JPH11118800A - Method for producing antiphospholipid antibody measurement reagent - Google Patents

Abstract

(57) [Summary] [PROBLEMS] To provide a method for producing an antiphospholipid antibody measuring reagent which can be applied to general biochemical automatic analysis, and thereby can quickly and easily measure a large amount of antibodies. A method for producing an anti-phospholipid antibody measuring reagent in which an anti-phospholipid antibody is supported on a latex made of a synthetic polymer. Step of preparing a lipid antigen, centrifuging the prepared lipid antigen, suspending in a buffer by sonication, and mixing the lipid antigen suspension and latex,
And the lipid antigen comprises cardiolipin, lecithin, cholesterol, and the latex 10
Cardiolipin is composed of 0.01 to 1 μg, lecithin is 5 to 15 times the amount of cardiolipin, and cholesterol is 10 to 40 times the amount of cardiolipin.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing 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.

JP-A-6-148193 discloses an immunoassay for an anti-phospholipid antibody using a carrier treated with a steroid such as cholesterol and a phospholipid such as cardiolipin. Adsorb to microtiter plate, ELISA
A method for performing detection by the method is described. However, 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 But the results cannot be obtained quickly. Also, JP-A-5-212808 discloses that
Anti-phospholipid antibodies are detected by a method using magnetic particles, but a device for applying a magnetic force is required, and the versatility is poor.

[0006]

SUMMARY OF THE INVENTION The present invention has been made to solve the above problems, and has an object to be applicable to general biochemical automatic analysis, whereby rapid and simple measurement of a large amount of antibodies can be performed. An object of the present invention is to provide a method for producing a possible antiphospholipid antibody measurement reagent.

[0007]

The method for producing an anti-phospholipid antibody measuring reagent of the present invention is a method for producing an anti-phospholipid antibody measuring reagent in which an anti-phospholipid antibody is supported on a latex made of a synthetic polymer. A step of preparing a lipid antigen, a step of centrifuging the prepared lipid antigen, suspending the lipid antigen in a buffer by sonication, and a step of mixing the suspension of the lipid antigen with latex. And the lipid antigen is composed of cardiolipin, lecithin, and cholesterol, and 0.01 to 1 μg of cardiolipin and 10 to 5 μg of the latex are 5 to 5 parts of cardiolipin per 10 μg of the latex.
15 times as much cholesterol as cardiolipin 10-4
It is composed of 0 times the amount.

As the lipid of the above-mentioned lipid antigen, three types of cardiolipin, lecithin (phosphatidylcholine) and cholesterol are usually used, but it is not necessary to include all three types, and at least cardiolipin is included. Generally, a lipid antigen that reacts with an anti-syphilis antibody is called a VDRL antigen, and is composed of the above three types of lipids. Therefore, as the lipid antigens, the above three kinds of lipids are cardiolipin 0.01 to 1 μg and lecithin 5 to cardiolipin 5 to 10 μg of the latex.
15 times as much cholesterol as cardiolipin 10-4
Preferably, the amount is 0 times.

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 the amount is large, nonspecific aggregation occurs and precipitation occurs. The cholesterol is added as a nucleus for forming a VDCL antigen. If the amount of the cholesterol is small, it is difficult to form a lipid antigen mass, and if the amount is large, nonspecific aggregation occurs and precipitation occurs.

[0010] Cardiolipin, lecithin and cholesterol used in the present invention are preferably as pure as possible to suppress non-specific reactions. Cardiolipin and cholesterol are preferably 90%.
% Or more, and lecithin preferably contains 70% or more of phosphatidylcholine as a main component.

In order to further improve the accuracy, compounds having a cholesterol skeleton such as phospholipids such as phosphatidylinositol, phosphatidylserine, phosphatidic acid, phosphatidylethanolamine and phosphatidylglycerol, and bile acids. And the like may be used as a mixture with the lipid.

Next, an example of a method for preparing a VDRL antigen, which is a lipid antigen, will be described below. 0.4 ml of buffered saline is placed in a vial, and 0.5 ml of an ethanol solution containing cardiolipin, lecithin, and cholesterol is mixed dropwise. To this is added an additional 4.1 ml of buffered saline and stirred vigorously for 10 seconds. As a result, a VPRL antigen (lipid antigen) can be prepared.

The lipid antigen prepared as described above is
In this state, the particles are too large to be applied to an automatic analyzer. In addition, self-aggregation is so severe that it cannot be carried on latex in the next step. Therefore, in the present invention, the lipid antigen prepared above is then centrifuged and suspended in a buffer by sonication. That is, 15,000
rpm, 15 minutes (5-3 at 5000-20,000 rpm)
(0 min) to obtain a precipitate. Buffer is added to the precipitate. This is suspended by sonication. The ultrasonic crushing is preferably performed for 1 minute or more per 1 ml of the lipid antigen, and if the amount is too small, the lipid antigen is not sufficiently crushed. It is preferable that the VPRL antigen prepared as described above has a particle size of 0.1 μm or less. If the particle size is large, the latex cannot be adsorbed on the latex, and conversely, the latex aggregates with the DDRL antigen as a nucleus, or the absorbance becomes too large to be applied to an automatic analyzer. Examples of the buffer include phosphate buffer, phosphate buffered saline, and Tris-HCl buffer.

Next, the suspension of the lipid antigen and the latex are mixed. After sonication as described above, latex is quickly added to carry the VDRL antigen on the latex to obtain a reagent for measuring anti-phospholipid antibodies. To suppress non-specific aggregation in the buffer when suspending the above lipid antigen,
It is preferable to perform blocking by adding a biologically inactive protein. Examples of the inactive protein include albumin such as bovine serum albumin and ovalbumin, and animal serum such as human, cow, sheep, goat, rabbit and mouse. The blocking may be performed after the VTRL antigen is supported on the latex. In this case, the buffer containing the inactive protein is added after the support and stirred for several hours.

