JPS6089430A - Non-a-non-b-type hepatitis relating antigen - Google Patents

Abstract

PURPOSE:To provide the titled antigen composed of the protein recovered from the urine, blood, etc. of a non-A.non-B-type hepatitis patient, and useful for the remedy and diagnosis of non-A.non-B-type hepatitis. CONSTITUTION:The urine, blood (preferably blood plasma), etc. of a non-A.non- B-type hepatitis patient is used as a raw material, is subjected to a proper combination of the concentration, the fractionation taking advantage of the solubility difference, the ion exchange material treatment, the gel filtration, etc. to obtain the objective SO antigen having the following characteristics: (i) electrophoretic analysis, moves toward the beta-region; (ii) molecular weight, about 250,000, decomposed to a fraction having a molecular weight of about 38,000 by electrophoresis; (iii) electron microscopic observation, granular structure having a diameter of 11nm; (iv) exhibiting the antigenecity to animal; and (v) causing strong agglutination reaction with the serum and urine of only the non-A.non-B-type hepatitis patient by the RPHA reagent prepared from the refined antigen. A reagent to detect the relating antibody can be produced by bonding said antigen with a carrier, and a vaccine is prepared by treating the antigen at 60 deg.C for 10hr or treating with formalin.

Description

[Detailed description of the invention] 〔Technical field〕 The present invention relates to a novel non-A/non-B hepatitis-related antigen.

[Technical level]

Of the types of post-transfusion hepatitis, type B has been conquered through the discovery of the hepatitis virus, the development of test reagents for it, and research into prevention methods using immunoglobulin preparations and vaccines. However, regarding non-A and non-B hepatitis, the actual state of the virus has not yet been investigated, and therefore testing methods and prevention methods have not yet been established.

Under these circumstances, the inventors have been researching hepatitis viruses for many years, and have discovered a substance that is closely related to non-A and non-B hepatitis, and have succeeded in extracting and purifying it.
Since this substance has a close relationship with non-A/non-B hepatitis, it is possible to treat and diagnose non-A/non-B hepatitis by using it as a raw material for vaccines, reagents, and reagents. This finding led to the completion of the present invention.

(Disclosure of the Invention) The present invention is a protein that can be recovered from the urine of non-A/non-B hepatitis patients, and when analyzed by electrophoresis, migrates to the β region,
Molecular weight is approximately 25,001: When electrophoresis is performed in the presence of SDS, the molecular weight is approximately 38,000, and it is
! RPHA using a purified antigen shows a particle shape with a diameter of about 1 nm as observed by 11, has antigenicity to animals, and uses purified antigen.
This invention relates to a non-A/non-B hepatitis-related antigen (hereinafter referred to as HO antigen), which is characterized by causing a strong agglutination reaction with serum and urine only from patients with non-A/non-B hepatitis in a reagent.

(Cleavage of HO antigen) Raw material: Urine, blood (preferably plasma), etc. of non-A/non-B hepatitis patients are used as raw materials.

Purification method: HO antigen is recovered by, for example, an appropriate combination of concentration, fractionation based on solubility difference, ion exchanger treatment, gel filtration, and the like.

It is preferable that the raw material is first concentrated. As a concentration method, ultrafiltration treatment can be suitably used.

For example, it can be carried out using a membrane with fractionated molecules ff1lo,000. For concentration, it is preferable to further perform fractionation based on solubility difference, and in this case, for example, ammonium sulfate, polyethylene glycol, ethanol, etc. can be suitably used. Through this treatment, the HO antigen is recovered as a precipitate.

Thus, for example, HO antigen becomes precipitated by saturation with 50% ammonium sulfate. The precipitate is separated into a precipitate and a supernatant by centrifugation, and these are collected.

Excess salt is removed by redissolving this precipitate in an aqueous solution of p116-8 and dialysis.

The aqueous solution containing the HO antigen is then subjected to treatment with a cation exchanger. HO antigens are separated by cation exchanger adsorption. As the cation exchanger, one having a carboxymethyl (CM-), phosphoryl (P-), or sulfophosphoryl (SPi) functional group is preferably used. 2-1QmM phosphate buffer) (pH 6-
8) It is preferable to equilibrate by l, N. Elution is carried out according to sound techniques, for example by increasing the salt concentration.

