JP3647210B2 - Method for producing antiphospholipid antibody measuring reagent and reagent - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an immunological diagnostic reagent using a lipid antigen. In particular, it relates to an immunological diagnostic reagent used for syphilis testing.
[0002]
[Prior art]
When Treponema Pallidum, a pathogen of syphilis, infects a living body, a Wasselman antibody reactive with phospholipid is produced together with an antibody 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.
[0003]
Currently, methods using lipid antigens include VDRL method, cord method, RPR (Rapid Plasma region) method, glass plate method, etc., but RPR method and glass plate method are mainly used for qualitative screening. .
Such a test method using a lipid antigen has a defect that it exhibits a positive reaction even in diseases other than syphilis, but the Wasselman antibody has an advantage that well reflects the infection state of syphilis. It is also applied to follow the progress of syphilis treatment using this.
[0004]
The RPR method is a method in which a lipid containing cardiolipin and lecithin adsorbed on kaolin or carbon powder is mixed with a specimen on a white plate, and the presence or absence of aggregation of kaolin or carbon powder is determined.
The glass plate method is a method for determining the presence or absence of aggregation of cholesterol crystals by mixing a lipid antigen solution containing cardiolipin, lecithin and cholesterol on a glass plate with a specimen.
[0005]
Since the above two methods are manual techniques, they are not suitable for testing a large amount of specimens. Further, in the case of the RPR method, the carbon powder aggregation is visually determined, and in the case of the glass plate method, since the formation of cholesterol crystals is observed under a microscope, the determination requires skill. . Therefore, it has a defect that the determination result may be different depending on the determiner.
[0006]
Japanese Laid-Open Patent Publication No. 4-204257 discloses a method for producing a measurement reagent in which lipid antigen micelles containing cardiolipin are prepared and adsorbed on a microtiter plate, which is an insoluble carrier, at room temperature. In this anti-phospholipid antibody detection method using a measuring reagent, the degree of aggregation is measured by absorbance, so the determination of yin and yang can be performed objectively. However, reagent dispensing or sample dispensing on a microtiter plate is not possible. There were problems such as manual operations and complicated operations.
[0007]
JP-A-5-312808 discloses an antigen-antibody reaction between a lipid antigen immobilized on a carrier and a target antibody contained in a specimen, and then the target antibody bound to the lipid antigen in the presence of a magnetic field. When an anti-phospholipid antibody is detected by performing an antigen-antibody reaction with a magnetic particle sensitized with an antibody against the target antibody to form a pattern on a carrier and examining syphilis, 1 to Although a detection method using a lipid antigen containing 50% by weight of cardiolipin has been disclosed, there is a drawback that a device for applying a magnetic force is necessary and the versatility is poor.
As described above, there has been no antiphospholipid antibody measurement reagent that can be applied to a general biochemical automatic analyzer.
[0008]
[Problems to be solved by the invention]
In view of the above, the present invention provides a method and a reagent for producing an antiphospholipid antibody measuring reagent that can measure a large amount of specimens quickly and easily and can be used in a highly objective detection method for syphilis. The purpose is to do.
[0009]
[Means for Solving the Problems]
The method for producing an antiphospholipid antibody measurement reagent according to claim 1 of the present invention is a method for producing an antiphospholipid antibody measurement reagent in which a lipid antigen is supported on an insoluble carrier made of latex , wherein the lipid antigen comprises cardiolipin, In the step of supporting the lipid antigen on the insoluble carrier comprising lecithin and cholesterol, the lipid antigen is 0.01 to 1.0 μg of cardiolipin relative to 10 μg of the insoluble carrier, lecithin is 3 to 15 times the amount of cardiolipin, and cholesterol is It is blended at a ratio of 1 to 5 times the amount of cardiolipin, the reaction temperature is 30 to 55 ° C., and the reaction time is 1 to 10 hours.
[0010]
In the method for producing an antiphospholipid antibody measuring reagent according to claim 2 of the present invention, the insoluble carrier is latex made of polystyrene or a styrene-styrenesulfonic acid copolymer. It is a manufacturing method.
[0011]
The antiphospholipid antibody measurement reagent according to claim 3 of the present invention is obtained by the method for producing an antiphospholipid antibody measurement reagent according to claim 1 or 2.
The production method of the antiphospholipid antibody measurement reagent of the present invention is described in detail below.
[0012]
In the present invention, a lipid antigen is supported on an insoluble carrier.
