JPS58108458A - Reagent for examination of syphilis - Google Patents

Abstract

PURPOSE:To eliminate non-specific reaction of TPHA test almost completely by absorbing any non-specific antibody employing an antigen derived from bacteria of treponema group always presenting in a human body or an animal. CONSTITUTION:A culture medium containing one kind or more of bacteria of treponema group is treated centrifugally at 10,000 G to pelletize a bacterial body and non-soluble culture medium components. The supernatant thus formed is incuvated at 100 deg.C and nonsoluble denatured protein is removed to make a cultured supernatant. A sediment containing the bacterial body and the non- soluble culture medium components is suspended in a dilution solvent and combined with the cultured supernatant to make a reaction liquid. The reaction liquid thus obtained is combined with a standard serum, an antigen sensitized carrier, an antigen non-sensitized carrier, a recovery liquid, a dilution liquid and the like to provide the kit of a reagent for examination of syphilis.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the treatment of Treponema pallidum (pathogenic Treponema pallidum).
Syphilis is produced by sensitizing the disrupted bacterial cells of P&l idum Nlcols strain (hereinafter abbreviated as TP) to a carrier such as animal red blood cells, and agglutinating the resulting antigen-sensitized carrier in the presence of the corresponding antibody. A tool for diagnosing infections called Treponema Pallidum Hem
agglutinationTest (hereinafter referred to as TPHA
(referred to as tests).

More specifically, the present invention relates to a syphilis testing reagent characterized in that a liquid containing a fungus-derived antigen is used as the reaction liquid of the syphilis testing reagent.

The TPHA test, developed by Tomizawa et al. in 1966, is now widely used due to its advantages such as sensitivity, specificity, and ease of operation, and its excellence is recognized around the world as a representative clinical testing method for syphilis. It is being

Although this TPHA test is a testing method with excellent sensitivity and specificity, it is impossible to deny the possibility that non-specific reactions may occur in rare cases, and it requires equipment, cost, and a high level of skill. There may be rare cases where a fluorescent antibody method (PTA-ABS method) is required (Fukuoka Nagao, Japan Clinical, 3
8.1021 (1980)).

In addition, the present inventors' new syphilis test reagent (patent application No. 56-168646, hereinafter referred to as "
In the new TPiIA J), the present invention was completed based on the discovery that the non-specific reaction can be almost completely eliminated using an antigen derived from a Trebonema bacterium.

The Treponema genus bacteria used in the present invention may be any bacteria as long as they are resident in the human body or animals.
ticola), Treponema mycosum (Tre
ponema mucosum ) %) Reponema 8
Orale (Treponema orale) s
) Treponem 0 Scoliodentum
acoliodontum), Treponema guince/tii (Treponemavincentii)
), Treponema macrodentium (Trepo
nema macrodentium), Treponema trimero/Tiram (Treponema trimero)
erontiuJ Treponema pucalis (Trepo
nema buccalis) 11Treponema ΦPatsukale (Treponema buccale) s
Trehonema enteronoyai 9 Evening A (Trepon
emaenterogyratum) etc. or Trehonema baraluisucniculi (Tr.
eponema paraluia - cuniculi
), Treponem +1
a hyos ) 1) Reponema IIA! Nosa (T
reponema penortha) and the like are suitable.

One type of these Treponema bacteria may be used, but it is preferable to use two or more types.
eni@#Relter strain). The reaction solution containing antigens derived from these Treponema bacteria can be obtained by any method, but the current TPH
Just follow the method commonly used for A-tests.

For example, the following method may be used.

1 medium containing one or more types of Treponema bacteria
The cells and insoluble medium components are pelleted by centrifugation at 0.000 G, and the supernatant is incubated at 100° C. to remove insolubilized denatured proteins to obtain a culture supernatant. The precipitate containing the bacterial cells and insoluble medium components is suspended in a diluting solvent and combined with the culture medium to form the reaction solution of the present invention. In the method for obtaining the reaction solution described above, it is preferable to contain two or more types of Treponema, and it is even more preferable to contain a part of the culture solution rather than only Treponema cells.

Treponema can also be used after being crushed by homogenizer treatment, ultrasonic treatment, etc., if necessary.

It goes without saying that the reaction solution containing the Treponema-derived antigen in the present invention can be used as a reaction dilution solution in carrier agglutination reactions, but it can also be used, for example, as a reconstitution solution for lyophilized antigen-sensitized carriers. The desired effects of the present invention can also be obtained when used in advance as a diluent or absorption liquid for a test liquid (serum, plasma, etc.) for a carrier agglutination reaction.

The absorption of non-specific antibodies by the reaction solution of the present invention can also be carried out simultaneously with the carrier agglutination reaction.

The reaction solution obtained according to the present invention can be prepared using standard serum,
A syphilis test reagent (kit) is made by combining an antigen-sensitized carrier, an antigen-unsensitized carrier, a restoring solution, a diluent, etc.

