JPH09304391A - Immunological treponema pallidum diagnostic reagent and its manufacture - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide the same kind of performance as a case prepared from a fungus body despite of using an antigen prepared by a genetic engineering method by mixing an insoluble carrier and a specific treponema pallidum antigen protein in an aqueous medium containing water soluble high polymer. SOLUTION: A treponema pallidum antigen protein obtained by cloning using a genetic engineering method is carried by an insoluble carrier so as to prepare an immunological diagnostic reagent. In this case, the insoluble carrier and the treponema pallidum antigen protein are mixed in an aqueous medium containing water soluble high polymer. An antigen protein of molecular weight 17kDa or an antigen protein of molecular weight 15kDa is preferable to be used for this treponema pallidum antigen protein. Polyethylene glycol or polyglycosilethyl methacrylate are preferable for the water soluble high polymer Latex, composed of synthetic high polymer is preferable as the insoluble carrier.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention optically measures the degree of aggregation caused by an antigen-antibody reaction between an anti-treponemal antibody contained in a test substance and a carrier carrying (sensitized) an antigen. Thus, the present invention relates to a diagnostic reagent used for measuring an anti-treponemal antibody in a test substance and diagnosing an infection with treponema pallidum. For more details,
The present invention relates to a method for producing a diagnostic reagent obtained by allowing an insoluble carrier to carry a Treponema pallidum antigen cloned by a genetic engineering technique.

[0002]

2. Description of the Related Art Syphilis is an infectious disease caused by Treponema pallidum, and whether or not it is infected by syphilis is judged by the presence of anti- Treponema pallidum antibody in the blood. In the field of clinical testing, an immunoassay method has been conventionally used in which an insoluble carrier is loaded with T. pallidum syphilis, and the antibody is measured by detecting the degree of aggregation of the insoluble carrier produced by an antigen-antibody reaction with an anti-Treponema syphilis antibody. Has been. Hemagglutination and latex agglutination methods are known as such measurement methods.

Conventionally, the Treponema pallidum antigen used in these methods has been obtained by a method of extracting from syphilis cells (see JP-A-2-234063).

However, culturing S. syphilis in vitro is not possible by the current technology except for culturing in the testes of rabbits, and it has been difficult to obtain a large amount of antigen. Moreover, even when syphilis cells are cultured in the testes of rabbits, there are many unstable factors such as the culture being influenced by the physical condition of the rabbit, the purification process from the cells being complicated, and necessary for the preparation of diagnostic reagents. Those with various antigenic activities were not always constantly obtained.

It is known that there are various molecular weight surface antigen proteins of Treponema pallidum,
Molecular weight 4 is the major factor in the antigen-antibody reaction
An antigen protein having a molecular weight of 7 kDa, an antigen protein having a molecular weight of 17 kDa, and an antigen protein having a molecular weight of 15 kDa are known. Since the genes encoding these surface antigens have already been cloned and their amino acid sequences have been determined, it is possible to produce them by genetic engineering (Infection and Immunity 57 (1), 196-203 (1989), In
fection and Immunity 61,1202-1210 (1993), Molecula
r 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.

[0006] As methods for producing diagnostic reagents using these proteins, those disclosed in Japanese Patent Publication No. 59-502131 and Japanese Patent Publication No. 2-500403 are known. There are no reports available yet.
This may be due to lack of measurement sensitivity, inconsistency in specificity, or the like.

Further, in the method disclosed in Japanese Patent Laid-Open No. 7-287017, glutathione-S-
Transferase and 15kDa antigen or 17kDa
I am making a diagnostic reagent with a complex protein with an antigen,
Since the obtained antigen binds glutathione-S-transferase, it cannot be said to be a pure syphilis antigen, and it is considered that glutathione-S-transferase is present in the reaction system and adversely affects the sample. To be

[0008]

DISCLOSURE OF THE INVENTION The present invention is intended to solve the above problems, and its object is to provide a diagnostic reagent using a genetically engineered syphilis treponemal antigen. It is an object of the present invention to provide a diagnostic reagent having the same performance as a diagnostic reagent obtained by using an antigen produced and purified from bacterial cells.

[0009]

The method for producing a diagnostic reagent for immunological Treponema pallidum syphilis of the present invention comprises carrying an immunological diagnosis of an antigen protein of Treponema pallidum syphilis, which can be cloned using a genetic engineering technique, on an insoluble carrier. In producing the reagent, an insoluble carrier and the Treponema pallidum antigen protein are mixed in an aqueous medium containing a water-soluble polymer. There are usually several types of Treponema pallidum antigen proteins.