As the latex comprising the synthetic polymer, those generally used in immunological agglutination reactions and agglutination inhibition reactions can be used.
For example, polystyrene, styrene-sulfonic acid copolymer, styrene-methacrylic acid copolymer, acrylonitrile-butadiene-styrene copolymer, vinyl chloride-acrylate copolymer, vinyl acetate-acrylate copolymer and the like. In particular, polystyrene and styrene-sulfonic acid copolymers are preferable because they have excellent lipid adsorbability and can stably maintain biological activity for a long period of time. The average particle size of the latex varies depending on the measuring method and the measuring instrument, but is usually from 0.1 to 1.0 μm, preferably from 0.1 to 0.5 μm.

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. Further, various sensitizers may be used for improving the measurement sensitivity and promoting the antigen-antibody reaction. Examples of the sensitizer include, for example,
Examples include alkylated polysaccharides described in 173567, pullulan and polyvinylpyrrolidone described in JP-A-5-180838. 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 caused by the immune reaction 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 a particle size distribution or an 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 assay, at least two measurements are taken at different time points, a rate test for determining the degree of agglutination based on the increment of the measurement value between these time points (ie the rate of increase), and a certain time point ( One measurement 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 measurement value can be used. It is preferable to conduct a speed test by a turbidity method.

[0019]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described with reference to embodiments. (Example 1) 1) Preparation of lipid antigen-carrying latex Cardiolipin (manufactured by Sigma) was added to anhydrous ethanol.
A lipid ethanol solution was prepared by dissolving so that the concentration was 03% by weight, lecithin (manufactured by Nacalai Tesque) 0.3% by weight, and cholesterol (manufactured by Nacalai Tesque) 0.9% by weight. 0.5 ml of the above lipid ethanol solution was slowly dropped into a vial containing 0.4 ml of buffered saline while stirring. Further, 4.1 ml of buffered saline was added to make the total volume 5 ml, and the vial was stoppered and vigorously stirred.
This solution was transferred to a centrifuge tube, and centrifuged at 15000 rpm for 15 minutes to precipitate lipid antigen. 1% by weight
10 ml of a 100 mM phosphate buffer containing bovine serum albumin was added, and an ultrasonic crusher (manufactured by BRANSON) was added.
For 10 minutes. Immediately after ultrasonic crushing, polystyrene latex (average particle size 0.4
μm, solid content 10% (w / v), manufactured by Sekisui Chemical Co., Ltd.) 1
After adding 00 μl and stirring at room temperature for 30 minutes, the lipid antigen was carried on the latex.

2) Preparation of a sample diluent A bovine serum albumin was added to a 100 mM phosphate buffer to a concentration of 1% by weight, and pullulan (molecular weight: 200,000, manufactured by Hayashibara) was added to a concentration of 1% by weight and dissolved. Thus, a sample diluent was prepared. 3) Measurement of sensitivity Serum with an RPR value of 0, 1, 2, 4, or 8 was used as a sample. 60 μl of the lipid antigen-carrying latex, 180 μl of the sample diluent, and 10 μl of each sample were mixed, and the amount of change in absorbance was measured. The measurement was performed using a biochemical automatic analyzer (Hitachi 71
The measurement wavelength was 700 nm using an automatic analyzer (type 70). Table 1 shows the results.

Example 2 The same procedure as in Example 1 was repeated except that the concentration of lecithin was changed to 0.18% by weight. Table 1 shows the results.

Example 3 The preparation of the lipid ethanol solution of Example 1 was carried out in the same manner except that the lecithin concentration was changed to 0.4% by weight. Table 1 shows the results.

Example 4 The same procedure as in Example 1 was repeated except that the concentration of lecithin was changed to 0.4% by weight and the concentration of cholesterol was changed to 0.35% by weight. Table 1 shows the results.

Example 5 The same procedure as in Example 1 was repeated except that the concentration of lecithin was 0.4% by weight and the concentration of cholesterol was 1.1% by weight. Table 1 shows the results.

(Comparative Example 1) The procedure of Example 1 was repeated except that the concentration of lecithin was changed to 0.1% by weight. Table 1 shows the results.

Comparative Example 2 The preparation of the lipid ethanol solution of Example 1 was carried out in the same manner except that the concentration of lecithin was changed to 0.3% by weight and the concentration of cholesterol was changed to 0.2% by weight. Table 1 shows the results.

Comparative Example 3 Preparation of a lipid ethanol solution of Example 1 was carried out in the same manner except that the concentration of lecithin was 0.3% by weight and the concentration of cholesterol was 1.5% by weight. Table 1 shows the results.

(Comparative Example 4) The same procedure as in Example 1 was carried out except that ultrasonic crushing of the lipid antigen was not carried out. Table 1 shows the results.

[0029]

[Table 1]

[0030]

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 antiphospholipid antibody measurement reagent can be provided.

Claims (1)

[Claims] 1. A method for producing an anti-phospholipid antibody measurement reagent in which an anti-phospholipid antibody is supported on a latex made of a synthetic polymer, comprising the steps of: preparing a lipid antigen; and centrifuging the prepared lipid antigen. Suspending in a buffer by sonication, and mixing the lipid antigen suspension and latex,
And the lipid antigen is composed of cardiolipin, lecithin and cholesterol, and cardiolipin is 0.01 to 1 μg, lecithin is 5 to 15 times the amount of cardiolipin, and cholesterol is 10 to 40 times the cardiolipin for 10 μg of the latex. A method for producing a reagent for measuring an anti-phospholipid antibody, characterized in that the reagent is constituted by an amount.