JPH09288110A - Reagent for immunologically diagnosing syphilis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain reagent having equal sensitivity to that prepared by using conventional Treponema pallidum obtained from bacteria by using Treponema pallidum surface antigen protein obtained by DNA recombination. SOLUTION: This reagent for immunologically diagnosing syphilis comprises insoluble carriers with at least two or more kinds of Treponema pallidum surface antigen protein obtained by DNA recombination. Treponema pallidum surface antigen protein used may be selected among Treponema pallidum surface antigen protein with a molecular weight of 47kDa, Treponema pallidum surface antigen protein with a molecular weight of 17kDa, Treponema pallidum surface antigen protein with a molecular weight of 15kDa or the like for example. The insoluble carrier used may be organic macromolecule particles, microorganisms, blood cells, cytomembrane pieces, plastic micro-tighter plate or the like for example.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an immunological syphilis diagnostic reagent for measuring the antibody of Treponema pallidum surface antigen protein bound to an antigen-antibody reaction with anti- Treponema pallidum antibody. Regarding

[0002]

2. Description of the Related Art Syphilis is Treponema pallidum (Treponema Pa).
llidum) is an infectious disease caused by syphilis, and it is judged by the presence of anti- Treponema pallidum antibody in the blood. In clinical tests for the diagnosis of syphilis, conventionally, an immunoassay is carried out in which the Treponemal syphilis antigen is carried on an insoluble carrier and the degree of aggregation of the insoluble carrier produced by the antigen-antibody reaction with the anti- Treponema pallidum antibody is detected to measure the antibody. Method is used. Hemagglutination and latex agglutination methods are known as such measurement methods.

Conventionally, the Treponema pallidum antigen used in these methods has been extracted from syphilis cells (Japanese Patent Laid-Open No. 2-234063). However, in vitro culture of S. syphilis is impossible with current technology, and even in vivo, it is only possible to culture in rabbit testes, making it difficult to obtain a large amount of antigen. . In addition, there are many unstable factors such as the condition of rabbits and the complicated purification process from bacterial cells, and it is not always possible to obtain the one having the antigenic activity necessary for preparing a diagnostic reagent for each purification.

[0004] It is known that surface antigen proteins of Treponema pallidum of various molecular weights exist, and the major one in the antigen-antibody reaction is the surface antigen protein of Treponema pallidum of 47 kDa (hereinafter referred to as "surface antigen protein"). , 47kDa antigen), 17kD
a. Treponema pallidum surface antigen protein (hereinafter, 17 kD)
a) antigen, 15 kDa syphilis treponemal surface antigen protein (hereinafter sometimes referred to as 15 kDa antigen), etc. are known. Since the genes encoding these surface antigen proteins have already been cloned and their amino acid sequences have been determined, it is possible to produce these surface antigen proteins using gene recombination techniques (Infection and Immunity 57 (1 ), 196-203 (1989). Infe
ction and Immunity 61, 1202-1210 (1993). Molecular
Microbiology 4,1371-1379 (1990)).

In particular, a production method using the gene recombination technique is proposed for the 47 kDa antigen, and a method for detecting an anti-syphilis treponemal antibody using the obtained 47 kDa antigen is also proposed (Patent Document 1). 2-500403
Issue). By using this method, it is considered possible to stably obtain a large amount of Treponema pallidum antigen. However, the details of the method for detecting anti- Treponema pallidum antibody using this 47 kDa antigen are not described in JP-B-2-500403.

The above-mentioned 47 kDa antigen and 17 kDa antigen,
The 15 kDa antigens have different amino acid sequences and are completely different surface antigen proteins. That is,
The anti- Treponema pallidum antibody is produced against these antigens, and in order to more accurately test for the presence or absence of the antibody, it should be a reagent containing not only one type of these antigens but also two or more types. Is desirable.

[0007] In Japanese Patent Laid-Open No. 7-287017, a recombinant protein of glutathione-S-transferase and a 17 kDa antigen and a fusion protein of glutathione-S-transferase and a 15 kDa antigen are produced using a gene recombination technique, and they are produced. A diagnostic reagent for measuring anti- Treponema pallidum antibody using the same is disclosed. However, this reagent still has insufficient sensitivity, and since glutathione-S-transferase is present in the reaction system of the reagent, there is concern that it may adversely affect the sample.

[0008]

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problems, and its object is to enable mass production.
Using the surface antigen protein of Treponema pallidum, which has a stable antigenic activity and is obtained by gene recombination technique, and has the same sensitivity as the reagent produced using the conventional Treponema pallidum antigen obtained from bacterial cells. It is to provide an immunological syphilis diagnostic reagent having.

