JP2004325413A - Antiphospholipid antibody measuring reagent, and antiphospholipid antibody measuring method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To make it possible to apply to various biochemical auto-analyzers, to quickly and easily measure a lot of specimens, and to hardly generate a determination failure caused by nonspecific agglutination. <P>SOLUTION: Agglutination by an antigen-antibody reaction between an antiphospholipid antibody and an antigen is measured based on a change of absorbance, using this antiphospholipid antibody measuring reagent. The antiphospholipid antibody measuring reagent comprises an antigen reagent containing an insoluble carrier adsorbed with a lipid antigen comprising cardiolipin, lecithin and cholesterol, and containing a water-soluble polymer for blocking the lipid antigen adsorbed onto the insoluble carrier, and a buffer solution for dilution containing the water-soluble polymer. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【００４８】
【発明の効果】
本発明に係る抗リン脂質抗体測定試薬は、カルジオリピン、レシチン及びコレステロールからなる脂質抗原が吸着された不溶性担体と、該不溶性担体に吸着された脂質抗原をブロッキングするための水溶性高分子とを含む抗原試薬と、水溶性高分子を含む希釈用緩衝液とからなり、ブロッキングに上記水溶性高分子が用いられているため、抗リン脂質抗体の測定に際しての非特異的凝集を抑制することができる。従って、より確実に梅毒感染状態を検出することができる。また、本発明に係る測定試薬は、上記抗原試薬と希釈用緩衝液とからなるものであり、様々な生化学自動分析機に適用することができる。従って、迅速かつ簡便に大量の検体を測定することができ、しかも上記のように非特異的凝集による判定不良が生じ難いため、客観性に優れた梅毒判定結果を得ることができる。
【００４９】
本発明に係る抗リン脂質抗体測定方法では、本発明に係る抗リン脂質抗体測定試薬と検体とを混合し、抗原抗体反応による凝集を生じさせ、該凝集の程度を特定波長の吸光度を測定することにより行われる。従って、様々な生化学自動分析機において測定を行うことができ、迅速かつ簡便に大量の検体を測定することができる。また、本発明に係る抗リン脂質抗体測定試薬を用いるものであるため、非特異的凝集が生じ難く、従って、梅毒の感染状態を高精度に検出することができる。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an antiphospholipid antibody measuring reagent and a measuring method for immunologically measuring an antiphospholipid antibody contained in a sample.
[0002]
[Prior art]
When Treponema pallidum, which is a pathogen of syphilis, infects a living body, a Wasselmann antibody reactive with phospholipids 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]
At present, methods using lipid antigens include the VDRL method, the cord method, the RPR method, and the glass plate method. For the qualitative screening, the RPR method and the glass plate method are mainly used.
[0004]
Test methods using these lipid antigens have the disadvantage that they exhibit a positive reaction even for diseases other than syphilis, but they have the advantage that the Wasselman antibody satisfactorily reflects the infection status of syphilis. Therefore, the RPR method and the glass plate method are used to monitor the progress of treatment for syphilis.
[0005]
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 or not kaolin or carbon powder has aggregated.
[0006]
The glass plate method is a method in which a lipid antigen solution containing cardiolipin, lecithin and cholesterin is mixed with a specimen on a glass plate, and the presence or absence of aggregation of cholesterol crystals is determined.
[0007]
Since the above two methods are both manual methods, they are not suitable for testing a large number of samples. In the RPR method, the determination is made by visually observing the aggregation of the carbon powder, and in the glass plate method, by observing the formation of cholesterol crystals under a microscope. Therefore, in any of the methods, there is a problem that the judgment requires skill and the judgment result may be different depending on the judge.
[0008]
In order to solve such a problem, Patent Literature 1 listed below discloses a syphilis antiphospholipid antibody measuring reagent applicable to a biochemical automatic analyzer. Here, an inactive protein is used as a blocking agent.
