JPH04188066A - Manufacture of antibody - Google Patents

Abstract

PURPOSE:To make it possible to manufacture an antibody which is specific for Treponema pallidum simply in high purity by immunizing an animal with refined antigen obtained from the extract of Treponema pallidum by using hydroxyapatite gel. CONSTITUTION:The extract of Treponema pallidum is coarsely manufactured by ion exchange chromatography. Protein containing antigen is adsorbed with hydroxyapatite gel. Then, the adsorbed material in the ranges of pH of 5.5 - 10.0 and ion strength of 50 - 150mM is eluted. Thus, the refined antigen is obtained. Thereafter, an animal is immunized with the refined antigen together with a material (adjuvant) having the immunity reinforcing action. The animal which can be utilized as an ordinary immunity animal such as a house rabbit and a rat can be used. The adjuvant is made to be the adjuvant whose Freund is complete or incomplete, aluminum hydroxide or the like. The antibody obtained by the immunization is used as the intact state of antiserum or in the refined state.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is directed to the use of Treponema pallidum (pathogenic Treponema pallidum).
apallidum (hereinafter abbreviated as TP), specifically, the method for producing an antibody that specifically reacts only with antigenic components derived from TP without reacting with non-pathogenic treponemes. Regarding the method.

[Conventional technology]

Conventionally, various detection methods and measurement methods that utilize antigen-antibody reactions have been used in research fields, diagnostic fields, and the like. When developing these detection and measurement methods, an important element is the creation of specific antibodies or antisera involved in antigen-antibody reactions.

Conventionally, the following methods have been known as methods for producing antiserum against TP.

(a) Inoculating live infectious TP bacteria into animals such as domestic rabbits,
A method of artificially infecting syphilis and obtaining antiserum against TP.

(b) A method for obtaining antiserum by immunizing a suitable animal with killed TP bacteria or TP extract, either directly or with an adjuvant.

(C) A hybridoma is created using myeloma and the lymphocytes of an animal such as a mouse stimulated by immunization using the method described in (a) or (b), and a TP-specific antibody is extracted from the hybridoma. A method for obtaining specific antibodies by selecting clones that produce them.

[Problem to be solved by the invention]

However, the conventional method as described above has various problems as described below.

First, the method mentioned in (a) or (b), namely T
In the method of obtaining antiserum by infecting live bacteria of P. or immunizing with killed bacteria or extracts of TP, antibodies against the common antigen with non-pathogenic Treponema are also produced5, so the obtained antiserum is It could not be used as an antiserum, that is, an antibody that specifically reacts only with .

Second, the antiserum prepared by the method described in (a) or (b) is subjected to an absorption operation using a non-pathogenic treponema or an extract thereof, and reacts with at least the non-pathogenic treponema used for the absorption operation. By removing antibodies that
Attempts have also been made to obtain antibodies specific for P.

However, even in such an attempt, the following problems were encountered.

(i) TP in non-pathogenic Treponema used for absorption procedure
TP does not necessarily contain all non-specific antigenic components, and there is no guarantee that all non-specific antibodies to TP will be removed by the absorption procedure.

(ii) Due to the absorption operation, the titer generally decreases due to reasons such as non-specific adsorption of even the specific antibody component to the substance used for the absorption operation.

Furthermore, in the conventional method described in (C), that is, the method for producing monoclonal antibodies, because TP contains many components, a clone that produces the desired specific antibody can only be obtained with a low probability. I can't.

Furthermore, when screening for clones that produce antibodies that recognize TP-specific antigens, it was necessary to screen for both non-pathogenic treponemal and TPO antigens, making the procedure extremely complicated.

Therefore, the purpose of the present invention is to solve the various problems of the conventional methods as described above, and to provide a method for easily producing TP-specific antibodies with high purity. do.

[Means to solve the problem]

The present invention comprises a step of purifying an extract of Treponema pallidum by chromatography using hydroxyapatite gel to obtain a purified antigen, and a step of obtaining an antibody specific to Treponema pallidum by immunizing an animal with the purified antigen. It is characterized by having the following.

Hereinafter, the configuration of the present invention will be explained in detail.

Purification of Antigen In the present invention, a purified antigen is obtained using an extract of Treponema pallidum, and the extract of Treponema pallidum is prepared as follows.