[0009]

The immunological syphilis diagnostic reagent according to claim 1 (hereinafter referred to as the present invention 1) is at least two kinds of Treponema pallidum surface antigen protein obtained by using a gene recombination technique. Is comprised of an insoluble carrier.

The Treponema pallidum surface antigen protein used in the present invention 1 was obtained using a gene recombination technique.
It is not particularly limited as long as it is a T. pallidum surface antigen protein, for example, T. pallidum surface antigen protein having a molecular weight of 47 kDa, T. pallidum surface antigen protein of 44 kDa (Journal of Genetical Microbiology 133,
1793-1803 (1987)), 42 kDa syphilis treponemal surface antigen protein (Infection and Immunity 57 (9), 2612-2623).
(1989)), a 35 kDa syphilis treponemal surface antigen protein (Infection and Immunity 59 (10), 3536-3546 (199).
1)), 34kDa Treponema pallidum surface antigen protein (I
nfection and Immunity 57 (11), 3314-3323 (1989)), 1
7kDa Treponema pallidum surface antigen protein, 15kD
a syphilis treponemal surface antigen protein of a.
It is preferably selected from the group consisting of a 47 kDa surface antigen protein, a 17 kDa surface antigen protein and a 15 kDa surface antigen protein, which are considered to be major surface antigen proteins of Treponema pallidum.

The syphilis Treponema pallidum surface antigen protein used in the present invention 1 is limited to those obtained by a gene recombination technique, but if it is cloned from the syphilis bacterium gene, the cloning method is Any method will do. As an example of the method for obtaining the surface antigen protein of Treponema pallidum, in the case of the 47 kDa antigen, the DNA sequence of the 47 kDa antigen is known as described above. Amplify, purify, and incorporate this into an appropriate expression vector. A method of introducing this vector into Escherichia coli and causing Escherichia coli to mass-produce it is mentioned. In order to efficiently purify the 47 kDa antigen from Escherichia coli, it is preferable to use a vector containing a glutathione-S-transferase (hereinafter, referred to as GST) gene as described in Japanese Patent Publication No. 6-81596. When this method is used, the 47 kDa antigen gene is inserted after the GST gene, and E. coli is produced as a fusion protein of GST and the 47 kDa antigen. After sufficient production, the bacterial cells are destroyed, and the fusion protein is separated by contacting the lysoid with a fixed glutathione column. Then
Cleavage of this fusion protein yields the 47 kDa antigen. To obtain the surface antigen protein of other Treponema pallidum, instead of the DNA for the 47 kDa antigen described above,
An antigen having a desired molecular weight can be obtained in the same manner by using a DNA for each antigen having a desired molecular weight.

Examples of the insoluble carrier used in the present invention 1 include organic polymer particles, microorganisms, blood cells, cell membrane pieces, plastic microtiter plates and the like.

Examples of the organic polymer particles include natural polymer particles such as insoluble agarose, cellulose and insoluble dextran; polystyrene, styrene-methacrylic acid copolymer, styrene-styrene sulfonate copolymer, acrylonitrile-butadiene. -Styrene copolymers, vinyl chloride-acrylic acid ester copolymers, vinyl acetate-acrylic acid ester copolymers and the like synthetic polymer particles and the like can be mentioned, especially latex in which synthetic polymer particles are uniformly suspended. preferable.

When the above latex particles are used, the particle size is preferably 0.1 to 1.0 μm, and 0.1 to 0.5 μm.
m is more preferred.

The method for supporting the Treponema pallidum surface antigen protein on the insoluble carrier may be either a chemical binding method or a physical adsorption method.

When supported by the above-mentioned chemical bonding method,
As the insoluble carrier, one having an active group capable of forming a covalent bond with the surface antigen protein, for example, a carboxyl group, an amino group, a thiol group, or the like is used. In this case, when the insoluble carrier originally has the above-mentioned active group, it may be used as it is, but when it does not have the above-mentioned active group, the insoluble carrier may undergo reactions such as oxidation, reduction and modification. Alternatively, the active group may be introduced.

When latex particles having a carboxyl group are used as the insoluble carrier, the surface antigen protein can be bound by a condensation reaction with a water-soluble carbodiimide. In this case, the amount of carboxyl groups in the latex particles is such that the surface charge is 0.03 to 0.75 me.
qCOO ⁻ / g is preferable, and 0.05 to 0.40 meq
COO ⁻ / g is more preferable.