[0009]
On the other hand, Patent Literature 2 below discloses a syphilis antiphospholipid antibody measurement reagent that can be measured by a counting immunoassay method. This measurement reagent is obtained by mixing an antigen solution containing 2 to 15 times the amount of phosphadylcholine, 0 to 6 times the amount of cholesterol, and 30 to 95 mg of these lipid antigens per 1 g of the carrier particles with respect to the weight of the cardiolipin. Thereby, the lipid antigen is sensitized on the carrier particles, and the carrier particles are further blocked with polyvinyl alcohol.
[0010]
[Patent Document 1]
Patent No. 3290520 [Patent Document 2]
JP 2001-242171 A
[Problems to be solved by the invention]
In Patent Document 1, in bovine serum albumin and the like shown as a typical example of a protein used as a blocking agent, a non-specific reaction occurs due to the influence of other proteins and the like contaminating during the extraction process, and a syphilis-negative sample is regarded as positive. There was a drawback that false positives occurred.
[0012]
Further, the measurement reagent described in Patent Document 2 is used for a device employing the counting immunoassay method, but cannot be measured when used for other devices.
[0013]
An object of the present invention, in view of the above-mentioned state of the art, is that it can be used for various biochemical automatic analyzers, makes it possible to measure a large amount of samples quickly and easily, and has a highly objective detection result. It is an object of the present invention to provide an anti-phospholipid antibody measuring reagent and an anti-phospholipid antibody measuring method which can obtain the following.
[0014]
[Means for Solving the Problems]
The anti-phospholipid antibody measurement reagent according to the present invention is an anti-phospholipid antibody measurement reagent for measuring aggregation due to an antigen-antibody reaction between an anti-phospholipid antibody and an antigen by a change in absorbance, and comprises cardiolipin, lecithin and cholesterol. An antigen reagent containing an insoluble carrier to which a lipid antigen is adsorbed, a water-soluble polymer for blocking the lipid antigen adsorbed to the insoluble carrier, and a dilution buffer containing a water-soluble polymer. It is characterized by.
[0015]
In a specific aspect of the antiphospholipid antibody measurement reagent according to the present invention, the water-soluble polymer for blocking a lipid antigen is selected from the group consisting of polyvinylpyrrolidone, polyethylene glycol, water-soluble polysaccharides, and derivatives thereof. It is at least one of the following.
[0016]
In another specific aspect of the antiphospholipid antibody measurement reagent according to the present invention, the water-soluble polysaccharide is at least one selected from the group consisting of pullulan, dextran, dextran sulfate, and sodium alginate.
[0017]
The antiphospholipid antibody measurement method according to the present invention is a measurement method using an antiphospholipid antibody measurement reagent configured according to the present invention, wherein the antigen reagent, the dilution buffer, and a sample are mixed. A step of causing aggregation by an antigen-antibody reaction, and a step of measuring the degree of the aggregation based on the absorbance at a specific wavelength, thereby detecting an antiphospholipid antibody in the sample.
[0018]
Hereinafter, details of the present invention will be described.
As described above, the anti-phospholipid antibody measurement method using the anti-phospholipid antibody measurement reagent according to the present invention is such that the antigen reagent, the dilution buffer, and the sample are mixed, and aggregation by the antigen-antibody reaction is performed. This is performed by measuring the absorbance at the wavelength of the degree of aggregation and detecting the antiphospholipid antibody based on the absorbance.
[0019]
The cardiolipin is not particularly limited, but cardiolipin purified from bovine heart is preferably used. The main component of cardiolipin is phosphatidylglycerol, and preferably has high phosphatidylglycerol purity, more preferably 90% or more.
[0020]
The lecithin used is purified or chemically synthesized from egg yolk. The component of lecithin is phosphatidylcholine, preferably having high phosphatidylcholine purity, and more preferably having a purity of 70% or more.
[0021]
The cholesterol used may be purified from animal fat or chemically synthesized. Cholesterol also preferably has a high purity, and preferably has a purity of 98% or more.
