JP3225468B2 - Antibody measurement reagent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a reagent for measuring an antibody, in particular, to measure an antibody in a sample by an immunological method using an antigen having a sensitive thiol group or a peptide having substantially the same action as the antigen. The present invention relates to a measurement reagent for measuring an antibody with high sensitivity without being affected by the storage state of the reagent and the like, and for more accurately measuring the antibody. In particular, the invention relates to hepatitis C virus (HCV)
The present invention relates to a reagent for measuring an antibody against, particularly a reagent for measuring an HCV antibody in a sample with high sensitivity by an immunological method and for more accurately measuring the same.

[0002]

2. Description of the Related Art As a method for diagnosing infection by hepatitis C virus (HCV), in 1988, Chiron, USA, reported NS3 and N3, which are nonstructural regions on the HCV genome.
H using C100-3 antigen encoded in S4 region
A CV antibody measurement system has been developed. In 1991, HCV
Core region and C100-3, which are structural regions on the genome
The use of the core antigen encoded by the NS3 region which does not overlap with the antigen and the 33C antigen has led to the development of an HCV antibody measurement system with even higher sensitivity and detection rate. As a method for measuring these HCV antibodies, an agglutination method using erythrocytes or latex particles as an antigen-carrying carrier, an immunometric method using beads, tubes, or plates as an antigen-immobilized carrier are used. However, during the step of immobilizing the antigen on the carrier or during the storage of the prepared reagent, the activity of the antigen rapidly decreases in the reaction solution, and the antigen-antibody reaction cannot proceed sufficiently. Not go up,
Further, there is a problem that the reproducibility of sensitivity is deteriorated because the activity of the antigen changes with time. As described above, in the case of a reagent using a certain antigen, the activity of the antigen rapidly decreases in the reaction solution during storage of the reagent, and the antigen-antibody reaction does not proceed sufficiently, and the measurement sensitivity does not sufficiently increase. There has been such a problem that the reproducibility of sensitivity is deteriorated because the activity of the antigen changes with time.

[0003]

The present inventors have [0005] As a result of intensive studies to solve the problem of sensitivity in the conventional HCV antibody detection system, this poor sensitivity problems, sensitive thiol
It has been elucidated that the cysteine contained in the HCV antigen having a group , particularly the cysteine contained in the protein encoded in the NS3 region on the HCV genome is naturally oxidized and becomes a disulfide bond or the like. The present inventors can prevent the problem of a decrease in the sensitivity of the HCV antibody measurement system by adding a reducing agent, particularly a thiol protective agent, to the HCV antibody measurement system based on these findings. The present invention has been completed by clarifying that the measurement system is not adversely affected.

[0004]

SUMMARY OF THE INVENTION The present invention relates to a method for detecting HCV in a specimen.
Sensitive thiols for measuring antibodies by immunological methods
A reagent for HCV antibody measurement, wherein the reagent contains a reducing agent, or the solid-phased antigen-containing reagent is treated with a reducing agent. The sample used in the present invention may be any component derived from a living body containing an HCV antibody, such as blood, serum, semen, spinal fluid, lymph,
Examples include body fluids such as sputum, tears, saliva, milk, leukocytes, digestive organ mucus, and urine, and tissue fluids, and further include, but are not limited to, in vitro cell culture solutions.

[0005] The present invention uses an antigen having a thiol group sensitive to a fine particle carrier such as erythrocytes or latex particles or a sensitizing carrier sensitized with a peptide having substantially the same action as that of the antigen, and agglutination reaction in the specimen. In producing a reagent to be used for immunologically measuring a specific antibody, after sensitizing the carrier to the antigen, the obtained sensitized carrier is dispersed in a buffer containing a reducing agent, and the sensitization is performed. This can be achieved by freeze-drying the carrier-containing buffer. In particular, the present invention relates to the use of HC on fine particle carriers such as red blood cells and latex particles.
In producing a reagent used for immunologically measuring HCV antibodies in a sample by agglutination reaction using a sensitized carrier sensitized with V antigen, the carrier is obtained after sensitizing the carrier with HCV antigen. The sensitizing carrier is dispersed in a buffer containing a reducing agent, and the buffer containing the sensitizing carrier is freeze-dried. Examples of the reducing agent that can be used here include those known as thiol protecting agents.
As the reducing agent, those which are antioxidants of thiol groups are preferable, and include, for example, dithiothreitol, dithioerythritol, thioglycolic acid, cysteine, glutathione, 2-mercaptoethanol, 2-mercaptoethylamine and mixtures thereof. And at least one selected from the group. Particularly, dithiothreitol, glutathione, 2-mercaptoethanol and the like are preferable.

