JPH03190898A - Synthetic polypeptide and reagent for hcv antibody analysis using same polypeptide - Google Patents

Abstract

NEW MATERIAL:A synthetic polypeptide having an amino acid sequence of the formula, etc., and having an antigen-decision base common to hepatitis C virus. EXAMPLE:A reagent for hepatitis C virus (HCV) analysis. PREPARATION:A C-terminal amino acid alpha-amino group of which is protected, Boc-leucine (Boc is t-butyloxycarbonyl) is reacted in the presence of carbodiimide, e.g. by solid-phase synthesis using an oxymethyl resin as a carrier and the protective group for alpha-amino group is subsequently removed. Protected amino acids are then bonded in accordance with the amino acid sequence of a polypeptide and removal of the protective groups for alpha-amino groups are subsequently repeated so as to synthesize a polypeptide resin. The resultant polypeptide resin is treated with HF in anisole so that elimination of the polypeptide from the carrier resin and removal of the whole protective groups may be carried out, thus obtaining the objective polypeptide of the formula, etc.

Description

[Detailed Description of the Invention] [Field of Industrial Application] The present invention has a common antigenic determinant with hepatitis C virus (hereinafter referred to as HCV), which is known as the causative virus of non-A, non-B hepatitis. Synthetic polypeptides and their use7
', relates to an HCV antibody measurement reagent.

[Conventional technology]

In Japan, hepatitis is said to be the national disease of the 21st century because there are many patients and it easily progresses to liver cirrhosis and hepatocellular carcinoma, which has a high mortality rate.

In recent years, the causes of hepatitis are gradually being elucidated, and viruses account for a large proportion of the cases.

The causative viruses are hepatitis B virus (HBV) and A
hepatitis virus (HA-V), cytomegalovirus,
Epstein-Barr virus, etc. are known, and many detection and measurement systems for these virus particles, protein antigens derived from these viruses, or antibodies against them have been developed using serological testing methods based on antigen-antibody reactions. 6 On the other hand, the most common route of infection for viral hepatitis is through blood transfusion, and other known routes include intravenous drug abuse, infection within medical institutions, hemodialysis, and vertical transmission within families. .

1 = 4 - Therefore, in our country, donor blood is immunoserologically tested and blood containing HAV, HBV, or any of these antibodies is excluded from transfusion. A hepatitis B vaccine has also been developed and is now in use.

Therefore, the incidence of developing hepatitis A or B after blood transfusion has decreased significantly.

However, there are many cases in which hepatitis develops after blood transfusion, and since these hepatitis cases are neither hepatitis A nor hepatitis B, they have been called non-A, non-B hepatitis (NANBH).

Recently, the virus responsible for non-A, non-B hepatitis was isolated, confirmed to be different from HBV, HAV, cytomegalovirus, and Epstein-Barr virus, and named hepatitis C virus (trcV). It was done.

It has been pointed out that many cases of hepatitis C have a history of blood transfusion, and as it takes a relatively long time for symptoms to develop, such as onset occurring more than 6 months after a blood transfusion, many cases become chronic. The rate of progression to liver cirrhosis and hepatocellular carcinoma is also not low.

The amount of HCV antigen in the blood of HCV-infected people is extremely low compared to HBV, so it is difficult to detect HCV in the blood, and it is difficult to detect HCV in the blood.
Since the extent to which the host causes an immune response due to infection with CV is weak, the antibody titer in the blood is also lower than that of HBV antibodies.

[Problem to be solved by the invention]

Previously, HCV-derived antigens used for detecting ICV antibodies were produced using genetic and cell engineering methods, isolated from culture fluid, and
Because it was refined, special equipment and skilled technicians were required to obtain the refined product, which required a long period of time and a great deal of effort.

Therefore, purified antigens derived from HCV are expensive, and reagents for measuring HCV antibodies produced using them are also expensive, and mass production is also difficult. Therefore, the IC
Reagents for measuring V antibodies have not yet become widely available.

The present invention makes it possible to obtain a polypeptide having an antigenic determinant common to HCV-derived vcJX by peptide synthesis, thereby making it possible to mass-produce it at a low cost, and to obtain an excellent HCV
The purpose is to establish a highly sensitive HCV antibody measurement system by imparting antigen specificity.