[0022]
After adsorbing the lipid antigen consisting of cardiolipin, lecithin and cholesterol to an insoluble carrier, the water-soluble polymer used for blocking is not particularly limited, but may be a water-soluble polymer represented by polyvinylpyrrolidone. Polyvinyl derivatives; water-soluble polysaccharides represented by pullulan, dextran, dextran sulfate and sodium alginate and derivatives thereof; copolymers of alkyl glycols represented by polyethylene glycol, and derivatives thereof can be used.
[0023]
The molecular weight of the water-soluble polymer varies depending on the water-soluble polymer, but is preferably in the range of about 1,000 to 70,000. More specifically, for example, in the case of polyvinylpyrrolidone, the molecular weight is preferably in the range of 5,000 to 40,000, and in the case of pullulan, the molecular weight is preferably in the range of 10,000 to 70,000. A range of 30,000 is preferred.
[0024]
If the molecular weight of the water-soluble polymer is too low, a sufficient blocking effect may not be obtained, and if it is too large, the solubility may be poor.
Examples of the insoluble carrier used in the present invention include organic polymer powders, microorganisms, blood cells, cell membrane fragments, and plastic microtiter plates.
[0025]
As the organic polymer powder, for example, insoluble agarose, cellulose, natural polymer powder such as insoluble dextran, polystyrene, styrene-styrene sulfonic acid copolymer, acrylonitrile-butadiene-styrene copolymer, vinyl chloride-acrylate Examples include a synthetic polymer powder such as a copolymer and a vinyl acetate-acrylate copolymer, and a synthetic polymer latex is particularly desirable.
[0026]
When the above latex is used, the particle size is preferably from 0.1 to 1.0 μm, more preferably from 0.1 to 0.5 μm.
[0027]
As the buffer for dilution, any buffer can be used as long as the buffer has a pH of 5.0 to 9.0. Examples of the buffer that can be used include a phosphate buffer, a glycine buffer, a veronal buffer, a borate buffer, and a citrate buffer.
[0028]
The above-mentioned dilution buffer contains various water-soluble polymers for the purpose of improving the measurement sensitivity and promoting the antigen-antibody reaction. Examples of such a water-soluble polymer include alkylated polysaccharides described in JP-A-2-173567 and pullulan or polyvinylpyrrolidone as described in JP-A-5-180838. it can. The water-soluble polymer contained in the dilution buffer may be the same as or different from the above-described blocking water-soluble polymer.
[0029]
The concentration of the water-soluble polymer in the buffer for dilution is preferably in the range of 0.01 to 10 (w / v)%. If it is less than 0.01 (w / v)%, the measurement sensitivity may not be sufficiently high.
[0030]
Further, proteins such as bovine serum albumin and ovalbumin may be added to the dilution buffer for the purpose of suppressing non-specific reactions caused by other substances present in the specimen.
[0031]
In obtaining the antiphospholipid antibody measurement reagent according to the present invention, first, the lipid antigen is adsorbed on an insoluble carrier, but the adsorption method is not particularly limited. For example, by mixing the insoluble carrier with a buffer containing a lipid antigen, the lipid antigen is adsorbed on the insoluble carrier. Thereafter, a water-soluble polymer for blocking the lipid antigen adsorbed on the insoluble carrier is mixed with the insoluble carrier having the lipid antigen adsorbed thereon to obtain an antigen reagent.
[0032]
The buffer for dilution containing a water-soluble polymer can be obtained by mixing and dissolving the water-soluble polymer in the buffer.
The anti-phospholipid antibody measurement reagent according to the present invention is a two-component measurement reagent having the antigen reagent and a dilution buffer.
[0033]
In the anti-phospholipid antibody measurement method according to the present invention, the antigen reagent, the dilution buffer, and the specimen are mixed, whereby aggregation due to the antigen-antibody reaction occurs. The degree of aggregation is then measured optically. That is, the absorbance at a specific wavelength is measured, and the degree of aggregation is detected based on the change in the absorbance, whereby the anti-phospholipid antibody in the sample is detected. In measuring the change in absorbance in this case, various automatic biochemical analyzers can be used.
