JPH08338842A - Production of syphilis antigen - Google Patents

Abstract

PURPOSE: To produce a reagent for inspecting syphilis in which initial infection can be detected with high specificity and sensitivity by carrying an obtained syphilis antigen on an insoluble carrier. CONSTITUTION: A suspension of Treponema pallidum is admixed with a bacteria dissolving agent and then the insoluble components are removed. Subsequently, and the fraction having molecular weight of 150,000 or above is demarcated, as a syphilis antigen, from the soluble solution.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing an antigen used as a reagent for syphilis test. More specifically, it relates to a simple method for producing an antigen of a syphilis test reagent having high specificity and high purity.

[0002]

BACKGROUND OF THE INVENTION Syphilis is an infectious disease caused by Treponema pallidum. For syphilis test reagents, Wasselman reaction reagents, microtiter reagents, latex agglutination reagents,
There are fluorescent antibody method reagents and the like. The mainstream method is a method of detecting an antibody in patient serum or plasma using an antigen-antibody reaction. Typically, the TPHA test developed by Tomizawa et al. Is used as an antigen prepared from Treponema pallidum,
It is a test reagent for detecting Treponema pallidum antibody (hereinafter abbreviated as TP antibody) in patient serum.

Conventional antigens used as reagents for syphilis testing are used by crushing Treponema pallidum syphilis by ultrasonic treatment, homogenizer treatment, etc., and directly or by solubilizing it with a surfactant or the like.

Antigens produced by conventional methods contain various contaminants in addition to antigens derived from Treponema pallidum. When a syphilis test reagent is prepared using an antigen containing contaminants in this way, the contaminants cause nonspecific reactions other than antigen-antibody reactions, causing the cause of misdiagnosis using syphilis reagents. Becomes Furthermore, the antigen-initial antibody (IgM) based on the antigen-antibody reaction in which the contaminants are relatively weak
It has been pointed out that there is a problem in that the initial antibody cannot be detected due to the inhibition of the reaction of E. coli and the initial antibody cannot be detected appropriately. Therefore, it is essential that the antigen used for the reagent for syphilis test is high in purity and free of contaminants in order to obtain high diagnostic accuracy and detect initial infection with high efficiency.
Especially in syphilis, for which an appropriate treatment method with antibiotics etc. has been established, early detection of infection is important.

A protein containing antigenicity obtained from Treponema pallidum has a molecular weight of 15,500, 17,000, 44, which has major antigenicity as analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting.
4 subunits of 500, 47,000 and molecular weights of 30,000, 33,000-3 with relatively weak antigenicity
It is known that there are 3 subunits of 5,000 and 38,000 to 40,000. Among them, the molecular weight is 15,500, 17,000, 44,500, 47,0
It is known that a diagnostic accuracy of 93.8% is obtained by using 3 subunits among 4 subunits having major antigenicity of 00 as an antigen (Robert E. et.
al, Journal of Clinical M
icology, 30, vol. 30, p11
5 (1992)). Furthermore, to obtain a diagnostic accuracy close to 100%, include all subunits having antigenicity,
It is important for the antigen of the diagnostic reagent for syphilis to be free of contaminants. However, at present, a method for producing a high-purity antigen used for a syphilis test reagent containing all of these subunits and containing no contaminant has not yet been established.

As a technique similar to the present invention, Japanese Patent Laid-Open No. 58-71457 discloses an example of using an antigen-containing substance obtained by removing a fraction having a specific gravity of 1.01 or less from Treponema pallidum. Although the specific gravity fractionation is a simple method for producing an antigen, it is difficult to produce a high-purity antigen containing no contaminants only by the specific gravity fractionation.

As another similar technique, Japanese Patent Application Laid-Open No. 2-
234063 and JP-A-3-267760 have a method of removing contaminants from a culture of Treponema pallidum with a cation exchanger and a method of removing contaminants from a culture of Treponema pallidum with hydroxyapatite. The method for producing an antigen with a cation exchanger and hydroxyapatite is complicated, and fractionation with a cation exchanger, hydroxyapatite, etc. may partially eliminate the protein having antigenicity. is there.

[0008] Yet another technique is disclosed in Japanese Patent Laid-Open No. 4-18
In 8067, an immobilized antibody was prepared using an antibody obtained by immunizing an antigen purified with hydroxyapatite,
There is a method of producing by affinity column chromatography. This production method is a method in which a highly specific antigen may be recovered. However, the disadvantage of this method is that the purity of the produced antigen is greatly influenced by the specificity of the immobilized antibody used for affinity column chromatography.

[0009]

DISCLOSURE OF THE INVENTION The present invention has a high specificity in a method for producing an antigen used for a reagent for syphilis test,
Moreover, it is an object of the present invention to provide a simple method for producing a highly pure antigen.