Examples of the fine particle carrier which can be used here include those widely known as fine particle carriers, for example, those made of natural or synthetic polymers such as synthetic resins and nitrocellulose, and those made of latex. Particles or erythrocytes. According to the present invention,
The reducing agent may be added to a buffer used for suspending the sensitizing carrier during the agglutination reaction. Further, the present invention provides an enzyme immunoassay (EIA) using an immobilized antigen obtained by immobilizing an antigen having a sensitive thiol group or a peptide having substantially the same action as that of the antigen on an insoluble carrier. When manufacturing a reagent used for immunologically measuring a specific antibody in a sample by a method such as radioimmunoassay (RIA) or fluoroimmunoassay (FIA), an antigen having a sensitive thiol group on an insoluble carrier or substantially the same as the antigen. After immobilizing a peptide having substantially the same action as the solid phase, immersing the obtained antigen-binding solid phase in a buffer containing the reducing agent or a mixture thereof, and drying the solid phase. You can do it. In particular, in the present invention, a method such as an enzyme immunoassay (EIA), a radioimmunoassay (RIA), or a fluoroimmunoassay (FIA) is performed using an immobilized antigen obtained by immobilizing an HCV antigen on an insoluble carrier. In producing a reagent used for immunologically measuring HCV antibodies in a sample,
This can be achieved by immobilizing the HCV antigen on an insoluble carrier, immersing the obtained antigen-binding solid phase in a buffer containing the above-mentioned reducing agent or a mixture thereof, and then drying the solid phase. I can do it.

As the reducing agent that can be used here, those known as thiol protecting agents similar to those described above can be mentioned. According to the present invention, the above-mentioned reducing agent or a mixture thereof may be added to a reaction solution used when reacting a specific antibody in a sample with an antigen-binding solid phase. Examples of the insoluble carrier that can be used herein include those widely known as carriers for solid phase immobilization, for example, synthetic resins such as polyethylene, polypropylene, polystyrene, and polyacrylate, nitrocellulose, polymerized amino acids, and polysaccharides. Alternatively, there is a shape such as a particle, a film, a bead, a tube, or a plate made of a synthetic polymer, glass, or the like. For solid-phase formation, there is a physical adsorption method or a method of chemically solidifying with a binder. The chemical binder can be selected from those commonly known to those skilled in the art, for example, 6-maleimidocaproic acid, 2-bromoacetic acid, 2-bromoacetic acid,
Examples include active esters such as iodoacetic acid and succinic acid, active esters of triazine, sulfonic acid ester derivatives, and the like, but are not limited thereto.

[0008] Examples of the antigen used in the present invention include an antigen having a sensitive thiol group or a peptide having substantially the same action as the antigen. Here, the susceptible thiol group is a thiol group in cysteine contained in a protein or peptide, which is normally sensitive to natural oxidation or artificial oxidation and can greatly affect the activity of the antigen. Say. Here, the antigen activity refers to an activity capable of producing a specific antigen-antibody reaction, and particularly refers to an activity of a specific antigen that reacts with a specific antibody in a sample by an immunological method. As long as the antigen used in the present invention has a sensitive thiol group in this way, a recombinant antigen as an expression product produced by a genetic engineering technique, or a synthetic peptide can also be used without particular limitation. . In the present invention, even if a thiol group of cysteine or a disulfide bond derived therefrom is not a group that can affect the activity of the antigen, treatment with a reducing agent is performed in the present invention. It is not intended as an antigen. Particularly, the HCV antigen used in the present invention is a recombinant H which is an expression product produced by a genetic engineering technique.
CV antigen or HCV antigen peptide which is a synthetic peptide is exemplified. As the HCV antigen used in the present invention,
Preference is given to N in the nonstructural region on the HCV genome.
An example corresponding to the S3 region is given.

