JP2000105232A - PREPARATION OF MEASURING REAGENT FOR MEASURING ANTI-HBs ANTIBODY - Google Patents

Abstract

PROBLEM TO BE SOLVED: To prepare a highy sensitive anti-HBs antibody measuring reagent, in particular an anti-HBs antibody measuring latex-flocculated reagent, by using a small-granulated HBs antigen which is prepared by adding an additive selected from the group comprising a surfactant and a protein modifier into an HBs antigen solution to be treated to form particulates. SOLUTION: A particulate HBs antigen prepared by adding an additive selected from the group comprising a surfactant and a protein modifier into an HBs antigen solution and by conducting small-granulating treatment is carried on an insoluble carrier to prepare an anti-HBs antibody measuring reagent. The surfactant is required only to be lipid-removable without being specified, and a variety of anion surfactants, a cationic surfactant, an amphoteric surfactant, a nonionic surfactant and the like are used for the surfactant. The protein modifier is a modifier capable of loosing higher-order stucture of a protein to dissociate a hydrogen bond and a hydrophobic bond between polypeptide side chains, and hydrochloric acid salt of guanidine or the like is used for this purpose. The small-granulated HBs antigen holding an antibody activity is prepared by the small-granulating treatment.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing an anti-HBs antibody measurement reagent, and more particularly, to a method for producing an anti-HBs antibody measurement reagent using small-particle HBs antigen.

[0002]

2. Description of the Related Art In addition to a double-stranded DNA virus called Dane particles having a diameter of 42 nm, small spheroid particles having a diameter of 22 nm and lengths having the same outer diameter are included in the serum of a patient at the time of hepatitis B virus (HBV) infection. It is known that 50-700 nm tubular particles are present. These HBVs include HBV
There are a surface antigen (HBs antigen), a nuclear antigen (HBc antigen), and the like corresponding to the surface, the core, and the like.

[0003] Detection of such antibodies against each antigen is carried out as follows.
Important for clinical diagnosis. In particular, since the HBs antibody is a protective antibody against HBV, it is detected in the blood when HBV infection has been eliminated in the past and has already been eliminated or after HBV vaccination. Thus, a positive HBs antibody indicates that it is resistant to HBV and does not have a risk of re-infection.

As a method for quantitatively measuring HBs antibody in blood, an immunoassay utilizing an antigen-antibody reaction is usually used. For example, a hemagglutination method in which the HBs antigen is carried on an insoluble carrier such as blood cells or latex is used. And latex agglutination method.

In the hemagglutination method or latex agglutination method, a purified HBs antigen is carried on latex, blood cells, or the like, and this is mixed with an HBs antibody-containing specimen. As a result of the antigen-antibody reaction, latex and blood cells are cross-linked by the antibody to cause agglutination. By measuring the degree of agglutination, the amount of HBs antibody in the sample can be quantified.

The amount of antigen carried on latex or blood cells is
As the number increases, the reactivity increases, and the measurement sensitivity of the reagent improves. However, HBs antigens have a molecular weight of 3,000,000
Because it is a macromolecule of 4 million, the number of supported molecules per surface area of the carrier (latex or blood cell) decreases,
It was difficult to obtain sufficient reagent sensitivity. for that reason,
If the HBs antigen can be reduced in particle size and supported on an insoluble carrier, measurement sensitivity can be improved as compared with conventional reagents.

[0007] JP-A-6-138123 discloses HB
HBs obtained by cleaving the disulfide (-SS-) bond of the HBs antigen using a reducing agent, for example, 10% dithiothreitol, to obtain a microparticle having a molecular weight of 500,000 to 1,000,000.
A method for preparing an s-antigen and using the same to prepare an enzyme-labeled product is disclosed. However, since the HBs antigen obtained by such a method cleaves to a disulfide bond involved in antigen activity, the activity of the HBs antigen decreases, and the desired reagent sensitivity cannot be improved. Therefore,
There has been a demand for a method of preparing a small-particle HBs antigen having antigen activity and converting it into a reagent.