Syphilis testing is simple using the test reagent obtained in the present invention, and it has the excellent effect of showing a sensitive and stable positive result for syphilis, and having no or very few non-specific reactions. Therefore, there is no need to use the PTA-ABS method, which requires equipment and costs.

Hereinafter, the effects of the present invention will be explained in more detail using experimental examples.

Experimental Example 1 560 healthy serum samples obtained from the Japanese Red Cross Society were mixed with the 8 reaction solution mentioned in Example 1 (conventional method), the ES reaction solution obtained in Example 3, or the BES obtained in Example 4. After each was diluted 10 times with reaction solution, the current TPHA test using antigen-sensitized sheep red blood cells was performed. The results are shown in Table 1.

Table 1 The surgical technique used in the above experimental example is as follows.

(1) Reaction solution (B solution, Es1t or BES solution): Add 25 d of 10-fold diluted M test serum to each well.
1, 2, and 3, respectively.

(2) Pour 25d of the reaction solution into a 3.4-well tube and dilute it several times starting from well 3 using a gilutator. After diluting well 4, the 25 d attached to the guilluter
Throw away d.

(3) Add 25d of the reaction solution containing the sensitized carrier suspended at 0.625% (solution C) to well-2e3a4tlC.

(4) Add 25 d of a reaction solution containing unsensitized carrier suspended at 0.625% (solution B) to well-1.

(5) - After standing overnight, serum that shows (+) at x80 is considered a non-specific serum.

Experimental Example 2 560 serum samples from healthy individuals obtained from the Japanese Red Cross Society were diluted 10 times with the reaction solution in the same manner as in Experimental Example 1. A new TPHA test using sheep red blood cells (% 56-168
646) was carried out. The results are shown in Table 2e.

Table 2 Example 1 A diluted solution (solution A) having a pH of 7.2 and containing the following components in Form 1 is prepared.

Na2HPO4”12H2020,7g-KH2PO4
2,39 NaC64,39 Gum Arabic 25 no NaN5 1. Off Healthy Rabbit Serum 10ml 10% Tween 80 111Ll Separately A liquid! Atap Treponema phagedeni
0.0611- of wet bacteria of S (Reiter strain) is added (solution B).

Treponema mu provided by Japan Dental University
201+1J of liquid medium containing 1.4 x 108/d of cosumE-21 strain was incubated at 4°C at 10,000 G for 30
Centrifuge for one minute to remove the bacterial cells and insoluble medium components such as gelatin, and incubate the supernatant on a 100°C water bath for 30 minutes with occasional stirring. Thereafter, heat-denatured and insolubilized proteins are removed using Whatman A2 F paper to obtain a culture supernatant.

The sediment containing the bacterial cells and insoluble medium components was suspended in Solution A, and 2
The cells were disrupted by ultrasonication at 0 kHz for 10 minutes, and the culture dilution factor was defined as 2 times.

This E solution was combined with standard serum, antigen sensitization carrier, anti-sensitization carrier, restoring solution, diluent, etc. in a conventional manner to prepare a syphilis test reagent (kit).

Treat in the same manner as above using solution B instead of solution A.
kit).

Example 2 Treponemaden received from Japan Dental University
ticola S-door 3 plants 1.4 X 108/ml
A liquid culture medium containing 20- was treated with liquid A in the same manner as in Example 1, and the reaction liquid (hereinafter referred to as liquid B) was treated at 40 mA! I got it. Similarly, it was treated with B solution to obtain 40 mJ of a reaction solution (BS solution).

ζ[F], B solution, or 88 solution was combined with an antigen sensitization carrier and the like in the same manner as in Example 1 to prepare a reagent (kit) for syphilis testing.

Example 3 Solution E diluted 10 times with Solution A and Solution B diluted 10 times with Solution A were combined to form a reaction solution (hereinafter referred to as ES Solution 7).

This BS solution was combined with an antigen-sensitized carrier and the like in the same manner as in Example 1 to prepare a syphilis test reagent (kit).

Example 4 Solution E diluted 10 times with Solution B and Solution B diluted 10 times with Solution B were combined to form a reaction solution (hereinafter referred to as BF3 solution).

This BF3 solution was mixed with an antigen sensitized carrier etc. in the same manner as in Example 1.
The combination was used as a syphilis test reagent (kit).

Microorganism Treponema mucos used in Examples
um E-21 strain and Treponema den
ticola S-173 strain was shipped to the Institute of Microbial Technology, Agency of Industrial Science and Technology on December 18, 1981 for the purpose of deposit (registered postal code 160chi B694).

Claims (1)

[Claims] A poison test reagent that utilizes the agglutination of a carrier sensitized with an antigen derived from Treponema pallidum by the presence of the corresponding antibody, and contains an antigen derived from a Treponema bacterium that is resident in humans or animals as a reaction liquid. A syphilis test reagent characterized by using a liquid that