Next, molecular sieving is performed by gel filtration to recover the HO antigen. Examples of carriers used in gel filtration include gel filtration carriers suitable for molecular sieving of compounds with a molecular weight of 100,000 to 500,000, such as Sephadex, agarose, gel, Sephacryl, etc. These carriers Please use G in advance on p116~
It is preferable to use one equilibrated with a low ionic strength buffer solution of about 8).

During these purifications, it is important to keep track of the HO antigen, even if L, non-A, etc.
・Agglutination reaction with serum from convalescent patients with non-B hepatitis can be used as a marker.

The HO antigen thus obtained is closely related to non-A/non-B BY inflammation, and may be used as a clinical reagent or its raw material, and when used as a pharmaceutical,
Therefore, virus inactivation, sterilization filtration, freeze-drying, dispensing, and formulation may be performed according to the usual techniques for pharmaceutical manufacturing.

(Characteristics of HO antigen) The characteristics of the HO antigen of the present invention are as follows.

■Molecular weight: Sephadex G-75, G-200, Sephacryl S
When the molecular weight was measured by gel filtration using -300 or the like under the following conditions, the molecular weight was about 25o, ooo.

Conditions: Column size...24X900 Solvent: 0.011J acid buffer (pH 7.0) Deployment flow rate □...1.5 ml/h r.

The molecule (■) was determined by comparison with a standard protein of known molecular weight.

■Mobility by electrophoresis; Based on immunoelectrophoresis. 0.0 at pH 7.0. Electrophoresis was performed on an agar plate using OIM phosphate buffer. After electrophoresis at 100 volts for about 40 minutes, anti-human serum and anti-HO antigen serum were added to the groove, and after being left for one night, the mobility of the HO antigen was determined to be β position from the sedimentation line formed.

(2) Color reaction: After hydrolysis in a 6N hydrochloric acid solution at 110°C for 22 hours, a ninhydrin reaction test revealed a purple-blue color due to α-amino acids. Both the α-naphthol sulfuric acid reaction (Molish reaction) and the phenol sulfuric acid reaction for sugar tests were negative.

(2) 5DS-electrophoresis treatment: When the molecular weight of the HO antigen of the present invention was measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis, the molecular weight was determined to be approximately 38,000.

■Electron microscopy: It showed a particle shape with a diameter of about 11 nm.

■Specificity for non-A/non-B hepatitis: When normal serum and serum from patients with non-A/non-B hepatitis were tested using the RPHA reagent (example described below) using a specific antibody against HO antigen, non-A/non-B hepatitis was detected. It showed an agglutination reaction specifically only with hepatitis B patient serum. Thus, the HO antigen is considered to be an effective raw material for the diagnosis and treatment of non-A and non-B hepatitis.

(Application of HO antigen and anti-HO as reagents) In order to use HO antigen or anti-HO as a diagnostic reagent, the already known PHA method,
Examples include the latex agglutination reaction method and the in-agar immunodiffusion method.

Specifically, the PHA method reagent or R
A reagent for PHA method can be produced. Birds include chickens, geese, turkeys, etc., and mammals include sheep, cows, horses, goats, humans, etc., but are not limited to these species. A known method can be used to sensitize these red blood cells with HO antigen or anti-HO. For example, Imai (the history of medicine,
78.759-760.1971) can be adopted.

Anti-HO can be prepared by means known per se.

By binding HO antigen or anti-IO to an insoluble support such as a resin or an insoluble polysaccharide, it is possible to perform the ElA method or RIA method.
It is also possible to make reagents for the method.

Examples of the resin include bead-shaped, test layer-shaped, and plate-shaped solids made of acrylic, polyethylene, vinyl, and the like. Examples of insoluble polysaccharides include agar cellulose. After binding HO antigen to these insoluble supports by a known method and reacting with a sample containing anti-HO, I
Anti-HO can be detected by reacting a labeled substance such as 25 I, enzyme, etc. with the labeled HO antigen and then measuring the activity of the labeled substance such as 125 I, enzyme, etc.

Furthermore, the HO antigen can also be detected by binding anti-HO instead of the HO antigen.