[0010]

[Means for Solving the Problems] The present inventors have conducted extensive research to solve the above problems. As a result, a solubilizing agent was added to the suspension of Treponema pallidum bacillus for solubilization to remove insolubilized components, and then a fraction having a molecular weight of 150,000 or more was obtained from the resulting soluble solution. It has been found that the above-mentioned object can be achieved by using it as a syphilis antigen, and the present invention has been completed.

That is, according to the present invention, a solubilizing agent is added to a suspension of Treponema pallidum bacillus to solubilize it, the insolubilized components are removed, and then the soluble solution has a molecular weight of 150,000. A method for producing a syphilis antigen, characterized by fractionating the above fractions as a syphilis antigen.

The invention will be described in detail below.

Treponema pallidum referred to in the present invention (hereinafter,
The abbreviation "TP bacterium" refers to a pathogenic Treponema pallidum bacterium, which is a causative bacterium of syphilis and is a type of spirochete. As a culture method for obtaining a large amount of TP bacterium, there is an in vitro culture method in which a liquid artificially added with a nutrient, that is, a medium is used for culturing, but the in vitro culture method has not yet been established for TP bacterium. Absent. Therefore, an in-vivo culture method has been adopted in which the testes of rabbits are infected with TP bacteria and cultured. Specifically, the TP bacterium is subjected to the following method, that is, the method of Miller et al. (Milleret al., Journal of:
Immunology, vol. 96, p450, (1
It may be cultured according to 966)). Specifically, the rabbit testis was inoculated with TP bacteria, allowed to grow for 11 to 14 days, and then the rabbit testis was cut with scissors and suspended with a 0.15 M sodium chloride aqueous solution (hereinafter also referred to as physiological saline). Turbid and TP
Use the extract of the bacterium. The extract is centrifuged at 200 xg for 15 minutes to precipitate out large debris of the testis tissue. Then, it is centrifuged at 19,000 × g for 90 minutes to suspend the precipitate in physiological saline or the like to obtain a suspension of TP bacteria. At this point, the suspension of TP bacteria usually contains a large amount of rabbit testicle components and the like as contaminants. In general, most of the components having antigenicity (proteins, lipids, saccharides, glycoproteins, lipoproteins, etc.) are expected to be present in the surface layer of cells in an insoluble state in an aqueous solvent. Therefore, TP bacteria containing contaminants cultured in the testes of rabbits are subjected to subsequent solubilization treatment,
It is necessary to solubilize the component having antigenicity.

The solubilization treatment referred to in the present invention is an operation for making a component having an antigenicity present as an insoluble component on the cell surface of TP bacterium soluble in an aqueous solvent (hereinafter, this operation is also abbreviated as solubilization). Say). In the TP bacterium, most of the components having antigenicity are present in the cell surface in a state of being insoluble in an aqueous solvent. Therefore, in order to obtain a component having antigenicity, first, a solubilization treatment in which the component having antigenicity is solubilized and eluted in an aqueous solvent from the cell surface layer of the TP bacterium is essential. The solubilization treatment is carried out by adding a bacterial solubilizing agent to TP bacteria.

The solubilization treatment is carried out by adding T to a solution containing a microbial solubilizer.
It is carried out either by directly suspending the P bacterium to solubilize it or by suspending the TP bacterium once in a solvent containing no microbial solubilizer and then solubilizing it by adding the microbial solubilizer. It
In order to uniformly subject the TP bacterium to the solubilization treatment, it is desirable that the TP bacterium is sufficiently suspended in a solvent containing no bacterium solubilizing agent, and then the bacterium solubilizing agent is added to sufficiently suspend the solubilizing treatment.

The extraction efficiency of the component having the antigenicity obtained by sufficiently stirring the suspension containing the solubilizing agent for bacteria is improved. The stirring method is not particularly limited as long as it is a condition under which the microbial solubilizer and the TP bacterium are sufficiently mixed. For example, an aqueous solvent suspension of TP bacteria is placed in a beaker or the like, and a solution containing a bacterium solubilizer is added while stirring with a magnetic stirrer or the like. Alternatively, the TP bacterium may be directly suspended in a solution containing a microbial solubilizer, and then sufficiently stirred with a magnetic stirrer or the like. By performing the stirring as instantaneously as possible, the component having antigenicity can be obtained with high extraction efficiency.

The type of liquid for suspending the TP bacterium during the solubilization treatment is suitable for the solubilizing agent of the bacterium to be used, but generally an aqueous solvent having a pH of about 5.0 to 9.0 is desirable.
For example, phosphate buffer (pH 7.2), phosphate buffer (pH 7.2) -containing physiological saline (hereinafter also abbreviated as PBS), physiological saline and the like can be mentioned. The concentration of the TP bacteria in suspension may be arbitrary, but considering the efficiency of the solubilization treatment with the solubilizing agent, it is preferably about 10 ⁹ cells / ml.