Examples of the water-soluble carbodiimide include 1-ethyl-3- (3-dimethylaminopropyl)
Carbodiimide hydrochloride (hereinafter referred to as EDC), 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimidometh-p-toluenesulfonate, 1-ethyl-
Examples thereof include 3- (3-diethylaminopropyl) carbodiimide perchlorate, and EDC is preferable from the viewpoint of solubility, reactivity, and the like.

When the above condensation reaction is used, the latex particles and the antigen may be bound via a suitable spacer. The spacer is a substance that is interposed between the insoluble carrier and the surface antigen protein to bind the insoluble carrier and the surface antigen protein, and forms a covalent bond with the insoluble carrier and the active group on the surface antigen protein. It happens. As such a spacer, amino acids such as β-aminoalanine, γ-aminobutyric acid, ε-aminocaproic acid, glutamic acid and p-aminobenzoate are preferable. Particularly preferable spacers are γ-aminobutyric acid and ε-aminocaproic acid, since the antigen is more stably bound to the carrier via the spacer.

The reaction temperature of the coupling reaction by the EDC is preferably 0 ° C to 40 ° C because if the reaction temperature becomes low, the reaction does not proceed sufficiently, and if it becomes high, the surface antigen protein may be denatured and lose its antigenicity.

When the above insoluble carrier is loaded with Treponema pallidum surface antigen protein by a physical adsorption method, the insoluble carrier and the surface antigen protein are brought into contact with each other in a solution,
Adsorption easily occurs due to the charge of the surface antigen protein and the insoluble carrier. The reaction temperature of this adsorption reaction is preferably 0 ° C to 40 ° C because if the reaction temperature becomes low, the reaction does not proceed sufficiently, and if it becomes high, the surface antigen protein may be denatured and lose its antigenicity.

To prepare the immunological syphilis diagnostic reagent of the present invention 1, for example, γ-aminobutyric acid or ε-is prepared by using EDC as a coupling agent in an insoluble carrier having an active group.
A spacer such as aminocaproic acid is covalently bonded, and then EDC is used as a coupling agent, and the spacer is further reacted with a mixture of at least two kinds of Treponema pallidum surface antigen proteins obtained by gene recombination technique. And a method of covalently bonding.

As another production method, an insoluble carrier and a mixture of at least two kinds of Treponema pallidum surface antigen proteins obtained by using a gene recombination technique,
Examples include a method of contacting in a solution and physically adsorbing.

When the immunological syphilis diagnostic reagent of the present invention 1 is used in a mixture with a sample, the antibody can be measured based on the antigen-antibody reaction with the anti- Treponema pallidum antibody in the sample.

In the above antigen-antibody reaction, the sample is
It may be diluted with a buffer having a pH of 5.0 to 9.0.
Examples of the diluent for the above-mentioned specimen include a phosphate buffer,
Examples include glycine buffer, veronal buffer, borate buffer, citrate buffer, Tris buffer and the like.

Various sensitizers can be added to the above-mentioned sample diluent for the purpose of improving the measurement sensitivity and promoting the antigen-antibody reaction. Examples of the sensitizer include alkylated polysaccharides such as methyl cellulose and ethyl cellulose described in JP-A-2-173567;
And pullulan and polyvinylpyrrolidone described in JP-A-180838.

Further, bovine serum albumin, ovalbumin, etc. may be added to the above-mentioned sample diluent for the purpose of suppressing non-specific reactions.

The assay system for measuring anti- Treponema pallidum antibody using the immunological syphilis diagnostic reagent of the present invention includes, for example, the degree of aggregation of the insoluble carrier due to the reaction between the insoluble carrier and the anti- Treponema pallidum antibody. The latex agglutination reaction system using latex particles as an insoluble carrier is particularly preferable.

Examples of the method for measuring the agglomeration produced by the above reaction include a method of optically observing the degree of agglutination and a method of visually observing the degree.

Specifically, in the optical observation method, a liquid obtained by diluting a sample with a sample diluent contains the immunological syphilis diagnostic reagent of the present invention 1 (usually a phosphate buffer, Glycine buffer, veronal buffer, borate buffer, citrate buffer, Tris buffer, etc.) are mixed, and then the scattered light intensity, absorbance or transmitted light intensity of the mixed solution is measured. The measurement wavelength can be 300 to 2400 nm, and the measurement method is a known method, and the increase or decrease of scattered light intensity, absorbance, or transmitted light intensity is measured according to the particle size, concentration, and reaction time of the insoluble carrier used. These methods may be used alone or in combination.