(Application of HO antigen as a vaccine) A vaccine can be obtained by subjecting the purified HO antigen to treatment at 60° C. for 10 hours, which is generally known as a virus inactivation method, or treatment with Polmarin.

Hereinafter, the present invention will be explained in more detail with reference to Examples.
The present invention is not limited to this in any way.

Example 1 Non-A/non-B hepatitis patient urine 10β with a molecular weight cutoff of 10.00
Concentrate to 200 ml using a membrane of
After adding to 0% saturation, the precipitate that had separated out was collected by centrifugation. This precipitate was dialyzed against 0.01M phosphate buffer (pH 7.0) and passed through a column packed with CM-ion exchanger to collect both fractions containing the HO antigen. Further, the mixture was passed through a column of Sephadex G100 (manufactured by Pharmacia) to obtain two fractions containing a high concentration of HO antigen. The wood HO antigen showed a single band when electrophoresed in polyacrylamide containing 5DSIO%.

Example 2 Sheep, chicken or human 1-0 in which the HO antigen obtained in Example 1 was treated with glutaraldehyde and tannic acid
The cells were sensitized to type red blood cells to obtain HO antigen-sensitized red blood cells.

Using this HO antigen-sensitized red blood cell, non-A and
When testing non-B hepatitis, convalescent serum, hepatitis B patient serum, and normal human serum, a strong agglutination reaction was observed only with non-A/non-B hepatitis convalescent serum, and with the other two serums. It turned out that it didn't respond.

Example 3 A rabbit was immunized with the HO antigen obtained in Example 1 to prepare anti-HO serum. IgG was purified from this antiserum by a known method. This purified IgG was used to sensitize sheep, chicken or human type 0 red blood cells according to the method of Example 2 to produce anti-HO sensitized red blood cells.

Using this HO sensitized red blood cell, we tested non-A/non-B hepatitis patient serum, isobaric, hepatitis B patient serum, isobaric, normal human serum, and isobaric using the RPHA method. It showed a strong agglutination reaction only with hepatitis patient serum and their urine.

Example 4 The purified HO antigen obtained in Example 1 was coated on a styrene test tube or beads and reacted with non-A/non-B hepatitis convalescent serum, purified 1gG obtained in Example 2, and normal human serum. After this, the reaction mixture was reacted again with a certain amount of HO antigen previously labeled with 1251 or alkaline phosphatase, and the radioactivity of 1251 or the enzymatic activity of alkaline phosphatase was measured as determined by the trial court.

The result 1. Non-A/non-B convalescent serum and Example 2
It was found that it reacted strongly only with tHJigc obtained in .

Example 5 In place of the purified HO antigen in Example 4, purified 1gG obtained in Example 2 was used, and non-A
- When testing non-B hepatitis patient serum and urine, as well as normal human serum, a strong reaction was observed only with non-A/non-B hepatitis patient serum and urine.

Example 6 Albumin was added to the 1q sensitized red blood cells obtained in Examples 2 and 3 at a concentration of 2 to 5 w/v% and lyophilized. After storing the sensitized blood cells at 4° C. for 15 months, they were redissolved in distilled water and tested, and the properties were the same as in the test before freeze-drying.

Patent applicant Sendai Microbiology Research Institute procedural amendment (
Own ship November 24, 1981 1. Indication of the incident ■ 541 Address 4-53-53 Hirano-cho, Higashi-ku, Osaka-shi 3 Description of the amendment to specification 86 (correction) 1. Name of the invention Non-A/Non-B type Hepatitis-related antigen 2, claims + l) A protein that can be recovered from the convalescent stage of non-A/non-B hepatitis, and when analyzed by electrophoresis, it migrates to the β region, and has a molecular weight of approximately 250,000. When electrophoresis is performed in the presence of SDS, the molecular weight is broken down to about 38,000, and when observed using an electron microscope, it shows a particle shape with a diameter of about 1 nm, and has antigenicity to animals. A non-A/non-B hepatitis-related antigen characterized by causing a strong agglutination reaction (6) with serum and urine only from patients with non-Δ/nodial B hepatitis in G1.

(2. Claim No. 11: A reagent for detecting non-Δ, non-B hepatitis-related antibodies, wherein 11'& is formed by binding the non-A, non-B hepatitis-related antigen described in claim 11 to a carrier.

