JPH09101308A - Treponema pallidum antibody measuring method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a treponema pollidum antibody measuring method which enables accurate measurement with high versatility and without causing non- specific reaction. SOLUTION: In this treponema pallidum antibody measuring method of detecting the degree of agglutination of the insoluble carrier as caused by an antigen/antibody reaction with a treponema pallidum antibody using an insoluble carrier supporting a treponema pallidum antigen to measure the treponema pallidum, 0.001-0.5mol. concentration of an aminoethane sulfonic acid derivative and/or aminopropane sulfonic acid derivative is added to a reaction system for agglutinating the insoluble carrier.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an assay method for Treponema pallidum antibody based on the antigen-antibody reaction between the Treponema pallidum antigen carried on an insoluble carrier and the antibody against Treponema pallidum in a sample.

[0002]

2. Description of the Related Art The syphilis treponemal antibody measuring method for measuring syphilis treponemal antibody by detecting the degree of aggregation of the insoluble carrier generated by the antigen-antibody reaction with the syphilis treponemal antibody is carried by the syphilis treponemal antibody measuring method. For example, it is widely used as a method for quickly and accurately inspecting and diagnosing the presence or absence of syphilis infection using blood or the like as a sample. Such syphilis treponemal antibody measurement method,
A red blood cell agglutination reaction method, a latex agglutination method and the like are known depending on the form in which the agglutination reaction is performed.

In the measurement of these agglutination reactions, polyethylene glycol (JP-A-58-47256) and polyglycosylethyl methacrylate (JP-A-4-122) are used for the purpose of improving the measurement sensitivity or promoting the antigen-antibody reaction.
858), polyvinylpyrrolidone (JP-A-5-1)
A method of adding a water-soluble polymer compound such as JP-A No. 80383) to a reaction system is known.

However, addition of these polymer compounds causes non-specific aggregation, which is aggregation due to the reaction of coexisting substances other than the target substance, and may give erroneous measurement results. Therefore, it is known to add a polymer compound such as bovine serum albumin or equine serum albumin (Japanese Patent Application Laid-Open No. Sho 5).
No. 8-144748). However, it is impossible to completely prevent the non-specific reaction even by adding such albumin.

As a method of adding a low molecular weight compound as an aggregation accelerator, a method of simultaneously adding N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid and ethylenediaminetetraacetic acid salt is known. (Japanese Patent Publication No. 6-84972). However, since ethylenediaminetetraacetate inhibits the antigen-antibody reaction of Treponema pallidum in this method, it is not a suitable method for measuring the antibody against Treponema pallidum.

[0006]

SUMMARY OF THE INVENTION In view of the above, an object of the present invention is to provide a syphilis treponemal antibody assay method that is highly versatile, does not cause non-specific reactions, and enables accurate assay.

[0007]

Means for Solving the Problems The present invention uses an insoluble carrier carrying a Treponema pallidum antigen to detect the degree of agglutination of the insoluble carrier caused by an antigen-antibody reaction with a Treponema pallidum antibody. A syphilis treponemal antibody measuring method for measuring syphilis treponemal antibody, wherein an aminoethanesulfonic acid derivative and / or aminopropanesulfonic acid derivative has a molar concentration of 0.001 to 0.5 in a reaction system for aggregating the insoluble carrier. It is characterized by being added. Hereinafter, the present invention will be described in detail.

In the method for measuring Treponema pallidum antibody of the present invention, an antigen-antibody reaction between Treponema pallidum antigen carried on an insoluble carrier and Treponema pallidum antibody present in a sample is carried out in a reaction system to carry out agglutination reaction. To complete. Then, by measuring the degree of this agglutination reaction, the amount of Treponema pallidum antibody present in the sample is measured.

The above reaction system is usually carried out by mixing the above insoluble carrier with a sample in a buffer solution. In the present invention, in the above reaction, an aminoethanesulfonic acid derivative and / or an aminopropanesulfonic acid derivative is added to the reaction system. These are a buffer as a reaction medium in the above reaction, and also serve as an aggregation promoter.

The above-mentioned aminoethanesulfonic acid derivative and /
Alternatively, the aminopropanesulfonic acid derivative is not particularly limited, and examples thereof include 3- (N-morpholino) propanesulfonic acid (MOPS) and piperazine-N, N′-bis (2
-Ethanesulfonic acid) (PIPES), N-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HE
PES), N-hydroxyethylpiperazine-N'-3
-Propanesulfonic acid (EPPS), 3- (N-morpholino) -2-hydroxypropanesulfonic acid (MOPS
O), N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS), N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES), N-2-hydroxyethylpiperazine-
N'-2-hydroxypropane-3-sulfonic acid (HE
PSO), piperazine-N, N′-bis (2-hydroxypropanesulfonic acid) (POPSO), N-tris (hydroxymethyl) methyl-2-hydroxy-3-aminopropanesulfonic acid (TAPSO) and the like. Especially, since the effect of promoting aggregation is remarkable, MO
PS, PIPES and HEPES are preferred.

