin had been bound in the same manner were washed with PBS containing no Tween 20, and the reaction solution for the primary reaction was reacted with either healthy human serum (Comparative Example I-1) or without the addition of serum (Comparative Example I-2). Thereafter, the same procedures as in Example 1 were repeated to measure absorbance (450 nm) as an indicator of antibody titer. After the primary and secondary reaction steps, washing solutions corresponding to the above washing solutions were used in each experimental group.

In addition, 0.01% dried cardiolipin was added to a test tube.
Bovine heart cardiolipin was bound to each well of a 96-well microtiter plate in the same manner as in Method (II) above, except that it was suspended in PBS (pH 6.0) containing BSA. The wells were then filled with 0.05% PBS-Tween.
The reaction mixture was washed with 100 ml of ...
The absorbance (450 nm) was measured as an index of antibody titer. In addition, wells to which cardiolipin had been bound in the same manner were washed with PBS containing no Tween 20, and the reaction solution for the primary reaction was added with serum from a healthy person (Comparative Example II-1) or without (Comparative Example II-2). Thereafter, the procedure was the same as in Example 1, and the absorbance (450 nm) was measured as an index of antibody titer.
In addition, PBS containing no Tween 20 was used for washing after the first reaction step and the second reaction step in each experiment.

In all of the above experiments, As serum and Ya serum were used as test solutions, which were diluted to a constant antiphospholipid antibody titer as proposed by Harris (EN) et al. at the 3rd Kingston Antiphospholipid Antibody Symposium (KAPS).

The results of these experiments are shown in Table 2.

The values in the table represent absorbance at 450 nm, and the values in parentheses are values measured using a carrier without cardiolipin binding (control test).

As shown in Table 2, washing with Tween 20 causes antigenic expression of cardiolipin, enhances the binding of As antibody to immobilized cardiolipin, and suppresses nonspecific adsorption of immunoglobulin to the plate.
In the antiphospholipid antibody binding carrier prepared by method (I), physically adsorbed cardiolipin is thought to undergo lipid transfer to Tween 20 micelles. The formation of "surfactant-phospholipid micelles" is essential for expressing the antigenic three-dimensional structure of cardiolipin on the plate surface, and the Tween 20 micelles themselves have the effect of suppressing nonspecific adsorption of immunoglobulins. Immobilization of "surfactant-phospholipid micelles" can be achieved not only by method (I) but also by method (II). In method (II), cardiolipin micelles are first formed in a buffer solution, then incubated in a plate, allowing cardiolipin to be adsorbed to the plate surface via the lipid bilayer membrane of the micelles, followed by washing with Tween 20.

Example 4: Effect of pBSA Treatment on Maintenance of Cardiolipin Antigenic Expression Using cardiolipin-bound plates prepared in the same manner as in Method (I) above, the absorbance (450 nm) was measured as an index of anticardiolipin antibody titer for the plates treated with pBSA (present invention) and untreated (comparative example), with varying concentrations of Tween 20 added for plate treatment. The results are shown in Table 3.

The surfactant treatment was carried out using Tween 2 at various concentrations.
The measurement was performed using PBS (pH 7.4) containing 0. The samples measured were 24 units (24U) and 12 units (12U) (according to the units of Harris et al.) of As serum.

As is clear from Table 3, the effect of Tween 20 on antigenic expression was observed regardless of whether or not the cells were treated with pBSA. However, the antigenic expression of cardiolipin was not observed with 1% pBSA.
This was promoted with or without treatment with BSA at 4°C for 1 hour. In other words, after the treatment of drying the ethanol solution of cardiolipin in method (I), the formation of a physiological three-dimensional conformation with antigenicity occurs not only with Tween treatment but also with BSA treatment. Furthermore, the combined use of these two treatments promotes antigenic expression, increases stability, and prevents nonspecific adsorption of antibodies, resulting in the so-called "blocking promotion effect."

Example 5: Effect of Various Surfactants In the same method as in Example 4, the type of surfactant used to treat the plate was changed to detect As serum (2
The absorbance (450 nm) was measured as an index of antibody titer (4 U). The results are shown in Table 4. A 0.05% solution of each surfactant was used.

