JPH11258236A - Reagent for measuring anti phospholipid antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a reagent for measuring an anti phospholipid antibody which is applicable to general biochemical automatic analysis, thereby enabling quick, simple and highly objective measurement for a high volume of antibodies. SOLUTION: A lipid antigen of cardiolipin and lecithin is carried by an insoluble carrier, preferably via a denatured serum albumin such as alkylated serum albumin or the like. The lipid antigen is carried after the denatured serum albumin is carried by the insoluble carrier. Or the lipid antigen is carried by the insoluble carrier after forming a complex with the denatured serum albumin. The lipid antigen consists of 0.01-1 μg cardiolipin and lecithin of 5-15 times the cardiolipin with respect to 10 μg insoluble carrier.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a reagent for measuring anti-phospholipid antibodies.

[0002]

2. Description of the Related Art When a living organism is infected with Treponema Pallidum, which is a pathogen of syphilis, a Wasselmann antibody (anti-phospholipid antibody) that is reactive with phospholipids is produced together with antibodies against the pathogen. Conventional methods for testing syphilis include a method using an antigen itself extracted from syphilis cells to detect antibodies against syphilis cells and a method using lipids containing cardiolipin as antigens to detect Wasselman antibodies. It is roughly divided into two.

At present, as a method using lipid, VDR is used.
L (Veneral Disease Research Lab.) Method, cord method, RP
There are the R (Rapid Plasma Reagin) method, the glass plate method and the like, and the RPR method and the glass plate method are mainly used for qualitative screening. Test methods using these lipids have the drawback of producing a positive reaction even in diseases other than syphilis. However, on the other hand, antiphospholipid antibodies also have the advantage of reflecting the infection status of syphilis well, and are used to follow the course of syphilis treatment using this. Autoimmune diseases such as systemic lupus erythematosus are known as diseases that exhibit a positive reaction other than syphilis, and it is useful to measure antiphospholipid antibodies to diagnose these diseases.

The RPR method is a method in which lipid containing cardiolipin and lecithin is adsorbed on kaolin or carbon powder and mixed with a sample on a white plate to determine whether kaolin or carbon powder is aggregated. Further, the glass plate method is a method in which a lipid antigen solution containing cardiolipin, lecithin and cholesterin is mixed with a specimen, and the presence or absence of cholesterol crystal aggregation is determined. Since the above two methods are manual methods, they are not suitable for testing a large number of samples.
In addition, in the case of the RPR method, the aggregation of kaolin or carbon powder is visually observed, and in the case of the glass plate method, the formation of cholesterol crystals is observed under a microscope to make a judgment. Therefore, there is a disadvantage that the judgment result may be different depending on the judge. Furthermore, the above two methods have the drawback that although it is possible to determine qualitatively whether or not they are infected, it is difficult to quantify antibodies.

[0005] A method has been known in which a lipid antigen containing cardiolipin is adsorbed on a microtiter plate and detection is carried out by ELISA (NSPedersen et al.).
l., J. Clin. Microbiology, 25 (9), 1711-1716 (1987))
In this method, negative and positive judgments can be made objectively because the agglutination is determined by the absorbance.However, since the dispensing of reagents and samples to the microtiter plate is performed manually, the operation becomes complicated, and the reaction time is increased. Long and inability to get results quickly. In Japanese Patent Application Laid-Open No. Hei 5-212808, anti-phospholipid antibodies are detected by a method using magnetic particles. However, a device for applying a magnetic force is required, and versatility is poor.

[0006]

SUMMARY OF THE INVENTION The present invention has been made to solve the above problems, and has as its object to be applicable to general biochemical automatic analyzers, whereby rapid and simple measurement of a large amount of antibodies can be performed. And a highly objective anti-phospholipid antibody measurement reagent.