The lipid antigen is composed of cardiolipin, lecithin and cholesterol. The amount of cardiolipin is 0.01 to 1.0 μg, preferably 0.1 to 0.8 μg, and lecithin is 3 to 15 times the amount of cardiolipin, preferably 10 μg of insoluble carrier. 5 to 12 times the amount, cholesterol is 1 to 5 times the amount of cardiolipin, preferably 1 to 4 times the amount. When the amount of cardiolipin is less than 0.01 μg, sufficient sensitivity cannot be obtained, and when it exceeds 1.0 μg, non-specific aggregation occurs and precipitation occurs, so the amount is limited to the above range. If the amount of lecithin is less than 3 times the amount of cardiolipin, lipid antigens having the antigenicity necessary for measurement will not be formed, and if it exceeds 15 times the amount of cardiolipin, non-specific aggregation occurs and precipitation occurs. It is limited to. Cholesterol is added to improve the sensitivity of the reagent measurement system, non aggregation occurs precipitation exceeds 5 times the amount of mosquito Rujioripin is limited to the range for occurs.
[0013]
In the present invention, the reaction temperature in the step of supporting a lipid antigen on an insoluble carrier is 30 to 55 ° C. If the temperature is lower than 30 ° C., a measurement reagent having sufficient sensitivity cannot be prepared. If the temperature exceeds 55 ° C., the stability of latex cannot be maintained and a measurement reagent cannot be prepared. Limited to range. More preferably, it is 35 degreeC-50 degreeC.
[0014]
In the present invention, the step of supporting the lipid antigen on the insoluble carrier includes the step of mixing the lipid antigen with the insoluble carrier and performing physicochemical adsorption, and the step of stabilizing the lipid antigen-carrying insoluble carrier by blocking with a blocking agent. Consists of.
In the present invention, it is necessary that at least the step of performing the physicochemical adsorption among the two steps is the reaction temperature.
[0015]
In the present invention, the reaction time of the step of supporting the lipid antigen on the insoluble carrier is the step of performing the physicochemical adsorption and the step of stabilizing by the blocking (hereinafter, these two steps are collectively referred to as “sensitization step”). ) And the two steps are limited to 1 to 10 hours. Specifically, it is preferable that the physicochemical adsorption step is 0.5 hour or longer and the stabilization step is 0.5 hour or longer. In particular, it is desirable to make the stabilization process longer. If the sensitization process is less than 1 hour, it is not sufficient for physicochemical adsorption or stabilization, and if it exceeds 10 hours, it becomes difficult for the insoluble carrier particles to maintain their stability (particularly, the reaction temperature is Therefore, it is limited to the above time.
[0016]
The insoluble carrier is limited to latex in which a synthetic polymer powder is uniformly suspended.
The synthetic polymer is not particularly limited. For example, polystyrene, styrene-styrene sulfonic acid copolymer, styrene-methacrylic acid copolymer, acrylonitrile-butadiene-styrene copolymer, vinyl chloride-acrylic acid ester copolymer. And vinyl acetate-acrylic acid ester copolymer. Of these, polystyrene and styrene-styrene sulfonic acid copolymer are preferable.
In the present invention, the latex scan the particle size is preferably from 0.1 to 1.0 [mu] m, more preferably 0.1 to 0.5 [mu] m.
[0017]
The blocking agent is not particularly limited, and examples thereof include albumin such as bovine serum albumin and ovalbumin; and serum of animals such as human, cow, sheep, goat, rabbit, and mouse.
[0018]
The reagent prepared by the present invention can also be used by diluting with an appropriate specimen diluent.
The sample dilution solution is not particularly limited as long as it is a buffer solution having a pH of 5.0 to 9.0. For example, phosphate buffer solution, glycine buffer solution, veronal buffer solution, borate buffer solution, citrate buffer solution, and the like. Is mentioned.
[0019]
Various sensitizers can be added to the specimen diluent for improving the measurement sensitivity and promoting the antigen-antibody reaction.
Examples of the sensitizer include alkylated polysaccharides such as methylcellulose and ethylcellulose described in JP-A-2-173567; pullulan and polyvinylpyrrolidone described in JP-A-5-180838. It is done.
[0020]
Bovine serum albumin (BSA), egg albumin, etc. can also be added to the sample dilution liquid in order to suppress non-specific reactions caused by other substances present in the sample.
