JPH11258241A - Latex reagent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a latex reagent which can be preserved stably for a long period by a method wherein a dispersion stabilizer is added to the suspension of latex particles sensitized by an antigen or an antibody and their mixture is freeze-dried. SOLUTION: In order to obtain the long-term preservation stability of a letex sensitized by a hepatalis C virus(HCV) antigen, a dispersion stabilizer or an antioxidant is added to the suspension of latex particles sensitized by an antigen or an antibody, and their mixture is then free-dried. When the latex which is sensitized by the HCV antigen is used, an HCV infective disease can be captured quickly and surely. The latex particles which are to be used are not limited especially as long as they can carry an antigen or an antibody, and it is preferable that an organic substance which is insoluble in a sample solution is used. As fine particles which are composed of the organic polymer substance, polystyrene, a styrene-maleic acid copolymer, a styrene-metacrylic acid copolymer and the like can be enumerated.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a latex reagent used in a latex agglutination reaction system and a test method using the same, and more particularly, to a latex reagent that can be stably stored for a long period of time and a hepatitis C virus using the same. Regarding inspection methods.

[0002]

2. Description of the Related Art Post-transfusion non-A non-B hepatitis is a major cause of hepatitis.
Hepatitis B virus (HCV) gene was cloned in 1989 by Chiron, USA. After that, Kato et al. Of the National Cancer Center [Proceedings of National Academy of Sciences USS
A (Proc. Natl. Acad. Sci. U.S.A.).
S. A. ), 87, 9524-9528, 1990],
Takamizawa et al., Osaka University Micro Laboratory [Journal of Virology, 65, 1
105-1113, 1991)] published the sequences of all genes of the structural and non-structural proteins of the hepatitis C virus. The whole amino acid sequence of hepatitis C virus is described in Choo et al. [Procedings of National Academy of Sciences USA (Proc. Natl. Acad. Sci. U.
SA), 88, 2451-2455, 1991].

Chiron Co., Ltd. has proposed a fusion protein having a polypeptide in the region from NS3 to NS4 (363 residues), which is a part of the non-structural protein of hepatitis C virus, at the C-terminus (N-terminal is human superhuman). Oxide dismutase) is expressed in yeast (Japanese Unexamined Patent Publication No. 2-500880), and E. coli is used in cooperation with Ortho, using this recombinant antigen.
LISA [Enzyme-Linked Immunosorbent
Assay (enzyme-linked immuno)
sorbent assay)]. afterwards,
Various HCV antibody detection systems using antigens derived from non-structural or structural regions of the HCV gene have been developed. HC
For V antibody testing, N, a non-structural region of the HCV genome
The use of a protein antigen encoded by S3 is clinically essential. However, it is known that the antigen has a reducing property due to the presence of a cysteine residue, and rapidly loses its binding ability to an antibody when oxidized. Conventional detection systems have mainly been radioimmunoassay systems using a radioisotope as a measurement system. However,
The radioimmunoassay system is not suitable as a routine test at a clinical laboratory because the detection time is as long as several hours or more, and facilities that can handle radioisotopes are limited.

On the other hand, conventionally, an antibody or antigen carried on insoluble carrier particles such as latex is reacted with the antigen or antibody in a liquid medium, and the reaction proceeds (latex aggregation).
A method of quantifying the concentration of an antigen or an antibody in a sample based on a decrease in the transmittance of a reaction mixture, that is, an increase in absorbance associated with the reaction is known (described in Japanese Patent Publication No. 58-11575, Japanese Patent No. 2510551, etc.). . The latex agglutination method is a method that does not require a special facility and can quickly obtain a determination result, and a clinical test method using the method has been applied to the test for HCV antibodies. However, it is inherently difficult for the latex reagent to maintain agglutinating activity for a long period of time due to factors such as separation of the sensitized antigen or antibody from the latex surface. In addition, the latex reagent sensitized with the NS3 region-derived antigen has its agglutinating activity reduced more rapidly due to oxidation of the sensitized antigen, making accurate detection of HCV antibodies difficult.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a latex reagent capable of making an accurate determination over a long period of time during storage of the reagent and an HCV antibody test method using the same.