As a condition for the solubilization treatment, a solubilization temperature of 1 ° C. to 100 ° C., more preferably 20 ° C. to 60 ° C. is effective. The solubilization treatment is effective for 30 minutes or more, and preferably 30 minutes or more and 24 hours or less.

Before carrying out the solubilization treatment, physically crushing the TP bacterium by freeze-thaw crushing, ultrasonic crushing, homogenizer crushing, glass bead crushing, etc.
The cell surface of the bacterium is partially crushed to improve the efficiency of the solubilization treatment and improve the yield of the finally produced syphilis antigen, which is preferable, and is carried out as necessary.

Further, since the component having antigenicity is localized on the cell surface of the TP bacterium, the soluble component of the suspension of TP bacterium obtained by the method of Miller et al. Is unnecessary. Therefore, before carrying out the solubilization treatment of the present invention, the TP bacterium may be washed with an aqueous solvent in order to remove unnecessary soluble components which are contaminants derived from the testes of rabbits. The aqueous solvent used for washing is, for example, pH.
An aqueous solvent of about 5.0 to 9.0 is desirable. For example, a phosphate buffer solution (pH 7.2), PBS, physiological saline and the like can be mentioned. Washing with the aqueous solvent is usually performed by centrifugal washing. Centrifugal washing is a suspension of TP bacteria in an aqueous solvent, and then a suspension of TP bacteria is placed in a centrifuge rotor, and a centrifugal force field is created by rotating the rotor, whereby the soluble components in the suspension are To remove only insoluble components including TP bacteria. By carrying out the main washing operation before the solubilization treatment, the contaminants contained in the soluble component can be removed. Therefore, the washing operation can further improve the purity of the finally obtained syphilis antigen.

The bacterium solubilizing agent used in the solubilization treatment in the present invention is a treating agent for solubilizing an insoluble antigenic component present on the cell surface of TP bacterium in an aqueous solvent. Typical examples include the following surfactants and denaturing agents such as urea and guanidine hydrochloride.

As the surface active agent, a nonionic surface active agent or an ionic surface active agent can be used. Ionic surfactants include anionic surfactants, cationic surfactants, and amphoteric surfactants. Any of them can be suitably used for the solubilization treatment of the present invention.

Specific examples of the nonionic surfactant include octyl glucoside, decyl glucoside, heptyl thioglucoside, octyl thioglucoside, nonyl thioglucoside, octyl maltoside, dodecyl maltoside, decanoyl-N-methylglucamide, triton. X45, Triton X114, Nonidet P40, Triton X10
0, Triton X102, Triton X165, Triton X
305, bridge 76, bridge 96, bridge 56,
Bridge 58, Triton N101, sucrose monohexanoate, sucrose monolaurate,
Sorbitol monostearate, sorbitol monolaurate, etc. can be used.

Specific examples of the anionic surfactant include sodium dodecyl sulfate (SDS), sodium tetradecyl sulfate, sodium dodecylsulfonate, sodium tetradecylsulfonate, sodium cholate, sodium deoxycholate and sodium taurocholate.
Sodium taurodeoxycholate or the like can be used.

As the cationic surfactant, specifically cetyltrimethylammonium bromide, tetradicyltrimethylammonium chloride, dodecylpyridinium bromide, cetylpyridinium chloride and the like can be used.

Examples of the amphoteric surfactant include 3-[(3-colamidopropyl) dimethylammonio] -1-propanesulfonic acid and 3-[(colamidopropyl) dimethylammonio] -2-hydroxy-1-propane. Sulfonic acid or the like can be used.

Of these surfactants, octyl glucoside, decyl glucoside, heptyl thioglucoside, octyl thioglucoside, nonyl thioglucoside, octyl maltoside, dodecyl maltoside, among others, which have a sugar in the hydrophilic group of the nonionic surfactant. , Decanoyl-N-methylglucamide can be preferably used.

The concentration of the surfactant used for the solubilization treatment is the concentration at which micelles are formed when the surfactant is dissolved in an aqueous solvent such as physiological saline or PBS, that is, the critical micelle concentration (hereinafter also abbreviated as CMC). Yes) above is effective. Further, it is preferably used at a concentration of CMC or more or up to 10 times the concentration of CMC, and when solubilization is carried out at a concentration of CMC to 5 times the concentration of CMC, it works well. For the surfactant, HLB (hydrophile-lipoph) is used as a value indicating the balance between strength and weakness of hydrophilic and hydrophobic groups.
There is an ile balance). As the surfactant used in the present invention, HLB10 to 17 can be preferably used.

