JPH0486559A - Syphilis inspection reagent - Google Patents

Abstract

PURPOSE:To improve specificity and sensitivity to initial syphilis by verifying antigen purity for a sample with a column to eliminate variations in antigen quality using an antigen exceeding a specified purity for an inspection reagent. CONSTITUTION:An anti-syphilis treponema antibody is detected and a sample containing an antigen derived from a syphilis treponema germ as antigen in an inspection reagent for diagnosing syphilis is made to adsorb on a hydroxy apatite gel column and eluted to perform a verifying of antigen purity. Here, an antigen with the antigen purity exceeding 25% which is verified by a formula [{amount of recovered protein)/(amount of added protein x column recovery ratio)}] x 100% is used as reagent raw material. The antigen is supported on an animal erythrocyte or a carrier such as resin beads or latex to manufac ture an inspection reagent. The use of the inspection reagent enables the expan sion of a range of an applicable reagent form eliminating false positive as caused by non-specific reaction.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is directed to the use of Treponema pallidum (pathogenic Treponema pallidum).
pallidum N1cols, hereinafter abbreviated as TP. ) Supporting crushed bacterial cells or antigens derived therefrom on carriers such as animal red blood cells, EIA pieces, and latex;
The present invention relates to an improvement in a test for diagnosing syphilis infection by detecting the presence of antibodies in a test serum that react with the obtained antigen-sensitized carrier. More specifically, the present invention relates to a syphilis test reagent that has high specificity and can detect early infection with high sensitivity.

[Conventional technology]

Conventionally, the diagnostic method for syphilis has been a testing method that uses TP bacteria directly as an antigen and utilizes the antigen-antibody reaction with anti-Treponema pallidum antibodies (hereinafter referred to as TP antibodies) in the test serum. Among these, the TPHA test developed by Onyoshi et al. is currently widely used due to its advantages such as sensitivity, specificity, and ease of operation, and its excellence as a representative clinical test method for syphilis has been recognized around the world. It is being

The antigen derived from TP bacteria (hereinafter referred to as TP antigen) used in the above method is obtained by suspending bacterial cells collected from conventional TP bacteria cultured in rabbit testicles in an appropriate buffer, immediately treating them with a homogenizer, and applying ultrasonic waves. The TP cells were crushed or solubilized by treatment and used as a carrier sensitized antigen solution.

[Problem to be solved by the invention]

However, such conventional techniques have problems as described in (1) to (3) below.

(1) Difficulty in detecting early syphilis In syphilis antibody detection methods that utilize antigen-antibody reactions such as TPHA, detection sensitivity for early antibodies (Ig-M) is lower than for late antibodies (Ig-G). Ta. it is,
The cause was the purity of the antigen carried on the carrier. In other words, the TP antigen used to carry animal red blood cells in the conventional TPHA test inevitably contains many impurities due to the manufacturing method, and more than 90% of the impure protein is due to impurities and antigenicity derived from the domestic rabbit chestnut circle in which the TP bacteria were cultured. It is said to be a protein derived from TP bacterial cells.

In this way, since the impurity fraction is mixed in at a much higher amount than the antigen fraction, the initial antibody (I
g-M) was not detected.

(2) Difficulty in controlling the quality of reagents Conventionally, TP antigen has been obtained by crushing TP cells collected from rabbit testicles or by solubilizing the cells and directly using them as a carrier-sensitizing antigen solution. When culturing TP bacteria using Shikaji rabbits, the antigenicity of the bacteria tends to vary depending on the culture temperature, the condition of the rabbits during culture, and mutations during passage of the bacteria. However, since there was no simple and reliable method for recognizing the quality of TP antigen, quality control of syphilis test reagents was difficult.

(3) Limitations on the reagent preparation method As mentioned in (1), conventionally used antigens had low antigen purity, so it was necessary to use a method capable of highly sensitive measurement as a reagent preparation method.

Examples of such highly sensitive measurement systems include enzyme immunoassay and fluorescence immunoassay, and the hemagglutination reaction used for TPHA is also a method capable of relatively highly sensitive measurement. However, the above-mentioned high-sensitivity measurement method is not fully satisfactory in terms of simplicity, and a simpler method, such as latex agglutination reaction, has been desired. However, methods such as latex agglutination reaction, which have somewhat insufficient detection sensitivity, have the problem that the required sensitivity cannot be obtained with antigens of conventional purity.

Therefore, an object of the present invention is to solve the problem of variation in antigen quality in a test reagent for diagnosing syphilis by detecting anti-Treponema pallidum antibodies, and to
The objective is to provide a reagent for syphilis testing that is highly specific and sensitive for early syphilis as well as late syphilis, and does not cause false positives due to non-specific reactions, and also to expand the range of applicable reagent forms. .

[Means to solve the problem]

As a result of intensive research, the present inventors have solved the above problem by testing the antigen purity of samples containing antigens derived from Treponema pallidum cells using hydroxyapatite gel columns and using antigens with a certain purity or higher as syphilis test reagents. We have found a solution to this problem and have completed the present invention.

That is, the first invention of the present invention is to detect an anti-Treponemal pallidum antibody as an antigen in a test reagent for diagnosing syphilis by adsorbing and eluting an antigen-containing sample derived from Treponema pallidum cells onto a hydroxyapatite gel column. This syphilis test reagent is characterized by using an antigen whose antigen purity is 25% or more as determined by the following formula when assayed for antigen purity, thereby solving the above problem.

However, the amount of added protein in the above formula is determined by thoroughly equilibrating the hydroxyapatite gel column with 10 mM potassium phosphate buffer (pH 6.0, hereinafter referred to as solution A) containing 1% octyl glucoside, and This is the amount of protein in the antigen-containing sample when the solvent of the antigen-containing sample derived from bacterial cells is replaced with Solution A and then added to the column.The amount of recovered protein is the amount of protein recovered by adding Solution A to the column after adding the antigen-containing sample After washing the unadsorbed components thoroughly, add 350 mM potassium phosphate buffer (hereinafter referred to as solution B) containing 1% octyl glucoside as an elution buffer (hereinafter referred to as solution B).
pH 6.0), and the ratio of B solution to A solution is 0% to
The amount of protein recovered is between 8% and 40% in solution B when elution is carried out continuously or stepwise up to 100%, and the column recovery rate is - The figure shows the ratio of the amount of protein recovered the second time to the amount of protein recovered the first time when the same fraction was collected by adding the same protein to the column again under the same conditions.