[0034]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be clarified by giving specific examples and comparative examples of the present invention. Note that the present invention is not limited to the following examples.
[0035]
(Example 1)
1) Preparation of antigen reagent 3 mL of a 5 mg / mL ethanol solution of cardiolipin (Sigma), 10 mL of a 15 mg / mL ethanol solution of phosphatidylcholine (Sigma), and cholesterol (Nacalai Tesque) A 15 mg / mL ethanol solution was mixed with 4.5 mL to obtain a lipid antigen solution.
[0036]
1 mL of a polystyrene latex suspension [(average particle size: 0.4 μm, 10 w / v%) concentration, manufactured by Sekisui Chemical Co., Ltd.] was heated at 37 ° C. with gentle stirring in advance, and then 3 mL of the above lipid antigen solution Was added and stirred at 37 ° C. for 1 hour. Phosphate-buffered saline (36 mL phosphate buffer, pH 6.5, 0.74 wt% NaCl, hereinafter abbreviated as PBS buffer) containing 2% by weight polyvinylpyrrolidone (molecular weight 10,000, BASF, product number: K-15) ) 5 mL was added, and the mixture was further stirred at 37 ° C for 1 hour. Thereafter, the solution was centrifuged at a temperature of 4 ° C. and 15000 rpm for 30 minutes to remove the supernatant. After removing the supernatant, the precipitated latex was suspended in 3 mL of PBS buffer containing 1% bovine serum albumin. This operation was repeated three times to wash the latex, and finally suspended in 20 mL of PBS buffer containing 10 mL of EDTA · 2Na (manufactured by Dojindo) and 500 mM choline chloride (manufactured by Wako Pure Chemical Industries, Ltd.) to remove the antigen reagent. Obtained. This antigen reagent is for measuring syphilis antiphospholipid antibodies.
[0037]
2) Preparation of buffer for dilution A 100 mM phosphate buffer (pH 7.4) containing 1 w / v% of pullulan (manufactured by Hayashibara) and 1 w / v% of bovine serum albumin was prepared and used as a buffer for dilution. .
[0038]
3) Measurement Using the antigen reagent obtained as described above and the dilution buffer, measurement was carried out using an automatic biochemical analyzer 7170 manufactured by Hitachi, Ltd. The specific wavelength was 700 nm, and for the measurement of one sample, an antigen reagent (60 μL) and a dilution buffer (180 μL) were used for a sample of 20 μL. More specifically, 180 μL of the dilution buffer was added to 20 μL of the sample, and the mixture was stirred, kept at 37 ° C., added with 60 μL of the antigen reagent, stirred, and then absorbance was measured 1 minute and 5 minutes later. Was measured, and the change in absorbance during this time was determined. Sample No. shown in Table 1 below. About 1-10, the said measurement was performed. The results are shown in Table 1 below.
[0039]
(Example 2)
In the measurement of the antigen reagent, the procedure of Example 1 was repeated except that 1% by weight of polyvinylpyrrolidone (molecular weight: 30,000, manufactured by BASF, product number: K-30) was used instead of 2% by weight of polyvinylpyrrolidone K-15. A reagent was prepared and evaluated in the same manner.
[0040]
(Example 3)
Example 1 was repeated except that 2% by weight polyvinylpyrrolidone used in the preparation of the antigen reagent of Example 1 was replaced with 3% by weight pullulan (manufactured by Hayashibara, product number: PI-20). Reagents were made and evaluated.
[0041]
(Example 4)
A reagent was prepared in the same manner as in Example 1 except that 3% by weight of polyethylene glycol (manufactured by Wako Pure Chemical Industries, Ltd., molecular weight: 20,000) was used instead of 2% by weight of polyvinylpyrrolidone in preparing the antigen reagent of Example 1. Was prepared and evaluated.
[0042]
(Comparative Example 1)
A reagent was prepared and evaluated in the same manner as in Example 1 except that 1% by weight of bovine serum albumin was used instead of 2% by weight of polyvinylpyrrolidone in the preparation of the antigen reagent of Example 1.