[0006]

Means for Solving the Problems In view of the above-mentioned situation, the present inventors have conducted intensive studies and as a result, in order to obtain the long-term storage stability of a latex sensitized with HCV antigen, the dispersion stabilization of the latex was carried out. A method for freeze-drying by adding an agent or an antioxidant to an antigen was established. Furthermore, the use of this HCV antigen-sensitized latex has made it possible to quickly and accurately detect HCV infection. That is, the present invention resides in a latex reagent obtained by freeze-drying latex particles sensitized with an antigen or an antibody, and a method for detecting hepatitis C virus using the same.

[0007]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The latex particles used in the present invention are not particularly limited as long as they can carry an antigen or an antibody, but usually, it is preferable to use an organic polymer substance which is insoluble in a sample solution. As fine particles composed of such an organic polymer substance, polystyrene, styrene-
Maleic acid copolymer, styrene-methacrylic acid copolymer, acrylic acid-styrene copolymer, styrene-acrylic acid-acryl acrylate copolymer, vinyl acetate-acrylic acid copolymer, styrene-butadiene copolymer, poly Latex particles made of a synthetic polymer such as an acrylate or a polymethacrylate can be exemplified. Of these, polystyrene latex particles are particularly preferred. The diameter of the latex particles is 0.1 to 10 μm
Can be used, but usually within a range of 0.1 to 1 μm.

[0008] The antigen or antibody sensitizing the carrier is
There is no particular limitation as long as it can react with the antibody or antigen present in the test sample. Those used as sensitizing antibodies can be obtained, for example, by purifying from the blood of rabbits immunized with the antigen. The antibody can be purified and used by a known purification method, salting out with ammonium sulfate, protein A affinity chromatography, ion exchange chromatography, gel chromatography, electrophoresis and the like. The obtained antibody can be diluted with an appropriate buffer or physiological saline and used for latex sensitization. Buffers used include Tris-HCl buffer, phosphate buffer,
Borate buffer and the like.

[0009] The antigen carried on the carrier is
For example, the HCV antigen is mentioned. The HCV antigen used is H
There is no particular limitation as long as it can react with HCV antibodies present in the blood of a CV infected person. NS3, NS4 and C derived from the core region are used as HCV antigens.
25 antigens, C33c antigen from NS3 region, C100 antigen from NS4 region, C2 from NS3 and NS4 regions
And recombinant antigens such as the NS5 antigen derived from the NS5 region, the C5-3 antigen derived from the core region, and the like. (C2
5, C33c, C200, C22-3, and NS5 antigens: Isao Okuda et al .: Significance of HCV antibody measurement by CLEIA method, Medicine and Pharmacy, 37 (5), 1181-1.
188, 1997) These antigens can be obtained by a known method such as expression of a recombinant gene in yeast. The obtained antigen can be used as it is without purification or after purification by a known purification method. In order to prevent non-specific reactions in the latex agglutination system, the antigen may be modified as necessary. The obtained purified or unpurified HCV antigen can be diluted with an appropriate buffer or physiological saline and used for latex sensitization.

The above-mentioned sensitization of the antigen or antibody to the latex particles can be carried out by a commonly used method, for example, a physical adsorption method using physical adsorption caused by mixing the latex particles with a solution of the antigen or antibody, a carbodiimide or the like. By using a coupling agent, a carboxyl group or an amino group on the surface of latex particles can be chemically bonded to an antigen or an antibody by a chemical bonding method or the like. Further, an antigen or an antibody may be bound to latex particles via a spacer molecule. Further, after binding to a protein such as albumin using a chemical bonding method, the protein may be bound to latex particles. There is no particular limitation on the amount of antigen or antibody sensitized to latex particles,
In the case of latex particles, usually 1 ml of latex suspension
0.01 to 2 mg is suitable for
For the C25 antigen of V, 0.02 to 0.1 mg is preferable for 1 ml of the latex suspension.

The suspension of latex particles sensitized with the antigen or antibody may be pure water or an appropriate buffer.
For example, a MES buffer, a Tris-HCl buffer, a phosphate buffer, a borate buffer, or the like is used. A dispersion stabilizer may be added to the suspension of latex particles before freeze-drying. When the latex particles are freeze-dried, the latex particles may aggregate when condensed. Dispersion stabilizers are preferred to prevent this. Examples of the dispersion stabilizer used in the present invention include saccharides and / or surfactants. As saccharides, for example, sucrose, lactose,
Mannitol and the like, and as the surfactant, sodium dodecyl sulfate (SDS), Tween 80, T
riton X-100, sodium N-dodecanoyl sarcosine and the like. When using saccharides,
Usually, 1 to 20%, preferably 2 to 10% is added to the suspension of latex particles. When a surfactant is used, it is usually 0.005 to the suspension of latex particles.
To 0.2%, preferably 0.01 to 0.1%.