[0007]

The reagent for measuring antiphospholipid antibodies according to claim 1 of the present invention is characterized in that a lipid antigen comprising cardiolipin and lecithin is supported on an insoluble carrier via denatured serum albumin. Features. The anti-phospholipid antibody measurement reagent according to claim 2 is obtained by loading the lipid antigen after the anti-phospholipid antibody measurement reagent according to claim 1 is loaded on an insoluble carrier after denatured serum albumin is loaded. It is characterized by. The anti-phospholipid antibody measuring reagent according to claim 3 is characterized in that, after the anti-phospholipid antibody measuring reagent according to claim 1 forms a complex of denatured serum albumin and a lipid antigen, the denatured serum albumin is transferred to an insoluble carrier. It is obtained by being carried. Further, in the anti-phospholipid antibody measurement reagent according to claims 1 to 3,
As described in claim 4, the lipid antigen is an insoluble carrier 10
It is preferable that the amount of cardiolipin is 0.01 to 1 μg and the amount of lecithin is 5 to 15 times the amount of cardiolipin based on μg. Further, as described in claim 5, in the antiphospholipid antibody measurement reagent according to claims 1 to 4, it is preferable that the denatured serum albumin is an alkylated serum albumin.

[0008] In the reagent for measuring an antiphospholipid antibody of the present invention, a lipid antigen is carried on an insoluble carrier via denatured serum albumin. The lipid antigen is composed of cardiolipin and lecithin (phosphatidylcholine) as lipids, and these are supported on an insoluble carrier via denatured serum albumin. It is preferable to use cardiolipin and lecithin with as high a purity as possible in order to suppress a non-specific reaction. Cardiolipin purified from bovine heart is used. The main component is phosphatidyl glycerol, and the purity of phosphatidyl glycerol is 90.
% Or more is preferable. Lecithin purified from egg yolk or chemically synthesized is used.
The main component is phosphatidylcholine, and phosphatidylcholine having a purity of 70% or more is preferable.

When the amount of the cardiolipin decreases,
If the obtained reagent does not have sufficient sensitivity for the measurement, non-specific agglutination will occur and the precipitation will occur if the number of reagents increases. If the amount of lecithin is small, a lipid antigen having the antigenicity required for measurement cannot be obtained, and if it is large, nonspecific aggregation occurs and precipitation occurs. Therefore, the lipid antigen is insoluble carrier 10μ
It is preferable that the amount of cardiolipin is 0.01 to 1 μg and the amount of lecithin is 5 to 15 times the amount of cardiolipin based on g.

[0010] The denatured serum albumin can be obtained by denaturing serum albumin by any method. Examples of the above method include thermal denaturation, denaturation with a protein denaturant, denaturation by chemical modification, and the like. In the case of the thermal denaturation, the protein is preferably heated to a protein denaturation temperature of 40 ° C or higher, more preferably 50 to 60 ° C.
The heating time is preferably about 20 to 40 minutes, because if the time is long, serum albumin binds and the molecular weight becomes too large. In the case of denaturation with the above protein denaturant, examples of the protein denaturant include aldehyde derivatives such as formaldehyde, acetaldehyde and glutaraldehyde, and guanidine derivatives such as guanidine hydrochloride and nitrosoguanidine. As a method of denaturation with a protein denaturant, a method of mixing a protein denaturant and serum albumin as described above is usually used. in this case,
To prevent serum albumin degradation due to excessive denaturation,
The concentration of the protein denaturant is preferably 1% by weight or less,
% By weight or less is more preferable.

[0011] Further, denaturation by chemical modification can be obtained by modifying the functional groups of the constituent amino acids of serum albumin. Examples of the denatured serum albumin thus obtained include alkylated serum albumin such as methylated serum albumin and carboxymethylated albumin. The methylated serum albumin can be obtained by adding methanol to a serum albumin solution under acidic conditions of hydrochloric acid to form methyl ether.
Alternatively, denatured serum albumin can be obtained by reducing the SS bond in the serum albumin molecule with an SH group reducing agent such as 2-mercaptoethanol or dithiothreitol. Of the above, alkylated serum albumin is preferred as the modified serum albumin. As the above-mentioned serum albumin, those derived from animals such as cow, human, rabbit, rat and mouse can be used, and bovine serum albumin is preferable.