In the visual observation method, usually, a sample and a liquid containing the immunological syphilis diagnostic reagent of the present invention 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.

The above-mentioned antigen-antibody reaction is carried out under normal conditions,
As the reaction medium, for example, a phosphate buffer solution, a citrate buffer solution, a glycine buffer solution, a Tris buffer solution or the like is used as described above. The pH during the reaction is preferably from 4.5 to 10.0, more preferably from 5.8 to 8.0. The temperature during the reaction is preferably 0 to 50 ° C, more preferably 20 to 40 ° C. The reaction time is appropriately determined.

The immunological syphilis diagnostic reagent according to claim 2 (hereinafter referred to as the present invention 2) is an insoluble carrier carrying a syphilis treponemal surface antigen protein obtained by a gene recombination technique, and a gene recombination. It is characterized in that the above-mentioned T. pallidum surface antigen protein obtained by the method is mixed with an insoluble carrier carrying a T. pallidum surface antigen protein different from the above.

The materials for the Treponema pallidum surface antigen protein and the insoluble carrier used in the present invention 2 are the same as those in the present invention 1.

In the second aspect of the present invention, an insoluble carrier carrying a treponema pallidum surface antigen protein obtained by a gene recombination technique and a syphilis different from the above-mentioned treponema pallidum surface antigen protein obtained by a gene recombination technique are used. The Treponemal surface antigen protein is mixed with an insoluble carrier.

The insoluble carrier carrying the syphilis treponemal surface antigen protein obtained by the gene recombination method used in the present invention 2, and the above-mentioned syphilis treponemal surface antigen protein obtained by the gene recombination method. A method for obtaining insoluble carriers carrying different syphilis treponemal surface antigen proteins is such that at least one or more kinds of syphilis treponemal surface antigen proteins are used in the production of the respective surface antigen protein-supporting insoluble carriers, and The method is the same as the method for obtaining the insoluble carrier used in the first aspect of the present invention, except that different types of Treponema pallidum surface antigen proteins carried by the respective insoluble carriers are used.

In order to produce the immunological syphilis diagnostic reagent of the present invention 2, an insoluble carrier carrying the syphilis treponemal surface antigen protein obtained by the gene recombination technique obtained as described above, And an insoluble carrier carrying a T. pallidum surface antigen protein different from the above T. pallidum surface antigen protein obtained by using a gene recombination technique. In this case, not only two types of insoluble carriers carrying different syphilis treponemal surface antigen proteins but also three or more types may be mixed.

The method of using the immunological syphilis diagnostic reagent of the present invention 2 is the same as that of the present invention 1.

[0039]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described with reference to Examples and Comparative Examples. Reference Example 1 Preparation of 47 kDa Antigen (1) Preparation of Expression Vector Treponema pallidum (Treponema Pallidum) From the crushed rabbit testicles (manufactured by Joban Kagaku), Treponema pallidum cells were extracted and genomic DNA was extracted. . Since the DNA sequence of the 47 kDa antigen was already known, primers were prepared based on the DNA sequence using a DNA synthesizer.
Using the genomic DNA extracted above as a template and the above primers, the DN method of the 47 kDa antigen was
A fragment was obtained. Next, the DNA fragment described above was inserted into a vector pGEX-4T-3 (manufactured by Pharmacia) for expressing a fusion protein with GST.
It was named X-4T-3-47k.

(2) Production of 47 kDa antigen Next, pGEX-4T-3-47k was introduced into E. coli,
Cultured. The expression of the fusion protein of 47 kDa antigen and GST was induced by adding isopropyl-β-(-)-thiogalactopyranoside to the culture medium. Glutathione Sepharose 4B (manufactured by Pharmacia) was added to the supernatant obtained by disrupting the cells of the E. coli expressing the fusion protein sufficiently by sonication and removing the cell membrane, nucleus, etc. by centrifugation, and allowing the mixture to stand overnight for fusion. The protein was adsorbed on glutathione Sepharose 4B. Glutathione Sepharose 4B to which the fusion protein had been adsorbed was collected by centrifugation, and packed into a column. After washing several times, the fusion protein was released and eluted from glutathione sepharose 4B by flowing a glutathione solution. A thrombin solution was added to the thus purified 47 kDa antigen-GST fusion protein solution to cleave the bond between the 47 kDa antigen and GST. As a result, a solution containing the 47 kDa antigen, GST and thrombin was obtained. GST was applied to the Glutathione Sepharose 4B column by applying this solution.
Was removed. Furthermore, heparin sepharose CL-6B
Thrombin was removed by flowing through a column packed with (Pharmacia) to obtain a 47 kDa antigen.