First, TP is cultured and collected by a known method. Next, a TP-derived extract is obtained by suspending the bacterial cells in a buffer solution and solubilizing the cooled and crushed cells. A specific method for this method may be appropriately selected from generally known methods. As a crushing method, a homogenizer treatment, an ultrasonic treatment, a freeze-thaw method, or the like is used. As a solubilization method, a protein solubilization treatment using a surfactant, a chaotropic ion, urea or an alkali, an enzyme treatment, an autolysis method, etc. are used. Among these, a solubilization treatment using a solution containing a nonionic or amphoteric surfactant in a low ionic strength buffer is particularly suitable.

Preferably, in order to increase the amount of purified antigen, it is recommended to remove impurities in advance. Pretreatment methods for removing such impurities include: ■ Rough purification by ion exchange chromatography: a method in which both fractions other than the antigen fraction are removed using a cation exchange resin, and ■ Ammonium sulfate fractionation or Examples include rough purification by polyethylene glycol fractionation, but considering the complexity of the operation, the pretreatment method (2) is preferable.

When purifying the antigen, the pH is preferably 5.0 to 8.
0, ionic strength 2-30mM, more preferably pH
The TP-derived extract is brought into contact with the hydroxyapatite gel at an ionic strength of 5.5 to 7.0 and an ionic strength of 10 to 20 mM, and the protein containing the antigen is adsorbed onto the hydroxyapatite gel.

Next, preferably pH 5.0 to 11.0, ionic strength 10 to 360 mM, more preferably pH 5.5 to 10.
．． The fraction obtained by eluting the adsorbed substance at 0 and ionic strength in the range of 50 to 150 mM is used as the purified antigen.

■ Next, animals are immunized with the purified antigen alone or with a suitable adjuvant. Examples of animals that can be used include those commonly used as immunized animals, such as rabbits, rats, mice, guinea pigs, goats, horses, pigs, sheep, and chickens.

Furthermore, since the purified antigen is specific to TP, it can also be used to immunize humans for the purpose of vaccination.

Adjuvants include Freund's complete or incomplete adjuvant, aluminum hydroxide or N-Ace.
tylmuramyl-L-alanyl-D-iso
glutamine (muramyl dipeptide, hereinafter referred to as MDP)
) and other substances that have immunostimulatory effects.

As for the amount of antigen to be immunized, the number of times, and the interval, general conditions can be applied. For example, 10~1000μ
Conditions such as g7.7 inoculation 1 to 5 times every 1 to 4 weeks are applied.

How to obtain TP As the TP inoculum, for example, the WHO pathogenicity standard Nichols strain or the TP used by various testing institutions for syphilis testing may be used as is. The WHO pathogenicity standard Nichols strain is, for example, the CDC (Center for Disease Control) strain.
Control, Public Health 5
service.

U, S, Del) art of Heal
th, Education and Welfare,
Atlanta Georgia U,S, ). In addition, T used in the present invention
In obtaining the P extract, any known method can be used for culturing TP, collecting bacteria from the culture, and treating bacterial cells.

[Effect]

In the present invention, anti-T
Since this method obtains P antibodies, it is possible to obtain high purity antibodies specific only to TP while preventing contamination with antibodies that also react with non-pathogenic treponemes.

Further, even if the yield of purified antigen is low, a large amount of antiserum, ie, antibody, can be obtained by immunization, so that highly pure antibodies can be produced efficiently and easily as described above.

[Scope of Application of the Present Invention] The antibody obtained in the present invention recognizes an antigen specific to Treponema pallidum, and does not react with, for example, the antigen of Reiter strain, which is a non-pathogenic Treponema pallidum.

Therefore, if an antigen assay system is created using the antibody of the present invention, diagnostic agents (TPHA,
It can be used for quality control of syphilitic corpuscles and antigens used in FTA-ABS, etc.). As a method for creating an assay system for quantifying antigens, known and established techniques such as enzyme immunoassay, radioimmunoassay, hemagglutination, and the like can be used.

Furthermore, in syphilis diagnosis, detection of bacterial cells from the affected area is used as a definitive diagnosis of syphilis. Detection of bacterial cells is performed by observing and confirming bacterial cells contained in tissue collected from the affected area using a microscope. The assay system for the above antigen is TP
It is also used to identify antigens in affected tissue or tissue extracts in order to recognize antigens specific to syphilis. Ru.