The second invention of the present invention is to use a hydroxyapatite gel after treating an antigen-containing sample derived from Treponema pallidum cells with a cation exchange column as an antigen in a test reagent for diagnosing syphilis by detecting anti-Treponema pallidum antibodies. A syphilis test reagent characterized by using an antigen whose antigen purity is 25% or more as determined by the following formula when assayed for antigen purity by adsorption and elution on a column. This solves the above problem.

However, the amount of added protein in the above formula is determined by thoroughly equilibrating the cation exchange column and hydroxyapatite gel column with 10 mM potassium phosphate buffer (pH 6.0, hereinafter referred to as solution A) containing 1% octyl glucoside. It is the amount of protein in an antigen-containing sample derived from Treponema pallidum cells, which is obtained by replacing the solvent of the antigen-containing sample with Solution A and adding it to a cation exchanger column. After adding to the cation exchanger column, A
After flowing the liquid to obtain a flow-through fraction, add the flow-through fraction to a hydroxyapatite gel column, flow liquid A at least 3 times the column volume, and after sufficiently washing unadsorbed components,
The elution buffer (hereinafter referred to as solution B) was a 350mM potassium phosphate buffer containing 1% octyl glucoside (pH
6,0), and the ratio of B liquid to A liquid is 0% to 10.
The amount of protein recovered is between 8% and 40% when the percentage of solution B is 8% to 40% when elution is carried out continuously or stepwise down to 0%, and the column recovery rate is - The figure shows the ratio of the amount of protein recovered the second time to the amount of protein recovered the first time when the same fraction was collected by adding the same protein to the hydroxyapatite gel column again under the same conditions.

The present invention will be explained in more detail below.

In the present invention, first, an antigen to be used in a syphilis test reagent is prepared. Antigens used in syphilis test reagents are prepared in a variety of ways, examples of which are as follows. TP cells are cultured by a known method.

This is used as a syphilis antigen as it is, or if necessary, destroyed by cooling or solubilized. Further, if necessary, this is purified by an appropriate method or non-antigenic proteins are removed and used as a syphilis antigen. In addition, antigens derived from TP cells may be antigens produced in E. coli, yeast, etc. through genetic recombination.

As the syphilis antigen of the present invention, an antigen whose antigen purity has been determined to be 25% or more by the assay shown below is used, and this is carried on a carrier such as animal red blood cells, plastic beads, latex, etc., and the syphilis test reagent of the present invention is used. Manufacture.

Method for Obtaining and Purifying TP Antigen 1) Selection and Cultivation of Bacterial Cells As the TP inoculum, for example, the WHO pathogenic standard Nichols strain or the TP bacteria used by various testing institutions for syphilis testing may be used as is. In addition, WHO pathogenicity standard Nichols, for example, CDC (Center for Disease)
ase Control, Public He
alth 5service.

Ll, S, Department of Health
, Education and Welfare,
It is readily available from Atlanta Georgia. Furthermore, the method for culturing TP bacteria and the method for collecting bacteria from the culture in the present invention can be arbitrarily adopted from among known methods.

2) Method for obtaining TP antigen from TP cells The TP cells are used as they are or, if necessary, after being subjected to the following treatments. After culturing the TP bacteria in 1), collect the cells, suspend them in a buffer solution, cool and crush them, then fractionate them by centrifugation, and collect the antigen-containing fractions from each fraction to collect the TP bacteria. Culture-derived antigen is obtained and optionally solubilized. The method may be appropriately selected from generally known methods. As the crushing method, homogenizer treatment, ultrasonic treatment, freeze-thaw method, etc. are used. Examples of solubilization methods include protein solubilization treatment using surfactants, chaotropic ions, urea, and alkali, enzyme treatment, and autolytic methods.

3) Purification of TP antigen TP antigen is purified as necessary. Any method for purifying the TP antigen can be adopted from among known biochemical methods. For example, there are the following methods.

■ Purification using ammonium sulfate salting out fractionation, polyethylene glycol precipitation fractionation, and isoelectric precipitation.

■ Purification by chromatography; gel chromatography, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, reversed phase chromatography, chromatography using a hydroxyapatite carrier, etc. It is also possible to purify the antigen by the above chromatography using a high performance liquid chromatography device.

4) Other methods for obtaining TP antigen Other TP antigens may be samples containing TP antigens produced in E. coli, yeast, or other hosts through genetic recombination.

The company manufactures reagents for syphilis testing.

1) In the first aspect of the present invention, a sample containing a syphilis antigen is added to a hydroxyapatite gel column. At this time, as an adsorption buffer, 10 mM potassium phosphate buffer (1) H6 containing 1% octyl glucoside,
0, A solution) to sufficiently equilibrate the column, and samples containing syphilis antigens are subjected to solvent exchange with A solution by an appropriate method such as dialysis or gel filtration before being added to the column. At this time, the amount of protein in the sample containing the syphilis antigen added to the column is defined as the amount of added protein.

Next, the A solution was flowed at least 3 times the column volume, and after the unadsorbed components were sufficiently washed, a 350 mM potassium phosphate buffer (B solution) containing 1% octyl gel colcito was used as the elution buffer. The ratio of B liquid to A liquid is 0% to 1
Elute continuously or stepwise up to 00%. At this time, the amount of protein obtained between 8 and 40% of the B solution is defined as the amount of recovered protein. In the present invention, when the antigen purity is determined using the following formula, 25% or more of the TP antigen is used as the raw material.The column recovery rate in the formula refers to the protein once recovered by this method, which is then added to the column again under the same conditions. It is the ratio of the amount of protein recovered in the second time to the amount of protein recovered in the first time when the same fractions are collected.

The hydroxyapatite gel used in the present invention may have any properties such as its shape, Ca/P ratio, etc.
The recovery rate varies greatly depending on the properties of the gel. This means that depending on the hydroxyapatite gel, the adsorbed component may be 1
00% is because some items are not recovered. That is, in carrying out the present invention, it is necessary to correct the properties of the gel. Therefore, when the protein recovered once using that method is added to the column again under the same conditions and the same fraction is recovered, the ratio of the amount of protein recovered in the second time to the amount of protein recovered in the first time is determined, and this is calculated from the column recovery amount. must be corrected as a percentage.

