JP2000327698A - Peptide for measuring antibody against hepatitis c and reagent for measuring - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a reagent which is used for measuring an antibody against hepatitis C and can solve the problem of false negative reactions in hepatitis C screening tests. SOLUTION: This peptide used for measuring an antibody against hepatitis C comprises one or more peptides selected from six kinds of peptides in the sequence table, such as the sequence No.1: Val Lys Phe Pro Gly Gly Gly Asn Ile Val Gly Gly Leu Tyr Leu Leu Pro Arg Arg Gly, and the sequence No.2: Val Lys Phe Pro Ala Gly Gly Gln Ile Leu Gly Gly Val Tyr Leu Val Pro Arg Arg Gly. The reagent for measuring the hepatitis C comprises the peptide(s) carried on an insoluble carrier.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a peptide for measuring hepatitis C antibody and a reagent for measuring hepatitis C antibody using the same.

[0002]

2. Description of the Related Art In the field of clinical testing, it is desired to accurately measure the components of hepatitis C antibody. As a result, immunoassay reagents utilizing highly specific antigen-antibody reactions have been widely used. Has become. For example, JP-A-5-22
As described in Japanese Patent No. 2094, an immunoassay reagent for hepatitis C antibody using latex, which uses an antigen protein obtained by genetic modification, has been developed. However, this reagent has a problem of the matching rate with the PCR method which is a standard method, and a sample which is determined to be negative by this reagent is pointed out as a so-called false-negative problem which is determined to be positive by the PCR method. Have been. Such a false negative problem is that the specimen is judged to be negative even though it is positive, and it is inevitable especially in the test for hepatitis C, which can take a serious course if left untreated after infection. It was a must-have problem.

[0003]

SUMMARY OF THE INVENTION The present invention has been made to solve the above problems, and an object of the present invention is to provide a hepatitis C antibody measuring reagent which has solved the problem of false negative in a screening test. .

[0004]

According to the first aspect of the present invention,
It is a hepatitis C antibody measurement peptide composed of at least one peptide selected from the six peptides represented by SEQ ID NOs: 1 to 6.

[0005] The invention of claim 2 is a reagent for measuring hepatitis C antibody, wherein the peptide of claim 1 is carried on an insoluble carrier.

In the present invention, hepatitis C antibody in a biological sample is used as a substance to be measured.

The peptide for measuring hepatitis C antibody of the present invention comprises:
It is composed of one or more peptides among the six peptides represented by SEQ ID NOs: 1 to 6 in the sequence listing. The method for producing these peptides is not particularly limited, but generally, those obtained by solid phase synthesis can be used.

[0008] The hepatitis C antibody measurement reagent of the present invention comprises the above-mentioned hepatitis C antibody measurement peptide supported on an insoluble carrier.

The insoluble carrier includes, for example, organic polymer powder, microorganisms, blood cells or cell membrane fragments.

Examples of the organic polymer powder include natural polymer powders such as insoluble agarose, cellulose, and insoluble dextran; polystyrene, styrene-sulfonic acid (salt) copolymer, styrene-methacrylic acid copolymer, acrylonitrile- Synthetic polymer powders such as a butadiene-styrene copolymer, a vinyl chloride-acrylate copolymer, and a vinyl acetate-acrylate copolymer are exemplified.

Among the above insoluble carriers, it is particularly preferable to use latex in which a synthetic polymer powder is uniformly suspended. In this case, the particle size of the latex particles is 0.05 to
1.5 μm is preferred, and 0.1 to 0.8 μm is more preferred.

As the insoluble carrier, those having sulfonic acid groups, carboxyl groups or the like introduced on the surface can be used as appropriate.

When the peptide is carried on an insoluble carrier, the binding method may be either physical adsorption or chemical bonding.

When the peptide is supported by physical adsorption, the peptide can be supported by, for example, adding a suspension of the insoluble carrier to a solution containing the peptide and stirring.

When the peptide is supported by chemical bonding, the peptide can be supported by using an insoluble carrier having a sulfonic acid group or a carboxyl group introduced on the surface and adding an appropriate crosslinking agent. .

The pH of the solution at the time of carrying out the above supporting step is 3
-10 and the temperature is preferably 2-50 ° C. This is because if the pH is out of this range, problems such as denaturation of the protein occur. The temperature is 2 ° C
If it is less than 50 ° C., the reaction rate is low, and it is difficult to obtain one having the desired sensitivity. If it exceeds 50 ° C., there is a problem that the protein is denatured.

