JPH09304392A - Manufacture of immunological syphilis diagnostic reagent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for manufacturing an immunological syphilis diagnostic reagent, that utilizes a surface antigen protein of syphilis treponema which can be massproduced, has stable antigen activity, and is obtained using gene recombination techniques, and that provides sensitivity usable for practical purposes. SOLUTION: This manufacturing method comprises mixing a treponema palladium surface antigen protein (e.g. treponema pallidum surface antigen proteins with molecular weights of 47, 17, and 15 kDa), obtained using gene recombination techniques, with an insoluble carrier (e.g. microplate) within a solvent containing divalent metal ions (e.g. magnesium or calcium ions), and thereby causing the insoluble carrier to carry the surface antigen protein.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an immunological syphilis diagnostic reagent for measuring the antibody based on the antigen-antibody reaction with an anti- Treponema pallidum antibody using an insoluble carrier carrying the Treponema pallidum surface antigen protein. Manufacturing method.

[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 the clinical test of syphilis diagnosis, conventionally, a method of supporting the syphilis treponemal antigen on an insoluble carrier and detecting the degree of aggregation of the insoluble carrier generated by an antigen-antibody reaction with an anti-syphilis treponemal antibody, or a syphilis treponemal antigen was carried. Anti-syphilis treponemal antibody is obtained by reacting the reaction product generated by the antigen-antibody reaction with the anti- Treponema pallidum antibody with the enzyme-labeled second antibody using a plastic microplate and measuring the degree of enzyme activity of the reaction product. Enzyme immunoassays and the like have been used to quantify the.

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.

On the other hand, it is known that there are various molecular weight surface antigen proteins of Treponema pallidum, and the major one in the antigen-antibody reaction is a surface antigen protein of Treponema pallidum with a molecular weight of 47 kDa (hereinafter , 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)). The amino acid sequences of these three types of surface antigen proteins are different, and they are completely different proteins.

The production method of Treponema pallidum surface antigen protein using the gene recombination technique is described in Japanese Patent Publication No. 59-5021.
No. 31, Japanese Patent Publication No. 2-500403, etc. (of which, the Japanese Patent Publication No. 2-500403 discloses a method for producing a 47 kDa antigen), and the obtained antigenic protein A method for detecting an anti- Treponema pallidum antibody using the same has also been proposed. By using this method, it is considered possible to stably obtain a large amount of Treponema pallidum antigen. However, the special table Sho 59-5
No. 02131 and Japanese Patent Publication No. 2-500403 do not describe details of a method for detecting an anti- Treponema pallidum antibody using the obtained antigenic protein.

[0006] In addition, to date, using the Treponema pallidum surface antigen protein obtained by the gene recombination technique,
No practical immunological syphilis diagnostic reagent has been reported. Possible reasons for this are that the sensitivity of the measurement is insufficient and that the specificity does not match that of the conventional immunological syphilis diagnostic reagent, but the lack of sensitivity is presumed to be the major cause.

Further, in Japanese Patent Laid-Open No. 7-287017, a fusion protein of glutathione-S-transferase and 17 kDa antigen, and glutathione-S-transferase and 15 kDa are used by a gene recombination technique.
A diagnostic reagent for producing a fusion protein of a antigen and measuring anti- Treponema pallidum antibody using them is disclosed. However, this reagent is not suitable for glutathione-S-
Since it is bound to transferase, it cannot be said to be a pure Treponema pallidum surface antigen protein, and glutathione-S-transferase is present in the reaction system of the reagent, which may cause adverse effects on 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.
To provide a method for producing an immunological syphilis diagnostic reagent, which has a stable antigenic activity and uses a surface antigen protein of Treponema pallidum obtained by a gene recombination technique and which has practically usable sensitivity. Is.

[0009]

The method for producing an immunological syphilis diagnostic reagent of the present invention comprises the steps of using a T. syphilis surface antigen protein obtained by a gene recombination technique and an insoluble carrier.
It is characterized in that the surface antigen protein is supported on the insoluble carrier by mixing in a solvent containing a valent metal ion.