Reference Example 2 Preparation of 17 kDa Antigen (1) Preparation of Expression Vector Treponema pallidum (Treponema Pallidum) From the crushed rabbit testicles (manufactured by Joban Kagaku), Treponema pallidum cells were extracted, and genomic DNA was extracted. Was extracted. Since the DNA sequence of the 17 kDa antigen was already known, primers were prepared based on the DNA sequence using a DNA synthesizer.
Using the genomic DNA extracted as a template as a template and the above primers, the DN of 17 kDa antigen was
A fragment was obtained. Next, the DNA fragment described above was inserted into a vector pGEX-4T-3 (manufactured by Pharmacia) for expressing a fusion protein with GST.
It was named X-4T-3-17k.

(2) Production of 17 kDa antigen Next, pGEX-4T-3-17k was introduced into E. coli,
Cultured. Expression of a fusion protein of 17 kDa antigen and GST was induced by adding isopropyl-β-(-)-thiogalactopyranoside to the culture medium. In Escherichia coli in which the fusion protein was sufficiently expressed, 47 kD in the explanation of (2) Production of 47 kDa antigen in Reference Example 1
The procedure of (2) of Reference Example 1 was repeated except that the expression "a antigen" was replaced with the expression "17 kDa antigen".
The kDa antigen was obtained.

Reference Example 3 Preparation of 15 kDa Antigen (1) Preparation of Expression Vector Treponema pallidum (Treponema Pallidum) Passage rabbit testicle crushed material (manufactured by Joban Kagaku) was used to extract Treponema pallidum cells, and genomic DNA was extracted. Was extracted. Since the DNA sequence of the 15 kDa antigen was already known, primers were prepared based on the DNA sequence using a DNA synthesizer.
Using the genomic DNA extracted as above as a template and the above primers, the DN of 15 kDa antigen was
A fragment was obtained. Next, the DNA fragment described above was inserted into a vector pGEX-4T-3 (manufactured by Pharmacia) for expressing a fusion protein with GST.
It was named X-4T-3-15k.

(2) Production of 15 kDa antigen Next, pGEX-4T-3-15k was introduced into E. coli,
Cultured. The expression of a fusion protein of 15 kDa antigen and GST was induced by adding isopropyl-β-(-)-thiogalactopyranoside to the culture medium. In Escherichia coli in which the fusion protein was sufficiently expressed, 47 kD in the explanation of (2) Production of 47 kDa antigen in Reference Example 1
A 15-kDa antigen was obtained by the same procedure as in the production of (2) 47-kDa antigen of Reference Example 1, except that the expression of a-antigen was replaced with the expression of 15-kDa antigen.

Example 1 (1) Bonding of spacer to latex Styrene-methacrylic acid copolymer latex (carboxyl group 0.25 meqCOO ⁻ / g, particle size 0.4 μm,
Sekisui Chemical Co., Ltd.'s 5% (w / v) aqueous suspension (150 μl) was added to pH 5.0 phosphate buffer (hereinafter,
1 ml of a solution prepared by dissolving EDC at a ratio of 15% (w / v) in PB buffer) was added, and the mixture was stirred at room temperature for 5 hours.
Unreacted EDC was removed by centrifugation, washed twice with PB buffer, and then suspended in 1 ml of borate buffer (pH: 9.0) (hereinafter referred to as BB buffer). In this suspension, B
1 ml of a solution in which γ-aminobutyric acid was dissolved in B buffer at a ratio of 10% (w / v) was added, and the mixture was stirred at room temperature for 24 hours.
Unreacted γ-aminobutyric acid was removed by centrifugation to remove BB
After washing twice with a buffer solution, suspending in 1 ml of PB buffer solution,
A spacer-bonded latex was obtained in which the carboxyl group of the latex and the amino group of γ-aminobutyric acid were covalently bonded.