[0008]

SUMMARY OF THE INVENTION In view of the above circumstances, an object of the present invention is to provide a method for producing an anti-HBs antibody measurement reagent using a small-sized HBs antigen that retains antigen activity.

[0009]

SUMMARY OF THE INVENTION The present invention is directed to a small particle HB.
A method for producing an anti-HBs antibody measurement reagent using s antigen, wherein the small-particle HBs antigen is obtained by adding at least one selected from the group consisting of a surfactant and a protein denaturant to an HBs antigen solution. This is a method for producing an anti-HBs antibody measurement reagent which has been subjected to a small particle treatment.

[0010] The method for producing an anti-HBs antibody assay reagent of the present invention uses a small-particle HBs antigen. The above-mentioned HBs antigen having a small particle size is obtained by adding at least one selected from the group consisting of a surfactant and a protein denaturant to an HBs antigen solution, and then performing a particle-izing treatment. By performing the above-mentioned small particle size treatment, a small particle size HBs antigen retaining the antigen activity can be obtained. As used herein, the term “microparticled HBs antigen” refers to a HBs antigen having a reduced molecular weight obtained by treating the HBs antigen with at least one selected from the group consisting of a surfactant and a protein denaturant. I do.

The surfactant is not particularly limited as long as it can remove lipids. For example, sodium dodecyl sulfate (SDS, cmc = 8.2 mM) and sodium dodecyl sulfone sulfate (cmc = 9.8 mM) , Sodium cholate (cmc = 14 mM), sodium deoxycholate (cmc = 5 mM), sodium taurodeoxycholate (cmc = 4 mM), and other anionic surfactants; cetyltrimethylammonium bromide (C
Cationic surfactants such as 16TAB, cmc = 0.92 mM) and dodecylpyridinium chloride (cmc = 16 mM); 3-[(cholamidopropyl) dimethylammonio] -1-propanesulfonic acid (CHAPS,
amphoteric surfactants such as cmc = 8 mM) and 3-[(cholamidopropyl) dimethylammonio] -2-hydroxy-1-propanesulfonic acid (CHAPSO, cmc = 8 mM); octylglucoside (OG, cmc = 25 m)
M), octylthioglucoside (cmc = 9 mM), nonanoyl-N-methylglucamide (MEGA-9, cm
c = 25 mM), decanoyl-N-methylglucamide (MEGA-10, cmc = 7 mM), polyoxyethylene dodecyl ether (Briji, cmc = 0.0)
02-0.3 mM), polyoxyethylene i-octylphenyl ether (Triton X-100, cmc =
0.24 mM: Triton X-114, cmc = 0.
20 mM: Triton X-165, cmc = 0.43
mM), polyoxyethylene nonyl phenyl ether (Nonidet P-40, cmc = 0.29 m)
M), polyoxyethylene fatty acid ester (Spa
n), polyoxyethylene sorbitol ester (Tw
een, cmc = 0.012 to 0.059 mM).

[0012] The above-mentioned surfactants are selected depending on the concentration in the solution.
Its existence form is different, and exists as a monomer at a low concentration, but forms a micelle at a certain concentration or more. The micelle is an aggregate in which a plurality of monomers are compactly assembled, and has a structure in which a hydrophilic (polar) portion faces outward and comes into contact with water, and a hydrophobic (non-polar) portion is hidden inside. ing. In the micelle state, the interaction with the hydrophobic part of the protein becomes large. Therefore, when the HBs antigen is subjected to the particle reduction treatment, the concentration at which micelles are formed (critical micelle concentration: critical micelle concentration)
concentration, hereinafter also referred to as cmc. It is preferred that the above concentrations be present.

The above-mentioned protein denaturing agents include unfolding of higher-order structure of protein, hydrogen bonding between polypeptide side chains,
There is no particular limitation as long as it can dissociate the hydrophobic interaction, and examples thereof include guanidine hydrochloride and urea.
The concentration of the protein denaturant varies depending on the concentration of HBs antigen to be treated, but is preferably in the range of 0.1 to 8M. The surfactant and the protein denaturant may be used alone or as a mixture.