As is clear from Table 4, surfactants having hydrophobic group moieties of different lengths from those of Tween 20 also had the same effect as Tween 20.

Example 6 Detection of antiphospholipid syndrome-specific antibodies using a carrier treated with a combination of human serum, pBSA, and a surfactant The carrier for binding antiphospholipid antibodies of the present invention was obtained in the same manner as in method (1) of Example 1, except that in the method of Example 1 (1), the cardiolipin-bound plate was treated with PBS containing 5% normal human serum and 1% (W/V) pBSA instead of treatment with PBS containing 1% pBSA.

The absorbance (450 nm) as an index of the antibody titer of As serum was measured in the same manner as in Example 1, except that the above carrier (invention) and the carrier obtained by method (1) in Example 1 (control) were used and 1% PBS-BSA containing no healthy human serum was used as the reaction solution for the primary reaction. The results are shown in Table 5.

As is clear from Table 5, when the carrier for binding antiphospholipid antibodies was treated with a combination of healthy human serum, pBSA, and a surfactant, the same effect was obtained as when human serum was added in the primary reaction, even when no human serum was added.

Example 7 Measurement of serum from patients with antiphospholipid syndrome Using the antiphospholipid antibody binding carrier prepared by the method (I) of Example 1, the anticardiolipin antibody titers (units; unit (U)) of serum samples from healthy subjects and SLE patients (live birth group and miscarriage group) were measured in the same manner as in Example 1.
The results are shown in Figure 9. As shown in Figure 9, the absorbance (450 nm) as an index of anticardiolipin antibody titer in the serum of SLE patients was significantly higher than that of the serum group of healthy subjects. Furthermore, the habitual abortion group showed significantly higher antibody titers than the live birth group (group that successfully gave birth).

In addition, sera from patients with autoimmune diseases, including SLE patients, 24
Anticardiolipin antibody titers were measured for eight cases by the conventional method (method of Reference Example) using fetal bovine serum in the primary reaction and by the method of the present invention described above, and the correlation between the two was determined. The results are shown in Figure 10. In Figure 10, the vertical axis indicates the antibody titer (units) by the method of the present invention, the horizontal axis indicates the antibody titer (units) by the conventional method, and the dashed line indicates the cutoff value (average antibody titer in serum of healthy subjects + 3 x standard deviation (1 unit)). The correlation coefficient between the two measurement systems is 0.664, demonstrating high discrimination and quantification by the method of the present invention. In other words, the method of the present invention provides better discrimination between positive and negative. The reason for this is that, as mentioned above, antibodies derived from infectious diseases (i.e., Y
This is because the test results for type A antibodies become negative.

The dilution curves (showing the relationship between serum dilution and anticardiolipin antibody titer (units)) obtained by the method of the present invention for four SLE patient sera and two healthy subject sera are shown in Figure 11. As is clear from Figure 11, the method of the present invention exhibited excellent dilution linearity and high quantitative accuracy of antibody titer.

Example 8 Comparison of antiphospholipid antibody titers in the sera of patients with autoimmune diseases and patients with infectious diseases. Using antiphospholipid antibody-binding carriers prepared by binding various phospholipids to carriers in the same manner as in method (I) of Example 1, absorbance (450n) was measured as an index of antiphospholipid antibody titers in the sera of patients with autoimmune diseases (SLE) and patients with infectious diseases (tuberculosis and syphilis) in the same manner as in Example 1.
m) was measured.

The results of measurement of each test serum in the presence and absence of 5% healthy human serum are shown in Figure 12.
The serum was diluted to 24 U (1/200-fold concentration), and all other test sera were diluted to 1/100-fold concentration before use in the experiment.
L), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) were used as immobilized antigens.
L, PI, PS, and PA are the present invention, and PE and PC are controls).
The amount of antigen used was 2.5 μg/50 μl/well.

As is clear from Figure 12, autoimmune antibodies (antibodies specific to autoimmune diseases) showed serum-dependent enhancement of reactivity to negatively charged antigens (i.e., CL, PI, PS, and PA), whereas infectious disease antibodies (antibodies derived from infectious diseases) showed serum-dependent reduction in reactivity. This has made it possible to differentially quantify infectious disease and autoimmune antiphospholipid antibodies, which had previously been difficult to measure.