2) Next, the second aspect of the present invention will be explained. Depending on the culture, extraction, and purification conditions of the bacterial cells, or the origin of the antigen, a sample containing the antigen may contain components that interfere with antigen purity assay using hydroxyapatite. In such cases, it may be desirable to pass the sample through a cation exchanger before adsorption onto the hydroxyapatite gel column.

That is, such a component may be a substance (mainly protein) having an isoelectric point of 6 or higher, such as in a tissue or a bacterial cell. If these are present in large quantities in the sample,
In assays using hydroxyapatite, it may be eluted near the target fraction (8-40% of B solution) and interfere with the assay.

If it is eluted before the desired fraction, due to the tailing phenomenon,
Alternatively, if the protein is eluted after the target fraction, the amount of protein recovered in the target fraction increases due to the leading phenomenon, and in some cases, it is eluted within the target fraction, resulting in an apparent increase in antigen purity. This may make it difficult to obtain antigens of desired quality.

Therefore, in order to avoid this, it is necessary to use a cation exchanger to reduce the components that interfere with the measurement and increase the reliability of the assay using hydroxyapatite.

Hereinafter, a method for assaying an antigen using this cation exchanger will be specifically explained.

A column packed with a cation exchanger and a hydroxyapatite gel column are sufficiently equilibrated with the adsorption buffer solution (liquid A) in advance. In addition, samples containing syphilis antigens are exchanged with solution A by an appropriate method such as dialysis or gel filtration.

The sample containing the syphilis antigen is then passed through a column packed with a cation exchanger to obtain a flow-through fraction. This removes substances that would interfere with purity measurements using hydroxyapatite gel. This is then added to a column of hydroxyapatite gel. At this time, the amount of protein in the sample containing the syphilis antigen before being added to the cation exchanger is defined as the amount of added protein. The following is the same as 1) above.

Next, solution A is flowed at least three times the capacity of the hydroxyapatite column, and after the unadsorbed components are sufficiently washed, the ratio of solution B to solution A is reduced to 0 using the elution buffer (solution B).
% to 100% continuously or stepwise. At this time, the amount of protein obtained when the proportion of liquid B is between 8% and 40% is defined as the amount of recovered protein. Then, a syphilis test reagent is manufactured using the TP antigen, which has an antigen purity of 25% or more when determined by the following formula, as a raw material.

In addition, column recovery rate is the amount of protein recovered in the second time compared to the amount of protein recovered in the first time when the protein recovered by this method is added to the hydroxyapatite gel column again under the same conditions and the same fraction is recovered. It is the ratio of

There are various cation exchangers that can be used, but in the present invention, cellulose, agarose, or cross-linked agarose, which cause less non-specific adsorption of proteins, is preferably used as the gel matrix. As the ion exchange group, an ion exchanger into which a carboxyl group or a sulfonic acid group has been introduced is preferably used, and a strong ion exchanger into which a sulfonic acid group has been introduced is particularly preferable because of its wide applicable pH range. It is.

3) The syphilis test reagent of the present invention has an antigen purity of 25%.
The above TP antigen is used, and more preferably 30% or more. However, if the standard value regarding antigen purity becomes stricter than that, the number of TP antigens that can meet this standard will be limited, and an additional purification process for TP cells or TP antigen will be required, resulting in higher costs. .

A TP antigen with a purity of 25% or higher can be used to manufacture syphilis test reagents because it shows sufficient specificity and sensitivity when supported on a carrier, and no false positives due to non-specific reactions are observed. be.

In addition, in the above-mentioned elution from the hydroxyapatite gel column, as described in 1) and 2) above, the ratio of solution B to solution A is 8 to 40%. The ratio of liquid B may be changed continuously or stepwise.

Furthermore, the protein quantification method used in the antigen purity assay uses a method that is not affected by octyl glucoside since the sample contains it.

For example, a measurement method using bicinchoninic acid (Smith).

P, Ni, Krohn, R,1, etc. (198
5) Anal, Biochem.

150, 76-85. ) etc. can be used.

Form of the syphilis testing reagent of the present invention The syphilis testing reagent of the present invention can be in any of the following forms. As carriers, the antigen is immobilized using various animal red blood cells such as chicken red blood cells and sheep red blood cells, synthetic polymer carriers such as latex, and inorganic material carriers such as silica. In addition to the methods used for the antigen-antibody reaction, EIA (enzyme immunoassay) and FIA (fluorescence immunoassay) are also available, in which the antigen is immobilized on synthetic polymers such as plastic beads and plastic plates. Any method used for the syphilis test can be used as the syphilis test reagent of the present invention.

〔Example〕

In the following Examples, Example 1 describes an antigen purified using /S hydroxiabatite gel and its purity assay, and Example 2 describes an antigen purified by affinity chromatography and its purity assay. Ru. Using the antigens whose purity was tested in Examples 1 and 2, a self-syphilis test reagent was prepared using the TpHA method to examine the effects of the present invention. In Example 3, a reagent was prepared using the latex method and the present invention was tested. A study of the effects of the invention will be described as Example 4.

Hereinafter, the present invention will be explained in more detail with reference to Examples. However, the present invention is not limited to these Examples.

Example 1 Preparation of antigen and purity assay according to the present invention (TP antigen purified using hydroxyapatite gel) i. Preparation of antigen 1) Materials (1) Buffer (1-1) Phosphate buffer pH 6, 5 (hereinafter referred to as PBS) Potassium dihydrogen diphosphate and disodium hydrogen phosphate (12
hydrate) and sodium chloride, phosphoric acid concentration 0.036
Prepare a buffer solution with a NaCl concentration of 0.156M and a pH of 6.5, and add N to it to a concentration of 0.1% (W/V).
aN2 was added.

(1-2) 1% BSA/PBS: Bovine serum albumin (hereinafter referred to as BSA, manufactured by Miles) was added to PBS.
It was dissolved in BS to a concentration of 1% (W/V).

(1-3) 10mM potassium phosphate buffer (hereinafter KP
BI) H6,0 and pH 7,0): 10mM phosphorus, potassium dihydrogen phosphate solution, lomM potassium dihydrogen phosphate solution pH 6,0 and pH 7,
Adjusted to 0.