In addition to the above-mentioned surfactant and / or protein denaturant, a small amount of a reducing agent may be further added. The reducing agent is not particularly limited as long as it can dissociate the disulfide bond of the protein, for example,
β-mercaptoethanol, 2-mercaptoethylamine, dithiothreitol, dithioerythritol, sodium borohydride, monothiophosphoric acid, and the like.

The concentration of the reducing agent depends on the concentration of the HBs antigen to be treated, but it can usually be used in a concentration of 0.001 to 10 mM in the solution. If the amount is less than 0.001 mM, the effect may not be exhibited. If the amount exceeds 10 mM, dissociation up to an intramolecular disulfide bond required for antigen activity is achieved.

[0016] The HBs antigen of the present invention can be prepared by mixing a solution in which at least one selected from the group consisting of the above-mentioned surfactants and protein denaturants is dissolved, with an HBs antigen solution, and reacting. Obtainable.

The diluting solution of the HBs antigen solution is not particularly limited, and examples thereof include a phosphate buffer, a Tris buffer, a Good buffer, a glycine buffer, and a Tris-HCl buffer. The ionic strength of the diluent is not particularly limited as long as it is suitable for diluting the HBs antigen, but is preferably one in which NaCl is adjusted to 0.05 to 1M. The pH of the diluent is not particularly limited,
The range of 4.5 to 9.0 is preferred.

The buffer for dissolving at least one selected from the group consisting of the above-mentioned surfactants and protein denaturants, or a buffer to which a reducing agent is added, is not particularly limited. Examples thereof include those exemplified for the liquid. The treatment concentration of the HBs antigen is not particularly limited, and 0.001 to 10 mg / mL is usually used.

The above-mentioned particle-reducing treatment reaction is, for example, 10 to 6
Although the HBs antigen can be prepared as a small particle by simply incubating for 1 to 10 hours under a constant temperature condition such as 0 ° C., the particle size can be reduced by further performing a stirring operation, an ultrasonic treatment or a heat treatment. It can also accelerate the reaction.

In the present invention, an anti-HBs antibody measurement reagent is produced using the above-mentioned small-particle HBs antigen. It is preferable that the small-particle HBs antigen is carried on an insoluble carrier.

Examples of the insoluble carrier include an inert carrier having a hydrophobic surface and an inert carrier having a partially hydrophobic surface. Examples thereof include synthetic polymers such as latex, plastic beads, and plastic plates. Compounds; inorganic materials such as silica; erythrocytes treated with tannic acid; and polymer membranes such as nitrocellulose. Among them, latexes that can be mass-produced with industrially stable quality; plastic molded products such as plastic beads and plastic plates are preferable.

Various functional groups for the purpose of chemical bonding may be present on the surface of the insoluble carrier, and a spacer or the like for immobilizing a substance by utilizing affinity such as avidin-biotin. May be provided.

In particular, latex is more preferable because the inspection time can be shortened and labor can be saved by fully automatic inspection processing. The latex is not particularly limited, such as a polystyrene type or a synthetic rubber type, but is preferably a polystyrene type. The average particle diameter of the latex is preferably from 0.05 μm to 2.0 μm, more preferably from 0.1 μm to 0.5 μm.

The above-mentioned method of loading on an insoluble carrier includes, for example, a method in which the small-particle HBs antigen obtained by the present invention is used as it is, or diluted with a suitable buffer, or subjected to dialysis treatment to obtain a surfactant or the like. Is removed to obtain an HBs antigen solution. The HBs antigen solution and the insoluble carrier,
The HBs antigen can be immobilized on an insoluble carrier by contacting with an aqueous medium having a pH of 4.5 to 9.0 and incubating for a predetermined time.