[0043]
(Sample Nos. 1 to 10)
The sample No. Samples 1 to 10 were determined to be negative for syphilis in the RPR card test (manufactured by Sanko Junyaku Co., Ltd.), which is a method for use, but the specimens in Comparative Example 1 in which the amount of change in absorbance due to nonspecific aggregation increased.
[0044]
Sample No. 1 to 10 are determined to be negative in the RPR card test as described above, and thus the absorbance change amounts of the results of Comparative Example 1 and Examples 1 to 4 shown in Table 1 are non-specific. It shows the degree of aggregation.
[0045]
As is clear from Table 1, in Comparative Example 1, the degree of aggregation was high in all the samples. Therefore, as described above, although the sample was determined to be negative for syphilis in the RPR card test, When the reagent of (1) was used, the amount of change in absorbance increased significantly, and it was sometimes judged to be positive.
[0046]
In contrast, in Examples 1 to 4, the amount of change in absorbance was smaller than that of Comparative Example 1, that is, it was found that non-specific aggregation hardly occurred.
In Table 1, the value of Δabsorbance × 10 ⁴ is shown as the amount of change in absorbance.
[0047]
[Table 1]

[0048]
【The invention's effect】
The anti-phospholipid antibody measurement reagent according to the present invention contains an insoluble carrier to which a lipid antigen consisting of cardiolipin, lecithin and cholesterol is adsorbed, and a water-soluble polymer for blocking the lipid antigen adsorbed to the insoluble carrier. It consists of an antigen reagent and a buffer for dilution containing a water-soluble polymer, and the above-mentioned water-soluble polymer is used for blocking, so that non-specific aggregation in the measurement of anti-phospholipid antibodies can be suppressed. . Therefore, the syphilis infection state can be detected more reliably. The measuring reagent according to the present invention comprises the above antigen reagent and a buffer for dilution, and can be applied to various automatic biochemical analyzers. Therefore, a large amount of sample can be measured quickly and easily, and the determination failure due to non-specific aggregation is unlikely to occur as described above, so that a syphilis determination result excellent in objectivity can be obtained.
[0049]
In the antiphospholipid antibody measurement method according to the present invention, the antiphospholipid antibody measurement reagent according to the present invention is mixed with a sample, aggregation is caused by an antigen-antibody reaction, and the degree of the aggregation is measured by measuring absorbance at a specific wavelength. It is done by doing. Therefore, measurement can be performed by various biochemical automatic analyzers, and a large amount of sample can be measured quickly and easily. Further, since the anti-phospholipid antibody measurement reagent according to the present invention is used, non-specific aggregation hardly occurs, and therefore, the infection state of syphilis can be detected with high accuracy.

Claims (4)

An anti-phospholipid antibody measurement reagent for measuring aggregation due to an antigen-antibody reaction between an anti-phospholipid antibody and an antigen by a change in absorbance,
Cardiolipin, insoluble carrier adsorbed lipid antigen consisting of lecithin and cholesterol, and an antigen reagent containing a water-soluble polymer for blocking the lipid antigen adsorbed on the insoluble carrier,
A reagent for measuring anti-phospholipid antibodies, comprising a dilution buffer containing a water-soluble polymer. The antiphospholipid according to claim 1, wherein the water-soluble polymer for blocking a lipid antigen is at least one selected from the group consisting of polyvinylpyrrolidone, polyethylene glycol, a water-soluble polysaccharide, and a derivative thereof. Antibody measurement reagent. The reagent according to claim 2, wherein the water-soluble polysaccharide is at least one selected from the group consisting of pullulan, dextran, dextran sulfate, and sodium alginate. A method for measuring an antiphospholipid antibody using the antiphospholipid antibody measurement reagent according to claim 1,
Mixing the antigen reagent, the dilution buffer, and the sample to cause aggregation by an antigen-antibody reaction,
Measuring the degree of the aggregation based on the absorbance at a specific wavelength, thereby detecting the anti-phospholipid antibody in the sample.