Example 9 Effect of buffer solution The antiphospholipid antibody binding carrier prepared by the method (I) of Example 1 was used, and a 1% pH buffer solution was used as a diluent for the test solution.
HEPES buffer containing SA (10mM HEPES, 150mM NaC
The absorbance (450 nm) was measured as an index of the antibody titer of As serum in the same manner as in Example 1, except that a diluent containing HEPES was used. The results are shown in Figure 13. As is clear from Figure 13, when a reaction solution containing HEPES was used as the reaction solution for the primary reaction (a diluent for the test solution), a good calibration curve (anticardiolipin antibody titer (units) and absorbance (450 nm)) was obtained.
m) was obtained, and it was found to be useful for measuring antiphospholipid antibodies resulting from antiphospholipid syndrome.

Example 10 Purification of the Active Ingredient The active ingredient (anticardiolipin cofactor) in the serum fraction of healthy subjects in Example 2 was purified as follows.

(1) DEAE-cellulose (DE-5) in serum of healthy subjects
2) Ion-exchange column chromatography using a DEAE-cellulose (DE-52) column. In order to purify the active fraction from the serum of healthy subjects, ion-exchange column chromatography using DEAE-cellulose (DE-52) was carried out.
150 ml of ion exchange resin (Whatman) 2.5x
The column was packed into a 60 cm glass column and equilibrated with 14 mM sodium phosphate buffer (pH 7.4). After 2 days of dialysis against 3 L of the same buffer, 100 ml of human serum was added to the top of the column, and elution was carried out with 3 L of the same phosphate buffer. The eluate was collected in 10 ml aliquots using a fraction collector. The elution status of the protein was detected by measuring the absorbance of each fraction at a wavelength of 280 nm, and the results were analyzed using the (B) method described below.
Anticardiolipin cofactor activity was measured by the method (B). Furthermore, cofactor-independent anticardiolipin antibody activity was revealed by performing DEAE-cellulose column chromatography on serum from healthy individuals. Therefore, cofactor-independent anticardiolipin antibody activity was simultaneously measured by method (B). Fractions exhibiting cofactor activity were then recovered (Figure 14). The recovered active fractions were concentrated to approximately 100 ml by conventional ultrafiltration or salting out with 80% saturated ammonium sulfate, and then subjected to Protein A-Selfharose column chromatography as described below.

(2) Protein A-Sepharose column chromatography of the cofactor activity-containing fraction: To remove human IgG from the cofactor activity-containing fraction, protein A-Sepharose column chromatography was performed. Specifically, 20 ml of protein A-Sepharose (Pharmacia) was packed into a 1.5 x 20 cm glass column and equilibrated with 1.5 M glycine buffer (pH 8.9) containing 3 mM sodium chloride.
To this, 10 ml of the active fraction was added to the same glycine buffer solution.
The diluted solution (100 ml) was added to the top of the column, and elution was performed with 100 ml of the same glycine buffer. The eluate was collected in 10 ml aliquots using a fraction collector. The protein absorption of each fraction thus obtained was measured in the same manner as described above, and the peak absorption portion was collected (Figure 15).
The collected portion was diluted with 10 ml of 150 mM sodium chloride.
The mixture was dialyzed overnight against 2 L of 100 mM HEPES buffer (pH 7.4), concentrated to 5 ml using an ultrafilter, and subjected to affinity column chromatography as follows: The adsorbed IgG was eluted with 100 mM sodium citrate buffer (pH 3.0).

(3) Affinity chromatography using anti-human IgG antibody-Sepharose CL-4B column: To remove IgG that did not adsorb to Protein A, affinity column chromatography was carried out using a column prepared by coupling anti-human IgG antibody to Sepharose CL-4B column resin (Pharmacia) in a conventional manner. Specifically, the resin prepared in advance was packed into a 2.0 x 5 cm glass column, and the column was filled with 10 mM HEPES-sodium buffer (pH 7.4) containing 150 mM sodium chloride.
The column was equilibrated with 1 ml of the solution obtained in (2) above, and eluted with the same HEPES buffer. The eluate was collected in 1 ml aliquots using a fraction collector. The protein absorption of each fraction was measured in the same manner as described above, and the peak fractions were collected (Figure 16). The collected fractions were pooled together and subjected to sodium dodecyl sulfate (hereinafter abbreviated as SDS) polyacrylamide gel electrophoresis in the usual manner. After confirming that IgG had been completely removed (Figure 17, Fr
The adsorbed IgG was concentrated to 2 ml using a 0.1 M glycine hydrochloride buffer (pH 3.
0).