The above-mentioned Treponema pallidum antigen protein is an antigen protein having a molecular weight of 47 kDa (hereinafter, 47 kDa).
An antigen protein having a molecular weight of 17 kDa (hereinafter abbreviated as 17 kDa antigen) or an antigen protein having a molecular weight of 15 kDa (hereinafter abbreviated as 15 kDa antigen) is preferable.

The above water-soluble polymer is preferably polyethylene glycol or polyglycosyl ethyl methacrylate.

As the insoluble carrier, latex made of synthetic polymer is preferable.

By the above method, a highly sensitive and highly accurate diagnostic reagent for Treponema pallidum syphilis, in which an insoluble carrier carries treponema pallidum antigen cloned by a genetic engineering technique is provided.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The Treponema pallidum antigen used in the present invention may be any one cloned from a syphilis cell gene by a genetic engineering method, and any cloning method may be used. Since the gene of Treponema pallidum antigen is already known, the 47 kDa antigen is taken as an example. First, the DNA for the 47 kDa antigen is amplified and purified by the PCR method, and the obtained DNA fragment is inserted into an appropriate expression vector, and then this vector is used. Is introduced into Escherichia coli, and a large amount of E. coli is produced. In order to efficiently purify a 47 kDa antigen from E. coli, a vector containing a glutathione-S-transferase (hereinafter abbreviated as GST) gene is used as a vector (see Japanese Patent Publication No. 6-81596). In this method, the Treponema pallidum antigen gene is inserted after the GST gene, and this vector is introduced into E. coli to transform GS into E. coli.
A fusion protein of T and 47 kDa antigen is produced. After this is sufficiently produced, the bacterial cells are disrupted, and the fusion protein can be separated by contacting the supernatant obtained by centrifuging the bacterial cell with immobilized glutathione. This fusion protein can then be cleaved to give the 47 kDa antigen.

The Treponema pallidum antigens used in the present invention include 44 kDa (Journal of Genet) in addition to the 47 kDa antigen, 17 kDa antigen and 15 kDa antigen described above.
icalMicrobiology 133,1793-1803 (1987)), 42kD
a (Infection and Immunity57 (9), 2612-2623 (198
9)), 35 kDa (Infection and Immunity 59 (10), 35
36-3546 (1991)), 34 kDa (Infection and Immuni
ty 57 (11), 3314-3323 (1989)) and the like. 47k of these antigens
Da antigen, 17 kDa antigen and 15 kDa antigen are considered to be the main antigens and are preferably used.

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

Examples of the organic polymer powder include natural polymer powders such as insoluble agarose, cellulose and insoluble dextran, polystyrene, styrene-styrene sulfonic acid copolymer, styrene-methacrylic acid copolymer, acrylonitrile-butadiene. -Styrene copolymers, vinyl chloride-acrylic acid ester copolymers, synthetic polymer powders such as vinyl acetate-acrylic acid ester copolymers, and the like, in particular latexes in which the synthetic polymer powders are uniformly suspended. preferable.

When the above latex is used, the particle size is preferably 0.1 to 1.0 μm, more preferably 0.1 to 1.0 μm.
It is 0.5 μm.

The water-soluble polymer used in the present invention is, for example, polyethylene glycol or polyglycosyl ethyl methacrylate, that is, glycosyl ethyl methacrylate represented by the following formula.

Embedded image

The (co) polymer obtained by using
However, the invention is not particularly limited. The polyethylene glycol preferably has an average molecular weight of 1,000 to 50,000, more preferably 6,000 to 20,000. If the average molecular weight is 1,000 or less, the effects of the present invention described below may not be obtained. If the average molecular weight is 50,000 or more, the insoluble carrier may self-aggregate. Polyglycosyl ethyl methacrylate has an average molecular weight of 100,000 to 1,50.
50,000 is preferable, 500,000-1,2
More preferably, it is 100,000. Average molecular weight of 10
If it is less than 10,000, the effect of the present invention may not be obtained. Further, if the average molecular weight is 1,500,000 or more, the insoluble carrier may self-aggregate.

Various buffer solutions are preferable as the aqueous medium used for supporting the Treponema pallidum antigen protein on an insoluble carrier. 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,
Phosphate buffer, glycine buffer, Tris buffer and the like are used.