Furthermore, the antibody of the present invention can distinguish between antigens of pathogenic Treponema pallidum and non-pathogenic Treponema pallidum, and is therefore useful for various studies.

[Preferred embodiments of the invention]

Preferred embodiments of the present invention will be described below. However, the present invention is not limited to the following preferred embodiments.

Purification of antigens using hydroxyapatite gel Buffers used in general biochemical experiments such as phosphate buffer, Tris buffer, or glycine buffer can be widely used as solutions for purification using hydroxyapatite gel. Preferably, a phosphate buffer is used.

For antigen adsorption, the pH range is preferably 5.
It is in the range of 0 to 8.0, more preferably in the range of 5.5 to 7.0. If the pH is less than 5.0, the antigenicity is likely to be lost and the recovery rate of the purified immune antigen will be reduced. If the pH is more than 8.0, the adsorption efficiency of proteins to the gel will be reduced. This is because the yield decreases.

On the other hand, during antigen elution, the pH is preferably 5.0 to 5.0.
It is in the range of 11.0, more preferably in the range of 55 to 10.0. In particular, when using a buffer solution with a pH of 8.0 or higher, elution can be carried out with a buffer solution with an ionic strength of 10 to 60 mM. If the pH is less than 5.0, antigenicity is likely to be lost, which is undesirable. On the other hand, if the pH exceeds 11.0, impurities are likely to be eluted together with the antigen, which is not preferable.

Regarding the ionic strength of the solution, when the protein in the TP-derived extract is adsorbed onto the hydroxyapatite gel, the ionic strength is preferably 2 to 30 mM, more preferably 10 to 20 mM. This is because if the ionic strength is less than 2 mM, impurities are likely to be adsorbed to the hydroxyapatite gel, and the buffering capacity of the buffer solution will further decrease, which is not practical. On the other hand, if it exceeds 30 mM, the antigen becomes difficult to be adsorbed to the hydroxyapatite gel, resulting in a decrease in yield.

Furthermore, during antigen elution, the ionic strength is preferably 1
The range is 0-360mM, more preferably 50-150mM. If the ionic strength is less than 0mM, it will be difficult to elute at acidic pH, and if it exceeds 360mM, impurities will also be eluted, which is not preferable.

As additives added to the solution used for antigen adsorption and elution, any surfactant that can be used to solubilize TP, chaotropic ions, urea, etc., to which the hydroxyapatite gel is resistant can be used. be able to. However, considering the complexity of the operation, a buffer solution with an ionic strength appropriate for adsorption and elution, to which a nonionic or amphoteric surfactant is added, is suitable for purification using hydroxyapatite gel.

Note that it is preferable not to use a chelating agent such as EDTA because it degrades the hydroxyapatite gel.

The hydroxyapatite gel that can be used is not particularly limited, but there may be differences in the amount of protein adsorption and the recovery rate of the 'ff1M immune antigen depending on the gel manufacturing method, lot, etc. Therefore, it is necessary to set appropriate conditions for adsorption and elution depending on the characteristics of the gel used.

Common animals can be used to immunize with the purified immune antigen. Examples of experimental animals include rabbits, mice, rats, guinea pigs, goats, horses, pigs, sheep, and chickens. Practically speaking, rabbits, goats, and sheep are preferred because they are easy to raise, yield a relatively large amount of serum, and are easy to collect blood.

Furthermore, for the purpose of producing monoclonal antibodies, mice, rats, etc. are used, and mice are preferable because the procedures have been established and reagents for research are available.

As the immunization method, conditions for immunizing with general antigens can be employed. Generally, immunization is often carried out together with a substance (adjuvant) that has an immune-enhancing effect, but these adjuvants can also be used in the present invention. For example, Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide, etc. are typical, but isolated immunopotentiating substances such as MDP can also be used. In addition, multiple adjuvants may be used in combination, but if the adjuvant contains a different type of antigen component, such as Freund's complete adjuvant, in addition to antibodies against the purified antigen, antibodies against the antigen component contained in the adjuvant will also be produced. Therefore, it may not be appropriate depending on the purpose.