The denaturant used in the solubilization treatment is a substance that causes protein denaturation. Protein denaturation means that only the higher-order structure is destroyed without changing the inherent primary structure under physiological conditions. Say. Therefore, the denaturant is saline
It has the function of solubilizing insoluble components in an aqueous solvent such as PBS. As a specific example of the denaturing agent, 4M or more urea,
Guanidine hydrochloride of 2M or more is used. Preferably, 8 M or more urea and 6 M or more guanidine hydrochloride that completely denature the protein are used.

The bacterium-solubilizing agent can be used alone or in the presence of several bacterium-solubilizing agents, and can effectively solubilize a component having an antigenicity and insoluble from the surface layer of TP bacteria. For example, a surfactant and a modifier can be used together.

By carrying out the solubilization treatment, the component having the antigenicity is solubilized in the aqueous solvent and exists as the soluble component, and the residue which is the insoluble component has almost no antigenicity. Therefore, the suspension at the stage after the solubilization treatment is composed of a soluble component having a component having an antigenicity and a residue which is an insoluble component showing almost no antigenicity.

In the present invention, an insoluble component is removed from the suspension to obtain a soluble solution having antigenicity. By the step of removing this insoluble component, the purity and specificity of the syphilis antigen finally obtained by the present invention can be improved.
If this insoluble component is not removed, the purity of the syphilis antigen obtained will be significantly reduced, so the operation of removing this insoluble component is essential in this production method.

There is no particular limitation on the method for removing the insoluble component. For example, a method such as centrifugation is used, the suspension is put in a rotor for centrifugation, and a centrifugal force field is created by rotating the rotor to remove insoluble components contained in the suspension to dissolve soluble components. Only collect as a soluble solution. The centrifugal force used for the centrifugal separation in the above-mentioned insoluble component removing operation may be such that the insoluble component can be removed by precipitation. Usually 10 to remove insoluble components,
30 minutes at 000 × g is sufficient.

By removing the insoluble component, the component having antigenicity is dissolved in the solution containing the microbial solubilizer to be in a solution state. However, the soluble solution obtained by removing the insoluble component still contains contaminants other than the antigen component. Typical contaminants contained in the soluble solution are rabbit testis components and the like.

In the present invention, a highly specific and highly pure syphilis antigen to be used as a syphilis test reagent is produced by further fractionating a fraction having a molecular weight of 150,000 or more from the above-mentioned soluble solution having antigenicity. it can. That is, in the present invention,
Fractions having a molecular weight of 150,000 or more are used as syphilis antigens.

As a result of the assay by SDS-polyacrylamide gel electrophoresis, the molecular weight of the fractions having a molecular weight of 150,000 or more was 15,500, 17,000, 44,50.
4 subunits with strong antigenicity of 0,47,000, 3
30,000, 33,000-35,000, 38,000
It was confirmed that the substance has at least 1 subunit out of 3 subunits having an antigenicity of 0 to 40,000, which is relatively weak, and the content of contaminants is low. Accordingly, by fractionating a fraction having a molecular weight of 150,000 or more from a soluble solution, at least 1 subunit is recovered from 4 subunits having strong antigenicity and 3 subunits having relatively weak antigenicity. it is conceivable that. That is, the subunits having antigenicity are expected to form multimers and crosslinks in the native state, and have a molecular weight of 150,000 as a whole.
It is speculated that these major antigen subunits are fractionally purified to a high degree of purity by executing the fractionation operation of the above fractions.

The molecular weight in the present invention is a molecular weight in a native state. Although the molecular weight of the present invention is not limited, the gel filtration method is preferably used. For example, in the molecular weight assay by the gel filtration method, the molecular weight slightly changes depending on the three-dimensional structure of the antigen. Therefore, the molecular weight assay is performed as follows. The exclusion limit molecular weight of the stationary phase is 800
10,000 Sephacryl S-400 (manufactured by Pharmacia) is used, and the solvent has the same composition as the bacterial solubilizing agent used at the time of solubilizing the TP bacterial liquid. After assaying the elution time with a molecular weight marker, the molecular weight of the syphilis antigen produced according to the present invention is assayed. A calibration curve is prepared from the molecular weight of the molecular weight marker and the elution time, and the molecular weight of the syphilis antigen is calculated.

The syphilis antigen produced by this production method is highly specific and can be used as a highly pure reagent for syphilis test. The syphilis antigen produced by the production method of the present invention refers to an antigenic component that reacts with a specific antibody present in the body fluid of a patient with syphilis infection by an antigen-antibody reaction. The syphilis antigen is used as an antigen for a diagnostic test reagent for syphilis caused by the infection of Treponema pallidum.