The concentration of the water-soluble polymer in supporting the Treponema pallidum antigen protein on an insoluble carrier is preferably 0.05 to 10% (W / V), more preferably 1 to 5% (W / V). ). The concentration of water-soluble polymer is 0.
If it is at most 05% (W / V), the effects of the present invention described below may not be obtained. In addition, this concentration is 10% (W
/ V) or more, the Treponema pallidum antigen protein may self-aggregate due to the water-soluble polymer.

These water-soluble polymers can be produced by various methods, but commercial products may be used.

The loading of the Treponema pallidum antigen protein on the insoluble carrier is to bind the Treponema pallidum antigen protein to the insoluble carrier mainly by physical adsorption. At this time, the Treponema pallidum antigen protein and the insoluble carrier are easily adsorbed in an aqueous medium by the electric charge.

In the production method of the present invention, the water-soluble polymer only needs to be present in the aqueous medium when the Treponema pallidum antigen protein is supported on the insoluble carrier, and the aqueous solution containing the Treponema pallidum antigen protein or the insoluble carrier in advance. It may be contained in the medium.

The temperature of the aqueous medium for the physical adsorption is preferably 0 ° C to 50 ° C. Adsorption may not proceed sufficiently at a temperature lower than 0 ° C.
At a temperature higher than 50 ° C, the antigenic protein may be denatured and lose its antigenicity.

PH of the aqueous medium for the physical adsorption
Is preferably 4 to 10. If it is more acidic than pH 4 or more alkaline than pH 10, the antigenic protein may be denatured.

The measurement of anti-treponemal antibody using the diagnostic reagent prepared according to the present invention is performed according to a known method. That is, the anti-treponemal antibody in the test substance is mixed with the anti-treponemal antibody contained in the test substance and the diagnostic reagent prepared by the present invention is mixed, and the degree of agglutination produced is optically measured. taking measurement.

Specifically, the method of optically detecting the degree of aggregation of the insoluble carrier is performed by using an optical instrument for measuring the scattered light intensity, the absorbance and the transmitted light intensity. The measurement wavelength used is preferably 250 to 1,000 nm.
In addition, this detection method is carried out according to a known method by increasing or decreasing the scattered light intensity, the absorbance or the transmitted light intensity by selecting the particle size of the insoluble carrier to be used or the concentration of the reactant and the setting of the reaction time. Is performed, and these methods can be used in combination.

In the immunoassay by the antigen-antibody reaction, the reaction conditions 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, phosphate buffer, glycine buffer, Tris buffer, etc. are used. The pH of the reaction is preferably 4-10, particularly preferably 6-8. The reaction temperature is preferably 10
To 50 ° C, particularly preferably 30 to 40 ° C.

[0031]

EXAMPLES Examples of the present invention will be described below, and the effects thereof will be specifically described.

Example 1 1) Preparation of antigen As an example, a method for preparing a 47 kDa antigen will be described.

Treponema pallidum Treponema pallidum was extracted from the rabbit testis to obtain genomic DNA.
Since the DNA sequence of the 47 kDa antigen is already known,
Based on them, a primer was prepared using a DNA synthesizer. 47k by PCR using this primer
A DNA fragment of Da antigen was obtained. This DNA fragment was inserted into the vector pGEX43, which was created to express the fusion protein with GST. The recombinant thus obtained was designated as pGEX43-47k. This was introduced into Escherichia coli, and isopropyl-β-(-)-thiogalactopyranoside was added to the culture solution to give 47 kDa antigen and GST.
The expression of the fusion protein was induced. Escherichia coli in which the fusion protein was sufficiently expressed was sonicated to disrupt the cells, and glutathione sepharose 4B (Pharmacia) was added to the supernatant obtained by removing the cell membrane, nucleus, etc. by centrifugation,
The fusion protein was left overnight and adsorbed on glutathione sepharose. Glutathione sepharose to which the fusion protein was adsorbed was recovered by centrifugation and packed in a column. After washing several times, the fusion protein was released from the glutathione sepharose and eluted by flowing a glutathione solution.

47 kDa antigen-GS thus purified
Add the thrombin solution to the T-fusion protein solution,
The bond between the kDa antigen and GST was cleaved. As a result, 47
A solution containing the kDa antigen, GST and thrombin was obtained. GST could be removed by applying this solution to a glutathione sepharose column. Further, thrombin could be removed by applying this solution to a heparin column, and a purified 47 kDa antigen was obtained.