The syphilis treponemal surface antigen protein used in the present invention is not particularly limited as long as it is a syphilis treponemal surface antigen protein obtained by a gene recombination technique. For example, syphilis treponemal surface antigen protein having a molecular weight of 47 kDa. , 44kDa Treponema pallidum surface antigen protein (Journal of Genetical Microbiology 133,17
93-1803 (1987)), 42 kDa Treponema pallidum surface antigen protein (Infectionand Immunity 57 (9), 2612-623 (19)
89)), 35 kDa Treponema pallidum surface antigen protein (Infection and Immunity 59 (10), 3536-3546 (1991)),
34 kDa Treponema pallidum surface antigen protein (Infect
ion and Immunity 57 (11), 3314-3323 (1989)), 17kD
T. pallidum surface antigen protein of 15a and 15kDa trepidoma pallidum surface antigen protein, etc., which are considered to be major surface antigen proteins of T. pallidum 4
It is preferably at least one selected from the group consisting of a 7 kDa surface antigen protein, a 17 kDa surface antigen protein and a 15 kDa surface antigen protein.

The Treponema pallidum surface antigen protein used in the present invention is limited to those obtained by gene recombination techniques, but any cloning method can be used as long as it is cloned from the gene of S. syphilis. Such a method may be used. 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.

The insoluble carrier used in the present invention includes
Examples thereof include plastic microplates, glass fibers, organic polymer particles, microorganisms, blood cells, cell membrane pieces 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.

The method for producing an immunological syphilis diagnostic reagent of the present invention comprises mixing the above surface antigen protein of Treponema pallidum with an insoluble carrier in a solvent containing a divalent metal ion to form the surface antigen protein. It is supported on the insoluble carrier.

Examples of the above-mentioned divalent metal ion include magnesium ion, calcium ion, zinc ion and the like, with magnesium ion or calcium ion being particularly preferred. Also, these are usually chlorides, ie MgCl ₂ , CaCl ₂ , Zn.
Used as Cl ₂ . MgCl ₂ , CaCl ₂ , Z
Although nCl _{2 and the} like can be produced by various methods, commercially available products may be used.

As a solvent for dissolving the above metal ions,
Usually, water, especially buffers such as Tris buffer, glycine buffer, veronal buffer, borate buffer are preferred.

When the above-mentioned Treponema pallidum surface antigen protein and the insoluble carrier are mixed in a solvent containing a divalent metal ion, if the concentration of the divalent metal ion is low, the sensitivity of the obtained diagnostic reagent is sufficient. If not, the metal ion will have some effect on the surface antigen protein of Treponema pallidum and the sensitivity will not be sufficiently high.
0.1-300 mmol / l is preferable, 1-50 mm
ol / l is more preferred.

In the present invention, the mechanism by which the Treponema pallidum surface antigen protein is carried on the insoluble carrier is by physical adsorption. When Treponema pallidum surface antigen protein and the above insoluble carrier are mixed in a solvent containing a divalent metal ion,
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 50 ° C because if the reaction temperature becomes low, the reaction will not proceed sufficiently, and if it becomes high, the surface antigen protein may be denatured and lose its antigenicity. Whether the pH during the adsorption reaction is low or high, the surface antigen protein may be denatured and lose its antigenicity, so that it is preferably 4 to 10.

In the production method of the present invention, the divalent metal ion may be present in the solvent for mixing the surface antigen protein and the insoluble carrier. For example, the divalent metal ion may be contained in the surface antigen protein solution in advance. And a method of containing it in a buffer solution used at the time of mixing.

The method for measuring anti- Treponema pallidum antibody in a sample using the immunological syphilis diagnostic reagent obtained according to the present invention comprises the steps of mixing the sample with the reagent of the present invention to prepare syphilis carried on an insoluble carrier. An antigen-antibody complex is produced by reacting the surface antigen protein of treponemal with the anti-syphilis treponemal antibody in the sample. Next, an enzyme-labeled anti-human globulin antibody is bound to this antigen-antibody complex, then a chromogenic substrate that is decomposed by the enzyme is added, and the amount of the bound enzyme-labeled anti-human globulin antibody is changed according to the degree of color development. taking measurement. Since the degree of this color development correlates with the amount of anti- Treponema pallidum antibody in the sample, the amount of the antibody can be measured.

Any enzyme can be used as the above-mentioned assay as long as it can bind to the anti-human globulin antibody.
Any chromogenic substrate may be used as long as it can be decomposed by a binding enzyme and the amount thereof can be measured. Examples of the substitute for the enzyme include biotin and avidin, but any substance can be used as long as it can bind to the anti-human globulin antibody and can quantify the bound substance.

Explaining a concrete example of the above-mentioned measuring method, first, the surface antigen protein of Treponema pallidum is physically adsorbed on a microplate, and then a sample containing an anti- Treponema pallidum antibody is added to bind to the antigen protein. .
Next, peroxidase (hereinafter, referred to as POD) labeled anti-human globulin antibody is bound, and the POD activity of the bound labeled anti-human globulin antibody is determined as the chromogenic substrate o.
Measuring the amount of decomposition of phenylenediamine by a colorimetric method,
The amount of labeled antibody bound, in other words, the amount of anti-syphilis treponemal antibody in the sample is measured.