The above aminoethanesulfonic acid derivative and /
Alternatively, the addition amount of the aminopropanesulfonic acid derivative is 0.
001 to 0.5 molar concentration. When the concentration is low, a sufficient aggregation promoting effect cannot be obtained, and when the concentration is high, the buffer does not dissolve or crystals precipitate during low temperature storage, so the range is limited. Preferably, 0.0
It is 1 to 0.25 molar concentration.

In the method for measuring Treponema pallidum antibody of the present invention, the aminoethanesulfonic acid derivative and / or the aminopropanesulfonic acid derivative may be present in the reaction system. For example, in the case of an antigen-antibody reaction using a latex reagent, the latex carrying the Treponema pallidum antigen can be suspended in a buffer solution containing an aminoethanesulfonic acid derivative and / or an aminopropane derivative as a buffer.

It is also possible to use a latex reagent which does not contain these, and in this case, it is sufficient that these are present in the reaction system during the antigen-antibody reaction. For example,
There are a method of adding an aminoethanesulfonic acid derivative and / or an aminopropanesulfonic acid derivative to a sample, a method of using a buffer solution containing the above-mentioned buffer agent, and the like, which can be appropriately selected and used.

Therefore, in the method for measuring Treponema pallidum antibody of the present invention, the insoluble carrier carrying the Treponema pallidum antigen and the above-mentioned substance aminoethanesulfonic acid derivative and / or
Alternatively, a one-pack type reagent containing an aminopropanesulfonic acid derivative can be used, and further, a first reagent containing an insoluble carrier carrying a Treponema pallidum antigen, and the aminoethanesulfonic acid derivative and / or aminopropanesulfone. It is also possible to use a two-component type reagent composed of a second reagent consisting of a buffer solution containing an acid derivative.

The pH of the above reaction system is preferably 4.5 to 10.0, more preferably 5.8 to 8.0. If the pH is low or high, denaturation of the antigen protein, increase of non-specific reaction, etc. occur, which is not suitable.

The temperature of the reaction system according to the present invention is 0-5.
0 degreeC is preferable, More preferably, it is 20-40 degreeC. Further, the reaction time can be appropriately determined.

The insoluble carrier according to the present invention is not particularly limited, and examples thereof include organic polymer powder, inorganic substance powder,
Examples include microorganisms, blood cells and cell membrane pieces, and plastic microtiter plates. Examples of the organic polymer powder include natural polymer powders such as insoluble agarose, cellulose and insoluble dextran, polystyrene, styrene-styrene sulfonic acid copolymer, acrylonitrile-butadiene-styrene copolymer, vinyl chloride-acrylic acid ester. Examples thereof include synthetic polymer powders such as copolymers and vinyl acetate-acrylic acid ester copolymers, and latexes in which synthetic polymer powders are uniformly suspended are preferable.

As the above-mentioned inorganic substance powder, for example, gold,
Metal pieces such as titanium, iron, nickel, silica, alumina,
Carbon powder etc. are mentioned.

The average particle size of the insoluble carrier is measured by
Although it varies depending on the measuring device, a particle size of 0.05 to 1.0 μm is usually used. Examples of the method of supporting the Treponema pallidum antigen on the insoluble carrier include a method of sensitizing by chemical or physical binding. Examples of the chemical bonding method include a carbodiimide method, a carbodinyiimidazole method, a mixed acid anhydride method, an active ester method, and a water-soluble divalent reagent method.

As a reaction system for measuring Treponema pallidum antibody using the above-mentioned reagent, for example, latex agglutination reaction, hemagglutination reaction and the like are used. As a method for measuring agglutination caused by the above reaction, agglutination The method of observing the degree optically, the method of observing visually, etc. are mentioned.

Specifically, in the optical observation method, the measurement is carried out by mixing a sample, a sample diluent and an insoluble carrier carrying a Treponema pallidum antibody, and then measuring the scattered light intensity, absorbance or The transmitted light intensity is detected. The measurement wavelength may be 300 to 2400 nm, and the measurement method is according to a known method, such as the particle size and concentration of the insoluble carrier to be used,
The increase or decrease of scattered light intensity, absorbance or transmitted light intensity is measured by the reaction time. These methods may be used alone or in combination.

In the visual observation method, usually, a sample and a solution containing an insoluble carrier carrying an antigen are mixed on a determination plate and shaken, and then the presence or absence of aggregation is determined. The determination can be made not only by the naked eye but also by photographing with a video camera and performing image processing.

[0023]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 (Preparation of latex solution carrying Treponema pallidum antigen) 10
400 μl of Treponema pallidum antigen solution (protein concentration 15 μg / ml) was added to 0 mM phosphate buffer (pH 7.4) and polystyrene latex (solid content 10 μm) was used.
% (W / V), manufactured by Sekisui Chemical Co., Ltd.), and stirred at 4 ° C. for 1 hour. Then, 2 ml of 100 mM phosphate buffer (pH 7.4) containing 1% (W / V) bovine serum albumin (BSA) was added, and the mixture was stirred for 1.5 hours. This solution was mixed at 10 ° C. for 30 minutes, 18,000 rpm. p.
m. And centrifuged. 0.2 BSA was added to the obtained precipitate.
100 mM phosphate buffer containing 5% (w / v) (pH
7.4) 5 ml was added and the latex was suspended to prepare a syphilis treponemal antigen-bearing latex solution (first reagent).