Purified 17 kDa antigen and purified 15 kDa antigen could be obtained in the same manner as described above.

2) Preparation of 47 kDa Antigen Protein-Supporting Latex Solution 0.1 ml of 47 kDa antigen protein (protein concentration; 1 mg / ml) obtained in 1) above was added to 1 ml of 100 mM phosphate buffer (pH 7.4). . 0.5 ml of this antigen protein solution and 10% (W / V) polyethylene glycol 6,000 (hereinafter abbreviated as PEG 6,000, average molecular weight 7,500, manufactured by Nacalai Tesque)
Containing 100 mM phosphate buffer (pH 7.4) 0.5 m
1 was added and mixed.

To 0.5 ml of the polyethylene glycol-containing antigen protein solution, 0.1 ml of latex (solid content 10%, Sekisui Chemical Co., Ltd.) made of polystyrene having an average particle diameter of 400 nm was added, and the whole liquid was stirred at room temperature for 1 hour. did.

Next, 5 ml of 100 mM phosphate buffer (pH 7.4) containing 1% (W / V) bovine serum albumin (hereinafter abbreviated as BSA) was added, and the whole solution was stirred at room temperature for 1 hour. .

This mixed solution was added at 30 rpm at 15,000 rpm.
Centrifuged for minutes. 1% of BSA was added to the obtained precipitate.
(W / V) containing 100 mM phosphate buffer (pH 7.
4) 5 ml was added and the latex was resuspended, 47 kD
A latex solution carrying an antigen protein was prepared.

3) Preparation of sample dilution liquid 1 mM BSA was added to 100 mM phosphate buffer (pH 7.4).
% (W / V) and PEG 6,000 were dissolved at 2% (W / V) to prepare a sample diluent.

PEG 6,000 contained in this solution
Was used as a promoter for the antigen-antibody reaction.

4) Reagent for measuring anti-treponemal antibody for syphilis The reagent for measuring anti-treponemal antibody in this example is composed of a first reagent consisting of the above-mentioned antigen protein-carrying latex solution and a second reagent consisting of the above-mentioned sample diluent. It is a two-component system reagent.

5) Method for measuring anti-treponema pallidum antibody The sensitivity of the calibration curve and the amount of anti-treponemal antibody in human serum were measured with an automatic analyzer for biochemistry (Hitachi 7050, manufactured by Hitachi, Ltd.). The operation method is shown below.

To 20 μl of syphilis-positive standard serum (the amount of anti-treponemal antibody is known), sample dilution liquid (second reagent) 350 μl
l was added and mixed, and after a certain period of time, 50 μl of the antigen protein-carrying latex solution (first reagent) was added and mixed.

The amount of change in turbidity from 80 seconds to 320 seconds after the addition of the latex solution was taken as the reaction amount of the standard serum.

Human serum (15 samples, instead of standard serum)
The amount of anti-treponemal antibody was unknown) and the amount of reaction of the serum was determined in the same manner as described above, except that the amount was used. The amount of anti-treponemal antibody in human serum was determined from the reaction amount of standard serum and human serum. All reactions were carried out at 37 ° C., and the measurement wavelength was 570 nm.

In the sample measurement, a value measured using a commercial product (Mediace TPLA, manufactured by Sekisui Chemical Co., Ltd.) was used as a control (hereinafter referred to as a control product).

Example 2 Preparation of an anti- Treponema pallidum antibody measuring reagent and use thereof in the same manner as in Example 1) except that 15 kDa antigen was used in place of 47 kDa antigen in step 2) of Example 1. The anti- Treponema pallidum antibody was measured.

Example 3 Production of an anti-syphilis treponemal antibody measuring reagent and use thereof in the same manner as in Example 1) except that 17 kDa antigen was used in place of 47 kDa antigen in step 2) of Example 1. The anti- Treponema pallidum antibody was measured.

Example 4 In step 2) of Example 1, 10% (W / V) PEG was used.
Instead of 100 mM phosphate buffer containing 6,000,
Production of anti-syphilis treponemal antibody measuring reagent and measurement of anti-syphilis treponemal antibody using the same as in Example 1) except that 100 mM phosphate buffer containing 2% (W / V) PEG 6,000 was used. Was carried out.