(4) Purification of cofactors by cardiolipin liposomes The cofactors were completely purified from the purified fraction (hereinafter referred to as Fr. 1N) obtained in (3) from which IgG had been completely removed by affinity adsorption to cardiolipin liposomes.
2 ml of the 1/ml ethanol solution was placed in a 25 ml pear-shaped flask and slowly evaporated under vacuum to form a thin film on the wall of the flask.
The resulting liposomes were added to the purified cofactor solution Fr. 1N (1.2 mg/ml in the same HEPES buffer) obtained in (3) at the same volume, and stirred vigorously for 15 minutes to prepare cardiolipin liposomes. The liposomes were then added to the purified cofactor solution Fr. 1N (1.2 mg/ml in the same HEPES buffer) obtained in (3) at the same volume, and allowed to stand at room temperature for 1 hour to allow the cofactor to affinity adsorb to the liposomes. The liposomes were then recovered by centrifugation at 15,000 rpm at 4°C for 15 minutes, and then centrifuged and washed three times with the same HEPES buffer. The supernatants were recovered, and the absence of unadsorbed cofactor was confirmed by standard SDS polyacrylamide slab gel electrophoresis. If unadsorbed cofactor was present, it was adsorbed to the liposomes by repeating the same procedure and recovered. The liposomes with adsorbed cofactors in this manner were collected at 100-50%.
The cofactor-adsorbed liposomes were suspended in 0.0 μl of the same HEPES buffer and stored frozen at -20°C. Using the resulting cofactor-adsorbed liposomes, standard SDS-polyacrylamide slab gel electrophoresis yielded a protein that appeared as two closely spaced bands with a molecular weight of approximately 50,000 (Fig. 17, F-1). At the same time, cofactor purification was performed using 2 mM liposomes of dipalmitoylphosphatidylcholine (DPPC):cardiolipin (80:20, mol%) and 2 mM liposomes of DPPC:dipalmitoylphosphatidylethanolamine (DPPE) (80:20, mol%) in the same manner. The liposomes composed of DPPC and cardiolipin exhibited the same effect as liposomes composed of cardiolipin alone (Fig. 17, F-2), but the DPP
No components thought to be cofactors were adsorbed to the liposomes composed of C and DPPE (FIG. 17, F-3).

(5) Separation of liposome-adsorbed cofactors by HPLC. The cofactors purified by affinity adsorption to cardiolipin liposomes were separated by HPLC using a reversed-phase column.
That is, the liposome suspension to which the cofactor was adsorbed was centrifuged at 15,000 rpm for 15 minutes,
The liposomes were collected. A 1% SDS solution was added to the liposomes and the mixture was thoroughly stirred to dissolve the liposomes. The mixture was centrifuged again under the same conditions to precipitate the insoluble matter, and the supernatant was then applied to a reverse phase column (Waters Microbonder Spare C
HPLC was carried out using a 1-4 column (3.9 x 15 cm; manufactured by Waters) under the following conditions, while measuring the absorbance at 215 nm with an attached absorbance detector. In this way, a purified cofactor was obtained (first
Figure 8).