The molecular weight fractionation method in the present invention is not particularly limited as long as it is a method capable of fractionation with a molecular weight of 150,000 or more. For example, a gel filtration method, an ultrafiltration method and the like can be mentioned. A gel filtration method that can be repeatedly used is particularly preferably used. The fixed layer used for gel filtration has a molecular weight of 15
There is no limitation as long as it can fractionate over 50,000 fractions. If a component having an antigenicity at the exclusion limit molecular weight or more (void fraction) can be recovered using a fixed layer having an exclusion limit molecular weight of about 150,000, the operation is simple and useful. For example, when Sephacryl S-300HR (manufactured by Pharmacia) having an exclusion limit molecular weight of 150,000 and a PBS containing 2% octylglucoside are used as the stationary phase, a protein having a molecular weight of 150,000 or more is recovered in the void fraction. It

Further, in the fractionation by the ultrafiltration method, the fractionation operation is simple if a fractionation molecular weight of 150,000 is used. For example, the soluble solution is filtered through an ultrafiltration membrane having a molecular weight cut off of 150,000, and the syphilis antigen is recovered in the unfiltered fraction.

The component having an antigenicity contained in the soluble solution is insoluble in the TP bacterium, and the solvent for the fractionation operation is TP.
It is preferable to carry out the treatment using a bacterium containing a bacterium solubilizing agent having the same composition as that used for the solubilization treatment of the bacterium. In order to prevent adsorption of the syphilis antigen to the stationary phase in the gel filtration method and the filter in the ultrafiltration method, 0.1
Fractionation operation with a solvent having an ionic strength of 5 M or higher is suitable.

The syphilis test reagent is an immunological diagnostic agent for syphilis that applies an antigen-antibody reaction, and includes, for example, a microtiter reagent, a latex agglutinating reagent, an immunoturbidimetric reagent, an enzyme immunoreagent, and the like. The present invention is effective as a reagent that captures an antigen-antibody reaction by an agglutination reaction, such as a microtiter reagent or a latex agglutination reagent.

Usually, this solution state is used as a syphilis antigen and, if necessary, diluted or concentrated with physiological saline, PBS or the like and used for the sensitization operation of an insoluble carrier described later. The antigen solution may contain the microbial solubilizer used for solubilization, but if necessary, it may be removed by dialysis or the like.

The syphilis antigen obtained by the production method of the present invention can be carried on an insoluble carrier to produce a syphilis test reagent. As the insoluble carrier particles, carriers that can be used for a diagnostic reagent of a known immunological agglutination method can be used without limitation. For example, high specific gravity composite particles in which a dye is coated on an inorganic compound serving as a core (Japanese Patent Application Laid-Open No. 62-115366).
Reference, hereinafter also abbreviated as HDP), sheep red blood cells, polystyrene particles, gelatin particles, polyamino acid particles and the like. Among them, HDP and erythrocytes are preferably used because they have a large amount of syphilis antigen adsorption.

The particle size of the insoluble carrier is not particularly limited as long as it is within a known range that can be used as a diagnostic reagent for immunological agglutination method, but usually the particle size is 0.01 μm to 3 μm.
Those up to are preferably used. The specific gravity of the insoluble carrier may be 1.0 or more, but in particular, the carrier particles for microtiter reagents utilizing the precipitation reaction have a specific gravity of 10 or more because the higher the specific gravity, the faster the sedimentation speed and the shorter the determination time. Is preferably used.

Supporting the syphilis antigen produced in the present invention on an insoluble carrier means adsorbing an antigen agent or the like as a sensitizing substance on the insoluble carrier.

As a specific supporting method, the insoluble carrier is dispersed and mixed in a buffer solution in which the sensitizing substance is dissolved, the sensitizing substance is supported on the insoluble carrier, and then the excess sensitizing substance is removed by centrifugal washing. Then, a method of supporting a blocking agent in the same manner is used.

As a method for supporting the sensitizing substance on the insoluble carrier, a known method such as a physical adsorption method such as a hydrophobic adsorption method or a chemical adsorption method such as a chromium chloride method is adopted. The sensitizer must be supported under mild conditions as much as possible because it binds to the measurement target substance in the analyte by the antigen-antibody reaction after being supported on it, and therefore the hydrophobic adsorption method is particularly preferably used. .

The above-mentioned loading by the hydrophobic adsorption method is carried out in a state where the insoluble carrier and the adsorbent are dispersed in a buffer solution having a buffering action. The buffer solution can be used without limitation as long as it has a buffering action such as a phosphate buffer solution, a glycine buffer solution, a Tris buffer solution, an acetate buffer solution and the like. Regarding the pH of the buffer solution, an optimum range is selected and adopted depending on the adsorbing substance to be supported, but generally, the neutral range pH 6.0 to pH 8.
0 is desirable. The time for supporting the adsorbent on the insoluble carrier may be a time sufficient to allow the adsorbent to be uniformly supported on the surface of the insoluble carrier. For example, when the syphilis antigen is subjected to the HDP hydrophobic adsorption method, it is 30 minutes or more. Is enough. Further, regarding the temperature, a range in which the adsorbed material to be supported is not subjected to thermal denaturation, that is, 1 ° C. or more and 80 ° C. or less is adopted.