[Means to solve the problem]

The synthetic polypeptide invention of the present application relates to synthetic polypeptides having common antigenic determinants with hepatitis C virus, - for example, the formula I H-11e-11e-Pro-8sp-Arg-Glu
-Val-Leu-TyrArg-Glu-Phe-A
sp-Glu-Met-Glu-Glu-CysSer
-Gln-H1s-Leu-Pro-Tyr-I 1e
-G 1u-G inGly-Met-Met-Leu
-Ala-Glu-G 1n-Phe-LysGin-
Lys-Ala-Leu-Gly-Leu-OH-=
Some have the amino acid sequence shown in I.

As a method for synthesizing a polypeptide having the above amino acid sequence, a peptide synthesis method can be applied, such as a C-terminal activation method such as an activated ester method, a mixed acid anhydride method, or an azide method, or a coupling link method such as a carbodiimide method. Coupling method using agents, N-carboxy anhydride (NCA) method, redox method,
There are enzymatic methods, solid phase synthesis methods, etc.

The HCV antibody measurement reagent of the present invention comprises a solid phase reagent in which a synthetic polypeptide having a common antigenic determinant with hepatitis C virus is immobilized on a solid phase, and either Rin-sensing anti-human globulin antibody or the labeled synthetic polypeptide. It consists of a heel.

Examples of the solid layer include various synthetic resin microtiter plates, beads, sheets, porous films, magnetic latex, and the like.

Methods for immobilization on the solid phase include physical adsorption methods and chemical bonding methods using glutaraldehyde, water-soluble carbodiimide, and the like. When immobilized, the labeled anti-human polypeptide may be bound via a spacer such as hexamethylene diamine, or the synthetic polypeptide bound to a protein such as bovine serum albumin may be immobilized on the solid phase. The animal species of the anti-human globulin antibody used as the globulin antibody are goat,
Sheep, horses, rabbits, mice, guinea pigs, hamsters, dogs, etc. can be used, and anti-human IgG, anti-human I
gG<γ chain), anti-human IBM, or anti-human IgM (μ chain). Alternatively, a labeled anti-human globulin antibody prepared to react with both Hi1-IgG and Hi1-1gM may be used. Furthermore, not only polyclonal antibodies but also monoclonal antibodies can be used.

Labeling agents for labeled anti-human globulin antibodies or labeled synthetic polypeptides include radioisotopes such as 1251.1311.3H5''C, enzymes such as peroxitase, alkaline phosphatase, and β-D-galactoditase, biotin, or fluorescein. intiocyanate, tetramethylrhodamine intiocyanate, 3-carboe 1
- Fluorescent dyes such as xy-7-hydroxycoumarin, and further labeling agents such as Eu chelate-1- can be used. These labeling agents can be used to label the anti-human globulin antibody or synthetic polypeptide using known methods.

Furthermore, another HCV antibody measurement reagent of the present invention is one in which an insoluble particulate carrier is sensitized with a synthetic polypeptide having a common antigenic determinant with hepatitis C virus.

Insoluble particulate carriers include latex particles or silica;
High-density latex particles with a high sedimentation rate that have an inorganic core such as kaolin or metal and a synthetic resin layer on the outer surface, or immobilized red blood cells obtained by fixing red blood cells of chickens, sheep, etc. with formalin, etc., and various bacteria. It is possible to use immobilized bacteria having immobilized bodies, and further immunologically inactive particulate carriers such as gelatin particles.

As the sensitization method, there is a physical adsorption method as described above, or a chemical bonding method using glutaraldehyde, water-soluble carbodiimide, tannic acid, etc. In addition, in the case of sensitization, binding may be performed via a bifunctional spacer such as hexamethylene diamine, or the synthetic polypeptide may be bound to a protein such as bovine serum albumin and fixed on the solid phase. .

HCV antibodies are detected using an insoluble particulate carrier sensitized with the synthetic polypeptide.As a means of measurement, latex particles are used as a carrier, and the aggregation of the latex particles is caused by an antigen-antibody reaction with the HCV antibody in the test sample. A method of optically measuring changes in turbidity, absorbance, or scattered light intensity that are proportional to Furthermore, there is a method in which an agglutination reaction based on an antigen-antibody reaction between the synthetic polypeptide sensitized to high-density latex particles or immobilized red blood cells and an HCV antibody in a test specimen is determined by an image of the bottom of the tube after sedimentation. .

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to production examples and examples, but the present invention is not limited thereto.

Production Example 1 The α-amine group of Oisine is blocked with a tert-butylreoxysulfonyl (Boc) group by a conventional method.