In this immunoassay, the conditions of the antigen-antibody reaction are the same as in the usual case, and various buffers are used as the reaction medium. The buffer may have any ionic strength or pH that does not deactivate the test substance and does not inhibit the antigen-antibody reaction. For example, glycine buffer, Tris buffer, etc. are used.
The pH during the reaction is preferably 4 to 10, more preferably 6 to 8. The reaction temperature is preferably 10 to 50 ° C., and 15 to 4
5 ° C is more preferable.

[0024]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described with reference to embodiments, and its effects will be specifically described. 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 15 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 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 in the same manner as in the production of (2) 47 kDa antigen in Reference Example 1 except that the expression "a antigen" was replaced with the expression "15 kDa antigen".

Reference Example 3 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.

Example 1 1.0 m of 10 mM Tris-HCl buffer (pH 7.4) containing MgCl ₂ (manufactured by Wako Pure Chemical Industries, Ltd.) at a concentration of 1.1 mM
0.1 ml of the 47 kDa antigen (protein concentration: 1 μg / ml) obtained in Reference Example 1 was added to 1 (MgCl ₂
Final concentration of 1.0 mM). 0.1 ml of this 47 kDa antigen solution is added to the wells of a polystyrene 96-well microplate (Corning) and left at 25 ° C. for 2 hours. Then, the wells were washed 3 times with 10 mM Tris-HCl buffer (pH 7.4) in order to remove the 47 kDa antigen solution not bound to the microplate. Next, the well contains 3% (w / v) of bovine serum albumin (hereinafter, bovine serum albumin is referred to as BSA) 1.
200 μl of 0 mM Tris-HCl buffer (pH 7.4) was added, and the mixture was allowed to stand at 25 ° C. for 2 hours for blocking (adsorption of BSA on the surface of the microplate to prevent nonspecific reaction). The wells were then washed 3 times with 10 mM Tris-HCl buffer (pH 7.4) to remove BSA that was not bound to the microplate. Thus, a 47 kDa antigen-supporting microplate (immunological syphilis diagnostic reagent) was prepared.

Example 2 Instead of the 47 kDa antigen in Example 1, Reference Example 2
A 15-kDa antigen-supporting microplate (immunological syphilis diagnostic reagent) was prepared in the same manner as in Example 1 except that the 15-kDa antigen prepared in 1. was used.

Example 3 Instead of the 47 kDa antigen in Example 1, reference example 3 was used.
A 17-kDa antigen-supporting microplate (immunological syphilis diagnostic reagent) was prepared in the same manner as in Example 1 except that the 17-kDa antigen prepared in 1. was used.

Example 4 The MgCl ₂ (manufactured by Wako Pure Chemical Industries, Ltd.) in Example 1 was added to 1.1.
10 mM Tris-HCl buffer (pH)
Instead of 7.4), MgCl ₂ (manufactured by Wako Pure Chemical Industries, Ltd.)
10 mM Tris-HCl buffer solution (p containing 5 mM concentration)
H7.4) was used and the same procedure as in Example 1 was carried out to prepare a 47 kDa antigen-supporting microplate (immunological syphilis diagnostic reagent).

Example 5 The MgCl ₂ (manufactured by Wako Pure Chemical Industries, Ltd.) in Example 1 was added to 1.1.
10 mM Tris-HCl buffer (pH)
In the same manner as in Example 1 except that 10 mM Tris-HCl buffer (pH 7.4) containing CaCl ₂ (manufactured by Wako Pure Chemical Industries, Ltd.) at a concentration of 1.1 mM was used instead of 7.4). By operation, a 47 kDa antigen-supporting microplate (immunological syphilis diagnostic reagent) was prepared.

Comparative Example 1 MgCl ₂ (manufactured by Wako Pure Chemical Industries, Ltd.) in Example 1 was added to 1.1.
10 mM Tris-HCl buffer (pH)
47 kDa was operated in the same manner as in Example 1 except that 10 mM Tris-HCl buffer (pH 7.4) containing no MgCl ₂ (manufactured by Wako Pure Chemical Industries, Ltd.) was used instead of 7.4).
An antigen-carrying microplate was prepared.