HPLC conditions for separating cofactors from cofactor-adsorbed cardiolipin liposomes: Flow rate: 1 ml/min, Upper pressure limit: 300 kgf/ ^cm², Lower pressure limit: 0 kgf/ ^cm², Flow rate time: 60 min. Solutions used: Solution A: 0.1% trifluoroacetic acid aqueous solution; Solution B: acetonitrile:isopropanol (3:7, V)
/V) and 0.07% trifluoroacetic acid was added. Solution gradient conditions Solution A (%) Solution B (%) Time 0 minutes later 100 0 60 minutes later 40 60 (6) Molecular weight and isoelectric point of anticardiolipin cofactor To investigate the isoelectric point and molecular weight of the cofactor, An
ISODALT gel electrophoresis was performed according to the method of Derson et al. First, electrophoresis was performed on the solution Fr. 1N obtained in (3) (Fig. 19). As shown in Fig. 19, several spots appeared, but the cofactors adsorbed to cardiolipin liposomes were identified as spots No. 10 and No. 12 in Fig. 19 based on the results of SDS polyacrylamide electrophoresis of the purified cofactor obtained in (5) (Fig. 17, F-1). That is,
As shown in Figure 19, the cofactors had an isoelectric point of 6.75 and a molecular weight of 49,600 (No. 10) and an isoelectric point of 6.60 and a molecular weight of 50,000 (No. 12). These two components have the same amino acid sequence but different sugar chains, or correspond to proteins in which the functional groups of the amino acids have been partially modified in some way, and are considered to be subtypes of the cofactor.

(B) Method for Measuring Anticardiolipin Cofactor Activity and Cofactor-Independent Anticardiolipin Antibody Activity Anticardiolipin cofactor activity and cofactor-independent anticardiolipin antibody activity contained in the serum of healthy subjects were measured by the following method.

The measurement was carried out in substantially the same manner as in Reference Example 1.

That is, a 96-well microplate coated with cardiolipin was washed with 20 ml of PBS buffer (pH 7.4) containing 0.05% Tween (hereinafter referred to as washing solution) per well.
The plate is then washed with 50 μl of PBS three times to activate it.
Next, 10 mM HEPES buffer (pH 7.0) containing 150 mM sodium chloride and 1% BSA was added to the wells where the cofactor activity was to be measured.
4 (hereinafter referred to as dilution buffer) to a 200-fold dilution, and 50 μl of the As serum was added to the wells for measuring the activity of the independent cardiolipin antibody.
The wells were washed three times with washing solution, and then the plate was left to stand at room temperature for 30 minutes.
50 mM sodium chloride, 1% BSA and 1 mM EDTA
100 μl of horseradish peroxidase-conjugated anti-human IgG antibody, appropriately diluted with 10 mM HEPES buffer (pH 7.4) containing TA, was dispensed into each well and allowed to stand at room temperature for 30 minutes. After washing three more times with the washing solution,
0.3 mM TMBZ and 0.03% oxygen peroxide (10
0 μl) was added as a substrate solution, and after reacting for 10 minutes, the absorbance at 450 nm was measured using a plate reader.

Test Example The following test was carried out to examine the properties of the anticardiolipin cofactor.

(1) Preparation of biotinylated anticardiolipin cofactor: According to the method of Kumagai and Okumura et al., "Procedures for Immunological Experiments" (published February 20, 1980, Japanese Society for Immunology (Cell Antigen III, vol. 15-59, p. 2425)), 1 mg of the anticardiolipin cofactor purified in Example 10(3) was biotinylated to label the anticardiolipin cofactor.

(2) Binding of anticardiolipin cofactor to cardiolipin.
Cardiolipin ethanol solution was added at 5 μg/50 μl/well, dried under reduced pressure, and reacted with 1% BSA-containing PBS for 1 hour, followed by 0.05% Tween 20-containing PBS.
The plate was washed three times with PBS (200 μl). The cardiolipin-immobilized plate was loaded with 0 to 32 μg/ml of cardiolipin as shown in FIG.
ml of biotinylated anticardiolipin cofactor (10
The plate was incubated with 100 μl of 0.3 mM TMBZ and 0.003% hydrogen peroxide solution for 30 minutes at room temperature, and then washed three times. The plate was then incubated with avidin-conjugated peroxidase for 30 minutes at room temperature. After further washing three times, the plate was added with 100 μl of 0.3 mM TMBZ and 0.003% hydrogen peroxide solution and incubated for 10 minutes at room temperature. The reaction was then stopped by adding 100 μl of 2N sulfuric acid. The absorbance of the reaction solution was measured to determine the following:
The anticardiolipin cofactor bound to the immobilized cardiolipin was quantified.