Further, the blocking agent may be loaded on the insoluble carrier by the same method as the loading of the sensitizing substance.

[0051]

The present invention will be described below in detail with reference to examples. However, the technical scope of the present invention is not limited by these examples.

Reference Example 1 Method for Quantifying Protein Concentration The method for quantifying protein concentration was the Lowry method.
t al, J .; Bio. Chem. , Vol. 193
p265 (1951)). That is, 0.2 ml of a solution containing 5 to 100 μg of protein was placed in a test tube, and 1 ml of sodium carbonate-alkaline copper reagent was added and mixed. After standing at room temperature for 10 minutes, 0.1 ml of the phenol reagent diluted 2-fold with water was added and immediately mixed.
After standing for 0 minutes, the absorbance at 750 nm was measured. The standard curve was prepared using bovine serum albumin.

Reference Example 2 Method of Measuring Antigen Titer The antigenicity was evaluated by the dot blot method. The measurement procedure is as follows.

(1) The antigen solution which is the subject is 1, 2, 5, 10, 20, 50, 100, 200, 50 in PBS
It was diluted 0 times and spotted in 2 μl portions on a nitrocellulose filter (HAHY00010, manufactured by Millipore) (two sheets are prepared).

(2) After air-drying the filter, 10
% Skim milk containing 0.15 M sodium chloride 0.1
It was immersed in a blocking solution dissolved in M Tris buffer, pH 7.5 (hereinafter abbreviated as TBS), and blocked at 37 ° C. for 1 hour.

(3) Serum as a primary antibody and serum of a healthy individual were diluted 25 times with TBS to prepare a serum diluted solution, and one block of nitrocellulose was soaked in each of the diluted solutions at 37 ° C and 1 ° C. Reacted for hours.

(4) The nitrocellulose filter of (3) was washed with TBS containing 0.1% Tween 80 three times.

(5) Avidin-labeled anti-human IgG (Funakoshi) and avidin-labeled anti-human IgM as secondary antibodies
(Manufactured by Funakoshi Co., Ltd.) was diluted 400 times with TBS to prepare a secondary antibody solution, and the washed nitrocellulose filter of (4) was dipped and reacted at 37 ° C. for 1 hour.

(6) A washing operation was performed in the same manner as (4).

(7) Biotinylated peroxidase as an enzyme label was diluted 200 times with TBS, and the washed nitrocellulose filter of (6) was dipped and reacted at 37 ° C. for 30 minutes.

(8) Washing was performed in the same manner as (4).

(9) 1/20 of a 30% hydrogen peroxide solution was added to a 0.1% diaminobenzidine aqueous solution to act on a nitrocellulose filter to develop a color. The highest dilution ratio of the antigen solution whose color was confirmed in the nitrocellulose filter using patient serum as the primary antibody was defined as the antigen titer, which was used as an index of the antigenicity. In addition, the highest dilution ratio that developed the color in a nitrocellulose filter using the serum of a healthy person with the primary antibody was used as a control value, and was used as an index showing the degree of nonspecific reaction due to contaminants.

Example 1 (1) Cultivation of TP bacterium Culture of TP bacterium was carried out by the method of Miller et al.
er et al. , Journal of Immu
noology, Vol. 96, p450 (1966))
According to the above, the culture was collected. That is, the rabbit testicle has a T
P bacteria were inoculated for 11 to 14 days to grow, and the rabbit testis was cut with scissors and suspended in physiological saline to extract TP bacteria. The extract was centrifuged at 200 xg for 15 minutes to precipitate out large debris of testicular tissue. Then, 19,000 × g
The TP bacterium was precipitated by centrifugation at 90 ° C. for 90 minutes, and the TP bacterium was suspended in physiological saline to obtain a TP bacterium suspension.

(2) Crushing of TP bacteria by freeze-thawing The number of the above-mentioned TP bacteria was determined by a bacterial counter (CA Hausse).
It was 3 × 10 ⁹ cells / ml when measured with a dark field microscope (a bacterial counter manufactured by r & Son). TP bacterium liquid 1
ml was put in a Spitz tube (Eiken Equipment). Subsequently, the Spitz tube was brought into contact with liquid nitrogen for 30 seconds to freeze it, and then put in a 37 ° C. constant temperature water bath for 3 minutes to melt. This operation was repeated 3 times to crush the TP bacterium by freeze-thawing to obtain a crushed product suspension.

(3) Fractionation of TP Bacteria by Centrifugation The suspension of the crushed material was centrifuged at 10,000 × g for 30 minutes to fractionate soluble components containing contaminants to TP.
An insoluble component containing bacteria was obtained.