[0009]

EXAMPLES The present invention will be described more specifically with reference to the following examples. Example 1 Effect of DTT on Sensitized Blood Cells Human fixed erythrocytes were treated with phosphate buffer (pH 7.4) for 3 hours.
After washing twice, the suspension was suspended in an acetate buffer at pH 5.7 so as to be 1% by volume, and purified H produced by genetic recombination technology was used.
CV antigen (a mixture of core antigen, 33C antigen and C100 antigen) was added to a final concentration of 6 μg / ml, and the mixture was stirred in a greenhouse for 1 hour.
Washed three times with 7.4) and lyophilized in phosphate buffer (pH 7.4) containing 7.5% sucrose.
HCV antigen-sensitized red blood cells were prepared. The obtained HCV antigen-sensitized erythrocytes were reconstituted in Tris-HCl buffer (pH 7.8) containing 2 mM dithiothreitol (DTT) and Tris-HCl buffer containing no DTT as a control so as to be 1% by volume. Suspension was performed and the sensitivities were compared. Comparison of sensitivity
HCV antibody-positive human serum that had been serially diluted with serum that was negative for the antibody was used as serum for sensitivity control. To each well of a microtiter plate, 25 μl of a phosphate buffer (pH 7.4) and 25 μl of sensitivity-controlling serum at each concentration are dispensed, and 25 μl of sensitized blood cells is further dispensed, followed by stirring for 30 seconds with a mixer. After leaving at room temperature for 1 hour, the result was visually determined.

[Table 1] From the results in Table 1, it was shown that the sensitivity was increased when the sensitized blood cells were resuspended in the buffer containing DTT.

Example 2 Effect of 2-ME on Sensitized Blood Cells Sensitized blood cells prepared in the same manner as in Example 1 were treated with Tris-HCl buffer (pH 7) containing 40 mM 2-mercaptoethanol (2-ME). .8) to 1% by volume, and compared the storage stability after suspension with a Tris-HCl buffer without 2-ME as a control. The storage after lysis was stored at 2 to 8 ° C, and the sensitivity of sensitized blood cells was compared according to Example 1.

[Table 2] From the results in Table 2, it was confirmed that storage stability of the sensitized blood cells after suspension was improved by the addition of 2-ME.

Example 3 Influence of Glutathione on Sensitized Blood Cells [0011] Blood cells sensitized with HCV antigen according to Example 1
Glutathione (GS) was added to 5% sucrose-containing phosphate buffer (pH 7.4) so that the final concentration was 40 mM.
It was lyophilized in a lyophilization buffer to which H) had been added to obtain sensitized blood cells. As a control, the cells were freeze-dried in a buffer solution containing no GHS to obtain control-sensitized blood cells. Sensitized blood cells and control sensitized blood cells were added to Tris-HCl buffer (pH 7.8) at 1% by volume.
The stability after suspension at room temperature was compared by the sensitivity of the sensitized blood cells. The sensitivity comparison was performed according to Example 1.

[Table 3] From the results in Table 3, it was confirmed that the sensitized blood cells freeze-dried with the addition of GSH had good stability after suspension.