In carrying on the above-mentioned insoluble carrier, it is preferable that the concentration of the surfactant is not more than the cmc concentration. That is, the loading of the HBs antigen in the form of small particles on the insoluble carrier is physical adsorption based on the hydrophobic interaction between the two, so that when the surfactant is present in a micelle state, the hydrophobic interaction is hindered. That's because. When the concentration of the surfactant is less than the cmc concentration, the surfactant is present as a monomer. Therefore, the concentration of the surfactant can be reduced by a treatment such as dialysis.

After the completion of the incubation with the insoluble carrier, a blocking operation or the like is performed with a bovine serum albumin solution or the like for the purpose of suppressing nonspecific reaction of the reagent, and then the antigen-carrying insoluble carrier is centrifuged or the like. Can be recovered. After performing the washing operation several times,
Dilute and store using an appropriate buffer.

The solution for preserving the insoluble carrier carrying the above-mentioned small-particle HBs antigen and the aqueous medium used as the reaction solution for the antigen-antibody reaction are not particularly limited.
Commonly used buffers such as those exemplified as the dilution buffer for the HBs antigen are preferred.

When the above-mentioned enzyme-labeled small-particle HBs antigen and the small-particle HBs antigen supported on the insoluble carrier are used as immunoassay reagents, a highly sensitive immunoassay can be performed. For example, the amount of the HBs antibody in the sample can be measured by mixing the sample with the small-particle HBs antigen supported on the insoluble carrier and detecting the agglutination after the immunoagglutination reaction.

It is known that the HBs antigen is a macromolecule composed of proteins, lipids and phospholipids. Phospholipids are mainly involved in the formation of macromolecules, and it is thought that a stable structure is maintained by burying the hydrophobic part of the protein in the hydrophobic part of the phospholipid membrane. Furthermore, by stabilizing disulfide bonds and non-covalent bonds (hydrogen bonds, hydrophobic bonds, etc.) between proteins,
It is presumed that they form macromolecules.

The present invention removes non-covalent bonds between phospholipids and proteins, which are considered to be factors in the formation of macromolecules, by using a surfactant or a protein denaturant. The surfactant interacts with the hydrophobic part of the protein to separate the protein and lipid,
The protein denaturing agent can obtain a small-sized HBs antigen that retains antigen activity by dissociating non-covalent bonds such as hydrogen bonds and hydrophobic interactions between proteins.

[0031]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 Preparation of Small Particle HBs Antigen (1) (1) Reagents and Materials HBs antigen: Purified from human positive plasma. The purification method is
The small spherical particle antigen was recovered by a density gradient method using a cesium chloride solution and a sucrose solution, and further purified by a gel filtration method. The obtained HBs antigen was adjusted to 4 mg / mL using 50 mM sodium phosphate buffer (pH 7.5) +0.2 M NaCl, and this was used as an HBs antigen solution. Liquid for reducing particles: 50 mM sodium phosphate buffer (pH
7.5) Octylglucoside (manufactured by Dojindo Co.) was added to +0.2 M NaCl to a concentration of 60 mM, and this was used as a liquid for reducing particles. HBs antigen amount measurement kit: Enzygnost HBsAg monoclonal manufactured by Hoechst Japan was used.

(2) Particle size reduction method and disulfide reaction 2 mL of the HBs antigen solution and 2 mL of the particle size reduction solution were mixed and incubated at 25 ° C. for 2 hours. After diluting a small amount of the particle-containing antigen-containing solution to an appropriate amount and measuring the antigenicity of the solution using an HBs antigen measurement kit, it was found that the antigen activity was almost the same as that of the antigen solution before the particle size reduction operation. I confirmed that.

(3) Measurement of Molecular Weight In order to examine the molecular weight distribution of the small-particle HBs antigen obtained by this operation, analysis was performed by high performance liquid chromatography. High performance liquid chromatography is HPL manufactured by Shimadzu
The C-10A system was used, and a column for gel filtration chromatography TSKgelG4000SW _XL manufactured by Tosoh Corporation was used as a column, and 50 mM sodium phosphate buffer (p
H7.5) +0.1 M NaCl was used in which 30 mM octylglucoside was added. Detection was performed by measuring the absorbance at 280 nm, and a calibration curve was prepared using TSK standard polyethylene oxide manufactured by Tosoh Corporation as a molecular weight standard substance, and the molecular weight distribution was determined based on this curve. As a result, a peak was observed around 600,000 to 900,000 in the molecular weight distribution.