(1-4) 350mM KPB (pH 6,0)
: Adjusted to I) H6,O with 350 mM potassium dihydrogen phosphate solution and 350 mM potassium dihydrogen phosphate solution.

(2) Surfactant octyl glucopyranoside (lon
-Cutyl-β-D-glucopyranosite) hereinafter abbreviated as OG. A product for research on poorly soluble proteins (manufactured by Nacalai Tesque) was used.

(3) 611811m chromatography gel (3-1
) A cation exchanger S-3epharose Fast Flow (manufactured by Pharmacia) was used. This is a cation exchanger with sulfonic acid groups introduced onto the surface of cross-linked agarose gel.

(3-2) Hydroxyapatite gel biogel HT
P (manufactured by Bio-Rad) and HCA-200L (
(manufactured by Mitsui Toatsu Chemical Co., Ltd.) was used.

(4) TP cells: Cultured cells were used according to the method described below.

6.0X of the pathogenic standard Nichols strain Treponema pallidum
A suspension of 107 k/i was added to 17 n per rabbit.
1 was inoculated. After culturing for 10 days, chestnuts were collected from 10 rabbits, minced, and shaken with 500 i of 2.2% sodium citrate solution at 37°C for 30 minutes to extract TP bacteria. The extract was centrifuged at 200Xg for 5 minutes to remove the rabbit tissue precipitate, and the supernatant was centrifuged at 3000Xg for 30 minutes.
TP bacteria were precipitated. After repeatedly washing the obtained TP bacterial cells with PBS, they were suspended in PBS and the number of bacteria was calculated using a dark field microscope and adjusted to 1X10'' cells/ml.
A bacterial cell suspension was prepared. This bacterial cell suspension was observed under a dark field microscope, and it was confirmed that no rabbit sperm or rabbit tissue was present.

(5) Reagent for measuring protein concentration: B CA Protei
nAs5ay11%agent(PIERC
(manufactured by Company E) was used.

(6) Microtiter plate: Nunc's 96-well microwell plate (U bottom) was used.

(7) TP antigen-sensitized blood cells: Components of Serocrit TP (manufactured by Chemo and Serum Therapy Research Shin), a commercially available TP'HA kit, were used.

(8) Syphilis-positive rabbit serum: Serum was collected from a rabbit that had been cultured with Treponema pallidum in a rabbit komaru for 45 days and used. The antibody titer was measured using a commercially available TPHA kit and found to be 102.400. It was diluted with 1% BSA/PBS and used.

2) Method (1) Measuring protein concentration Bicinchoni is a modified method of the Lowry method.
Assay method using nicacid (Smith, P., Krohn, RoI.

etc, (1985) Anal, Biochem,
150.76-85. ) based on the principle of B CA Pr
otein As5ay Reagent (PI
(manufactured by ERCE). Standard is 1
BSA dissolved in 10mM KPB containing %OG was used, and the unit was expressed in (μg/mj).

(2) Measurement of antigen activity The test was carried out using a hemagglutination inhibition test.

■ Add 25 μβ of 1% BSA/PBS to each well of the microtiter plate.

(2) Add 25 μl of each antigen fraction to the front well of a microtiter plate, and dilute this 2-fold on the plate with a 1% BSA/PBS solution.

■ Add syphilis-positive rabbit serum diluted to an antibody titer of 50 to the antigen diluted with ■ and mix.

■ Allow to react at room temperature for at least 30 minutes. At this time, antibodies in wells with a high antigen concentration on the front side of the plate bind to the antigen and are consumed, or antibodies in wells with a low antigen concentration remain unconsumed.

■ Next, TP antigen-sensitized blood cells were added to each well, and the final dilution rate of the rabbit serum in the wells that caused an agglutination reaction with the remaining unconsumed antibodies (8, 16, 321,...) The antigen activity was defined as the antigen activity, and the unit was expressed as (titer/rnl).

(2) Antigen specific activity was expressed as the antigen activity divided by the protein concentration, and the unit was (titer/μg).

(3) Solubilization and extraction of antigen from TP cells Using PBS 50rnI for 50ml of TP cell suspension,
Centrifugal washing for 30 minutes at 00Xg three times, then PBS5
suspended at 0-. This suspension is treated with an ultrasonic crusher,
TP bacterial cells were crushed. This was centrifuged at 12,600×g for 30 minutes to remove the precipitate.

Precipitation was carried out using 10mM KPB pH 7.0.
After repeating centrifugal washing at 000Xg twice, 1% OG
(W/V) containing 10mM K P B 1) H7,0 5
The precipitate was thoroughly suspended and then solubilized by gentle ultrasonication. After leaving at 4°C for more than 16 hours,
Centrifuge at 0.000 x g for 1 hour, and the resulting supernatant
．． Filter with a 22μm filter (manufactured by Millepore, trade name Millex GS),
A TP bacterial cell-derived extract with a protein concentration of 365 μg/mj) was obtained. This is hereinafter referred to as extracted antigen.

(4) Purification of antigen ■ Dialysis of antigen The above extracted antigen was 10mM K P B p containing 1% OG.
) of which 3071L is dissolved in I7.0
1 was dialyzed against the same buffer (solution A) containing 1% OG and having a pH of 6.0.

The volume ratio of dialysate: extracted antigen solution was 4 volumes: 1 volume, and the external dialysate was exchanged three times or more. After the final dialysis, the p
H was measured and it was confirmed that the pH was in the range of 6.0±0.1.

■ Crude purification of antigen Pass the antigen in ■ through a column packed with S-Sepharose Gel 3 (W (Pharmacia, SR25/45), and collect the flow-through fraction of 60rnl (antigen activity 1024t 1ter/m).
/, protein concentration 101 μg/mN) was collected as an antigen fraction.

(Hereinafter, this will be referred to as crudely purified antigen.) ■Purification using hydroxyapatite gel Hydroxyapatite gel 8i was applied to a column (Pharmana, HRI
10mMK containing 1% OG
Equilibration was performed with P B pH 6.0 (hereinafter referred to as solution A).

Add 2 coats of the above crudely purified antigen to each hydroxyapatite column.
After adding 0, pour solution A into the column, add 0, D,
The column was washed until the absorption at 280 o, oio or less was reached. The fraction at this time was defined as the pass-through fraction.