That is, leucine (10 mmol) was dissolved in 30 mQ of dioxane-water (2:1), and while stirring under water cooling, I M NaOH (IDmQ) and (Boc
) 20 (2.4g 0.011mol). After stirring at room temperature for 30 minutes, concentrate under reduced pressure to obtain 5% KH.
Bring to pH 2:1 with 80.t and extract three times with ethyl acetate.

The entire ethyl acetate layer is washed with water, dried over Na2SO4, concentrated under reduced pressure, and the residue is crystallized.

B o c-leucine (3 equivalents), oxymethyl resin (
Add DMF (approximately 10 mQ per 1 g of resin) to DMF (1 equivalent) and carbodiimide (CDI) and stir overnight at room temperature. Transfer to a glass filter and add DMF, EtOH and CH.
, CI□ and then dried under reduced pressure at room temperature. The resin was then suspended in DMF and about 10 equivalents of A based on the unreacted hydroxyl groups were added.
c20 and NEt3 are added and shaken for 2 hours to acetylate unreacted hydroxyl groups, washed with DMF, E1-12-tOH and CH2Cl2, and dried. The amino acids introduced into the oxymethyl resin are quantified by the hydrolysis method.

The amino group of glycine is blocked with a 9-fluorenylmethyloxycarbonyl (Fmoc) group.

Put BOC-Leu-resin in a reaction vessel and add CH2 to it.
Add Cl2 and shake 5 times (1 minute each) to wash and swell the resin. Next, 60% TFA/CH2Cl2 was added and shaken for 20 minutes to remove Boc.

After washing 5 times (1 min each) with CH2Cl2, 5% DIEA/
Neutralization reaction is performed by shaking 3 times (2 minutes each) with C112C1□ and washing 5 times (1 minute each) with CH2Cl2.

Confirm positive Kaiser test.

C) I2C] of 3 equivalents of Fnoc-glycine to the amine component of the washed de-Boc-Leu-resin. Add the solution and shake for 10 minutes to fully disperse the Fmoc-glycine within the resin.

Without removing the solution from the vessel, add more DCC in CH2Cl2 to the mixture and perform a 90 minute coupling. After the reaction, wash with CH2Cl2 (3 times, 2 minutes each) and i-propatool (2 times, 1 minute).

The introduction of Gly was confirmed by a negative Kaisearch test, and Fm was removed by reacting with 20% piperidine/DMF for 10 minutes.
oc. Confirmation of Fmoc release is performed by a positive Kaiser test or by measuring the absorbance of free Fmoc.

Below, Leu, Ala, Lys, Gi without Fmoc
n, etc. are reacted in the same manner using an amino acid synthesizer.

Side chain amine groups, carboxyl groups, hydroxyl groups, etc. are protected with appropriate protecting groups.

After the final coupling of Fmoc-11e is completed, the dry polypeptide resin is placed in a reaction vessel, anisole is added and allowed to penetrate, HF is added and reacted at 0°C for 1 hour, and then HF is removed with O'C.

]% AcOH is added to the residue to extract the peptide, and the solution is washed with ether and then freeze-dried.

This lyophilized product is eluted with water and lyophilized again to obtain the synthetic polypeptide of formula (1).

Production Example 2 Ogishimethyl resin 0,4 in CH2C1210rnQ
meq, then Fmoc-Leu-OHI,
2 mmol of TOBt (hydrate) is added and completely dissolved, and 1.2 mmol of TOBt (hydrate) is also completely dissolved. Furthermore, 1.2 m of N,N''-diisopropylcarbodiimide
Add mol of Fmoc and react while stirring in ice for about 1 hour.
- Obtain Leu broken fat.

This Fmoc-Leu-resin was placed in an amino acid synthesizer, and 20% piperidine/DMF was reacted for 10 minutes to produce Fmoc-Leu-resin.
cleave the c group. After confirming that the Kaiser test is positive and that the Fmoc is completely extinct, it is washed with DMF and neutralized.

DM Floc-Gly was dissolved, added to the neutralized resin, and reacted for 30 minutes. Confirm introduction with positive Kaiser test.

Hereafter, Fmoc-amino acids are introduced sequentially, and the last Fmoc-amino acid is
c-I] After the coupling of e is completed, the synthetic peptide is cleaved from the resin, extracted and freeze-dried to obtain the synthetic polypeptide of formula I.

Example 1 Solution of polypeptide synthesized in Production Example 1 (50 μs/m
Add 1 amount of Q> to each well of a microtiter reaction plate.
After injecting 00 μQ and leaving it at 4°C overnight,
'//V) Wash with Tween 20-PBS (phosphate buffer with sodium chloride pH 6.4).