[0021]
The reagent for measuring antiphospholipid antibody of the present invention is obtained by the method for producing the reagent for measuring antiphospholipid antibody of the present invention.
The antiphospholipid antibody measurement reagent of the present invention can be applied to a general biochemical automatic analyzer that optically measures the degree of aggregation.
[0022]
【Example】
Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.
[0023]
Example 1
(1) Preparation of lipid antigen solution 2 ml of ethanol solution of cardiolipin (5 mg / ml, manufactured by Sigma), 10 ml of ethanol solution (10 mg / ml) of purified lecithin (manufactured by Nacalai Tesque) and ethanol of cholesterol (manufactured by Nacalai Tesque) 3 ml of the solution (10 mg / ml) was mixed to obtain a lipid antigen solution.
[0024]
(2) Preparation of lipid antigen-sensitized latex reagent 100 μl of polystyrene latex (average particle size 0.4 μm, 10% (w / v), manufactured by Sekisui Chemical Co., Ltd.) was warmed in advance at 37 ° C. with stirring. To this was added 250 μl of the lipid antigen solution prepared previously, and the mixture was stirred at 37 ° C. for 2 hours. Next, 2 ml of phosphate buffered saline (hereinafter referred to as “PBS”) containing 1% bovine serum albumin (hereinafter referred to as “BSA”) was added, and the mixture was further stirred for 1 hour at 37 ° C. Becomes). Centrifugation was performed at 15000 rpm at 4 ° C. for 15 minutes, the supernatant was removed, and the precipitated latex was again suspended in 3 ml of PBS containing 1% BSA. This operation was repeated three times to wash the latex, and finally suspended in 4 ml of PBS containing 1% BSA, 10 mM EDTA · 4Na, and 500 mM choline chloride. In this way, a lipid antigen-sensitized latex reagent was obtained.
[0025]
(3) Sensitivity measurement Using the lipid antigen-sensitized latex reagent obtained in (2) above and a sample diluent (attached to Mediace TPLA (manufactured by Sekisui Chemical Co., Ltd.)), Hitachi 7170 Biochemistry Measurements were made using an automated analyzer. The measurement wavelength was 700 nm, and for each sample measurement, 30 μl of the lipid antigen-sensitized latex reagent, 210 μl of the sample diluent, and 20 μl of the sample were used. For the measurement, samples having RPR method measurement values (RPR values) of 8, 4, 2, 1, 0.5, and 0 were used, and the amount of change in absorbance of each sample was measured. FIG. 1, FIG. 2, FIG. The results are shown in.
[0026]
Example 2
A reagent was prepared in the same manner as in Example 1 except that the sensitization temperature in Example 1 (2) was 50 ° C., and the sensitivity was measured. The results are shown in FIG.
[0027]
Example 3
In Example 1 (1), 2 ml of an ethanol solution of cardiolipin (5 mg / ml), 15 ml of an ethanol solution of purified lecithin (10 mg / ml) and 3 ml of an ethanol solution of cholesterol (10 mg / ml) were mixed, and the lipid antigen solution and Except for the above, a reagent was prepared in the same manner as in Example 1, and the sensitivity was measured. The results are shown in FIG.
[0028]
Example 4
In Example 1 (1), 2 ml of an ethanol solution of cardiolipin (5 mg / ml), 5 ml of an ethanol solution of purified lecithin (10 mg / ml) and 3 ml of an ethanol solution of cholesterol (10 mg / ml) were mixed together with a lipid antigen solution. Except for this, a reagent was prepared in the same manner as in Example 1, and the sensitivity was measured. The results are shown in FIG.
[0029]
Example 5
Example 1 (2) In preparation of lipid antigen-sensitized latex reagent of Example 1, except that 2 ml of PBS containing 1% BSA was added and further stirred at 37 ° C. for 3 hours (the sensitization process was 5 hours in total). A reagent was prepared in the same manner as in 1, and the sensitivity was measured. The results are shown in FIG.
[0030]
Example 6
Example 1 (2) Preparation of latex antigen-sensitized latex reagent of Example 1 except that 2 ml of PBS containing 1% BSA was added and further stirred at 37 ° C. for 6 hours (the sensitization process was 8 hours in total). A reagent was prepared in the same manner as in 1, and the sensitivity was measured. The results are shown in FIG.