In the present invention, the lipid antigen is supported on the insoluble carrier via the denatured serum albumin. The lipid antigen is bound to the denatured serum albumin after the denatured serum albumin is supported on the insoluble carrier. Alternatively, the complex may be carried by forming a complex of denatured serum albumin and a lipid antigen and then carrying the denatured serum albumin on an insoluble carrier.
In the former method, first, the method of supporting denatured serum albumin on an insoluble carrier includes a method of physically adsorbing and a method of chemically binding. The above-mentioned physical adsorption method is carried out by a so-called hydrophobic bond between a hydrophobic portion of an insoluble carrier and a hydrophobic portion of denatured serum albumin, and can be generally supported only by mixing both.

The above chemical bonding method is carried out by covalent bonding by a chemical reaction between a functional group on an insoluble carrier and a functional group on denatured serum albumin. For example, by adding a coupling agent such as a water-soluble carbodiimide, an amide bond is formed between the insoluble carrier and the denatured serum albumin, whereby the carrier can be supported.
Examples of the water-soluble carbodiimide include 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide meth-ρ-toluenesulfonate, and 1-ethyl- 3- (3-Emethylaminopropyl)
Carbodiimide perchlorate and the like.

Next, the denatured serum albumin supported on the insoluble carrier is bound to the lipid antigen, but the binding can usually be carried out by mixing the two. Although the detailed binding mode in this case is unknown, it is considered that the denaturation increases the hydrophobicity of serum albumin and binds to the hydrophobic portion of the lipid antigen.

Next, in the latter method, first, a complex of denatured serum albumin and a lipid antigen is formed in the same manner as in the case of binding denatured serum albumin and a lipid antigen as described above.
This is performed by mixing the two. Next, the denatured serum albumin is supported on the insoluble carrier. As the method, the same method as the method for supporting the denatured serum albumin on the insoluble carrier is used.

As the insoluble carrier, those generally used in immunological agglutination reactions and agglutination inhibition reactions can be used, for example, organic polymer powders, microorganisms, blood cells and cell membrane fragments, Examples include a plastic microtiter plate. As mentioned above,
When the denatured serum albumin is supported on an insoluble carrier by covalent bonding, a carrier having an active group such as a carboxyl group, an amino group, or a thiol group is used. As the organic polymer powder, for example, insoluble agarose, cellulose, natural polymer powder such as insoluble dextran, polystyrene, styrene-sulfonic acid copolymer, styrene-methacrylic acid copolymer, acrylonitrile-butadiene-styrene copolymer And synthetic polymer powders such as vinyl chloride-acrylate copolymers and vinyl acetate-acrylate copolymers, and the like. Latex in which a polymer powder is uniformly suspended is preferred. The average particle size of the latex varies depending on the measuring method and measuring instrument, but is 0.1 to 1.0 μm, preferably 0.1 to 1.0 μm.
Those having a thickness of 0.5 μm are usually used.

The reagent obtained as described above may be diluted with an appropriate sample diluent.
Any buffer solution of 5.0 to 9.0 can be used. Examples of the buffer include a phosphate buffer, a glycine buffer, a veronal buffer, a borate buffer, and a citrate buffer. In addition, various sensitizers may be used in the sample diluent for improving the measurement sensitivity and promoting the antigen-antibody reaction. Examples of the sensitizer include alkylated polysaccharides described in JP-A-2-173567 and JP-A-5-18083.
No. 8, such as pullulan and polyvinylpyrrolidone. Furthermore, bovine serum albumin, ovalbumin, etc. may be used to suppress non-specific reactions caused by other substances present in the sample.

The amount of anti-phospholipid antibody in the specimen is determined by optically measuring the degree of aggregation produced by the immunoreaction between the reagent obtained as described above and the anti-phospholipid antibody in the specimen. In the optical measurement method, scattered light intensity, transmitted light intensity, optical equipment that can detect the absorbance,
In particular, any general biochemical automatic analyzer can be used.