The suspension of latex particles of the present invention may further contain an antioxidant. The addition of antioxidants
It is particularly effective when a reducing antigen, for example, a C25 antigen or a C33c antigen of HCV is used as the antigen sensitizing the latex particles. Examples of the antioxidant include dithiothreitol (DTT), N-acetyl-L-cysteine, L-arginine, sodium sulfite, sodium ascorbate, glutathione, and the like. The amount of antioxidant added depends on the antigen used, etc.
Usually, it is 0.01 to 10 mM, preferably 0.1 to 5 mM. The freeze-drying of the suspension of latex particles sensitized with the antigen or antibody is performed by a usual freeze-drying machine in the same manner as the freeze-drying of an aqueous protein solution or the like, and the method is not particularly limited. For latex suspensions, sample temperature -30
After performing pre-freezing at a temperature of not more than 2 ° C. for 2 hours or more, the pressure in the freeze-dryer tank is reduced, and after the pressure is reduced to 0.1 Torr or less, the temperature of the sample shelf is raised to room temperature over about 20 hours to obtain an antigen or Dry the antibody-sensitized latex.

For the reconstitution of a freeze-dried product of latex particles sensitized with the antigen or antibody, pure water or a suitable buffer such as MES buffer, Tris-HCl buffer, phosphate buffer, borate buffer may be used. And so on. Further, in order to improve the dispersibility of the latex particles upon condensing, a surfactant such as sodium dodecyl sulfate (SDS), Tween 2
0, Tween 80, Triton X-100, N
-Sodium dodecanoyl sarcosinate may be added to the condensate. The biological components used in the present invention include serum, plasma, nasal discharge, urine, and the like separated from blood, and are generally serum or plasma separated from blood. Separation of serum or plasma can be performed by a known centrifugation method or the like without losing the antigen or antibody in the test sample.

The thus obtained latex particles sensitized with the antigen or antibody are mixed with a sample solution such as serum or plasma, and the desired antibody or antigen in the sample and the antigen or antibody sensitized to the latex particles are mixed. And the degree of aggregation of latex particles generated by the reaction is measured to detect the presence of antibody or antigen in the sample or to calculate the concentration. For example, when a latex sensitized with HCV C25 antigen is used, the anti-C level depends on the degree of aggregation of latex particles due to the reaction with the anti-C25 antibody in the test sample.
25 Antibody concentration is calculated to determine the presence or absence of HCV infection.
The above reaction is usually performed in a plastic cell or a glass cell. In this case, light in the near-infrared region from visible light to the outside of the cell, for example, light of usually 400 to 2400 nm, preferably 600 to 1000 nm is irradiated, and the degree of aggregation of latex particles is detected by detecting a change in absorbance or a change in intensity of scattered light. Is measured. At this time, the concentration of the antigen or antibody in the sample can be calculated by using a calibration curve prepared in advance.

As an apparatus for performing such agglutination measurement, a fully automatic immune serum test system LPIA of Mitsubishi Chemical Corporation is used.
-A700, COBAS MIRA from Roche Diagnostics Systems, and 7 from Hitachi, Ltd.
Model 070 Hitachi automatic analyzer. In addition, the agglutination reaction of the latex particles is usually performed in physiological saline, p
The reaction is carried out in a suitable buffer solution of about H5 to 10 such as Tris-HCl buffer, phosphate buffer, borate buffer and the like. In order to suppress the non-specific binding between the latex particles and the protein in the sample, a suitable surfactant such as sodium dodecyl sulfate (SDS), Tween 2
0, Tween 80, Triton X-100 or the like may be added to the reaction solution. A reagent kit for use in an immunological test including the above reaction and measurement steps,
It can be prepared by commonly used methods known per se.
Such a kit is provided in the same configuration as a kit using a normal immunoagglutination reaction. That is, the reagent kit of the present invention contains at least a latex reagent in a lyophilized state to which an antigen or an antibody has been sensitized, and further contains a sample diluent (condensate), a standard substance, and the like as optional elements. As a specific example, a kit containing three components of a lyophilized latex reagent, a buffer solution and a condensate solution may be mentioned. In this kit, a buffer is used as a stabilizer.