The amount of purified immunizing antigen to be inoculated, the number of inoculations, the interval between inoculations, etc. are appropriately selected depending on the animal to be immunized and the purpose. Generally, the inoculation amount is 10 to 1000 μg/time, the number of inoculations is 1 to 5 times, and the inoculation interval is 1 to 4 weeks.

Inoculation sites include subcutaneous, muscle, and abdominal cavity. In particular, when inoculating mice for the purpose of producing monoclonal antibodies, intraperitoneal inoculation is a convenient method.

Later evaluation of antibody titer To confirm whether antibodies are being produced, or
In order to confirm whether the titer of the obtained antiserum or antibody is sufficient, a commercially available measurement kit for diagnosing syphilis can be used.

As such kits, various TPHA test kits are commercially available from diagnostic drug manufacturers, and the antibody titer can be evaluated according to their instructions for use.

■ Live ■ Transfer ■ Antibodies obtained by immunization are used as antisera or purified depending on the purpose. Purification methods include ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography using a group-specific adsorbent such as protein A, or affinity chromatography using purified immune antigen immobilized on a gel. , these can be used alone or in combination.

In addition, if the obtained antibody is a monoclonal antibody, it can be grown as a culture supernatant of a hybridoma, or by inoculating the hybridoma into a mouse and growing it in the peritoneal cavity of the mouse.
Antibodies can be obtained as ascites fluid, and if necessary, antibodies can be purified using the above method.

[Explanation of Examples]

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

(1) Buffer ■ Phosphate buffer (pH 6.5) (hereinafter referred to as PBS) From potassium dihydrogen phosphate, disodium hydrogen phosphate (decahydrate) and sodium chloride, phosphoric acid concentration 0.036M, chloride A buffer solution with a sodium concentration of 0.156M and a pH of 6.5 was prepared.

■1% BSA/PBS Bovine serum albumin (BSA, manufactured by Miles Laboratories) was dissolved in PBS to a concentration of 1% (w/v).

■Potassium phosphate buffer (hereinafter referred to as KPB)・l OmM
KPB (pH 6,0) 10mM potassium dihydrogen phosphate solution and 10mM dipotassium hydrogenphosphate solution were mixed to adjust to 1) H6.0.

-I OmM KPB (pH 7.0) A 10 mM potassium dihydrogen phosphate solution and a 10 mM dipotassium hydrogen phosphate solution were mixed and adjusted to pH 7.0.

- 350mM KPB (pH 6,0) A 350mM potassium dihydrogen phosphate solution and a 350mM potassium dihydrogen phosphate solution were mixed to adjust the pH to 6.0.

(2) Surfactant octyl glucoside (hereinafter referred to as OG) for research on poorly soluble proteins (manufactured by Nacalai Tesque) was used.

(3) The cation exchanger S-Sepharose Fast Flow (Pharmacia) 301nI was added to a column (S
R25/45) was used.

(4) Hydroxyapatite gel Biogel HTP (Bio-Rad) 8- was packed in a Pharmacia column (HR10/10).

(5) TP suspension A TP suspension cultured and collected by the following method was used.

Corus strain on Treponema pallidum) in the domestic rabbit onibichu 1
After culturing for 0 to 14 days, the cells were extracted with a 22% sodium citrate solution at 37°C for 30 minutes. 20 extract liquid
The rabbit tissue precipitate was removed by centrifugation at 0×g for 5 minutes, and the supernatant was centrifuged at 3000×g for 30 minutes to precipitate TP. After repeatedly washing the obtained bacterial cells with PBS,
The number of bacterial cells was counted using a dark field microscope. After counting the number of bacterial cells, the number of bacterial cells was adjusted to 101 cells/yd.

(6) Protein concentration determination kit B CA Protein As5ay Reage
nt (Pierce) was used.

As a standard substance, 350mM BSA containing 1% OG was used.
A solution dissolved in KPB (pH 6,0) was used.

(7) Other Reagents below were JIS special grade or equivalent unless otherwise specified.

■Satomi! (1) Antigen extraction from bacterial cells: The TP suspension was diluted 2 times with PBS and disrupted using an ultrasonic disruptor. After crushing, the mixture was centrifuged at 12,600×g for 30 minutes to collect the precipitate.