(2) Binding of Treponema pallidum surface antigen protein 1 ml of a solution prepared by dissolving EDC in PB buffer at a ratio of 30% (w / v) was added to 1 ml of the above spacer-bound latex.
1, and the mixture was stirred at room temperature for 5 hours. Unreacted EDC was removed by centrifugation, washed twice with PB buffer, and then suspended in 2 ml of BB buffer. In this suspension, a mixed antigen-protein solution [phosphate buffer solution (pH 7.2) in which the 47 kDa antigen prepared in Reference Example 1 was dissolved to 500 μg / ml, phosphate buffer solution (pH 7.2) ), The solution in which the 17 kDa antigen prepared in Reference Example 2 was dissolved to 500 μg / ml and the phosphate buffer (pH 7.2) contained the 15 kDa antigen prepared in Reference Example 3 in an amount of 500 μg / ml.
100 μl of the solution obtained by mixing the solution dissolved so as to become 1: 1: 1] and stirred at room temperature for 24 hours. Unreacted 47kDa antigen by centrifugation, 1
After removing the 7 kDa antigen and the 15 kDa antigen and washing twice with BB buffer, a PBS buffer solution containing 0.9% by weight of bovine serum albumin (hereinafter, bovine serum albumin is referred to as BSA) is 1% (w / v). Sodium chloride-phosphate buffer, pH 7.4. The PBS buffer used below is also
Suspended in 2 ml of the same composition), stirred at room temperature for 1 hour, and blocked (to prevent nonspecific reaction,
Adsorption of BSA on the latex surface was performed.
Next, the unreacted BSA is removed by centrifugation to remove BSA.
Suspended in 4 ml of PBS buffer containing 1% (w / v) of 47kDa antigen, 17kDa antigen and 15kDa
An antigen-bound latex (immunological syphilis diagnostic reagent of the present invention 1) liquid was obtained.

Example 2 100 μl of the mixed antigen protein solution in the section (2) Treponemal syphilis surface antigen protein binding of Example 1 was dissolved so that the 47 kDa antigen prepared in Reference Example 1 became 500 μg / ml. Solution and phosphate buffer (pH
In 7.2), the 17 kDa antigen prepared in Reference Example 2 was 500.
The 47kDa antigen and 17kDa antigen-bound latex was operated in the same manner as in Example 1 except that the solution dissolved to give a concentration of μg / ml was replaced with 100 μl of the mixed antigen protein solution mixed at a ratio of 1: 1. (Immunological syphilis diagnostic reagent of the present invention 1) was obtained.

Example 3 100 μl of the mixed antigen protein solution in the section (2) Treponemal syphilis surface antigen protein binding of Example 1 was dissolved so that the 47 kDa antigen prepared in Reference Example 1 became 500 μg / ml. Solution and phosphate buffer (pH
In 7.2), the 15 kDa antigen prepared in Reference Example 3 was 500.
A 47 kDa antigen- and 15 kDa antigen-bound latex was prepared in the same manner as in Example 1 except that 100 μl of the mixed antigen protein solution, which was mixed at a ratio of 1: 1, was used to dissolve the solution so that the concentration became μg / ml. (Immunological syphilis diagnostic reagent of the present invention 1) was obtained.

Example 4 100 μl of the mixed antigen protein solution in the section (2) Treponemal syphilis surface antigen protein binding of Example 1 was dissolved so that the 17 kDa antigen prepared in Reference Example 2 became 500 μg / ml. Solution and phosphate buffer (pH
In 7.2), the 15 kDa antigen prepared in Reference Example 3 was 500.
A 17kDa antigen and a 15kDa antigen-bound latex were prepared in the same manner as in Example 1 except that 100l of the mixed antigen protein solution, which was mixed at a ratio of 1: 1, was used instead of the solution dissolved to have a concentration of 1g / ml. (Immunological syphilis diagnostic reagent of the present invention 1) was obtained.

Example 5 100 μl of the mixed antigen protein solution in the section of (2) Treponema pallidum surface antigen protein binding in Example 1 was dissolved so that the 47 kDa antigen prepared in Reference Example 1 became 500 μg / ml. A 47 kDa antigen-bound latex solution was obtained in the same manner as in Example 1 except that 100 μl of the surface antigen protein solution consisting of the solution alone was replaced. 100 μl of the surface antigen protein solution used to obtain the above 47 kDa antigen-binding latex solution was dissolved in 100 μl of the 17 kDa antigen prepared in Reference Example 2 to 500 μg / ml.
17kD
a Antigen-bound latex solution was obtained. 100 μl of the surface antigen protein solution used in obtaining the 47 kDa antigen-bound latex solution was replaced with 100 μl of the surface antigen protein solution consisting of the solution prepared by dissolving 15 kDa antigen prepared in Reference Example 3 to 500 μg / ml. Other than the above, the same procedure as above was carried out to obtain a 15 kDa antigen-bound latex solution. Obtained 47kDa antigen-binding latex liquid, 17kDa antigen-binding latex liquid and 15kD
The antigen-bound latex solution was mixed at a ratio of 1: 1: 1 to obtain a mixed solution of Treponema pallidum surface antigen protein-bound latex (immunological syphilis diagnostic reagent of the present invention 2).