As shown in FIG. 20, the biotinylated anticardiolipin cofactor exhibited binding to immobilized cardiolipin in a concentration-dependent manner.

(3) Binding specificity of biotinylated anticardiolipin cofactor to various phospholipids. Various phospholipids (i.e., cardiolipin, phosphatidylserine (PS), phosphatidylinositol (PI), dipalmitoylphosphatidic acid (DPPA), dipalmitoylphosphatidylcholine (DPPC), and dipalmitoylphosphatidylethanolamine (DPPE)) were incubated at 2.5 μg/50 μl in the same manner as in (2) above.
The binding specificity of biotinylated anticardiolipin cofactor was examined using avidin-conjugated peroxidase in a plate with 1/well of biotinylated anticardiolipin cofactor.
Anticardiolipin cofactors bind to negatively charged phospholipids (cardiolipin, PS, PI, DPPA)
It was specific to.

(4) Dependence of anticardiolipin cofactor in the anticardiolipin antibody reaction system Using the same ELISA method as in (2) above, anticardiolipin antibody (A) specifically present in antiphospholipid syndrome was detected.
Anticardiolipin cofactor (2 μg/well) was added to the reaction system between the anticardiolipin antibody and immobilized cardiolipin (serum) to examine the anticardiolipin cofactor dependency of the reaction between the anticardiolipin antibody and immobilized cardiolipin. As shown in Figure 22, the anticardiolipin antibody reacted in a manner dependent on the added antibody-cardiolipin cofactor.

(5) Species specificity of anticardiolipin cofactors: The activity of different species of anticardiolipin cofactors (human and bovine) was evaluated using the same ELISA method as in (2) above. The bovine cofactor enhanced the reactivity of both autoimmune (As serum) and infectious disease (Ya serum) anticardiolipin antibodies, whereas the human cofactor enhanced the reactivity of only the autoimmune antibody (As antibody) (Figure 23).

Example 11 Collection of fractions from serum of healthy subjects (1) Collection of fractions by DEAE-cellulose column chromatography 14 mM sodium phosphate buffer (pH 7.4) (hereinafter,
Serum (1 ml) from a healthy individual that had been dialyzed overnight against PB7.4 was applied to a DEAE-Sepharose column (10 ml, 1.0φ×13 cm, Whatman DE-Sepharose) previously equilibrated with PB7.4.
52), and 1 ml of the flow-through fraction was collected. 25 μl of each fraction was used to measure the activity of enhancing the reactivity of immobilized cardiolipin with anticardiolipin antibodies specifically present in the sera of patients with antiphospholipid syndrome (As) and the activity of inhibiting the reactivity of immobilized cardiolipin with anticardiolipin antibodies present in the sera of patients with syphilis (Sy) in substantially the same manner as in Reference Example 1 and Example 10 (A).

The results are shown in FIG.

(2) Collection of fractions by heparin-sepharose column chromatography: One ml of serum from a healthy individual, which had been dialyzed overnight against 10 mM sodium phosphate buffer containing 50 mM NaCl, pH 7.4, was adsorbed onto a heparin-sepharose column (6 ml, 1.0φ×8 cm, manufactured by Pharmacia) equilibrated with the same buffer. After thorough washing with the same buffer, the column was eluted with a concentration gradient of 50 mM to 1 M NaCl.
5 μl of serum from patients with antiphospholipid syndrome (A
The activity of enhancing the reactivity of anticardiolipin antibodies specifically expressed in syphilis patient serum (Sy) with immobilized cardiolipin and the activity of suppressing the reactivity of anticardiolipin antibodies present in syphilis patient serum (Sy) with immobilized cardiolipin were tested in Reference Example 1.
The measurement was carried out in accordance with the method of Example 10 (A).

The results are shown in FIG.