Example 2 Preparation of HBs Antigen of Small Particles (2) (1) Reagents and Materials Except for using the following as a liquid for small particles,
The same one as in Example 1 was used. Liquid for reducing particles: 50 mM sodium phosphate buffer (pH
7.5) To 70 mM of octylglucoside (manufactured by Dojindo Chemical Co., Ltd.) and 6 M of guanidine hydrochloride (manufactured by Wako Pure Chemical Industries) are added to +0.2 M NaCl so as to be 6 M, respectively. did.

(2) Method for reducing particle size The procedure was the same as in Example 1. After diluting a small amount of the particle-containing antigen-containing solution to an appropriate amount and measuring the antigenicity of the solution using an HBs antigen measurement kit, it was found that the antigen activity was almost the same as that of the antigen solution before the particle size reduction operation. I confirmed that.

(3) Measurement of molecular weight As a result of carrying out in the same manner as in Example 1, a peak was observed at about 400,000 to 600,000.

Example 3 Preparation of HBsAg in the form of small particles (3) (1) Reagents and materials
The same one as in Example 1 was used. Liquid for reducing particles: 50 mM sodium phosphate buffer (pH
7.5) MEGA-9 (manufactured by Dojin Chemical Co., Ltd.) was added to +0.2 M NaCl to 50 mM, and urea (manufactured by Wako Pure Chemical Industries) was added to 8 M, respectively, to obtain a liquid for reducing particles. .

(2) Method for Reducing Particle Size The same procedure as in Example 1 was carried out. After diluting a small amount of the particle-containing antigen-containing solution to an appropriate amount and measuring the antigenicity of the solution using an HBs antigen measurement kit, it was found that the antigen activity was almost the same as that of the antigen solution before the particle size reduction operation. I confirmed that.

(3) Measurement of molecular weight As a result of the same procedure as in Example 1, a main peak appeared at about 400,000 to 600,000.

Example 4 Preparation of miniaturized HBs antigen (4) (1) Reagents and materials Except that the following was used as the miniaturized liquid,
The same one as in Example 1 was used. Liquid for reducing particles: 50
mM sodium phosphate buffer (pH 7.5) + 0.2 M
40 mM CHAPS (manufactured by Dojindo) in NaCl,
Guanidine hydrochloride (manufactured by Wako Pure Chemical Industries, Ltd.) was added to a concentration of 8M, and dithiothreitol (manufactured by Wako Pure Chemical Industries, Ltd.) was added to a concentration of 1 mM.

(2) Method for Reducing Particle Size The procedure was the same as in Example 1. After diluting a small amount of the particle-containing antigen-containing solution to an appropriate amount and measuring the antigenicity of the solution using an HBs antigen measurement kit, it was found that the antigen activity was almost the same as that of the antigen solution before the particle size reduction operation. I confirmed that.

(3) Measurement of Molecular Weight As a result of the same procedure as in Example 1, a main peak appeared at about 200,000 to 400,000.

Example 5 Preparation of Latex Reagent for HBs Antibody Reagent (1) (1) Preparation of HBs Antigen-Sensitized Latex Reagent The small-sized HBs antigen obtained in Example 1 was treated with a 50 mM sodium phosphate buffer (pH 7.0). 1.0 mg using 5)
/ ML, and 7.5 mL of this solution was added to an average particle size of 0.3.
μm polystyrene latex (solid content 10% (W /
V), Sekisui Chemical Co., Ltd.) 0.5 mL and 0.1 M NaC
The mixture was added with 2 mL of 36 mM sodium phosphate buffer to which 1 l was added, and stirred at 30 ° C. for 60 minutes. Next, a phosphate-salt buffer (0.05 M phosphate buffer (pH: 1) containing 1% by weight of bovine serum albumin (BSA) in this solution was used.
7.0), 0.1 M NaCl, hereinafter also referred to as PBS), and stirred at 30 ° C for 60 minutes.
Washed by centrifugation for 1 minute at 18000 rpm. The washing operation was performed three times. BSA is added to the resulting precipitate.
Was added and 10% of PBS containing 1% by weight was added. After suspending the latex, dispersion treatment was performed with an ultrasonic crusher to prepare an HBs antigen-sensitized latex solution having a solid content of 0.1% (W / V). did.