As a method for optically measuring the degree of aggregation, a known method is used, for example, a turbidity method in which the formation of aggregation is regarded as an increase in turbidity, and the formation of aggregation is determined by particle size distribution or average particle size. , A change in forward scattered light due to the formation of agglomeration is measured using an integrating sphere, and the ratio with the transmitted light intensity is compared.
In the above-described assay, at least two measurements are taken at different time points, and a rate test (rate assay) that determines the degree of aggregation based on the increment of the measurement value (ie, the rate of increase) between these time points; One measured value is obtained at the time when the reaction is considered to be the end point of the reaction), and an endpoint test (endpoint assay) for determining the degree of aggregation based on the measured value can be used. It is preferable to conduct a speed test by a turbidity method.

[0020]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described with reference to embodiments. (Example 1) 1) Preparation of lipid antigen-carrying latex 2 ml of cardiolipin (5 mg / ml ethanol solution, manufactured by Sigma) and 10 ml of purified lecithin (10 mg / ml ethanol solution, manufactured by Nacalai Tesque) were mixed, and the lipid antigen solution was mixed. Prepared. 100 μl of polystyrene latex (average particle size: 0.4 μm, solid content: 10% (w / v), manufactured by Sekisui Chemical Co., Ltd.) was heated in advance at 37 ° C. with gentle stirring, and 1 w / v% methylated cattle was added thereto. Phosphate buffer containing serum albumin (Sigma) (hereinafter PBS)
1 ml), and the mixture was further stirred at 37 ° C. for 1 hour. To this solution was added 250 μl of the lipid antigen solution,
Stirred at 7 ° C. for 1 hour. Then 15000 rpm, 4 ° C
For 15 minutes, the supernatant was removed, and the precipitated latex was suspended again in 3 ml of PBS containing 1 w / v% methylated bovine serum albumin. This operation was repeated three times to wash the latex, and finally to 10 ml of PBS containing 1 w / v% methylated bovine serum albumin, 10 mM EDTA · 2Na (manufactured by Dojindo), and 500 mM choline chloride (manufactured by Wako Pure Chemical). The suspension was suspended to prepare a lipid antigen-carrying latex.

2) Measurement of Sensitivity As a sample diluent, a solution attached to a commercially available Mediace TPLA (manufactured by Sekisui Chemical Co., Ltd.) was used. Serum with an RPR value of 0, 1, 2, 4, or 8 was used as a sample. 30 µl of the lipid antigen-carrying latex, 210 of the sample diluent
μl and 20 μl of each sample described above were mixed, and the amount of change in absorbance was measured. The measurement was performed using a biochemical automatic analyzer (Hitachi Model 7170 automatic analyzer) at a measurement wavelength of 700 nm. Table 1 shows the results.

Example 2 1) Preparation of lipid antigen-carrying latex 2 ml of cardiolipin (5 mg / ml ethanol solution, manufactured by Sigma) and 10 ml of purified lecithin (10 mg / ml ethanol solution, manufactured by Nacalai Tesque) were mixed. An antigen solution was prepared. 250 μl of the above lipid antigen solution was added to 1 ml of PBS containing 1 w / v% methylated bovine serum albumin (manufactured by Sigma) and stirred at 37 ° C. for 1 hour to form a complex of lipid antigen and methylated bovine serum albumin. .
This solution was mixed with polystyrene latex (average particle size 0.4μ).
m, solid content 10% (w / v), manufactured by Sekisui Chemical Co., Ltd.) 10
0 μl was added, and the mixture was further stirred at 37 ° C. for 1 hour. Then, the mixture was centrifuged at 15000 rpm at 4 ° C. for 15 minutes, the supernatant was removed, and the precipitated latex was suspended again in 3 ml of PBS containing 1% w / v methylated bovine serum albumin. This operation was repeated three times to wash the latex, and finally 1 w / v% methylated bovine serum albumin, 10 mM
The suspension was suspended in 10 ml of PBS containing EDTA · 2Na (manufactured by Dojindo) and 500 mM choline chloride (manufactured by Wako Pure Chemical Industries) to prepare a lipid antigen-carrying latex.