(3) Collection of fractions by cardiolipin-acrylamide gel column chromatography i) Preparation of cardiolipin-polyacrylamide column Bovine heart cardiolipin (5 μmoles), cholesterol (5 μmoles), and dicetyl phosphate (0.5 μmoles) were dried under reduced pressure in a pear-shaped flask to form a film, and 500 μl of ethanol was added to form a 6-well column.
The mixture was heated to about 0°C to dissolve the lipids. Next, 5 ml of 15% acrylamide and 5% bisacrylamide solution was added and vigorously stirred with a vortex mixer. Then, 100 μl of ammonium persulfate (100 mg/ml) and 2 μl of TE
MED was added to polymerize the gel. The polymerized gel was homogenized and packed into a column (1.0φ×3 cm) containing 50 mM Na
The column was thoroughly washed and equilibrated with Cl-containing 10 mM sodium phosphate buffer, pH 7.4.

ii) Affinity column chromatography: 1 ml of serum from a healthy individual, dialyzed overnight against 10 mM sodium phosphate buffer containing 50 mM NaCl, pH 7.4, was passed through the column, thoroughly washed with the same buffer, and the adsorbed proteins were eluted with 10 mM sodium phosphate buffer containing 1.0 M NaCl, pH 7.4. Using 15 μl of each fraction, the activity of enhancing the reactivity of immobilized cardiolipin with anticardiolipin antibodies specifically present in the sera of patients with antiphospholipid syndrome (As) and the activity of inhibiting the reactivity of immobilized cardiolipin with anticardiolipin antibodies present in the sera of patients with syphilis (Sy) were measured according to the methods described in Reference Example 1 and Example 10(A).

The results are shown in FIG.

As described above, the fraction of the present invention can be obtained from the serum of healthy subjects using DEAE-cellulose column chromatography, heparin Sepharose column chromatography, or cardiolipin-acrylamide gel column chromatography.

Example 12: Activity of anticardiolipin cofactors The expression of activity that enhances the reactivity of anticardiolipin antibodies in the serum of patients with antiphospholipid syndrome (As) with cardiolipin (As↑ activity) and activity that inhibits the reactivity of anticardiolipin antibodies in the serum of patients with syphilis with cardiolipin (Sy↓ activity) was investigated.

The cardiolipin-immobilized plate was treated with Tween 20-containing PB
After washing with S, the sera of groups 3 and 4 shown in Table 6 were added to the cofactor (10 μg) obtained in Example 10 (3).
The control group was treated with cofactor lysis buffer.
The cells were washed three times with PBS containing 0, and serum As (1/400 dilution, 125 U/ml) and Sy (1/200 dilution) were added in the presence of a cofactor (10 μg/ml) (groups 2 and 4).
The reaction was carried out at room temperature for 30 minutes in the absence (groups 1 and 3). The subsequent procedures were the same as in Reference Example 1 and Example 10 (A).

As shown in Table 6, to obtain As↑ activity, a cofactor is either pretreated as described above, present in the reaction system with the antibody, or present in both. To obtain Sy↓ activity, a cofactor may be present during pretreatment, but must be present in the reaction system with the antibody.

Example 13 Using a method similar to that of Reference Example 1 and Example 10 (A), the serum of a patient with antiphospholipid syndrome (A
S) activity to enhance the reactivity of anticardiolin antibodies with cardiolipin (As↑ activity) and in syphilis patient serum (Sy)
The effect of purified bovine serum albumin on the inhibitory activity (Sy↓ activity) of the reactivity of anticardiolipin antibodies with cardiolin present in the serum was investigated.

When using highly purified lipid-free bovine serum albumin (BSA) and purified cofactor (the cofactor obtained in Example 10 (3)) in this assay, Sy↓ activity decreases depending on the BSA concentration in the reaction solution (see Table 27).
This is thought to be due to the fact that the Sy↓ activity site on the cofactor molecule is blocked by purified albumin (BSA). Therefore, the BSA concentration should be kept at 0-5% (especially 0
It is desirable to use it in the range of -1%).

Example 14 Determination of the amino acid sequence of the cofactor The cofactor obtained by the method of Example 10(3) was further subjected to cardiolipin-acrylamide gel column chromatography as used in Example 11(3) for adsorption, and then purified by elution with 1M NaCl (this cofactor contains the two subtypes identified in Example 10(6)).
The N-terminal amino acid sequence of the sample was determined. That is, 30 μl (300 pmol) of the sample was adsorbed onto a PVDF membrane (polyvinylidene difluoride; manufactured by Millipore; trade name: Immobilon), washed with 60% methanol, and then analyzed using a gas phase protein sequencer (PSQ-1 model, manufactured by Shimadzu Corporation).
The sequence of five amino acids from the N-terminus of the cofactor was determined.