The precipitate was added with 10mM KPB (pH 7) containing 1% OG.
. After being left at 4°C for 16 hours or more, the mixture was centrifuged at 50,000×g for 1 hour, the upper groove was taken, and the resultant was filtered through a 0.22 μm membrane filter (Millipore, Millex GS) and used as an extracted antigen.

(2) Cation exchange chromatography two extracted antigens
The cells were dialyzed into 10 mM KPB (pH 6,0) containing % OG, and the buffer was exchanged.

The cation exchanger was added to 10mM KPB containing 1% OG (pH
The extracted antigen, which had been equilibrated to 6.0) and the buffer solution exchanged, was added, and the 50-pass-through fraction (hereinafter referred to as crudely purified antigen) was collected.

(3) Hydroxyapatite gel chromatography; 10mM hydroxyapatite gel containing 1% OG
It was equilibrated with KPB (pH 6.0), and crudely purified antigen 50- was added. After addition, l Om containing 1% OG
M KPB (pH 6,0) as eluent 0, D, 28
The flow was continued until 0 nm became 0.010 or less, and unadsorbed components were washed away. 10mM KPB (p
H6,0), 350mM KP containing 1% OG
The ratio of B (pH 6,0) was increased linearly from O to 40% to elute the components adsorbed on the column.

350mM KPB (pH 6,0) containing 1% OG
The fractions eluted between 8% and 40% were collected and concentrated under reduced pressure using a dialysis tube to obtain an antigen fraction (hereinafter referred to as purified antigen).

After concentration under reduced pressure, the protein concentration of the purified antigen was measured and found to be 120 μg/−.

Immunization (1) Materials: Freund's incomplete adjuvant Freund's incomplete adjuvant from Difco was used.

-MDP MDP manufactured by Sigma was dissolved in sterilized physiological saline to a concentration of 511 g/MJ.

- Immunized animal A domestic rabbit was used as the immunized animal. Before immunization, blood was collected from the ear vein, and the absence of antibody titer was confirmed using a commercially available TPHA kit (described later).

(2) Method Purified antigen 1.5d, incomplete Freund's adjuvant) 1.
5711', MDPO, (16-) were mixed in a mixer in which two syringes were connected with a connecting needle to prepare an emulsion.

Two domestic rabbits were prepared, and one dose of the emulsion per rabbit was subcutaneously inoculated in several places on the back and flank.

After 4 weeks, a booster was given again under the same conditions, and after the booster,
Whole blood was collected from the carotid artery during the first week.

The collected blood was separated into serum, and a portion was frozen and stored at 4°C and the rest at -20°C.

Evaluation of antibody titer (1) Materials/TPH human kit To confirm the antibody titer of the antiserum, commercially available TP
E (As the A kit, Serocrit TP (Chemistry and Serum Therapy Research Institute) was used.

(2) Method The antibody titer of the collected antiserum was measured using a TPHA kit and operating according to the kit's instructions.

Confirmation of specificity (1) Materials ■Sample buffer 10mM) Lis-HCl buffer, 2mM EDTA (p
A sample buffer was prepared by adding 5% sodium dodecyl sulfate and 10% mercaptoethanol to H8,0).

■Reiter strain Reiter strain (distributed by the National Institute of Preventive Health) was used as a non-pathogenic treponemal strain.

Using thioglycollate medium containing 10% rabbit serum,
Cultured under anaerobic conditions.

■Reiter strain antigen sample Measure the number of bacteria using a dark field microscope, and collect approximately 1 (1”) bacterial cells.
After suspending in BS, it was centrifuged at 3000Xg and the precipitate was collected.

ld sample buffer was added to the precipitate, incubated at 100° C. for 5 minutes, returned to room temperature, and filtered through a 0° 22 μm membrane filter (Millibore, Millex GS) to obtain a lighter strain antigen sample.

■TP extraction antigen sample Using TP, extract T under the same conditions as the lighter strain antigen sample.
P-extracted antigen samples were prepared.

■ Using the electrophoresis device Pharmacia's Phast System, P
hast Get gradient media 1
Electrophoresis was performed using a combination of 0-15 and SDS BufferStripS.

■Western blotting device The Phast Transfer system manufactured by Pharmacia was used and operated according to the instruction manual.