350mMK P B p containing 1% OG for solution A
The ratio of H6,0 (hereinafter referred to as solution B) was gradually increased from 0 to 40% and elution was performed using a linear gradient, and then the ratio of solution B was increased to 100% and elution was performed.

The ratio of B liquid is 0% → 8%, 8 → 16%, 16 → 24%,
Elution fractions of 24→32% and 32→40% were collected, respectively.

Fractions eluted at 350mM K P 8100% were collected and defined as 40% or higher fractions.

■Each Fracunyone assay pass-through fraction, 0-8% fraction, 8-16% fraction, 1624
% fraction, 24-32% fraction, 32-40% fraction, 40%
The antigen activity and protein concentration of each of the above fractions were determined using the methods of 2) (2) and 2) (1), respectively, and the specific activity was calculated from this.

■Concentration of HAp antigen Among the fractions that showed antigenic activity, the specific activity was 12 titer/
Collect the fractions showing more than μg, and the protein concentration is 40 μg/r.
It was concentrated under reduced pressure using a cellophane tube (Wako Pure Chemical Industries) until it reached nI or higher. 10ml of antigen purified with each hydroxyapatite column (biogel-
The HTP-purified product has an antigenic activity of 2048 titer/
mf7, protein concentration 77μg/mN, purified with HCA-200L, antigen activity 1536t i ter/me
, a protein concentration of 42 μg/m2 was obtained. Hereinafter, the resulting fraction will be referred to as HAp purified antigen.

11. Antigen purity assay according to the present invention l) Assay according to the first invention ■ 2 ml of extracted antigen, 4 rnl of crudely purified antigen and F (Ap
Purified antigen (also known as biogel HTPSHCA-200L)
2 ml was dialyzed against A solution. External dialysis fluid: Antigen solution - 4 volumes, 1
The external dialysis solution was exchanged three times or more.

(2) A column packed with hydroxyapatite gel (Pharmacia, HRIO/10) was sufficiently equilibrated with Solution A.

(2) After adding the extracted antigen, crudely purified antigen, and HAp purified antigen to the column, the amount of protein before adding to the column was defined as the amount of added protein. ), solution A was flowed at least 3 times the column volume. 0
．． D. The column was washed until the absorption at 280 was below 0.010.

(2) Elution was performed while continuously changing the ratio of solution B to solution A from 0% to 40%, and then the column 1 was washed by increasing the ratio of solution B to 100%. The amount of protein recovered between 8% and 40% of the B solution (recovered protein amount) was calculated from the protein concentration.

■ Column recovery rate was calculated using the following method. In (2), each antigen is added to each hydroxyapatite column and a portion of the collected fraction is taken to give a protein amount P1 of 50 μg. ), the amount of protein recovered by repeating the operations ① and ② above after dialysis with solution A until the proportion of solution B is between 8 and 40% is defined as P2. ). The column recovery rate was calculated as column recovery rate = P2/P1.

■ The antigen purity of each antigen was calculated using the following formula.

2) Assay according to the second invention - 2 ml of extracted antigen, 4 ml of crudely purified antigen, and 2 ml of HAp purified antigen (both biogel HTP and HCA-200L)
1 was dialyzed against solution A. External dialysis solution: The volume ratio of antigen solution was 4 volumes to 1 volume, and the external dialysis solution was exchanged three times or more.

■ A column packed with S-Sepharose (Pharmacia, HRIO/10) and a column packed with hydroxyapatite gel (Pharmacia, HRIO/10)
) was sufficiently equilibrated with Solution A.

(2) The amount of protein after adding the extracted antigen, crudely purified antigen, and HAp purified antigen to the S-Sepharose column and before adding it to the column was defined as the amount of added protein. ), the flow-through fraction was obtained by flowing solution A.

(2) After adding the flow-through fraction of each antigen to a hydroxyapatite column, solution A was flowed at least three times the column volume. O,
D. The column was washed until the absorption at 280 was below 0.010.

(2) Elution was performed while continuously changing the ratio of solution B to solution A from 0% to 40%, and then the column was washed by increasing the ratio of solution B to 100%. The protein amount (recovered protein amount) of the protein recovered between 8% and 40% of the B solution was calculated from the protein concentration.

■ Column recovery rate was calculated using the following method. In (2), each antigen is added to each hydroxyapatite column and a portion of the collected fraction is taken to give a protein amount P1 of 50 μg. ), repeat the operations ① and ② above after dialyzing with solution A, and calculate the amount of protein recovered between 8% and 40% of solution B. The amount of protein obtained is defined as P2. ). The column recovery rate was calculated as column recovery rate - P2/P1.

■ The antigen purity of each antigen was calculated using the following formula.

ii.Results Antigen purification was performed using Biogel HTP and HCA-200L as hydroxyapatite gels in exactly the same manner. In addition to the fraction obtained by i.2) (4).2, the protein concentration and antigen activity were also measured for the extracted antigen and crudely purified antigen, and the antigen specific activity was calculated. The results are shown in Table 1.

Table 2 shows the results of calculating the column recovery rate using a portion of the extracted antigen, crudely purified antigen, and HAp purified antigen. There was no difference in column recovery rate depending on the measured sample. In the following calculations, the average values in Table 2 were used for column recovery rates.

Furthermore, the results of calculating the antigen purity using biogel HTP and HCA-200L for each of the extracted antigen, crudely purified antigen, and HAp purified antigen according to the first and second inventions of the present invention are compared with those according to the first invention. Table 3 shows the products according to the second invention, and Table 4 shows the products according to the second invention. As a result, it was found that antigens with high antigen purity tended to have high antigen specific activity.

(Margins below this page) Table 1 (1) Purification by Biogel HTP Table 1 (2) Purification Example 2 by HCA-20OL Preparation of antigen and purity assay according to the present invention (antigen purified by affinity chromatography) i. Preparation of Antigen 1) Materials Unless otherwise specified, reagents and samples with the same names as in Example 1 i-1) were prepared in the same manner as in Example 1 i-1).

(1) Buffer (1-1) Coupling buffer: sodium bicarbonate,
From sodium carbonate and sodium chloride, 0.5M NaC
A 0.1M carbonate buffer (pH 8.3) containing 1.

(1-2) After dissolving blocking buffer nigericin, N
A 0.2M glycine buffer (pH 8.0) was prepared by adjusting the pH with aOH.