Example 4 Effect of 2-ME Addition on Immobilized Antigen [0012] 1/4 inch polystyrene beads were placed in a phosphate buffer (pH 7.4) containing 7.5% sucrose, and the reaction was carried out. The HCV antigen used in Example 1 was used at a final concentration of 10 μm.
1 / ml, left at 37 ° C. for 1 hour, washed three times with a phosphate buffer (pH 7.4), and further contained a phosphate buffer containing 7.5% of sugar carose. (P
H 7.4), and then dried to obtain HCV antigen-immobilized beads. One of the HCV antigen-immobilized beads is
The mixture was placed in a wide well tray containing 200 μl of Tris-HCl buffer (pH 7.8) containing 20 mM 2-ME as a reaction buffer, left at room temperature for 1 hour, and 10 μl of serum for sensitivity control was added. The reaction was performed at 37 ° C. for 1 hour. After completion of the reaction, the well was washed three times with physiological saline, and 200 µl of peroxidase-labeled anti-human immunoglobulin G antibody was added, followed by reaction at 37 ° C for 30 minutes. After completion of the reaction, the mixture was washed three times with physiological saline, 1 ml of a substrate solution containing orthoethylenediamine and hydrogen peroxide was added, and the reaction was carried out at room temperature for 30 minutes. For comparison, the same operation was carried out using a Tris-HCl buffer containing no 2-ME as a reaction buffer, and a determination was made.

[Table 4] From the results in Table 4, the sensitivity was increased by adding 2-ME to the reaction buffer.

Example 5 Effect of 2-ME Addition HCV antigen-sensitized blood cells prepared according to Example 1 were suspended in Tris-HCl buffer (pH 7.8), left at 2-8 ° C. for 2 weeks, Anti-core (core region of HCV), anti-33C (NS3 region of HCV) and anti-C1
The sensitivity of the sensitized blood cells to three specimens mainly containing the respective antibodies of OO (NS3 to NS4 regions of HCV) was measured,
Investigation was conducted according to Example 1. 40 mM 2-M in suspension
E was added, and the sensitivity was similarly examined.

[Table 5] From the results, it was confirmed that the sensitivity of the sensitized blood cells, which had decreased in sensitivity for 2 weeks, was recovered by the addition of 2-ME for the 33C antigen encoded in the NS3 region on the HCV genome.

[0014]

According to the present invention, a reducing agent is contained in an antigen having a sensitive thiol group or a peptide-containing antibody measuring reagent having substantially the same action as the antigen, or the solid-phased reagent is treated with a reducing agent, When an antibody in a specimen is measured by an immunological method, the sensitivity of the reagent can be increased.

Continuation of the front page (72) Inventor Susumu Tokita 344 Minodai, Matsudo-shi, Chiba Dynabot Co., Ltd. (56) References JP-A-58-52228 (JP, A) JP-A-63-185996 (JP, A) (58) Field surveyed (Int. Cl. ⁷ , DB name) G01N 33/48-33/98 BIOSIS (DIALOG) WPI (DIALOG)

Claims (9)

(57) [Claims] 1. An HCV antigen-containing reagent having a sensitive thiol group for measuring an HCV antibody in a sample by an immunological method, wherein the reagent contains a reducing agent or is immobilized on a solid phase. A reagent for measuring HCV antibodies, characterized in that the reagent is treated with a reducing agent. 2. The method according to claim 1, wherein the reducing agent is present in the reaction solvent.
The reagent as described. 3. The reagent according to claim 1, wherein the reagent is an antigen obtained by immobilizing an HCV antigen having a sensitive thiol group on a carrier, and the antigen reagent is treated with a reducing agent. 4. The reagent according to claim 3, wherein the antigen having a sensitive thiol group is the NS3 region of a nonstructural region of the HCV genome. 5. The reagent according to claim 4, wherein the antigen having a sensitive thiol group is an expression product obtained by a genetic recombination technique. 6. The reagent according to claim 4, wherein the antigen having a sensitive thiol group is a synthetic peptide. 7. The reagent according to claim 1, wherein the reagent comprises a carrier, and the carrier is a bead, a tube, a plate, a red blood cell, or a latex particle. 8. The reagent according to claim 1, wherein the reducing agent is a thiol group antioxidant. 9. The method according to claim 1, wherein the reducing agent is at least one selected from the group consisting of dithiothreitol, dithioerythritol, thioglycolic acid, cysteine, glutathione, 2-mercaptoethanol, 2-mercaptoethylamine, and a mixture thereof. The reagent according to any one of claims 1 to 7.