(Preparation of Sample Dilution Solution) 0.1 at pH 7.0
0.25% BSA (W / V) in M MOPS buffer
Was dissolved (second reagent).

(Measurement of Treponema pallidum antibody titer) Using a biochemical automatic analyzer (Hitachi 7050 type, manufactured by Hitachi, Ltd.), T. U. Of the standard Treponema pallidum sera were measured. The measurement conditions are
The temperature was 37 ° C. and the wavelength was 570 nm. The measurement method is as follows: Dispense 20 μl of the sample or standard solution, and immediately after
(Reagent) was added and mixed in an amount of 350 μl, and then 50 μl of a latex solution (first reagent) was added and mixed. The reaction amount is the amount of change in absorbance from 80 seconds to 320 seconds after addition of the latex solution, and the amount of change in absorbance (n
The average value of 3) is shown in Table 1. The numerical values in Table 1 represent the amount of change in absorbance (1 / 10,000).

Example 2 Measurement was carried out in the same manner as in Example 1 except that 0.1M MOPS buffer at pH 7.0 was used as the sample diluent, and 0.1M PIPES buffer at pH 7.0 was used. I went. The results are shown in Table 1.

Example 3 Measurement was carried out in the same manner as in Example 1 except that 0.1M HEPES buffer of pH 7.0 was used instead of 0.1M MOPS buffer of pH 7.0 as the sample diluent. I went. The results are shown in Table 1. Example 4 The measurement was performed in the same manner as in Example 1 except that a 0.05 M MOPS buffer solution having a pH of 7.0 was used as the sample diluent instead of the 0.1 M MOPS buffer solution having a pH of 7.0. . The results are shown in Table 1.

Example 5 As a sample diluent, 0.05 M PIPES at pH 7.0 was used instead of 0.1 M MOPS buffer at pH 7.0.
The measurement was performed in the same manner as in Example 1 except that the buffer solution was used. The results are shown in Table 1. Example 6 As a sample diluent, 0.05M HEPES at pH 7.0 was used instead of 0.1M MOPS buffer at pH 7.0.
The measurement was performed in the same manner as in Example 1 except that the buffer solution was used. The results are shown in Table 1.

Comparative Example 1 Measurement was carried out in the same manner as in Example 1 except that a 100 mM phosphate buffer solution having a pH of 6.5 was used in place of the 0.1 M MOPS buffer solution having a pH of 7.0 as the sample diluent. went. The results are shown in Table 1. Comparative Example 2 The measurement was performed in the same manner as in Example 1 except that a 0.6M MOPS buffer solution having a pH of 7.0 was used as the sample diluent instead of the 0.1M MOPS buffer solution having a pH of 7.0. . The results are shown in Table 1.

Comparative Example 3 Measurement was carried out in the same manner as in Example 1 except that a 0.6 M HEPES buffer solution having a pH of 7.0 was used as the sample diluent instead of the 0.1 M MOPS buffer solution having a pH of 7.0. I went. The results are shown in Table 1. Comparative Example 4 The measurement was performed in the same manner as in Example 1 except that a 0.6 M PIPES buffer solution having a pH of 7.0 was used as the sample diluent instead of the 0.1 M MOPS buffer solution having a pH of 7.0. . The results are shown in Table 1.

[0032]

[Table 1]

As is clear from Table 1, in the comparative example, 0T.
U. And 500 T. U. Since the difference in the amount of change in absorbance is small, there is a problem that the difference from the negative sample is not clear when measuring a sample having a small antibody titer, but according to the example, 0T. U. And 500 T. U. Since the difference in the amount of change in absorbance was large and the sensitivity was high, it was found that even for a sample having a small antibody titer, negative and positive can be easily distinguished, and the Treponema pallidum antibody can be efficiently detected.

[0034]

The present invention is as described above, and by adding an aminoethanesulfonic acid derivative and / or an aminopropanesulfonic acid derivative to the reaction system, the syphilis treponemal antibody is highly versatile and has sufficient sensitivity. Since the measurement method can be obtained, it is possible to establish an effective diagnostic method for Treponema pallidum.

Claims (2)

[Claims] 1. A syphilis treponemal antibody is measured by detecting the degree of aggregation of the insoluble carrier generated by an antigen-antibody reaction with a syphilis treponemal antibody by using an insoluble carrier carrying a syphilis treponemal antigen. An antibody measuring method, wherein the aminoethanesulfonic acid derivative and / or the aminopropanesulfonic acid derivative is 0.001 in the reaction system for aggregating the insoluble carrier.
A method for measuring Treponema pallidum antibody, wherein the antibody is added at a concentration of 0.5 molar. 2. The aminoethanesulfonic acid derivative and / or aminopropanesulfonic acid derivative is 3- (N-morpholino) propanesulfonic acid, piperazine-N, N′-.
Bis (2-ethanesulfonic acid) and / or N-2-
The method for measuring Treponema pallidum antibody according to claim 1, which is hydroxyethylpiperazine-N'-2-ethanesulfonic acid.