1 5 Result: NH2 _- Gly-Arg-Thr-X-Pro-Lys-Pro (X is presumed to be Cys or His) INDUSTRIAL APPLICABILITY The present invention establishes a method for reproducibly distinguishing between antiphospholipid antibodies derived from antiphospholipid syndrome and antiphospholipid antibodies derived from infectious diseases, which could not be adequately distinguished by conventional methods, by using a carrier for binding antiphospholipid antibodies that has been treated with a surfactant alone, a surfactant in combination with purified serum albumin, or a combination of these with the blood component of the present invention, and/or by adding the blood component of the present invention (anticardiolipin cofactor) to the reaction solution in the first antigen-antibody reaction (primary reaction).The method of the present invention enables extremely accurate diagnosis of antiphospholipid syndrome.

Furthermore, the blood component of the present invention (anticardiolipin cofactor) has the effect of increasing the binding affinity between phospholipids and antibodies specific to antiphospholipid syndrome and decreasing the binding affinity between phospholipids and antiphospholipid antibodies derived from infectious diseases. Therefore, by using these serum or plasma, fractions, or proteins (anticardiolipin cofactors) in immunological assays for antibodies specific to antiphospholipid syndrome, it is possible to accurately diagnose antiphospholipid syndrome and to differentiate and detect antiphospholipid antibodies in antiphospholipid syndrome and infectious diseases.

──────────────────────────────────────────────────── Continued from the front page (56) References: Special Publication No. Hei 4-506415 (JP, A) ・Keio J Med, vol. 36, pp. 284-297 (1987) ・THROMBOSIS RESEARCH, vol. 52, pp. 609-619 (1988) ・The Journal of Rheumatology, vol. 15, pp. 80-86 (1988) ・IMMUNOLOGICAL INVESTIGATION, vol. 18, No. 9, 10, pp. 1121-1127 (1989) ・Br itish journal of ha ematology, vol. 73, P. 506-513 (1989) ・Uin. exp,I mmunol. , vol. 80, P. 171− 176 (1990)

Claims (13)

[Claims] [Claim 1] A method for differentially detecting or differentially quantifying anticardiolipin antibodies derived from antiphospholipid syndrome and anticardiolipin antibodies derived from an infectious disease by contacting a test liquid with a carrier for binding anticardiolipin antibodies to which cardiolipin has been bound, wherein when contacting the carrier for binding anticardiolipin antibodies with the test liquid, both a method of contacting in the presence of an anticardiolipin cofactor and a method of contacting in the absence of an anticardiolipin cofactor are performed to differentially detect or differentially quantify anticardiolipin antibodies derived from antiphospholipid syndrome and anticardiolipin antibodies derived from an infectious disease. 2. The method of claim 1, wherein the cardiolipin is immobilized on a microtiter plate. 3. The method according to claim 1, wherein the anticardiolipin cofactor is derived from the same or similar species of animal as the test solution. 4. The method according to claim 1, wherein the anticardiolipin cofactor is of human origin. Claim 5: As an anticardiolipin cofactor,
The method according to claim 1, wherein serum, plasma, or a fraction thereof containing an anticardiolipin cofactor is used. 6. The method of claim 1, wherein the anticardiolipin cofactor is contained in the specimen diluent. 7. The method according to claim 1, wherein the test fluid is human blood or a fraction thereof. 8. A kit for carrying out the method of claim 1, comprising anticardiolipin cofactor and cardiolipin as constituent reagents. 9. The kit of claim 8, wherein the cardiolipin is immobilized on a microtiter plate. 10. The anticardiolipin cofactor used in the test solution is derived from the same or similar species of animal.
The kit of claim 8. 11. The kit according to claim 8, wherein the anticardiolipin cofactor is derived from a human. 12. The kit according to claim 8, wherein serum, plasma, or a fraction thereof containing an anticardiolipin cofactor is used as the anticardiolipin cofactor. 13. The kit according to claim 8, wherein the anticardiolipin cofactor is contained in the specimen diluent.