Example 6 The 47 kDa antigen-bound latex solution and the 17 kDa antigen-bound latex solution obtained in Example 5 were mixed in a ratio of 1: 1 to prepare a mixed solution of Treponema pallidum surface antigen protein-bound latex (the present invention). 2 immunological syphilis diagnostic reagents)
I got

Example 7 The 47 kDa antigen-bound latex solution and the 15 kDa antigen-bound latex solution obtained in Example 5 were mixed at a ratio of 1: 1 to prepare a mixed solution of the Treponema pallidum surface antigen protein-bound latex (the present invention). 2 immunological syphilis diagnostic reagents)
I got

Example 8 The 17 kDa antigen-bound latex solution and the 15 kDa antigen-bound latex solution obtained in Example 5 were mixed at a ratio of 1: 1 to prepare a mixed solution of syphilis treponemal surface antigen protein-bound latex (the present invention). 2 immunological syphilis diagnostic reagents)
I got

Comparative Example 1 The 47 kDa antigen-bound latex solution obtained in Example 5 alone is used as the immunological syphilis diagnostic reagent of Comparative Example 1. Comparative Example 2 The 17 kDa antigen-bound latex solution alone obtained in Example 5 is used as the immunological syphilis diagnostic reagent of Comparative Example 2. Comparative Example 3 The 15 kDa antigen-bound latex solution alone obtained in Example 5 is used as the immunological syphilis diagnostic reagent of Comparative Example 3.

Evaluation of Performance of Immunological Syphilis Diagnostic Reagents In order to evaluate the performance of the immunological syphilis diagnostic reagents obtained in Examples 1 to 8 and Comparative Examples 1 to 3, the above reagents and commercially available Treponema pallidum diagnostics were diagnosed. Using the reagents, T. pallidum positive samples and T. pallidum negative samples were measured and the correlation of the measurement results was examined.

The measurement of the Treponema syphilis positive specimen and the Treponema pallidum negative specimen by the immunological syphilis diagnostic reagents obtained in Examples 1 to 8 and Comparative Examples 1 to 3 was specifically carried out as follows. . 0, 50, 1 by automatic biochemical analyzer (Hitachi 7050 type, manufactured by Hitachi Ltd.)
00, 250, 500 titer units (hereinafter, referred to as T.
U. Abbreviated) standard solution consisting of standard syphilis treponemal positive serum, 30 samples of syphilis treponemal positive samples (sample numbers 1 to 30), and 20 samples of syphilis treponemal negative samples (sample numbers 31 to 50). The measurement conditions were a temperature of 37 ° C. and a wavelength of 570 nm. The measuring method was as follows: Dispense 20 μl of the standard solution or the sample, and immediately add 350 μl of the sample diluent (the one contained in the reagent for syphilis diagnosis “Mediaace TPLA” (manufactured by Sekisui Chemical Co., Ltd.)). -Mix and then 50μ of the above immunological syphilis diagnostic reagent
1 was added and mixed. The reaction amount was the change in absorbance between 80 seconds and 320 seconds after the addition of the immunological syphilis diagnostic reagent. The amount of change in absorbance obtained for each standard solution and the T.V. U. A calibration curve is prepared from the sample, the amount of change in absorbance obtained by measuring each sample is applied to the obtained calibration curve, and the T. U. Was obtained and the results are shown in Tables 1 to 4.

Next, the Treponema pallidum positive specimen 30
T. Specimens, T. syphilis negative 20 specimens U.
Was measured using a commercially available diagnostic reagent for Treponema pallidum "Mediace TPLA" (manufactured by Sekisui Chemical Co., Ltd.), and the results are shown in the columns of commercially available products in Tables 1 to 4.

[0058]

[Table 1]

[0059]

[Table 2]

[0060]

[Table 3]

[0061]

[Table 4]

Next, for each of the reagents of Examples 1 to 8 and Comparative Examples 1 to 3, the correlation coefficient between the obtained measurement results and the measurement results obtained using a commercially available Treponema pallidum diagnostic reagent was investigated. The results are shown in Table 5.