(3) Using the non-A/non-B hepatitis-related antigen obtained by immunizing animals with the non-A/non-B hepatitis-related antigen described in claim (1), 1. A reagent for detecting non-A/non-B hepatitis-related antigens, comprising a hepatitis-related IJL antigen bound to a carrier.

3. Detailed description of the invention 〔Technical field〕 The present invention relates to a novel non-A/non-B hepatitis-related antigen.

[Technical level]

Of the types of post-transfusion hepatitis, type B can be said to have been largely conquered through the discovery of hepatitis viruses, the development of test reagents for the virus, and research into prevention methods using immunoglobulin preparations and vaccines. However, regarding non-A and non-B hepatitis, the actual state of the virus has not yet been investigated, and therefore testing methods and prevention methods have not yet been established.

Under these circumstances, the inventors have been researching hepatitis viruses for many years, and have discovered a substance that is closely related to non-A and non-B hepatitis, and have succeeded in extracting and purifying it.
Since this substance has a close relationship with non-A/non-B hepatitis, it is possible to treat and diagnose non-A/non-B hepatitis by using it as a raw material for vaccines, reagents, and reagents. This finding led to the completion of the present invention.

[Disclosure of the invention]

The present invention is a protein that can be recovered from the urine of non-A/non-B hepatitis patients, and when analyzed by electrophoresis, migrates to the β region,
It has a molecular weight of approximately 250,000, and when electrophoresed in the presence of SDS, it is decomposed into a molecular weight of approximately 38,000, and when observed with an electron microscope, it appears in the form of particles with a diameter of approximately 1 nm. It has antigenicity to animals and is purified. This is related to non-A/non-B hepatitis-related antigen (hereinafter referred to as SO antigen), which is characterized by a strong agglutination reaction with serum and urine only from non-A/non-B hepatitis patients in RPHA reagents using antigens. be.

(Preparation of SO antigen) Raw material: Urine, blood (preferably plasma), etc. of non-A/non-B hepatitis patients are used as raw materials.

Purification method: Recovery of the SO antigen is performed by appropriately combining, for example, concentration, fractionation based on solubility difference, ion exchanger treatment, gel filtration, and the like.

It is preferable that the raw material is first concentrated. As a concentration method, ultrafiltration treatment can be suitably used.

For example, it can be carried out using a membrane with fractionated molecules ff1lo,000. For concentration, it is preferable to further perform fractionation based on solubility difference, and in this case, for example, ammonium sulfate, polyethylene glycol, ethanol, etc. can be suitably used. Through this treatment, the SO antigen is recovered as a precipitate.

That is, for example, SO antigen becomes precipitated by saturation with 50% ammonium sulfate. The precipitate is separated into a precipitate and a supernatant by centrifugation, and these are collected.

This precipitate is redissolved in an aqueous solution of 1116-8 and subjected to dialysis to remove excess salt.

The aqueous solution containing the SO antigen is then subjected to treatment with a cation exchanger. The so antigen is separated by cation exchanger adsorption. As the cation exchanger, carboxymethyl (CM-), bosboryl (P-),
Those having a sulfophosboryl (Sp-) functional group are used. The ion exchanger is prepared in advance in a low ionic strength buffer (e.g. 2-10mM phosphate buffer) (pH
It is preferable to equilibrate according to steps 6 to 8). Elution is carried out according to current application techniques, for example by increasing the salt concentration.

Next, molecular sieving is performed by gel filtration to recover the so antigen. Examples of carriers used in gel filtration include gel filtration carriers suitable for molecular sieving of compounds having a molecular weight of ioo, ooo to 500,000, such as Sephadex, agarose, biogel, and Sephacryl. It is preferable that these 1-isomers be equilibrated in advance with a low ionic strength buffer solution of about p116 to 8 before use.

During these purifications, the SO antigen can be tracked by using, for example, a certain coagulation reaction with serum from a convalescent non-A/non-B hepatitis patient as a marker.

The SO antigen thus obtained has a close relationship with non-A and non-B hepatitis, and may be used as a clinical reagent or its raw material, and when used as a pharmaceutical, it can be used in accordance with the usual technology for pharmaceutical manufacturing. Therefore, inactivation of the virus,
Sterile filtration, lyophilization, dispensing, and formulation may be performed.