Example 5 In step 2) of Example 1, 10% (W / V) PEG was used.
Instead of 100 mM phosphate buffer containing 6,000,
10% (W / V) polyethylene glycol 20,000
Production of an anti-syphilis treponemal antibody measurement reagent and anti-syphilis treponemal antibody measurement using the same were carried out in the same manner as in Example 1) except that a 100 mM phosphate buffer containing the above was used.

Example 6 In step 2) of Example 1, 10% (W / V) PEG was used.
Instead of 100 mM phosphate buffer containing 6,000,
Production of an anti- Treponema pallidum antibody measuring reagent and an anti- Treponema pallidum antibody using the same as in Example 1) except that a 100 mM phosphate buffer containing 5% (W / V) polyglycosylethyl methacrylate was used. The measurement was carried out.

Comparative Example 1 In step 2) of Example 1, 100 mM phosphate buffer (pH 7.4) containing no PEG 6,000 and BSA was used instead of 100 mM phosphate buffer (pH 7.4) containing PEG 6,000 and BSA. In the same manner as in Example 1 except that was used, an anti-syphilis treponemal antibody measurement reagent was prepared and an anti-syphilis treponemal antibody measurement was performed using the same.

Comparative Example 2 In step 2) of Example 2, a 100 mM phosphate buffer solution (pH 7.4) containing no PEG 6,000 and BSA instead of the 100 mM phosphate buffer solution (pH 7.4) was used. In the same manner as in Example 2 except that was used, an anti-syphilis treponemal antibody measurement reagent was prepared and an anti-syphilis treponemal antibody measurement was performed using the same.

Comparative Example 3 In step 2) of Example 3, instead of the 100 mM phosphate buffer (pH 7.4) containing PEG 6,000 and BSA, a 100 mM phosphate buffer (pH 7.4) not containing them was used. In the same manner as in Example 3 except that was used, an anti-syphilis treponemal antibody measurement reagent was prepared and an anti-syphilis treponemal antibody measurement was performed using the same.

Performance Test Results 1) Calibration Curve Sensitivity The results of the calibration curve sensitivity measurements carried out in the above Examples and Comparative Examples are shown in Tables 1 and 2.

[0057]

[Table 1]

[0058]

[Table 2]

In the table, the standard is the syphilis-positive standard serum of the above-mentioned commercial product (Mediace TPLA). Also, T.I.
U. Is a unit showing the amount of anti-treponemal antibody when serum was measured with this commercial product (Mediace TPLA).

From the above table, it can be seen that the calibration curve sensitivity was obtained in the examples, whereas the calibration curve sensitivity was hardly obtained in the comparative examples.

2) Specimen Measurement Regarding the measurement of anti-treponemal antibody in human serum, the calibration curve sensitivity was hardly obtained in Comparative Examples 1 to 3, and therefore the specimen measurement was not carried out. In Examples 1 to 6 and the control product, antibody measurement was performed. The measurement results are shown in Table 3.

[0062]

[Table 3]

From the above table, it can be seen that in the Examples, the measured values of anti-treponemal antibody in serum also agree well with the measured values of commercially available products.

[0064]

INDUSTRIAL APPLICABILITY According to the present invention, a diagnostic reagent is produced using a genetically engineered Treponema pallidum antigen,
It is possible to provide a diagnostic reagent having the same performance as a diagnostic reagent obtained by using an antigen purified from bacterial cells.

Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI Technical display area C12R 1:19) (C12N 15/09 C12R 1: 645) (C12P 21/02 C12R 1:19)

Claims (5)

[Claims] 1. A method for producing an immunological diagnostic reagent, which comprises supporting an insoluble carrier of treponema pallidum antigen protein, which can be cloned using a genetic engineering method, in an aqueous medium containing a water-soluble polymer. A method for producing an immunological treponemal syphilis diagnostic reagent, which comprises mixing an insoluble carrier with the T. syphilis antigen protein. 2. The method according to claim 1, wherein the Treponema pallidum antigen protein is at least one selected from the group consisting of an antigen protein having a molecular weight of 47 kDa, an antigen protein having a molecular weight of 17 kDa, and an antigen protein having a molecular weight of 15 kDa. Method. 3. The method according to claim 1, wherein the water-soluble polymer is polyethylene glycol or polyglycosyl ethyl methacrylate. 4. The production method according to claim 1, wherein the insoluble carrier is a latex made of a synthetic polymer. 5. An immunological Treponema pallidum diagnostic reagent produced by the method according to any one of claims 1 to 4.