Add 2% (w/v) BSA-PBS (pH 6) to each well.
, 4) and store at 4°C.

The plate was soaked in 0.05% Tween 2O-PBS (pH
Wash once with 6,4> and inject 50 uQ of the test serum diluted 100 times into each well, leave it at room temperature for 1 hour,
Anti-HCV antibodies in the test serum are reacted with the immobilized synthetic polypeptide.

0.05% Tween 20-PBS (pH 6,4
), then add 50 μQ of horseradish peroxidase-labeled anti-human 8G antibody or horseradish peroxidase-labeled anti-human Igl/l antibody to each Ufuyuru, and incubate for 1 hour at room temperature.
H bound to the immobilized synthetic polypeptide after standing for a period of time.
React with CV antibody.

15-16 0.05% Tween 20-PBS (pH6,4
), 40 μQ of substrate solution (orthophenylenediamine, H2O2) was added to each well, and after standing at room temperature for 10 minutes, 200 μQ of IN sulfuric acid solution was added to stop the reaction. The absorbance at 490 nm was measured.

The absorbance of the HCV antibody antibody serum sample is 0.01-0.0
IgG to the plate, which is significantly lower than 5 and typically found
No increase in background absorbance due to non-specific adsorption of IgM or labeled antibodies occurred.

The absorbance of HCV antibody-positive serum samples changes sharply depending on the antibody titer, and the background absorbance is low, making it clear that the sample is weakly positive for HCV antibodies.

HCV antibody antibody positive cases The luminous intensity is also high and the measurement range is wide.

Example 2 In the same operation as in Example], a sample diluted 50 times was injected into a synthetic polypeptide-immobilized plate, and after reaction, 2
After washing twice, 50 μQ of horseradish peroxidase-labeled synthetic polypeptide was added to each well, and the mixture was left standing at room temperature for 1 hour to remove HC in the test serum bound to the immobilized synthetic polypeptide.
A labeled synthetic polypeptide is reacted with the V antibody.

After washing in the same manner, 40 μQ of the substrate solution is added to stop the reaction after color development, and the absorbance at 490 nm is measured.

In the case of this example as well, the absorbance of HCV antibody-negative cases was extremely low, and the absorbance of HCV antibody-positive cases was high.

(N
Add the polypeptide synthesized in step 3-, react overnight at 4°C, and block the active site with monoethanolamine.

Unreacted monoethanolamine is removed by dialysis.

This synthetic polypeptide-BSA complex was placed in a dry state on an immunodyne affinity membrane (Nippon Ball Co., Ltd., pore size 1.21 Im) in which a carboxyl group and a covalent ligand were introduced into the surface of a porous nylon membrane. Drop 2μ of the solution and air dry for 5 minutes. The covalent bonding reaction of the synthetic polypeptide-BSA complex to Menpuran is completed in about 1 minute.

The unbound synthetic polypeptide-BSA complex is washed away by immersing Menpuran in PBS (pi-17,0) and changing the solution several times while shaking.

The menpuran was immersed in 0.3% casein-PBS (pt(7,0) heated solution or 02-1.0 M glycine for 30 minutes to flock the covalent active sites of the menpuran. Then, at room temperature. Air dry for at least 30 minutes.

A sample prepared by diluting the test serum was applied to the complex binding site of the synthetic polypeptide-BSA complex-binding Menplan.
Dropped and left for 55 minutes, then immersed in 0.1% (V/'V > 1 to Lydon 15
Rinse the membrane for 1 minute with M P B (pH 7,0>).

10 μQ of horseradish peroxidase-labeled synthetic polypeptide solution is added dropwise, and after being allowed to stand for 5 minutes, the plate is immersed in PB (pH 7.0) containing 0.1% (hal) Triton X-100 for washing. After repeating the washing operation again, rinse with distilled water.
Rinse the membrane for a minute.

0.05% (W/'V) [)-chloronaphthol and H
Add 50 μ9 drops of substrate solution containing 20 □ and react for 10 minutes = 19 = 0 Stop the reaction by rinsing with water, air dry the membrane, visually observe the intensity of the developed color, or Measure with an I meter.

Almost no color development was observed in the HCV antibody-negative cases, and clear color development was observed in the HCV antibody weakly positive cases, making them easily distinguishable with the naked eye. In cases where HCV antibodies were strongly positive, the color development was strong, and the color development was not proportional to the antibody titer in the test serum.