[0031]
Example 7
In the preparation of the latex antigen-sensitized latex reagent of Example 1 (2), 250 μl of the lipid antigen solution was added and stirred as it was at 37 ° C. for 0.5 hour. Next, 2 ml of PBS containing 1% BSA was added, and a reagent was prepared and the sensitivity was measured in the same manner as in Example 1 except that the mixture was further stirred at 37 ° C. for 0.5 hour (the sensitization step was 1 hour in total). . The results are shown in FIG.
[0032]
Comparative Example 1
A reagent was prepared in the same manner as in Example 1 except that the sensitization temperature in Example 1 (2) was 25 ° C., and the sensitivity was measured. The results are shown in FIG.
[0033]
Comparative Example 2
In Example 1 (1), 2 ml of an ethanol solution of cardiolipin (5 mg / ml), 1 ml of an ethanol solution of purified lecithin (10 mg / ml) and 3 ml of an ethanol solution of cholesterol (10 mg / ml) were mixed together with a lipid antigen solution. Except for the above, a reagent was prepared in the same manner as in Example 1, and the sensitivity was measured. The results are shown in FIG.
[0034]
Comparative Example 3
In Example 1 (1), 2 ml of an ethanol solution of cardiolipin (5 mg / ml), 20 ml of an ethanol solution of purified lecithin (10 mg / ml) and 3 ml of an ethanol solution of cholesterol (10 mg / ml) were mixed together with a lipid antigen solution. Except for this, a reagent was prepared in the same manner as in Example 1, and the sensitivity was measured. The results are shown in FIG.
[0035]
Comparative Example 4
In the preparation of the latex antigen-sensitized latex reagent of Example 1 (2), 250 μl of the lipid antigen solution was added and stirred as it was at 37 ° C. for 0.25 hour. Next, 2 ml of PBS containing 1% BSA was added, and a reagent was prepared in the same manner as in Example 1 except that the mixture was further stirred at 37 ° C. for 0.25 hours (the sensitization process was 0.5 hours in total). It was measured. The results are shown in FIG.
[0036]
【The invention's effect】
An antiphospholipid antibody measurement reagent that can measure a large amount of specimens quickly and easily by the method for producing an antiphospholipid antibody measurement reagent of the present invention and that can be used in a highly objective detection method for syphilis Can be obtained.
[Brief description of the drawings]
FIG. 1 is a graph showing the sensitivity of a lipid antigen-sensitized latex reagent in an example. The horizontal axis represents the RPR value, and the vertical axis represents the change in absorbance (Δabs × 10000). White squares are measured values in Example 1, black triangles are measured values in Example 2, and white circles are measured values in Comparative Example 1.
FIG. 2 is a graph showing the sensitivity of a lipid antigen-sensitized latex reagent in Examples. The horizontal axis represents the RPR value, and the vertical axis represents the change in absorbance (Δabs × 10000). □ for whitening is the measurement value of Example 1, □ for blackening is the measurement value of Example 3, x is the measurement value of Example 4, and ○ for whitening is a comparative example 2 is a measured value of Comparative Example 3.
FIG. 3 is a graph showing the sensitivity of a lipid antigen-sensitized latex reagent in Examples. The horizontal axis represents the RPR value, and the vertical axis represents the change in absorbance (Δabs × 10000). □ for whitening is the measurement value for Example 1, □ for blackening is the measurement value for Example 5, △ for whitening is the measurement value for Example 6, and ○ for whitening These are measured values of Example 7, and Δ of blackening is a measured value of Comparative Example 4.

Claims (3)

A method for producing an antiphospholipid antibody measurement reagent in which a lipid antigen is supported on an insoluble carrier made of latex ,
The lipid antigen consists of cardiolipin, lecithin and cholesterol,
In the step of supporting the lipid antigen on the insoluble carrier, cardiolipin is 0.01 to 1.0 μg, lecithin is 3 to 15 times the amount of cardiolipin, and cholesterol is 1 to 5 times the cardiolipin. A method for producing an antiphospholipid antibody measuring reagent, characterized in that the reaction temperature is 30 to 55 ° C. and the reaction time is 1 to 10 hours. The method for producing an antiphospholipid antibody measuring reagent according to claim 1, wherein the insoluble carrier is a latex made of polystyrene or a styrene-styrenesulfonic acid copolymer. An antiphospholipid antibody measurement reagent obtained by the method for producing an antiphospholipid antibody measurement reagent according to claim 1 or 2.

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