2) Measurement of Sensitivity The amount of change in absorbance was measured in the same manner as in Example 1 except that the above-mentioned lipid antigen-carrying latex was used. Table 1 shows the results
Shown in

(Example 3) 1) Preparation of lipid antigen-carrying latex 2 ml of cardiolipin (5 mg / ml ethanol solution, manufactured by Sigma) and 10 ml of purified lecithin (10 mg / ml ethanol solution, manufactured by Nacalai Tesque) were mixed. An antigen solution was prepared. 100 μl of polystyrene latex (average particle size: 0.4 μm, solid content: 10% (w / v), manufactured by Sekisui Chemical Co., Ltd.) was heated at 37 ° C. with gentle stirring in advance, and 1 w / v% bovine serum albumin was added thereto. (Sigma) and 1 ml of PBS containing 0.1 w / v% formaldehyde were added, and the mixture was further stirred at 37 ° C. for 1 hour. To this solution was added 250 μl of the lipid antigen solution,
Stirred at 7 ° C. for 1 hour. Then 15000 rpm, 4 ° C
For 15 minutes, the supernatant was removed, and the precipitated latex was again washed with 1% w / v bovine serum albumin, 0.1% w / v.
The suspension was suspended in 3 ml of PBS containing 5% formaldehyde. This operation was repeated three times to wash the latex. Finally, 1 w / v% bovine serum albumin, 0.1 w / v% formaldehyde, 10 mM EDTA · 2Na (manufactured by Dojindo), 500 mM choline chloride (manufactured by Wako Pure Chemical Industries, Ltd.) ) Was suspended in 10 ml of PBS to prepare a lipid antigen-carrying latex.

2) Measurement of Sensitivity The amount of change in absorbance was measured in the same manner as in Example 1 except that the above-mentioned lipid antigen-carrying latex was used. Table 1 shows the results
Shown in

Example 4 1) Preparation of Lipid Antigen-Supported Latex 2 ml of cardiolipin (5 mg / ml ethanol solution, manufactured by Sigma) and 10 ml of purified lecithin (10 mg / ml ethanol solution, manufactured by Nacalai Tesque) were mixed. An antigen solution was prepared. 250 μl of the above lipid antigen solution was added to 1 ml of PBS containing 1 w / v% bovine serum albumin (manufactured by Sigma) and 0.1 w / v% formaldehyde.
The mixture was stirred at 7 ° C. for 1 hour to form a complex of lipid antigen and denatured bovine serum albumin. 100 μl of polystyrene latex (average particle size 0.4 μm, solid content 10% (w / v), manufactured by Sekisui Chemical Co., Ltd.) was added to the solution, and the mixture was further stirred at 37 ° C. for 1 hour. Then 15000 rpm, 4 ° C for 15
After centrifugation for 1 minute, the supernatant was removed, and the precipitated latex was suspended again in 3 ml of PBS containing 1 w / v% bovine serum albumin and 0.1 w / v% formaldehyde. This operation is repeated three times to wash the latex.
/ V% bovine serum albumin, 0.1 w / v% formaldehyde, 10 mM EDTA · 2Na (manufactured by Dojindo),
PB containing 500 mM choline chloride (Wako Pure Chemical Industries, Ltd.)
The suspension was suspended in S10 ml to prepare a lipid antigen-carrying latex.