Example 4 1 volume of a 1% (W/V) water suspension of latex with a particle size of 0.26 μm having a carboxyl group and 1 volume of a 0.1M 1-ethyl-3(3-dimethyl-aminopropyl)-carbodiimide/HCI aqueous solution and leave to stand at room temperature for 2 hours or at 4°C overnight. After centrifugation, the pellet was adjusted to pH 5.
water and centrifuge again.

The pellet was suspended in 1 volume of PBS (pH 6,4), and 1 mg/m of the polypeptide synthesized in Production Example 2 was added to it.
1/15M P B (pH 7,0
) and leave it at 4°C overnight.

Centrifuge and add 0.05% Tween 20-1/15MP
Wash twice with B (pH 7,0) and add 0.2% B.
Wash twice with SA-4/15MPB (pH 7,0),
1%BSA-1/15MPB (pH7.0
) to a latex concentration of 0.4%.

Mix 10 μa of a diluted test serum sample with 350 μQ of 1/15M PB (pH 7,0), then add 50 μΩ of a synthetic peptide-bonded latex suspension,
The absorbance at a wavelength of 600 nm (450 to 800 nm) is measured for 3 minutes (1 to 5 minutes), and the amount of increase in absorbance is calculated.

In HCV antibody negative cases, almost no change in absorbance was observed, and HC
V antibody weakly positive cases showed a significant increase in absorbance, and HC
In cases of strong V antibody positivity, an increase in absorbance of 0.100 or more was obtained.

Example 5 To 0.5 to several tens of μg of the polypeptide synthesized in Production Example 1, 1 mg of a high-density latex suspension is added, and the mixture is incubated at room temperature for 2 hours with stirring.

By centrifugation, O61% BSA~i/15MP B(
Wash 4 times with pt(7,0) and add 1% BSA-1,/'1 to obtain a latex suspension with a concentration of 0.25%.
5MP B ([)) Floating at (7,0).

A sample of 25 μΩ prepared by diluting the test serum and 25 μt of the synthetic polypeptide sensitized high-density latex suspension was
Add to the bottom well of reaction plate I, leave to stand at room temperature for 1 hour, and visually observe the image of the bottom of the tube after sedimentation to judge.

If there is no HCV antibody in the diluted sample, no agglutination reaction will occur between the synthetic polypeptide-sensitized high-density latex and the antigen-antibody reaction, and the synthetic polypeptide-sensitized high-density latex will form dots or small rings on the bottom of the tube. Since it precipitates, this is determined to be HCV antibody negative.

If HCV'vL is present in the diluted sample, HCV
An antigen-antibody reaction occurs between the CV antibody and the synthetic polypeptide sensitized on the surface of the high-density latex, and the high-density latex aggregates and settles to the bottom of the tube in the form of a medium to large ring or a smooth mat with no rings. So, change this to H
It is determined that the patient is CV antibody positive.

In all HCV'vc negative cases, dot-like or small ring-like bottom images were obtained. In the weakly positive HCV antibody cases, a medium to large ring-shaped canal bottom image was obtained, and in the strongly HCV antibody positive cases, a smooth mat-like, completely condensed canal bottom image with no rings was obtained.

Example 6 One volume of 0.025 μg/dQ tannic acid solution is mixed with one volume of formalin-fixed 3% sheep red blood cell suspension and incubated at 56°C for 30 minutes. 1. /1
After centrifugal washing twice at 5M P B (pH 7.0>), 1
/15M PH (pH 7,0) at a concentration of 3%.

Synthetic polypeptide prepared in the same manner as in Example 3-
One volume of the BSA complex solution and one volume of the tannic acid-treated red blood cells are mixed and incubated at 56°C for 2 hours. 1
/15M P B < pH 3-24 7,0) After centrifugal washing twice, add 0.1% B S A -1/15 M P B to a concentration of 0.25% (V/V).
(It floats in a pH 7.0 solution.

Add 25 μp of diluted test serum and 25 μQ of synthetic polypeptide-sensitized red blood cells to the wells (■ or U bottom) of the reaction plate, incubate at room temperature for 30 minutes, and visually observe the image of the bottom of the tube after sedimentation. Make a judgment.

In the case of this example, determination was made in the same manner as in Example 5 above, and HCV
In all of the antibody-negative cases, dot-like or small ring-like sedimentation images were obtained, and they were determined to be HCV antibody negative. In addition, in cases that are weakly positive for HCV antibodies, a medium to large ring-shaped sedimentation image is obtained;
It was determined that the patient was positive for CV antibody. In cases of strong HCV antibody positivity, a smooth mat-like, completely aggregated sedimentation image with no rings was obtained.