(1-3) Acetate buffer: Mix 0.1 M acetic acid and 0.1 M sodium acetate, add NaCl to this and add 0.5
0.1M acetate buffer (pH 4,0) containing M NaCl
was prepared.

(1-4) 50mM Tris-HCI-0,15M
K C1 (pH 8,0X or below, abbreviated as TKB) (2) Affinity chromatography gel CN B
r-activated-3apharose 4
B (manufactured by Pharmacia) was used.

(3) Anti-TP antibody: Example 1 i, 2) - (4) ■
The antigen purified by biogel HTP in Freund
Antiserum produced by immunizing a rabbit with complete adjuvant (antibody titer 40.960)
The meaglobulin fraction purified by 1.4M ammonium sulfate fractionation twice was used after dialysis against coupling buffer2)
, Method (1) Immobilization of anti-TP antibody: ■CNBr-activated-3apha
ose 4B dry powder at 1 ml in 1 mM HCl.
After swelling for 5 minutes, transfer the gel to 1 m on a glass filter.
MHC].

■Wash the gel with the coupling buffer and then suspend it in the same buffer. An anti-TP antibody solution was added so that 7 ml of anti-TP antibody was contained per rnl of gel, and the mixture was stirred at 4°C for 166 hours.

(2) The gel was suspended in blocking buffer and mixed for 2 hours at room temperature.

(2) After washing the gel with a coupling buffer, it was washed with an acetate buffer, and further washed with a coupling buffer. After repeating this three times, the cells were washed with TKB, suspended in TKB, and stored.

(2) Purification by affinity chromatography ■ Preparation of anti-TP antibody column Gel IO- on which anti-TP antibody was immobilized was packed into a column (Pharmacia, HRIO/10).

■ Preparation of TP Antigen The extracted antigen 10- obtained in Example 1 was added to TP antigen containing 1% OG.
It was used after dialysis with KB. Dialysate: Extracted antigen solution = 4 volumes: 1
Dialysis was performed in a volume-to-volume ratio, and the dialysis fluid was exchanged three times.

(2) Adsorption of TP antigen onto anti-TP antibody column The anti-TP antibody column was equilibrated in advance with TKB containing 1% OG, and then the TP antigen was added to the column. T containing 1% OG after addition
KB was passed through the column and absorbed at O, D, 280 or 0.
It was washed until it became 0.010 or less.

(2) Elution of TP antigen TP antigen was eluted by flowing TKB containing 3M guanidine hydrochloride and 1% OG through the column. The obtained TP antigen was immediately added to a Sephadex G-25 column (trade name: PDIO, manufactured by Pharmacia) equilibrated with 10mM KPB containing 1% OG, and guanidine hydrochloride was removed from the TP antigen by buffer exchange. did.

■ Concentrate the TP antigen under reduced pressure using a cellophane tube (Wako Pure Chemical Industries) until the protein concentration reaches 50 μg/- or more.
A fraction of the phlegm was obtained. The resulting fraction is referred to as AC purified antigen.

(3) Antigen Assay Method The antigen activity and protein concentration of the AC purified antigen were measured in the same manner as in Example 1.

ii1 Antigen purity assay according to the present invention Using 2- of the AC purified antigens, the test was carried out in the same manner as in ii of Example 1. The average value in Table 2 was used for the column recovery rate.

Results After performing antigen purification by affinity chromatography using the extracted antigen of Example 1 according to the above method, protein concentration and antigen activity were measured, and total antigen activity, total protein amount, and antigen specific activity were determined. was calculated. As a result, the antigen activity was 2048 titer/ml, and the protein concentration was 82.2 ug.
An AC purified antigen with a specific activity of /m11 antigen of 24.9 titer/μg was obtained.

Purity assays were conducted for each of the extracted antigen and the AC purified antigen according to the first and second inventions of the present invention. The results are shown in Table 5 for the first invention and in Table 6 for the second invention.

(Margins below this page) Example 3 TPHA The TP antigen whose purity was confirmed by the present invention was carried on sheep red blood cells to confirm the effect of the present invention in the TPHA method.

1) Materials Unless otherwise specified, reagents and buffers with the same names as in Example 1.2 were prepared in the same manner as in Example 1.2.

(1) Buffer (1-1) 0.15M phosphate buffer (1) 87.4
): 0.15M sodium dihydrogen phosphate (dihydrate)
and O, 15M disodium hydrogen phosphate (decahydrate) are mixed together in an aqueous solution and adjusted to pH 7.4.

(1-2) Add 9.0g of physiological saline to 10g of sodium chloride.
Dissolved in 00g of purified water.

(1-3) McIlvaine buffer (hereinafter abbreviated as McI) = 0.1M citric acid and 0.2M sodium hydrogen phosphate dodecahydrate) mixed and adjusted to pH 6.5 .

(1-4) 1% OG/Me I: 1% (w/v) OG dissolved in Me I.

(1-5) PHA buffer: Prepared by mixing the following solutions and reagents (buffer 10
amount per 00rnl).

■ Normal rabbit serum 3W ■ Sheep red blood cell stroma 107nl ■ Sodium azide 1g ■ 0.15M sodium phosphate buffer pH 7,400rnI ■ Physiological saline 86 (W) (2) Reagents, etc. (2-1) Tannic acid From Nacalai Tesque ■ I bought it.

(2-1) Fixed Sheep Serum Sheep red blood cells fixed with glutaraldehyde were used.

(2-3) Commercially available TPHA kits: Celodia TP (Fujirebio) and Cerocrit T
P (Kaketsuken) was used.

(3) Specimens (3-1) Syphilis positive specimens: Three fully treated late stage syphilis specimens containing many IgG antibodies (Cz, G1, G,
) and three specimens (M, , M2, and Ml) of early syphilis 3 to 5 weeks after infection, in which little IgG antibodies were produced and many Ig-M antibodies were produced.

(3-2) Syphilis negative specimens: 3 specimens (N,,N,,N,)
was used. All of these samples showed non-specific reactions in the commercially available TPHA test, and were confirmed to be syphilis-negative by PTA-ABS.

(3-3) Anti-rabbit tissue antiserum: Normal rabbit Suimaru was minced and solubilized with 1% OG, which was obtained by immunizing a goat.

(3-4) Anti-Relter strain antiserum: Trepone
ma phagedenisbiotype Re1t
A goat was immunized with the solubilized bacterial cell components of er in 1% OG.