[0063]

[Table 5]

From Table 5, there is a high correlation between the measurement results obtained with the reagents of Examples 1 to 8 and the measurement results obtained using the commercially available reagent for Treponema pallidum syphilis, and Comparative Examples 1 to 3
It can be seen that it is higher than the correlation between the measurement results obtained with the reagent of 1. and the measurement results obtained with the commercially available Treponema pallidum diagnostic reagent. In addition, in the reagents of Comparative Examples 2 and 3, even the negative samples were subjected to T. U. There are some that are high, and some that are difficult to distinguish between negative and positive.

[0065]

The composition of the immunological syphilis diagnostic reagent according to claim 1 is as described above, from an insoluble carrier carrying at least two kinds of Treponema pallidum surface antigen protein obtained by gene recombination technique. Therefore, the present invention provides an immunological syphilis diagnostic reagent having the same sensitivity as a reagent produced using a conventional Treponema pallidum antigen obtained from bacterial cells. As described above, since a surface antigen protein of Treponema pallidum obtained by using a gene recombination method, which can be mass-produced and has stable antigenic activity, was used and a reagent with the same sensitivity as the conventional product was obtained, The immunological syphilis diagnostic reagent according to Item 1 is a reagent that is produced with high sensitivity and stability.

The composition of the immunological syphilis diagnostic reagent according to claim 2 is as described above, using an insoluble carrier carrying the Treponema pallidum surface antigen protein obtained by the gene recombination technique and the gene recombination technique. Since an insoluble carrier carrying a T. pallidum surface antigen protein different from the T. pallidum surface antigen protein obtained by the above method is mixed, a reagent prepared using a conventional T. pallidum antigen obtained from bacterial cells An immunological syphilis diagnostic reagent having equivalent sensitivity is provided. As described above, since a surface antigen protein of Treponema pallidum obtained by using a gene recombination method, which can be mass-produced and has stable antigenic activity, was used and a reagent with the same sensitivity as the conventional product was obtained, The immunological syphilis diagnostic reagent according to Item 2 is a reagent that is produced with high sensitivity and stability.

The composition of the immunological syphilis diagnostic reagent according to claim 3 is as described above, wherein the Treponema pallidum surface antigen protein is:
Treponema pallidum surface antigen protein with a molecular weight of 47 kDa,
The syphilis immunological diagnostic reagent according to claim 1 or 2, which is selected from the group consisting of Treponema syphilis surface antigen protein having a molecular weight of 17 kDa and Treponema syphilis surface antigen protein having a molecular weight of 15 kDa. Provided is an immunological syphilis diagnostic reagent which is even more sensitive to a reagent produced using an antigen. Thus, by using the surface antigen protein of Treponema pallidum obtained by the gene recombination technique, which can be mass-produced and has stable antigenic activity, a reagent having a sensitivity much closer to that of the conventional product was obtained. Therefore, the immunological syphilis diagnostic reagent according to claim 3 is a reagent that is highly sensitive and stably produced.

The composition of the immunological syphilis diagnostic reagent according to claim 4 is, as described above, the immunological syphilis diagnostic reagent according to claim 1, 2 or 3 in which the insoluble carrier is a latex composed of synthetic polymer particles. Provided is an immunological syphilis diagnostic reagent having a sensitivity even closer to that of a reagent produced using a conventional Treponema pallidum antigen obtained from cells. Thus, by using the surface antigen protein of Treponema pallidum obtained by the gene recombination technique, which can be mass-produced and has stable antigenic activity, a reagent having a sensitivity much closer to that of the conventional product was obtained. Therefore, the immunological syphilis diagnostic reagent according to claim 4 is a reagent that is produced with high sensitivity and stability.

Claims (4)

[Claims] 1. An immunological syphilis diagnostic reagent comprising an insoluble carrier carrying at least two kinds of Treponema pallidum surface antigen proteins obtained by a gene recombination technique. 2. An insoluble carrier carrying a T. syphilis surface antigen protein obtained by a gene recombination technique and a T. pallidum surface antigen different from the above T. pallidum surface antigen protein obtained by a gene recombination technique. An immunological syphilis diagnostic reagent characterized by being mixed with an insoluble carrier carrying a protein. 3. The syphilis treponemal surface antigen protein is selected from the group consisting of a syphilis treponemal surface antigen protein having a molecular weight of 47 kDa, a syphilis treponemal surface antigen protein having a molecular weight of 17 kDa, and a syphilis treponemal surface antigen protein having a molecular weight of 15 kDa. 2. The immunological syphilis diagnostic reagent described in 2. 4. The immunological syphilis diagnostic reagent according to claim 1, 2 or 3, wherein the insoluble carrier is a latex composed of synthetic polymer particles.