The nitrocellulose membrane used had a pore size of 0.45 μm.

(1) First antibody An antiserum diluted with 1% BSA/PBS was used. The dilution ratio was determined according to the titer of the antiserum, and the antiserum was diluted to a titer of about 40 titers.

■Anti-rabbit 1gG (derived from goat) (Miles Laboratories) labeled with labeled second antibody peroxidase was added to 1% BSA.
/PBSI: Used after being diluted 1000 times.

■Enzyme substrate 4-chloro-1-naphthol (Nacalai Tesque M) l
After adding 3.33 - of ice-cold methanol to Oa+g and dissolving it, PB316.67yd! and 10 μm of 30% hydrogen peroxide solution was added immediately before use. This substrate was prepared fresh.

(2) Method ■ Preparation of sample The purified antigen and sample buffer were mixed in equal amounts and incubated at 1° OoC for 5 minutes. Hereinafter, the purified antigen subjected to this treatment will be referred to as a purified antigen sample.

Furthermore, the lighter strain antigen sample and the TP extracted antigen sample were also incubated at 100°C for 5 minutes before electrophoresis.

(2) Electrophoresis Using Phast System, 1 μm of each of the Reiter strain antigen sample, TP extracted antigen sample, and purified antigen sample was electrophoresed.

■Western blotting Using the Phast Transfer system, the electrophoresed sample was plotted on a nitrocellulose membrane.

■ 1% B of nitrocellulose membrane after staining and plotting.
It was immersed in SA/'PBS and left at room temperature for 2 hours to perform blocking.

The blocked nitrocellulose membrane was immersed in the first antibody and left at room temperature for 1 hour.

It was thoroughly washed with physiological saline and immersed in the labeled second antibody for 1 hour at room temperature.

It was thoroughly washed with physiological saline, immersed in enzyme substrate, and left at room temperature for 3 to 6 minutes while checking the degree of color development.

When a suitable color was developed, the enzyme substrate was sufficiently washed with purified water to stop the enzyme reaction.

(3) Results The titers of the collected antiserum were 20480 titer and 40960 titer according to the commercially available TPHA kit.

Staining after Western blotting was performed using the first antibodies diluted 500-fold and 1000-fold, respectively.

For the TP-extracted antigen sample and the purified antigen sample, a dark band was observed around the molecular weight of about 47,000, and a thin band was observed around the molecular weight of about 41,000.For the Reiter strain antigen sample, no stained band was observed. Not detected.

Comparative Example (1) Method As a comparative example, 1 TP was added to PBS instead of the purified antigen.
When collecting bacterial cells from rabbits that had been immunized and cultured with antiserum and TP, the other conditions were the same as in the examples. The antibody titer was evaluated in the same manner as in Example 1 using serum collected at the same time to confirm specificity.

(2) Results The titer of antiserum prepared by immunizing with TP was 40 for both birds.
, 960 titre. In addition, the titer of serum collected from a rabbit cultured with TP was 20°480 titer.

Regarding the antiserum prepared by immunization, the specificity was confirmed using a first antibody diluted 1.000 times and a first antibody prepared by diluting blood serum collected from a rabbit cultured with TP 500 times. For the Reiter strain antigen sample and the TP-extracted antigen sample, about 10 or more bands and about 20 to 30 bands were completely stained, respectively, over a wide molecular weight range, and for the purified antigen sample, the bands were stained at the same positions as in the example. A similar band was stained.

〔Effect of the invention〕

As described above, according to the present invention, antibodies that specifically react only with TP can be obtained with high purity and in large quantities.

Furthermore, complicated steps such as absorption operations in conventional methods can be omitted.

The present invention is based on the principle of recognizing only the TP antigen, purifying the TP-derived antigen used in conventional syphilis diagnostics, controlling the quality of TP and the TP-derived antigen, and diagnosing syphilis by detecting the TP antigen. It is extremely useful for creating syphilis diagnostic agents and for researching TP itself.

Claims (1)

[Claims] (1) A step of purifying a Treponema pallidum extract by chromatography using hydroxyapatite gel to obtain a purified antigen, and a step of obtaining an antibody specific to Treponema pallidum by immunizing an animal with the purified antigen. A method for producing an antibody, comprising: