JPH1090264A - Immunological diagnostic reagent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve the sensitivity by supporting a fused protein of diagnostically useful antigen protein and thioredoxine by an insoluble carrier. SOLUTION: Pallidum bacterium body is extracted form the crushed matter of a Treponema Pallidum passage rabbit testicle, and genom DNA is extracted to form a library. The DNA sequence of 47kDa antigen is known, and the formed library is used to provide a DNA fragment of 47kDa. The DNA fragment is inserted to a plasmid vector pTrxFus formed in order to manifest the fused protein with thioredoxine to name pTrxFus-47k. The-47k is introduced to colibaccilus and cultivated. Typtophan is added to the culture solution to guide the manifestation of the fused protein of 47kDa antigen with tioredoxin. The colibaccilus in which this protein is manifested is ultrasonically treated, and the supernatant form which cell membranes are removed by centrifugal separation is passed to a trade mark range column to adsorb the fused protein.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to an immunological diagnostic reagent comprising an insoluble carrier carrying an antigenic protein useful in diagnostics, particularly a Treponema pallidum surface antigen protein.

[0002]

2. Description of the Related Art In general, infectious diseases are determined by the presence of antibodies against bacteria, viruses and the like in blood. Further, it is known that an antibody against a protein secreted by cancer cells appears in a certain kind of cancer, and is used for cancer diagnosis. In the field of clinical examination, conventionally, these infectious disease antigen proteins and cancer cell secreted proteins are supported on an insoluble carrier, and the antibody is detected by detecting the degree of aggregation of the insoluble carrier caused by the antigen-antibody reaction with the antibody. Immunoassay methods for measuring have been used. Such measurement methods include:
Hemagglutination and latex agglutination methods are known.

One of such methods is disclosed in Japanese Patent Application Laid-Open No.
No. 500403 discloses a molecular weight of 4 for Treponema pallidum.
It is described that a 7 kDa antigen is prepared by genetic engineering, and an anti-treponema antibody is immunologically measured using the antigen.

Japanese Patent Application Laid-Open No. 7-287017 discloses a surface antigen protein of Treponema pallidum and glutathione-S-transferase (hereinafter referred to as GST).
A diagnostic reagent for measuring an anti-Treponemal antibody using a fusion protein with the above is disclosed. However,
This reagent has the disadvantage that if the molecular weight of the surface antigen protein of Treponema pallidum is large, the water solubility of the fusion protein with GST is reduced due to the hydrophobicity of the antigen.

[0005]

DISCLOSURE OF THE INVENTION The present invention has been made to solve the above problems, and an object of the present invention is to use a fusion protein having a higher water solubility as a fusion protein supported on an insoluble carrier, and to combine an antigen protein and glutathione. An object of the present invention is to provide a more sensitive immunological diagnostic reagent than when a fusion protein with -S-transferase is used.

[0006]

The immunological diagnostic reagent according to claim 1 is characterized in that a fusion protein of an antigen protein and thioredoxin, which is diagnostically useful, is carried on an insoluble carrier.

[0007] The immunological diagnostic reagent according to claim 2 is the immunological diagnostic reagent according to claim 1, wherein the antigen protein is a surface antigen protein of Treponema pallidum.

[0008] In the immunological diagnostic reagent according to the third aspect, the insoluble carrier is polystyrene, a styrene-styrene sulfonate copolymer or a styrene-methacrylic acid copolymer. Diagnostic reagent.

The antigenic protein useful in diagnostics used in the present invention is not particularly limited as long as it is an antigenic protein such as a pathogen for the purpose of the present invention. For example,
Bacterial antigen proteins include antigenic proteins such as Treponema pallidum, Helicobacter pylori, mycoplasma, and gonococci. Examples of virus antigen proteins include antigen proteins of human immunodeficiency virus, hepatitis B virus, hepatitis C virus, hepatitis G virus, genital herpes virus, Japanese encephalitis virus, rabies virus, and the like. Further, it may be an antigen protein specifically expressed by cancer cells. The present invention is particularly effective in the case of Treponema pallidum surface antigen protein.

In the case of the surface antigen protein of Treponema pallidum, the antigen protein may have a molecular weight of 47 kD.
a) Treponema pallidum surface antigen protein (Infection
andImmunity 57 (1), 196-203 (1989)), 44 kDa Treponema pallidum surface antigen protein (Journal of Genet)
ical Microbiology 133, 1793-1803 (1987)), 42 kDa
Treponema pallidum surface antigen protein (Infection an
d Immunity 57 (9), 2612-2623 (1989)), 35 kDa Treponema pallidum surface antigen protein (Infection and Im
munity 59 (10), 3536-3546 (1991)), 34 kDa Treponema pallidum surface antigen protein (Infection and Immuni
ty 57 (11), 3314-3323 (1989)), 17 kDa Treponema pallidum surface antigen protein (Infection and Immunity 6
1,1202-1210 (1993)), 15 kDa Treponema pallidum surface antigen protein (Molecular Microbiology 4,1371-1).
379 (1990)), and any of them can be used. Among these, 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, are preferred.

In the immunological diagnostic reagent of the present invention, a fusion protein of a single antigen protein and thioredoxin may be bound to an insoluble carrier to form a reagent. As described above, when a plurality of antigen proteins are known for one bacterium, a virus or a cancer cell, a fusion protein with thioredoxin for each of the plurality of antigen proteins is mixed to form the same insoluble protein. The reagent may be bound to a carrier, or a reagent in which each of the fusion proteins with thioredoxin separately bound to an insoluble carrier may be mixed to form a reagent.

The fusion protein of the antigen protein and thioredoxin used in the present invention is generally obtained by a genetic engineering technique. To explain this method in detail, if the antigen protein gene is known,
The DNA of the antigen protein is amplified and purified by the PCR method, and this is introduced into a plasmid vector containing the thioredoxin gene. The obtained plasmid vector is introduced into Escherichia coli, and Escherichia coli is caused to produce a large amount of the fusion protein.

The above plasmid vector may be any plasmid vector having a thioredoxin gene and capable of producing a fusion protein.
Accordingly, any promoter or drug resistance gene for expressing the fusion protein may be used.

The insoluble carrier used in the present invention includes:
For example, organic polymer particles, microorganisms, blood cells, cell membrane fragments and the like can be mentioned.

Examples of the organic polymer particles include natural polymer particles such as insoluble agarose, cellulose, and insoluble dextran; polystyrene, styrene-styrene sulfonate copolymer, styrene-methacrylic acid copolymer, acrylonitrile-butadiene. -Synthetic polymer particles such as styrene copolymer, vinyl chloride-acrylate copolymer, vinyl acetate-acrylate copolymer and the like, and especially latex in which the synthetic polymer particles are uniformly suspended. Polystyrene, styrene-styrene sulfonate copolymer particles or styrene-methacrylic acid copolymer particles are particularly preferred.

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

In the present invention, the method for supporting the fusion protein on the insoluble carrier includes a method using physical adsorption,
A method of bonding by a chemical reaction and the like can be mentioned.

As a method using the above-mentioned chemical reaction, for example, there is a method in which a water-soluble carbodiimide is used for bonding by a condensation reaction. In this case, a mixture of the suspension in which the insoluble carrier is suspended and the fusion protein solution is
Add water-soluble carbodiimide solution. By stirring the mixture, a crosslinking reaction for easily forming a peptide bond occurs, and the insoluble carrier and the fusion protein can be bound. In this case, a carboxyl group must be present on the surface of the insoluble carrier. As such an insoluble carrier, for example, latex particles composed of a styrene-methacrylic acid copolymer are preferable.

When the latex particles comprising the styrene-methacrylic acid copolymer are used, the amount of the carboxyl group is determined by the surface charge of 0.03 to 0.75 meqCOO ⁻
/ G is preferable, and 0.05 to 0.40 meqCOO ⁻ /
g is more preferred.

When the above-mentioned condensation reaction is used, the latex particles and the fusion protein can be bound via an appropriate spacer. As such a spacer, amino acids such as β-aminoalanine, γ-aminobutyric acid, ε-aminocaproic acid, and glutamic acid are preferable.

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

When the above EDC is used, ED in the reaction solution
When the C concentration is low, the reaction does not proceed sufficiently, and when the C concentration is high, EDC precipitates without dissolving.
(W / v) is preferred.

When the reaction temperature of the above EDC reaction is low, the reaction does not proceed sufficiently, and when the reaction temperature is high, the antigen protein may be denatured and lose antigenicity.
40 ° C. is preferred.

In the case of the method utilizing physical adsorption of the fusion protein to the insoluble carrier, by bringing the insoluble carrier and the fusion protein into contact in a solution, the adsorption easily occurs due to the charge of the antigen protein and the insoluble carrier.

The reaction temperature of the above-mentioned adsorption reaction is preferably 0 ° C. to 40 ° C., because when the temperature is low, the reaction does not proceed sufficiently, and when the temperature is high, the steric structure of the antigen protein or thioredoxin may change or decompose.

The immunological diagnostic reagent of the present invention is used by being mixed with a sample, whereby the immunodiagnostic reagent based on the antigen-antibody reaction between the antibody in the sample to be measured and the antigen protein of the reagent of the present invention is used. Antibodies can be measured.

In the above-mentioned antigen-antibody reaction, the specimen 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,
Glycine buffer, veronal buffer, borate buffer, citrate buffer and the like can be mentioned.

Various sensitizers can be added to the 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 and the like can be added to the sample diluent for the purpose of suppressing non-specific reactions caused by other substances present in the sample.

As a measurement system for measuring an antibody in a sample using the immunological diagnostic reagent of the present invention, for example, the degree of aggregation of the insoluble carrier due to the reaction between the insoluble carrier and the antibody in the sample is measured. In particular, a latex agglutination reaction system using latex particles as an insoluble carrier is preferred.

As a method of measuring the agglutination generated by the above reaction, a method of optically observing the degree of agglutination or a method of visually observing the degree of agglutination can be used.

Specifically, in the method of optical observation, a solution containing the immunological diagnostic reagent of the present invention (usually phosphate buffer, glycine buffer) is added to a solution obtained by diluting a sample with a sample diluent. Liquid, veronal buffer, borate buffer, citrate buffer, etc.), and then the scattered light intensity of the mixture,
Measure absorbance or transmitted light intensity. Measurement wavelength is 300
２2400 nm can be used, and the increase or decrease in scattered light intensity, absorbance, or transmitted light intensity is measured according to the particle size, concentration, and reaction time of the insoluble carrier to be used, according to a known method. These methods may be used alone or in combination.

In the method of visual observation, usually, a sample and a solution containing the immunological 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 antigen-antibody reaction is performed under normal conditions.
As the reaction medium, for example, a phosphate buffer, a citrate buffer, a glycine buffer and the like are 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 0
To 50 ° C, more preferably 20 to 40 ° C. The reaction time is appropriately determined.

[0035]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described with reference to embodiments, and its effects will be specifically described. Example 1 (Preparation of Diagnostic Reagent by Chemical Bonding Method) (1) Preparation of Fusion Protein of Treponema pallidum 47kDa Antigen and Thioredoxin Treponema pallidum cells were extracted, and genomic DNA was extracted to prepare a DNA library. Already DNA of 47kDa antigen
Since the sequence is known, primers were prepared based on the sequence using a DNA synthesizer. A DNA fragment of a 47 kDa antigen was obtained by PCR using the primers and the DNA library prepared above. Next, a plasmid vector pTrxFus (Invitrog) was constructed to express a fusion protein with thioredoxin.
The above DNA fragment was inserted into pTrxF
us-47k.

Next, pTrxFus-47k was introduced into E. coli and cultured. By adding tryptophan to the culture solution, the expression of a fusion protein of the 47 kDa antigen and thioredoxin was induced. Escherichia coli in which the fusion protein was sufficiently expressed was disrupted by sonication, and the supernatant from which cell membranes, nuclei, etc. had been removed by centrifugation was collected using ThioBo.
The protein was passed through an nd ™ resin column (manufactured by Invitrogen) to adsorb the fusion protein. After washing the column sufficiently, the fusion protein was eluted with a 5% by weight aqueous solution of β-mercaptoethanol. Next, the eluate was dialyzed against a phosphate buffered saline (hereinafter, referred to as PBS; a salt concentration of 0.9% by weight, pH 7.4) to remove excess β-mercaptoethanol, and to prepare treponema pallidum 47 kDa. A fusion protein of the antigen and thioredoxin was obtained.

(2) Preparation of diagnostic reagent for immunological syphilis Styrene-methacrylic acid copolymer latex [carboxyl group 0.25 meq COO ⁻ / g, particle size 0.4 μm,
Sekisui Chemical Co., Ltd., 10% (w / v)] 0.2 ml,
One ml of a solution in which EDC was dissolved at a ratio of 30% (w / v) in 50 mM phosphate buffer (hereinafter referred to as PB buffer) at pH 6.8 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 suspended in 2 ml of 100 mM borate buffer (pH 9.0) (hereinafter referred to as BB buffer). To this suspension was added 100 μl of the fusion protein solution obtained in the above (1) (fusion protein concentration: 100 μg / ml), and the mixture was stirred at room temperature for 12 hours. Unreacted fusion protein was removed by centrifugation, washed twice with BB buffer, and suspended in 4 ml of PBS containing 1% (w / v) of bovine serum albumin to obtain an immunological syphilis diagnostic reagent of the present invention. .

Example 2 The immunological syphilis diagnostic reagent of the present invention was obtained in the same manner as in Example 1 except that the Treponema pallidum 15 kDa antigen was used in place of the Treponema pallidum 47 kDa antigen in Example 1. Was.