(4) TP antigen The extracted antigen, crudely purified antigen, HAp purified antigen obtained in Example 1, and AC purified antigen obtained in Example 2 were used.

The antigen activity and protein concentration of each antigen solution are shown in Table 7.

2) Methods (1) Blood cell preparation method ■ Fixed sheep blood cells were centrifugally washed four times with physiological saline at 700×g for 5 minutes, and then suspended in physiological saline to a solid content of 6%. Dissolve tannic acid in physiological saline,
This was added to the above blood cell suspension and stirred.

(2) After stirring, the mixture was centrifugally washed twice with physiological saline and once with Mcl, suspended in Mcl to a solid content of 6%, and immediately used for antigen sensitization.

(2) The TP antigen solution (extracted antigen solution, crudely purified antigen solution, HAp purified antigen solution) was dialyzed in advance with 1% OG/McI.

This antigen solution was prepared as shown in Table 8 and used as an antigen sensitization solution. Mix equal amounts of the above blood cell suspension and antigen sensitization solution, and
The mixture was stirred at ℃ for 1 hour.

The sensitizing solution was prepared so that the antigen activity was 1 ootiter/mj. Sensitization solution B has a protein amount of 10μ
g/ml.

(2) The sensitized blood cells were washed twice with physiological saline and then suspended in PHA buffer to a solid content of blood cells of 0.2%.

After leaving it at room temperature for 3 hours, used for assay.

(Leaving space below the essence) (2) TPHA assay method■ Pour 100 μp of PHA buffer into the front well of the microtiter plate and 25 μp into the other wells.
It was dispensed in liter portions.

(2) 25 μβ of each sample was dispensed into the front well, and diluted several times on the plate. On the other hand, 75 μβ of the diluted sample liquid was taken out from the well on the front side, so that 25 μl of the diluted sample liquid remained in the well on the front side.

(2) The blood cell solution prepared in (1) was shaken well to make an even suspension, and 75 μl of the suspension was dropped into each well.

■ Place the corner of the plate on a light (portic mixer) and mix thoroughly, then cover with an empty plate to prevent evaporation, and leave to stand at room temperature being careful not to shake.

Judgment was made after 2 hours.

■ Place the plate on white paper and observe the hemagglutination image.
Judgment was made with the naked eye. The highest dilution that showed agglutination (20,
40,80,,,,) was taken as the antibody titer.

3) Results Using the sensitized blood cells obtained by the above method, the presence or absence of hemagglutination reaction and antibody titer of each specimen were measured, and the results shown in Table 9 were obtained.

(margin below essence) Table 9 Results of TPHA of the present invention +・Positive, −−Negative, ±−False positive, (1) Sensitization f1 prisoner Antigen amount constant sensitization The numbers below each result represent the antibody titer.

(2) Sensitization solution ■ Sensitization with constant protein amount Note) Antigen purity is the antigen purity by biogel HTP (HC
Antigen purity using A-20OL).

Note) Antigen purity is antigen purity CHC by biogel HTP.
Antigen purity at 8-20OL).

From Table 9, for the three early syphilis samples in which all sensitized blood cells carrying extracted antigen with antigen purity of 25% or less were negative,
All sensitized blood cells carrying antigens with an antigen purity of 25% or more (crude purified antigen, HAp purified antigen, AC purified antigen) are positive. In addition, in the three syphilis-negative samples in which a non-specific reaction was observed with sensitized blood cells carrying commercially available TPHA and extracted antigens, the sensitized blood cells carrying antigens with a purity of 25% or more showed an agglutination reaction due to a non-specific reaction. Not shown. In addition, purity 2
The sensitized blood cells carrying 5% or more of the antigen showed no agglutination reaction with the anti-rabbit tissue antiserum and the anti-Relter strain antiserum. In addition, the conventional TPHA method, RPR method, glass plate method,
Table 10 shows the test results using the PTA-ABS method.

(Leaving space below the essence) 4) Conclusion As is clear from the results, the TPHA method using blood cells carrying antigens that meet the purity standards of the present invention is superior to the conventional TPH
With method A, early syphilis that could not be detected becomes detectable, and false positives due to non-specific reactions are not observed.

Example 4 Latex reagent (measurement method using a fully automatic analyzer) The effectiveness of the present invention in quantifying antibodies against Treponema pallidum antigen was confirmed by measuring agglutination of a latex diagnostic reagent using a fully automatic analyzer. . Unless otherwise specified, reagents and specimens with the same names as those in Examples 1 to 3 are used in Example 1.
- Prepared in the same manner as in 3.

■) Preparation of reagents and specimens (1-1) Latex: 0.40 manufactured by Sekisui Chemical ■
0 μm polystyrene latex (solid content 10%) was used.

(1-2) PBS pH 7.4 0.02λ (phosphoric acid, 0.15M sodium chloride was adjusted from monosodium diphosphate (dihydrate), disodium phosphate (dihydrate), and sodium chloride) .Contains 0.1% NaNs.

(1-3) NaCl-PBS pH 6,5 phosphate monosodium IJ From rum (dihydrate), disodium phosphate (dihydrate), sodium chloride, 0.02M phosphoric acid, 1.
00M sodium chloride was prepared. 0.1% NaN 3
including.

(1-4) 100mM NaPB 100mM NaPB (pH 7,50) was prepared from sodium hydrogen diphosphate (anhydrous) and sodium dihydrogen phosphate (decahydrate). Contains 0.1% NaNx.

(1-5) 1%BSA-NaPB: 100m
MNaPBi: Adjusted so that BSA is 196.

(1-6) 5%BSA-NaPB: 100m
MNaPBf: Adjusted to have a BSA of 5%.

(1-7) Diluent = 5% BSA-NaPB in which polyethylene glycol (average molecular weight 500,000: Wako Pure Chemical Industries, Ltd.) was dissolved at a concentration of 0.25% (W/W).

(1-8) Apparatus: Measurement was performed using a Hitachi model 7050 fully automatic analyzer.

2) Method (1) Preparation of antigen sensitization solution Each antigen solution with the antigen activity and protein concentration shown in Table 7 was prepared by
NaCl-PBS, 10mMK containing 1%○G
A mixture of P and B in the amounts shown in Table 11 was used as an antigen sensitization solution. The sensitizing solution has an antigen activity of 250 tit.
er/ml. The sensitizing solution (■) was prepared to have a protein content of 25 μg/rn (!).