(Characteristics of So antigen) The characteristics of the SO antigen of the present invention are as follows.

■Molecular M: Cephadesox G-75, G-200, Cephacryl S
When the molecular weight was measured by gel filtration using -300 or the like under the following conditions, the molecular weight was about 25o, ooo.

Conditions: Column size: 24X900 Solvent: ・ ・ ・0. OIM phosphate buffer (
pl+ 7.0) Development flow rate: 1.5 ml/hr The molecular weight was determined by comparison with a standard protein of known molecular weight.

■Mobility by electrophoresis: Based on immunoelectrophoresis. Electrophoresis was performed on an agar plate using 0.01M phosphate buffer at pH 7.0. After electrophoresis at 100 volts for about 4θ minutes, put anti-human serum and anti-SO antigen serum into the groove and leave it overnight. From the sedimentation line formed, the mobility of SO antigen Color reaction: 6N hydrochloric acid solution After being hydrolyzed at 110° C. for 22 hours, the product was tested by ninhydrin reaction and exhibited a purple-blue color due to α-amino acids. Both the α-naphthol sulfuric acid reaction (MOI s h reaction) and the phenol sulfuric acid reaction for saccharide tests were negative.

(2) 5DS-electrophoresis treatment: When the molecular weight of the SO antigen of the present invention was measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis, the molecular weight was determined to be approximately 38,000.

■Electron microscopy: It showed a particle shape with a diameter of about 11 nm.

■Specificity for non-A and non-B hepatitis: R P HA reagent using a specific antibody against SO antigen (
When Seishi serum and serum from patients with non-A/non-B hepatitis were tested using Example (described later), an agglutination reaction was specifically observed only with serum from patients with non-A/non-B hepatitis.

“Thus, we believe that SO antigen is an effective raw material for the diagnosis and treatment of non-A and non-B hepatitis.

(Used as a reagent for So antigen and anti-SO) In order to use SO antigen or anti-SO as a diagnostic reagent, the already known PHA method,
Examples include the latex agglutination reaction method and the in-agar immunodiffusion method.

Specifically, by sensitizing red blood cells of birds, mammals, etc. with the present SO antigen or anti-SO, reagents for PHA method or RP can be prepared.
A reagent for the HA method can be produced. As birds,
Chickens, geese, turkeys, etc. are mentioned, and examples of the micropores include chickens, cows, horses, goats, humans, etc., but the species are not limited to these species. A known method can be used to sensitize these red blood cells with SO antigen or anti-SO. For example, joint ventures (the history of medicine,
The method of A.E., 759-760.1971) can be adopted.

Anti-SO can be subjected to gIliIH using a known method.

By binding SO antigen or anti-SO to an insoluble support such as a resin or an insoluble polysaccharide, it is possible to use the ElA method or PHA method.
It is also possible to make reagents for the method.

Examples of the resin include bead-shaped, test tube-shaped, and plate-shaped solids made of acrylic, polyethylene, vinyl, and the like. Examples of insoluble polysaccharides include agar cellulose. After binding SO antigen to these insoluble supports by a known method and reacting with a sample containing anti-SO, 1
Anti-SO can be detected by reacting a labeled substance such as a 251 enzyme with a labeled SO antigen and then measuring the activity of the labeled substance such as a 251 enzyme.

Furthermore, SO antigen can also be detected by binding anti-SO instead of SO antigen.

(Usage of So antigen as vaccine) Purified SO antigen can be made into a vaccine by subjecting it to treatment at 60° C. for 10 hours or formalin treatment, which is generally known as a virus inactivation method.

Hereinafter, the present invention will be explained in more detail with reference to Examples.
The present invention is not limited to this in any way.

Example 1 Non-A/non-B hepatitis convalescent stage 10A with a molecular weight cutoff of 10°00
Concentrate to 200 ml using a membrane of
After adding to 0% saturation, the precipitate that had separated out was collected by centrifugation. This precipitate was dialyzed against 0.01M phosphate buffer (pH 7.0) and passed through a column packed with CM-ion exchanger to collect both fractions containing the SO antigen. Further, the mixture was passed through a column of Cefγ Desox G100 (manufactured by Pharmacia) to obtain two fractions containing a high concentration of SO antigen. This SO antigen showed one band when electrophoresed in polyacrylamide containing 10% SDS.