Comparative Example 1 (1) Preparation of Fusion Protein of Treponema pallidum 47 kDa Antigen and GST In the same manner as in (1) of Example 1, DN of the 47 kDa antigen was
A fragment was obtained. Next, a plasmid vector pGEX-4T made to express a fusion protein with GST
-3 (manufactured by Pharmacia), and the DNA fragment was inserted and named pGEX-4T-3-47k.

Next, pGEX-4T-3-47k was introduced into E. coli and cultured. Isopropyl-β-
By adding (−)-thiogalactopyranoside,
The expression of a fusion protein of the 47 kDa antigen and GST was induced. Escherichia coli in which the fusion protein has been sufficiently expressed is disrupted by sonication, and the supernatant from which cell membranes, nuclei, etc. have been removed by centrifugation is added to glutathione Sepharose 4B.
(Manufactured by Pharmacia) and gently shake at 4 ° C. overnight, and glutathione Sepharose 4
B. 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. The eluate was phosphate-buffered saline (hereinafter, referred to as PBS; a salt concentration of 0.9% by weight, pH
Excess glutathione was removed by dialysis in 7.4) to obtain a fusion protein.

(2) Preparation of immunological syphilis diagnostic reagent In place of the fusion protein of 47 kDa antigen and thioredoxin in (2) of Example 1, fusion of the 47 kDa antigen obtained in (1) above with GST Except for using the protein, the same operation as in (2) of Example 1 was performed.
An immunological syphilis diagnostic reagent was obtained.

Comparative Example 2 An immunological syphilis diagnostic reagent was obtained in the same manner as in Comparative Example 1, except that the Treponema pallidum antigen of 15 kDa was used instead of the 47 kDa antigen of Treponema pallidum in Comparative example 1.

Comparative Example 3 (1) Purification of 47 kDa Antigen Treponema pallidum 47 prepared in (1) of Example 1
Fusion protein solution of kDa antigen and thioredoxin 5
ml (fusion protein concentration 100 μg / ml)
Add bovine enterokinase from UNITS and add 37
The mixture was reacted at 12 ° C. for 12 hours to cleave the 47 kDa antigen and thioredoxin. Next, it was passed through a ThioBond ™ resin column (manufactured by Invitrogen) to remove thioredoxin. Thus, the 47 kDa antigen was purified.

(2) Preparation of a diagnostic reagent for immunological syphilis The use of the 47 kDa antigen obtained in (1) above in place of the fusion protein of 47 kDa antigen and thioredoxin in (2) of Example 1 Otherwise, the procedure was the same as in Example 1, (2) to obtain an immunological syphilis diagnostic reagent.

Comparative Example 4 In place of the fusion protein of 47 kDa antigen and thioredoxin prepared in (1) of Example 1 in (1) of Comparative Example 3, fusion of 15 kDa antigen and thioredoxin prepared in Example 2 was performed. An immunological syphilis diagnostic reagent was obtained in the same manner as in Comparative Example 3, except that the protein was used.

Example 3 (Preparation of Diagnostic Reagent by Physical Adsorption Method) 100 μl of a solution (fusion protein concentration: 100 μg / ml) of a fusion protein of 47 kDa antigen and thioredoxin prepared in (1) of Example 1 was added to polystyrene latex ( Average particle size 0.4 μm, solid content 10% (w / v),
(Manufactured by Sekisui Chemical Co., Ltd.) and stirred at 4 ° C. for 1 hour. Next, 100 m of pH 7.4 containing 1% (w / v) of bovine serum albumin (hereinafter referred to as BSA).
M phosphate buffer (hereinafter referred to as 100 mM PB buffer)
Was added and stirred for 1.5 hours. This solution is 10 ° C
For 30 minutes at 18000 rpm. The resulting precipitate contained 0.25% (w / v) BSA.
5 ml of 00 mM MPB buffer was added to suspend the latex particles to prepare a fusion protein-carrying latex, thereby obtaining an immunological syphilis diagnostic reagent of the present invention.

Example 4 In Example 3, 47k prepared in (1) of Example 1 was used.
The immunological method of the present invention was carried out in the same manner as in Example 3, except that the fusion protein of the 15 kDa antigen and thioredoxin prepared in Example 2 was used instead of the fusion protein of the Da antigen and thioredoxin. A syphilis diagnostic reagent was obtained.