(Leaving space below essentials) Table 11 Composition of antigen sensitizing solution for latex reagent Sensitizing solution (a) Sensitization with constant antigen amount Sensitization solution with constant protein amount (2) Immobilized solid content of syphilis antigen 10% polystyrene latex lOOμp 4
While stirring with a magnetic stirrer in an incubator at 4°C, 400 μl of the syphilis antigen solution from (1) was quickly mixed and stirred at 4°C for 1 hour.

After that, add 5 ml of 1% BSA and add 1.5 ml of 1% BSA at 4°C.
Stir for hours. Thereafter, centrifugation was performed at 15,000 rpm for 1 hour. Add 1% BSA-Na to the obtained precipitate.
5 ml of PB was added and centrifuged in the same manner to wash the precipitate.

1% BSA-NaPB was added to this precipitate,
It was well dispersed to form a latex reagent with a solid content of 0.2%.

Latex reagents were stored at 4°C.

(3) Measurement conditions The measurement conditions were set as follows.

Sample volume 20μl (sample) R1 volume 50μl (latex reagent) R2 volume
350 μl (diluted solution) Wavelength 570 nm Measurement temperature 37°C (4) Measurement method Wavelength 570 nm 80 seconds and 320 seconds after the start of measurement
Take the difference between 0. D, the amount of change was 570.

3) Results Using the latex reagent prepared from the TP antigen solution by the above method, the reaction with each sample was measured as the change in turbidity at O, D, and 570. The results are shown in Table 12. I got it.

(Left below the essence) From Table 12, firstly, the three early syphilis samples have an antigen purity of 25%.
Latex reagents prepared using the following extracted antigens do not show enough sensitivity to make a positive test, or the antigen purity is 25%.
Latex reagents prepared using the crudely purified antigen, HAp purified antigen, and AC purified antigen described above all exhibit sufficient sensitivity for positive determination. In particular, the sensitivity of highly purified HAp-purified antigen and AC-purified antigen is significantly improved. A similar improvement in sensitivity was also observed in the three late-stage syphilis samples.

In the three syphilis-negative samples, no agglutination reaction due to non-specific reaction was observed in the latex reagent prepared using the purified antigen that met the specifications of the present invention.

Furthermore, latex reagents prepared using purified antigens that meet the specifications of the present invention include anti-rabbit tissue antiserum and anti-Re1te antiserum.
The r strain antiserum did not show a value that would be judged as positive.

4) Conclusion As is clear from the results, test reagents can be manufactured by latex agglutination reaction by using antigens that meet the specifications of the present invention. Furthermore, a latex reagent using an antigen that meets the specifications of the present invention is capable of detecting early syphilis, and false positives due to non-specific reactions are not observed.

〔Effect of the invention〕

The syphilis test reagent of the present invention uses an antigen with high antigen purity, so it is highly specific for early syphilis as well as late syphilis, and there are no false positives due to non-specific reactions. It is easy to control and maintain a constant level of quality, and variations in quality can be suppressed, making it extremely useful as a syphilis test reagent.

Claims (1)

[Scope of Claims] 1. As an antigen in a test reagent for diagnosing syphilis by detecting anti-Treponemal pallidum antibodies, an antigen-containing sample derived from Treponema pallidum cells is adsorbed onto a hydroxyapatite gel column and eluted. A syphilis test reagent characterized in that an antigen whose purity is 25% or more as determined by the following formula is used. Formula {Amount of recovered protein/(Amount of added protein x Column recovery rate)} x 10
0 (%) However, the amount of added protein in the above formula is determined by fully equilibrating the hydroxyapatite gel column with a 10 mM potassium phosphate buffer (pH 6.0, hereinafter referred to as solution A) containing 1% octyl glucoside, and determining the amount of protein added in the above formula. The amount of protein in the antigen-containing sample derived from Treponema cells is the amount of protein obtained by replacing the solvent of the antigen-containing sample with Solution A and adding it to the column. After flowing at least 3 times the volume and thoroughly washing unadsorbed components, add 1% elution buffer (hereinafter referred to as solution B).
When elution was carried out using 350mM potassium phosphate buffer (pH 6.0) containing octyl glucoside while changing the ratio of solution B to solution A continuously or stepwise from 0% to 100%, The ratio is the amount of protein recovered between 8% and 40%, and the column recovery rate is the amount of protein recovered when the same fraction is recovered by adding the recovered protein to the column again under the same conditions. The ratio of the second recovered protein amount to the first recovered protein amount is shown. 2. As an antigen in a test reagent for diagnosing syphilis by detecting anti-Treponemal pallidum antibodies, an antigen-containing sample derived from Treponema pallidum cells is treated with a cation exchange column and then adsorbed onto a hydroxyapatite gel column.
When assaying antigen purity by elution,
A syphilis test reagent characterized by using an antigen whose antigen purity is 25% or more as determined by the following formula. Formula {Amount of recovered protein/(Amount of added protein x Column recovery rate)} x 10
0 (%) However, the amount of added protein in the above formula is determined by preparing the cation exchange column and hydroxyapatite gel column in advance with 10 mM potassium phosphate buffer (pH 6.0, hereinafter referred to as solution A) containing 1% octyl glucoside. The amount of protein in the antigen-containing sample obtained when the sample containing the antigen derived from Treponema pallidum cells is sufficiently equilibrated and the solvent is exchanged with solution A and then added to the cation exchanger column, and the amount of recovered protein is the amount of protein recovered from the antigen. After adding the containing sample to the cation exchanger column, flowing solution A to obtain a flow-through fraction, adding the flow-through fraction to a hydroxyapatite gel column, flowing solution A at least 3 times the column volume, After the unadsorbed components have been thoroughly washed, use 350 mM potassium phosphate buffer (pH 6.0) containing 1% octyl glucoside as the elution buffer (hereinafter referred to as solution B), and adjust the ratio of solution B to solution A. 0
The column recovery rate is the amount of protein recovered between 8% and 40% when elution is performed while changing the elution rate continuously or stepwise from % to 100%. The figure shows the ratio of the amount of protein recovered in the second time to the amount of protein recovered in the first time when the same fraction was collected by adding the same amount of protein to the hydroxyapatite gel column again under the same conditions.