〔Effect of the invention〕

As described above, the synthetic polypeptide of the present invention and the HCV antibody measurement reagent using the same can provide various excellent effects as described below.

Polypeptides that share antigenic determinants with HCV can be obtained by peptide synthesis, whereas conventionally HCV-derived antigens were produced using genetic and cell engineering methods and purified using extensive equipment and labor. Highly pure polypeptides can be produced in large quantities at low cost.

2 The synthetic polypeptide of the present invention can be used to treat HCV infection in HCV-infected individuals.
It shows good reactivity with antibodies and has immunological reactivity similar to purified antigen from HCV. Conventionally, it has been believed that the immunological reactivity of synthetic polypeptides is lower than that of extracted antigens.

3. The synthetic polypeptide of the present invention can be easily immobilized on various solid phases used in immunoassays by physical adsorption or chemical bonding, and even after immobilization, it is compatible with HCV antibodies. An HCV antibody measurement reagent consisting of a solid-phase reagent that does not immobilize the synthetic polypeptide and either a labeled anti-human globulin antibody or a labeled synthetic polypeptide exhibits a reactivity that immobilizes the synthetic polypeptide. IgG to the solid phase
, IgM, woven antibodies, or labeled synthetic polypeptides are significantly reduced, and background labeling activity is significantly reduced. Furthermore, because the labeling activity increases sharply in accordance with the antibody titer in the test sample, the cut-off level is clear and measurements can be made with high sensitivity and in a wide range from weakly positive to strongly positive HCV antibodies. A similar measurement system using an extracted and purified antigen derived from HCV had low sensitivity and specificity due to strong nonspecific adsorption and high background labeling activity.

5. The HCV antibody measurement reagent obtained by sensitizing the synthetic polypeptide to an insoluble particulate carrier is easy to prepare and can be easily prepared since the synthetic polypeptide can be easily sensitized and immobilized on various insoluble particulate carriers. Moreover, it is convenient because an appropriate measurement system can be used depending on the application, and it is easy to operate and has high sensitivity.

hand Continued Supplementary Positive book January 12, 1990

Claims (15)

[Claims] (1) A synthetic polypeptide having a common antigenic determinant with hepatitis C virus. (2) The synthetic polypeptide according to claim (1), which has the amino acid sequence of formula I [has a gene sequence]...I. (3) An HCV antibody measurement reagent comprising a solid phase reagent in which the synthetic polypeptide of claim (1) is immobilized on a solid phase, and either a labeled anti-human globulin antibody or a labeled synthetic polypeptide of claim (1). (4) The HCV antibody measurement reagent according to claim (3), wherein the solid phase is any one of a microtiter reaction plate, beads, sheet, porous membrane, and magnetic latex. (5) Claim (3) wherein the synthetic polypeptide of claim (1) is immobilized on the solid phase by either physical adsorption or chemical bonding.
) described HCV antibody measurement reagent. (6) The HVC antibody measurement reagent according to claim (3), which is a solid phase reagent in which the synthetic polypeptide of claim (1) is immobilized on a solid phase via a spacer. (7) The HCV antibody measurement reagent according to claim (3), wherein the labeling agent is any one of a radioisotope, an enzyme, biotin, a fluorescent dye, and a Eu chelate. (8) Anti-human globulin antibodies are anti-human IgG, anti-human I
The HCV antibody measuring reagent according to claim (3), which is any one of gM. (9) An HCV antibody measuring reagent obtained by sensitizing the synthetic polypeptide of claim (1) to an insoluble particulate carrier. (10) The HCV antibody measurement reagent according to claim (9), wherein the insoluble particulate carrier is any one of latex particles, high-density latex particles, immobilized red blood cells, gelatin particles, and immobilized bacteria. (11) The HC according to claim (10), wherein the high-density latex particles have an inorganic core and a synthetic resin layer on the outer surface.
V antibody measurement reagent. (12) The HCV antibody measurement reagent according to claim (9), which comprises a synthetic polypeptide bound to an insoluble particulate carrier via a spacer. (13) H according to claim (9), wherein the quantitative measurement is performed by either latex agglutination turbidimetry or latex agglutination light scattering method.
CV antibody measurement reagent. (14) The HCV antibody measurement reagent according to claim (9), which determines the image of the bottom of the tube sedimented by an agglutination reaction. (15) The HCV antibody measurement reagent according to claim (9), wherein the agglutination image is determined visually by latex agglutination reaction.