(4) Washing of TP bacteria by centrifugation washing 2 ml of PBS was added to this insoluble component to obtain 10,000
Centrifugation was performed at 0 × g for 30 minutes, and the soluble components containing TP bacteria were washed by centrifugation. This centrifugal washing operation was repeated 3 times to obtain a washed insoluble component. (5) Solubilization of TP Bacteria with Surfactant 2 ml of 1% octyl glucoside dissolved in PBS was added to the washed insoluble component to solubilize it for 1 hour at 37 ° C. After solubilization, centrifugation was performed at 10,000 × g for 30 minutes to obtain 2 ml of the soluble component of the centrifugation supernatant as a syphilis antigen-soluble solution.

(6) Preparation by gel filtration column The stationary phase resin used for gel filtration was Sephacryl S-3.
00 (manufactured by Pharmacia, exclusion limit molecular weight 150,000)
0). As the solvent, 1% octyl glucoside used as a solubilizing agent for TP bacteria in Example 1 was dissolved in PBS.

Sephacryl S-300 was φ10 mm × 5
A 00 mm glass column was packed and thoroughly washed with a solvent. Flow rate is 1 ml / hr.

(7) Molecular Weight Fractionation of Soluble Solution The soluble solution containing the syphilis antigen was concentrated 10 times and 200 μl
Was gently layered on top of the column to start fractionation. The eluate eluted from the gel filtration column was collected in 1 ml fractions with a fraction collector. The concentration of the eluted protein was measured by ultraviolet absorption (see Table 1).

(8) Analysis of Fractionated Fractions The protein concentration of the samples fractionated in (2) is shown in Reference Example 1,
Antigenicity was measured according to Reference Example 2. As a result of protein quantification according to Reference Example 1, the protein was fraction No. No. 11 started to be detected in the vicinity. 14 and the fraction No. It was divided into two around 31.
As a result of assaying the antigenicity according to Reference Example 2, the antigenicity was found to be Fraction No. I was concentrated around 14. Fraction No. 11 to No. Fifteen were collected and concentrated to 2 ml to obtain the syphilis antigen.

(9) Evaluation of Obtained Syphilis Antigen The protein was quantified according to Reference Example 1, and the result was 0.17.
It was mg / ml. Furthermore, when the antigen titer was measured according to Reference Example 2, the antigen titer was 100 times. The control value, which is an index of non-specific reaction, was 1 time.

(10) Reagentization and performance evaluation HDP (Tokuyama product) with a diameter of 1.8 μm was added to PBS.
Was suspended at 2.5 (w / w)% to obtain an HDP suspension. The syphilis antigen prepared in Examples (1) to (9) was diluted 10 times to prepare an antigen solution. The above HDP suspension 1
ml and 1 ml of the antigen solution were mixed in a test tube and left at room temperature for 1 hour to be hydrophobically supported on the HDP surface (hereinafter, this adsorption operation is also referred to as sensitization).

Then, in order to remove the surplus syphilis antigen, the above mixture was centrifuged at 2,500 rpm for 5 minutes, and the centrifugation supernatant was removed. To the centrifugal precipitate, 2 ml of PBS was added for washing, and after suspension, 2,500 rpm, 5
After centrifugation for a minute, the supernatant was removed. HDP adsorbed with the above-mentioned syphilis test reagent antigen was used as syphilis antigen-sensitized particles.

2 ml of 1% bovine serum albumin (hereinafter also referred to as BSA) was added to syphilis antigen-sensitized particles (50 mg), and blocking was performed at 37 ° C. for 3 hours. After blocking, the above suspension was centrifuged at 2,500 rpm for 5 minutes to remove the centrifugation supernatant. The blocking operation was completed by the above operation, and a syphilis test reagent was prepared. 0.5% (w / w) of this syphilis test reagent was added to PBS containing 3 (vol / vol)% normal rabbit serum (hereinafter also referred to as solution A).
vol)% to obtain a suspension of syphilis test reagent, and the suspension was subjected to the following measurement operation.

On the other hand, the sample used for the test was serially diluted with the solution A from 5 times. Next, 25 μl each of the diluted solution of the sample was dropped from 1 to 12 holes on a 96-well microtiter plate. Then, 25 μl of each well of the syphilis test reagent suspension was added dropwise. After dropping, the mixture was shaken with a plate mixer and allowed to stand for 30 minutes, and then a tube bottom aggregation image was observed.