Performance Evaluation of Immunological Syphilis Diagnostic Agent The performance of the immunological syphilis diagnostic reagents obtained in Examples 1 to 5 and Comparative Example 1 was evaluated as follows. 100 μl of anti- Treponema pallidum antibody-positive standard serum was placed in the well of the microplate carrying the surface antigen protein of Treponema pallidum.
After addition, the mixture was allowed to stand at 25 ° C. for 2 hours and then washed 3 times with 10 mM Tris-HCl buffer (pH 7.4). 1 in this well
POD-labeled anti-human globulin antibody (manufactured by Kappel) diluted 100 times with 0 mM Tris-HCl buffer (pH 7.4)
100 μl was added, and the mixture was allowed to stand at 25 ° C for 2 hours, then 10 mM
It was washed 3 times with Tris-HCl buffer (pH 7.4). 50 μl of 30% hydrogen peroxide solution was added to the well after washing, and then o-phenylenediamine (manufactured by Wako Pure Chemical Industries, Ltd.)
Further, 50 μl of 10 mM Tris-HCl buffer (pH 7.4) containing 20 mM of the above was added, and the mixture was added at 25 ° C. for 2 hours.
After standing for 1 hour, 100 μl of 1N sulfuric acid is used as a reaction terminator.
1 was added and the absorbance at 580 nm was measured using a microplate reader.

The above-mentioned anti- Treponema pallidum antibody-positive standard serum is a commercially available diagnostic reagent kit for Treponema pallidum,
Sekisui Chemical Co., Ltd. product name "Mediace TPLA" attached to each concentration 0,109,242,460 titer unit (hereinafter abbreviated as TU. TU is the diagnostic reagent kit " The unit representing the anti-syphilis treponemal antibody amount measured using "Mediace TPLA".) Was used. Thus obtained, 0, 109,
242, 460 for each T.I. U. The absorbance at 580 nm for the anti-syphilis treponemal antibody-positive standard serum of Table 1 is shown in Table 1.

[0038]

[Table 1]

From Table 1, the immunological syphilis diagnostic reagents of Examples 1 to 5 have the sensitivity that can be used as a calibration curve, but the immunological syphilis diagnostic reagent of Comparative Example 1 is used as a calibration curve. I couldn't get the sensitivity I could.

[0040]

As described above, the method for producing the immunological syphilis diagnostic reagent according to claim 1 can be mass-produced and has stable antigenic activity. Treponema syphilis obtained by the gene recombination technique. Since it consists of an insoluble carrier carrying a surface antigen protein, it can be stably produced as compared with a reagent produced using a conventional Treponema pallidum syphilis antigen obtained from cells. Since the obtained immunological syphilis diagnostic reagent has a practically usable sensitivity, it can be effectively used for syphilis diagnosis in clinical diagnosis.

The structure of the method for producing an immunological syphilis diagnostic reagent according to claim 2 is as described above, wherein the T. pallidum surface antigen protein has a molecular weight of 47 kDa, the T. pallidum surface antigen protein has a molecular weight of 47 kDa, and the T. pallidum surface antigen protein has a molecular weight of 17 kDa. The method for producing an immunological syphilis diagnostic reagent according to claim 1, which is at least one member selected from the group consisting of a 15-kDa Treponema pallidum surface antigen protein, which is clinically useful because a more sensitive immunological syphilis diagnostic reagent can be obtained. It can be effectively used for diagnosis of syphilis.

The structure of the method for producing the immunological syphilis diagnostic reagent according to claim 3 is as described above, and the divalent metal ion is magnesium ion or calcium ion. Since it is a method for producing a reagent and a more sensitive immunological syphilis diagnostic reagent can be obtained, it can be effectively used for syphilis diagnosis in clinical diagnosis.

Claims (3)

[Claims] 1. A syphilis treponemal surface antigen protein obtained by a gene recombination technique and an insoluble carrier are mixed in a solvent containing a divalent metal ion to give the surface antigen protein as the insoluble carrier. A method for producing an immunological syphilis diagnostic reagent, which comprises carrying the reagent. 2. The Treponema pallidum surface antigen protein is at least one member selected from the group consisting of Treponema pallidum surface antigen protein having a molecular weight of 47 kDa, Treponema pallidum surface antigen protein having a molecular weight of 17 kDa, and Treponema pallidum surface antigen protein having a molecular weight of 15 kDa. 1
A method for producing the immunological syphilis diagnostic reagent described. 3. The method for producing an immunological syphilis diagnostic reagent according to claim 1, wherein the divalent metal ion is magnesium ion or calcium ion.