2) Measurement of Sensitivity The amount of change in absorbance was measured in the same manner as in Example 1 except that the above-mentioned lipid antigen-carrying latex was used. Table 1 shows the results
Shown in

Example 5 1) Preparation of lipid antigen-carrying latex 2 ml of cardiolipin (5 mg / ml ethanol solution, manufactured by Sigma) and 10 ml of purified lecithin (10 mg / ml ethanol solution, manufactured by Nacalai Tesque) were mixed. An antigen solution was prepared. Styrene-methacrylic acid copolymer latex (carboxyl group 0.25 meq COO ⁻ /
g, average particle size 0.4 μm, solid content 10% (w / v), manufactured by Sekisui Chemical Co., Ltd.) 100 μl, 30% (w / v) 1-
50 mM phosphate buffer containing ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (pH
6.0) 1 ml was added and stirred at room temperature for 5 hours. Unreacted 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was removed by centrifugation.
M phosphate buffer (pH 6.0) twice,
The suspension was suspended in 2 ml of mM borate buffer (pH 7.5). 2 ml of 1 w / v% methylated bovine serum albumin (Sigma) was added thereto, and the mixture was stirred at room temperature for 24 hours. Remove unreacted methylated bovine serum albumin by centrifugation,
After washing twice with a 50 mM borate buffer (pH 7.5), the cells were suspended in 1 ml of PBS. 250 μl of the lipid antigen solution was added to 100 μl of this solution, and the mixture was stirred at 37 ° C. for 1 hour. Subsequently, the mixture was centrifuged at 15000 rpm at 4 ° C. for 15 minutes, the supernatant was removed, and the precipitated latex was again washed with 1 w /
3 ml of PBS containing v% methylated bovine serum albumin
Suspended in water. This operation was repeated three times to wash the latex. Finally, 1 w / v% methylated bovine serum albumin,
0mM EDTA · 2Na (manufactured by Dojin Kagaku), 500m
PBS 10m containing M choline chloride (Wako Pure Chemical Industries, Ltd.)
1 to prepare a lipid antigen-carrying latex.

2) Measurement of Sensitivity The amount of change in absorbance was measured in the same manner as in Example 1 except that the above-mentioned lipid antigen-carrying latex was used. Table 1 shows the results
Shown in

Comparative Example 1 In Example 1, 1 w / v
A lipid antigen-carrying latex was prepared in the same manner except that 1 % w / v bovine serum albumin was used in place of% methylated bovine serum albumin, and the sensitivity was measured. Table 1 shows the results.

Comparative Example 2 In Example 2, 1 w / v
A lipid antigen-carrying latex was prepared in the same manner except that 1% w / v bovine serum albumin was used in place of% methylated bovine serum albumin, and the sensitivity was measured. Table 1 shows the results.

Comparative Example 3 In Example 5, 1 w / v
A lipid antigen-carrying latex was prepared in the same manner except that 1% w / v bovine serum albumin was used in place of% methylated bovine serum albumin, and the sensitivity was measured. Table 1 shows the results.

[0033]

[Table 1]

[0034]

The method for producing the antiphospholipid antibody measurement reagent of the present invention is as described above, and is applicable to general biochemical automatic analysis, whereby rapid and simple measurement of a large amount of antibody is possible. In addition, a highly sensitive and highly objective antiphospholipid antibody measurement reagent can be provided.

Claims (5)

[Claims] 1. A reagent for measuring an antiphospholipid antibody, wherein a lipid antigen comprising cardiolipin and lecithin is carried on an insoluble carrier via denatured serum albumin. 2. The method according to claim 1, wherein the reagent for measuring anti-phospholipid antibody is carried on a carrier in which denatured serum albumin is insoluble.
A reagent for measuring an anti-phospholipid antibody, which is obtained by carrying a lipid antigen. 3. The reagent for measuring anti-phospholipid antibody according to claim 1, which is obtained by forming a complex of denatured serum albumin and a lipid antigen and then supporting the denatured serum albumin on an insoluble carrier. Characteristic reagent for measuring anti-phospholipid antibodies. 4. The lipid antigen according to claim 1, wherein the amount of cardiolipin is 0.01 to 1 μg and the amount of lecithin is 5 to 15 times the amount of cardiolipin per 10 μg of the insoluble carrier. 4. The reagent for measuring an antiphospholipid antibody according to 4. 5. The reagent according to claim 1, wherein the modified serum albumin is an alkylated serum albumin.