Performance evaluation of the syphilis test reagent prepared as described above was carried out using 3 healthy subject samples, 1, 2, 3 and 3 syphilis patient samples, A, B and C. The results are shown in Table 2. No positive image was detected in any of the three healthy samples even with a 20-fold dilution. Positive sample A in syphilis patient samples
The positive image could be detected up to 160-fold dilution, with the positive specimen B to 320-fold dilution, and with the positive specimen C to 1280-fold dilution. As a result of the examination of reagents, no agglutination reaction was observed in the healthy subject samples, and appropriate sensitivity was obtained in the syphilis patient samples. It is considered that the molecular weight fractionation by gel filtration improved the purity of the syphilis antigen and removed the contaminants.

Comparative Example 1 (1) Culturing of TP bacteria, (2) Crushing of TP bacteria by freeze-thawing, (3) Fractionation of TP bacteria by centrifugation, (4) Washing of TP bacteria by centrifugal washing, (5) ) Solubilization of TP bacteria with a surfactant was carried out according to Example 1. However, fractionation by a gel filtration column was not performed.

(6) Evaluation of antigenicity When the protein was quantified according to Reference Example 1, it was 0.70.
It was mg / ml. Furthermore, when the antigen titer was measured according to Reference Example 2, the antigen titer was 100 times. The control value, which is an index of non-specific reaction, was doubled. From this, it was suggested that contaminants exist. Compared with the syphilis antigen prepared in Example 1, the antigenicity did not change, and the protein concentration was about 4 times. It can be said that the syphilis antigen is four times as pure as the soluble solution due to the molecular weight fraction of the syphilis antigen.

(7) Reagentization and performance evaluation The reagent was used according to (10) Reagentization and performance evaluation of Example 1.

Performance evaluation of the syphilis test reagent prepared as described above was carried out using 3 healthy subject samples, 1, 2 and 3 syphilis patient samples, A, B and C. The results are shown in Table 2.
In all of the healthy subjects, a positive image was detected in all three samples at a magnification of 20 times. In the syphilis patient specimen, positive images could be detected up to 160-fold dilution with positive specimen A, 320-fold dilution with positive specimen B, and 1280-fold dilution with positive specimen C. One of the healthy subjects showed an agglutination reaction at a low dilution ratio, and some non-specific reaction was observed. This is considered to be due to the inclusion of some contaminants contained in the syphilis antigen.

It was shown that the purity of the syphilis antigen was improved by carrying out the molecular weight fractionation with a molecular weight of 150,000.

Comparative Example 2 (1) Culturing of TP bacteria, (2) Crushing of TP bacteria by freeze-thawing, (3) Fractionation of TP bacteria by centrifugation, (4) Washing of TP bacteria by centrifugal washing, (5) ) Solubilization of TP bacteria by a surfactant, (6) Preparation by gel filtration column (7) Molecular weight fractionation of the soluble solution was carried out according to Example 1.

(8) Analysis of Fractionated Fraction The protein concentration of the fractionated sample was measured according to Reference Example 1, and the antigenicity was measured according to Reference Example 2. When the protein was quantified according to Reference Example 1, the protein was fraction No.
Protein was started to be detected from 11 and fraction N
o. 14 and the fraction No. It was divided into two around 31. As a result of assaying the antigenicity according to Reference Example 2, the antigenicity was found to be Fraction No. I was concentrated around 14.

Since the fraction having a molecular weight cutoff of 150,000 or less is used as an antigen, the fraction No. No. 15 to No. Three
Up to 2 were collected and concentrated to 2 ml to serve as an antigen.

(4) Evaluation of Obtained Syphilis Antigen The protein was quantified according to Reference Example 1, and the result was 0.51.
It was mg / ml. Furthermore, when the antigen titer was measured according to Reference Example 2, the antigen titer was doubled, and the control value, which was an index of nonspecific reaction, was also doubled. Almost no antigenicity was present and 75% of the protein in the soluble solution was recovered.

Of the following methods for preparing syphilis test reagents,
The sensitization, washing operation, blocking operation, and measurement operation were carried out according to (7) to (10) of Example 1.

(11) Evaluation Results of Syphilis Test Reagents The performance evaluation of the syphilis test reagents prepared as described above was carried out using three healthy subject samples, 1, 2, 3 and three syphilis patient samples.
It carried out using A, B, and C. The results are shown in Table 2. Among the three healthy specimens, two specimens showed no positive image even when diluted 20-fold, whereas one specimen showed a weak agglutination reaction at 20-fold. In the syphilis patient specimen, no positive image was detected in all of the positive specimens A, B, and C even when diluted 1-fold. Since no reaction was obtained in the syphilis patient sample in the examination of the reagent, the fraction having a molecular weight of 150,000 or less has no antigenicity.

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[Table 1]

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[Table 2]

Claims (1)

[Claims] 1. A suspension of Treponema pallidum bacillus is solubilized by adding a solubilizing agent to a solubilizing agent, insoluble components are removed, and then a fraction having a molecular weight of 150,000 or more is obtained from the soluble solution. A method for producing a syphilis antigen, which comprises fractionating as a syphilis antigen.