(2) Preparation of Sample Diluent 0.9% by weight of polyethylene glycol having an average molecular weight of 500,000 (hereinafter also referred to as PEG: manufactured by Wako Pure Chemical Industries, Ltd.) was added to PBS containing 1% by weight of BSA. It dissolved so that it might become a density | concentration.

(3) HBs Antibody Measurement Reagent The HBs antibody measurement reagent of this example is the HBs antibody measurement reagent of the above item (1).
a first reagent comprising a s antigen-sensitized latex, (2)
And a second reagent composed of the PEG solution described in the above section.

(4) Standard HBs Antibody Solution 0, 300, 600, 1200, and 1800 HBs antibodies
Human serum at a concentration of mIU / mL was used as a standard.

(5) Measuring method 20 μL of the sample and 120 μL of the sample diluent described in the above item (2) are mixed and kept at 37 ° C. for an appropriate time.
120 μL of s antigen-sensitized latex solution was added and stirred. Thereafter, the absorbance at a wavelength of 750 nm after 1 minute and 5 minutes was measured, and this difference was defined as the change in absorbance (△ Abs). The measurement was performed using Hitachi Automatic Analysis Model 7150. A standard HBs antibody solution was measured to prepare a calibration curve. The obtained results are shown in Table 1 and FIG.

Example 6 Preparation of Latex Reagent for Measurement of HBs Antibody (2) A latex reagent for measurement of HBs antibody was prepared in the same manner as in Example 5, except that the small-sized HBs antigen obtained in Example 3 was used. It was prepared and a calibration curve was created. The obtained results are shown in Table 1 and FIG.

Comparative Example 1 The same procedure as in Example 5 was carried out except that the antigen sensitizing the latex was replaced with a purified HBs antigen that had not been subjected to particle reduction treatment in place of the particle-sized HBs antigen prepared in Example 1. Was. The obtained results are shown in Table 1 and FIG.

Comparative Example 2 The antigen sensitizing the latex was obtained by using the HBs antigen prepared in Example 1 by using 100 mM dithiothreitol instead of 70 mM octylglucoside and 8 M urea in the liquid for forming particles. Except having used, it carried out similarly to Example 5. The obtained results are shown in Table 1 and FIG.

[0052]

[Table 1]

[0053]

The method for producing a reagent for measuring anti-HBs antibody of the present invention has the above-mentioned constitution. Therefore, by using a small HBs antigen which retains its activity, a highly sensitive reagent for measuring anti-HBs antibody, particularly, It has become possible to prepare a latex agglutination reagent for anti-HBs antibody measurement.

[Brief description of the drawings]

FIG. 1 is a graph showing the relationship between the amount of anti-HBs antibody and the change in absorbance in Examples 5 and 6 and Comparative Examples 1 and 2. The vertical axis indicates the amount of change in absorbance, and the horizontal axis indicates the unit (mIU / mL) indicating the amount of the anti-HBs antibody.

Claims (2)

[Claims] 1. A method for producing an anti-HBs antibody measurement reagent using a small HBs antigen, wherein the small HBs antigen comprises at least one selected from the group consisting of a surfactant and a protein denaturant. A method for producing a reagent for measuring an anti-HBs antibody, characterized in that the reagent has been subjected to a small particle treatment by being added to an HBs antigen solution. 2. The method for producing a reagent for measuring an anti-HBs antibody according to claim 1, wherein the small-particle HBs antigen is carried on an insoluble carrier.