Example 2 Sheep, chicken or human type 0 red blood cells treated with glutaraldehyde and tannic acid were sensitized with the SO antigen obtained in Example 1 to obtain SO antigen-sensitized red blood cells.

Using this SO antigen-sensitized red blood cell, we tested non-A/non-B hepatitis, convalescent serum, hepatitis B patient serum, and normal human serum using the PHA method. It was found that a strong agglutination reaction was observed only with the sera of the other two subjects, and no reaction was observed with the sera of the other two subjects.

Example 3 A rabbit was immunized with the SO antigen obtained in Example 1 to prepare anti-SO serum. IgG was purified from this antiserum by a known method. This purified 1gG was sensitized to sheep, chicken, or human +-0 red blood cells according to the method of Example 2, and anti-SO
Sensitized red blood cells were produced.

Using this SO-sensitized red blood cell, we tested non-A/non-B hepatitis patient serum, isobaric, hepatitis B patient blood ln, isobaric, normal human serum, and isobaric using the RP HA method. A strong agglutination reaction was observed only with hepatitis B patient serum and their urine.

Example 4 The purified SO antigen obtained in Example 1 was coated on a styrene test tube or beads and reacted with non-A/non-B hepatitis convalescent serum, purified 1gG obtained in Example 2, and normal human serum. After incubation, the mixture was reacted again with a certain amount of SO antigen previously labeled with 125I or alkaline phosphatase, and the radioactivity of 125I or the enzymatic activity of alkaline phosphatase was measured by a conventional method.

As a result, it was found that it strongly reacted only with non-A/non-B type convalescent serum and purified 1gG obtained in Example 2.

Example 5 In place of the purified SO antigen in Example 4, purified 1gG obtained in Example 2 was used, and non-A
- When testing non-B hepatitis patient serum and urine, as well as normal human serum, a strong reaction was observed only with non-A/non-B hepatitis patient serum and urine.

Example 6 Albumin was added to the sensitized red blood cells obtained in Examples 2 and 3 at a concentration of 2 to 5 w/v% and lyophilized.

After storing the sensitized blood cells at 4° C. for 15 months, they were redissolved in distilled water and tested, and the properties were the same as in the test before freeze-drying.

Patent Applicant Sendai Microbial Research Discontinuation Continued Tadashi Neichi (Voluntary) 1981 [11th 1, Incident Indication 1988 Patent Application No. 197356 2, Name of Invention Non-A/Non-B Hepatitis Related Antigen 3, Relationship with the person making the amendment Patent applicant name Sendai Microbiology Research Institute 4, Agent ■541 Address 406 New Life Hirano-cho, 4-53-3 Hirano-cho, Higashi-ku, Osaka Telephone: (06) 227-1156 6. Number of inventions increased by amendment 7. Column 8 of "Detailed Description of the Invention" of the specification subject to the amendment. Contents of the amendment (1) "Detailed Description of the Invention" of the specification. Column (Description (correction) attached to the procedural amendment submitted on November 24, 1982)
(Page 5, lines 2 and 12), "heikyo" is corrected to "equilibrium".

that's all

Claims (1)

[Scope of Claims] 11) A protein that can be recovered from the urine of non-A/non-B hepatitis patients, migrates in the β region when analyzed by electrophoresis, has a molecular weight of about 250,000, and has a molecular weight of about 250,000 in the presence of SDS. When subjected to electrophoresis, it is resolved into a molecular weight of approximately 38,000, and when observed using an electron microscope, it shows the form of particles with a diameter of approximately 11 nm, and has antigenicity to animals. A non-A/non-B hepatitis-related antigen that causes a strong agglutination reaction with serum and urine only from patients with hepatitis. (2. A non-A/non-B hepatitis-related antibody detection reagent characterized by binding the non-A/non-B hepatitis-related antigen described in claim (1) to a carrier. (3) Claim No. 1 (claim 11) Anti-non-A, non-B hepatitis-related antigen obtained by immunizing an animal with the non-A, non-B hepatitis-related antigen 1. A non-A/non-B hepatitis-related antigen detection reagent, characterized in that it is bound to a carrier.