[0016]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto. Example 1 Preparation of Lyophilized HCV Antigen Latex Reagent HCV C25 antigen was sensitized to the surface of a polystyrene latex (10% suspension; manufactured by JSR) having a diameter of 0.48 μm by the following method to obtain a latex reagent. 1%, 20
0.1 mg / mL C25 antigen was added to the mL latex suspension, and reacted at room temperature for 30 minutes. After centrifugation of the latex, blocking was performed for 30 minutes with 0.1% bovine serum albumin (BSA). The suspension was made into a latex reagent solution. Dispense 3 mL of the above latex reagent solution into a 10 mL glass vial, freeze-dry, and freeze-dry HC
It was stored as a V antigen latex reagent at 4 ° C.

Comparative Example 1 As a control, the above HCV antigen latex was suspended only in MES buffer (pH 6), and was suspended in a state without freeze-drying.
The reagent stored at ℃ was adjusted.

Example 2 and Comparative Example 2 Automatic Measurement of HCV Antibody HCV antibody-positive samples were examined. Sample 30μ
L, 140 μL of BSA-containing Tris-HCl buffer, Example 1
The lyophilized HCV antigen latex reagent obtained in
20 μL of water reconstituted with MES buffer (pH 6) containing 2% SDS was mixed and automatically dispensed into a reaction cuvette.
The immune response was measured by measuring the increase in near infrared light (800 nm) absorption for 10 minutes. Similarly, the control latex reagent shown in Example 1 was measured in the same manner. The measurement was performed using a fully automatic immune serum test system LPIA-A700. The measurement was carried out over a period of 8 weeks from the day when the lyophilized HCV antigen latex reagent and the control latex reagent were each prepared. The results are shown in FIG. The reactivity of the HCV antigen latex that had been freeze-dried did not change for up to 8 weeks in the reactivity with the HCV antibody-positive specimen.
On the other hand, in the case of HCV antigen latex that has not been freeze-dried, the reactivity of the HCV antigen latex significantly decreased in one week, and the sensitivity effective for measurement was lost.

[0019]

According to the present invention, by freeze-drying a latex reagent sensitized with an antigen or an antibody, it is possible to measure the antibody or the antigen easily and accurately over a long period of time during storage of the reagent. The method of the present invention having the above characteristics,
It is also effective for long-term storage of a latex reagent sensitized with an antigen or the like which is liable to lose its activity, which has been difficult in the past, for improving stability.

[Brief description of the drawings]

FIG. 1 is a graph showing the effect of freeze-drying of a HCV antigen latex reagent on the change over time in reactivity. The vertical axis is a value obtained by multiplying a change in measured absorbance per time by a coefficient.

 ──────────────────────────────────────────────────の Continuing on the front page (72) Inventor Masayoshi Washinasu 8-5-1 Chuo, Ami-cho, Inashiki-gun, Ibaraki Prefecture Inside Yuka Medias Development Center Co., Ltd.

Claims (8)

[Claims] 1. A latex reagent obtained by freeze-drying latex particles sensitized with an antigen or an antibody. 2. A latex reagent obtained by adding a dispersion stabilizer to a suspension of latex particles sensitized with an antigen or an antibody and freeze-drying. 3. The latex reagent according to claim 2, wherein the dispersion stabilizer is a saccharide and / or a surfactant. 4. The latex reagent according to claim 1, wherein an antioxidant is added to a suspension of latex particles sensitized with an antigen or an antibody, followed by freeze-drying. 5. The latex reagent according to claim 1, wherein the antigen is hepatitis C virus antigen. 6. The latex reagent according to claim 5, wherein the antigen is hepatitis C virus C25 antigen. 7. A hepatitis C virus antibody test method using the latex reagent according to claim 5 for a latex agglutination system. 8. A kit comprising three components A, B and C, wherein component A comprises the latex reagent according to any one of claims 1 to 6, component B comprises a buffer, A kit, wherein the component C contains a condensate.