Comparative Example 5 In Example 3, 47k prepared in (1) of Example 1 was used.
Instead of the fusion protein of Da antigen and thioredoxin, the 47 kDa antigen prepared in (1) of Comparative Example 1 and GS
An immunological syphilis diagnostic reagent was obtained in the same manner as in Example 3, except that a fusion protein with T was used.

Comparative Example 6 In Example 3, 47k prepared in (1) of Example 1 was used.
An immunological syphilis diagnostic reagent was prepared in the same manner as in Example 3 except that the fusion protein of 15 kDa antigen and GST prepared in Comparative Example 2 was used instead of the fusion protein of Da antigen and thioredoxin. I got

COMPARATIVE EXAMPLE 7 In Example 3, 47k prepared in (1) of Example 1 was used.
An immunological syphilis diagnostic reagent was obtained in the same manner as in Example 3 except that the 47 kDa antigen prepared in (1) of Comparative Example 3 was used instead of the fusion protein of Da antigen and thioredoxin. Was.

COMPARATIVE EXAMPLE 8 In Example 3, 47k produced in (1) of Example 1 was used.
An immunological syphilis diagnostic reagent was obtained in the same manner as in Example 3, except that the 15 kDa antigen prepared in Comparative Example 4 was used instead of the fusion protein of Da antigen and thioredoxin.

Measurement of Sensitivity The sensitivity of the immunological syphilis diagnostic reagent of the present invention obtained in Examples 1 to 4 and the immunological syphilis diagnostic reagents obtained in Comparative Examples 1 to 8 was determined as follows. It was measured. Using a biochemical automatic analyzer (Hitachi 7050, manufactured by Hitachi, Ltd.), a standard solution consisting of a standard treponema-positive serum of 0, 50, 100, 250, 500 titer units (hereinafter abbreviated as TU) was prepared. It was measured. The measurement conditions were a temperature of 37 ° C. and a wavelength of 570 nm. As a measurement method, after dispensing 20 μl of the above-mentioned standard solution, 350 μl of a sample diluent (the one contained in a reagent for diagnosis of syphilis “MEDIACE TPLA” (manufactured by Sekisui Chemical Co., Ltd.) was used) was immediately added and mixed Then, 50 μl of the immunological syphilis diagnostic reagent 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 value obtained by multiplying the absorbance change by 10,000 times and the T.V.
U. 1 (Example 1, Comparative Example 1 and Comparative Example 3), FIG. 2 (Example 2, Comparative Example 2 and Comparative Example 4), and FIG.
(Example 3, Comparative Example 5, and Comparative Example 7) and FIG. 4 (Example 4, Comparative Example 6, and Comparative Example 8).

[0053]

The composition of the immunological diagnostic reagent according to claim 1 is as described above. Since the fusion protein of the antigen protein and thioredoxin is supported on an insoluble carrier, the antigen protein and glutathione-S-transferase The present invention provides an immunological diagnostic reagent having a higher solubility in a fusion protein and a higher sensitivity as compared with an immunological diagnostic reagent in which a fusion protein with the above is carried on an insoluble carrier.
The composition of the immunological diagnostic reagent according to claim 2 is as described above, and the fusion protein of treponema pallidum surface antigen protein and thioredoxin is carried on an insoluble carrier. The present invention provides an immunological diagnostic reagent having higher solubility in fusion protein and higher sensitivity than an immunological diagnostic reagent in which a fusion protein with S-transferase is supported on an insoluble carrier. The configuration of the immunological diagnostic reagent according to claim 3 is as described above, and the insoluble carrier is a polystyrene, a styrene-styrene sulfonate copolymer or a styrene-methacrylic acid copolymer, so that the sensitivity is higher. An immunological diagnostic reagent is provided.

[Brief description of the drawings]

FIG. 1 is a graph showing the results of sensitivity measurements of Example 1, Comparative Examples 1 and 3.

FIG. 2 is a graph showing the results of sensitivity measurement of Example 2, Comparative Example 2, and Comparative Example 4.

FIG. 3 is a graph showing the results of sensitivity measurement of Example 3, Comparative Example 5, and Comparative Example 7.

FIG. 4 is a graph showing the results of sensitivity measurement of Example 4, Comparative Example 6, and Comparative Example 8.

Claims (3)

[Claims] 1. An immunodiagnostic reagent characterized in that a fusion protein of an antigen protein and thioredoxin, which is diagnostically useful, is carried on an insoluble carrier. 2. The immunological diagnostic reagent according to claim 1, wherein the antigen protein is a surface antigen protein of Treponema pallidum. 3. The immunological diagnostic reagent according to claim 1, wherein the insoluble carrier is polystyrene, styrene-styrene sulfonate copolymer or styrene-methacrylic acid copolymer.