The peptide may be directly carried on the insoluble carrier as described above, or may be carried on the insoluble carrier via a carrier protein such as BSA. In this case, for example, after a peptide is chemically bonded to a carrier protein in advance, the carrier protein can be supported on an insoluble carrier by physical adsorption. Alternatively, a suspension of a carrier protein supported on an insoluble carrier is added to a peptide-containing solution and stirred, whereby the peptide is bound to the carrier protein by physical adsorption. Furthermore, it can also be obtained by chemically bonding a peptide to a carrier protein by using an amino group or a carboxyl group on the surface of the carrier protein and adding an appropriate crosslinking agent. The pH during the binding reaction is 3-10, and the temperature is 2-5.
0 ° C. is preferred.

[0018]

The present invention has solved the problem of false negatives in screening tests, which has been a problem so far, by using a peptide having a sequence similar to the amino acid sequence of hepatitis C antigen. In other words, if only a peptide having the same sequence as a naturally occurring antigen is used, oversight will occur.By using a peptide with a similar sequence, the reagent detection region can be expanded, and the problem of false negatives can be increased. Is solved.

[0019]

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 (1) Preparation of Latex Solution a) Preparation of Maleimide Group-Introduced BSA A 20 mM 100 mM phosphate buffer (pH 7.4) containing 1% (w / v) BSA was added to SMCC (N -Succiimidyl-4- (N-arrayimidomethyl) -1-carboxylate) 8 mg / mL dimethylformamide solution 3 m
L, and stirred at 30 ° C. for 10 minutes. BSA and SMCC
And a maleimide group was introduced into BSA. Then
Using a dialysis membrane (UC20-32-100: size 20/32, manufactured by Sanko Junyaku Co., Ltd.), the above solution was treated with a physiological saline solution (0.
Dialysis was performed with 30 L of 9% NaCl for 24 hours. Thereafter, a latex having a particle size of 0.400 μm (solid content 10% (w /
v), 10 μl of Sekisui Chemical Co., Ltd. N-400) was added, and the mixture was stirred at 25 ° C. for 2 hours.

B) Carrying of Peptide Six kinds of peptides of SEQ ID Nos. 1 to 6 in the sequence listing (respectively obtained by solid phase synthesis) were added to a 100 mM phosphate buffer (p
H7.4) A total of 300 μg of 50 μg was added to 1 mL and dissolved. 150 μL of this solution was added to 6 mL of the maleimide-introduced BSA of the above (1) a) and stirred at 25 ° C. for 1 hour. This solution was added at 18,000 r for 30 minutes at 10 ° C.
Centrifuged at pm. 0.25% of BSA was added to the obtained precipitate.
(W / v) 100 mM phosphate buffer (pH 7.
4) 5 mL was added, and the latex was suspended to obtain a hepatitis C peptide antigen-sensitized latex solution.

(2) Preparation of Sample Diluent BSA was 0.25% (w / v), pGEMA (glycosylethyl methacrylate homopolymer, average molecular weight 1)
10% containing 140,000 (manufactured by Nippon Seika) 1% (w / v)
Sodium ethylenediaminetetraacetate was dissolved in 0 mM phosphate buffer (pH 7,4) to a concentration of 5 mM to prepare a sample diluent. .

(3) Hepatitis C antibody assay reagent The hepatitis C antibody assay reagent of the present invention is a first reagent comprising the hepatitis C peptide antigen-sensitized latex solution of the above item (1); And a second reagent comprising a buffer.

(4) Standard substance Human serum containing 0.2, 4, 8, 10 C.OI of hepatitis C antibody was used as a standard hepatitis C antibody solution.

(5) Measurement of Hepatitis C Antibody Titer Using an Automatic Analyzer A method for measuring hepatitis C antibody in a sample using an automatic biochemical analyzer “Hitachi 7050” (manufactured by Hitachi, Ltd.) is described below. Measurement mode: Original Abs Parameters: Sample volume 20 μL Latex liquid (first reagent) 50 μL Sample diluent (second reagent) 350 μL Measurement wavelength: 570 nm Measurement time: Sample diluent is added and mixed immediately after sample dispensing, and thereafter The latex liquid is added and mixed. After the addition of the latex solution, the amount of change in absorption from 80 seconds to 320 seconds was determined, and this was defined as the reaction amount. The pH of the reaction system was 7.4. In this measurement, 10 C.OI or more was regarded as a hepatitis C antibody positive specimen.

In order to determine the number of false negatives, 50 samples were measured by Amplicor HCV (manufactured by Nippon Roche) by the PCR method, and the relationship with the judgment according to the present invention is shown in Table 1.

[0027]

[Table 1]

[Comparative Example] The procedure was the same as in Example, except that the step (1) b) of loading the peptide in Example was carried out as follows. The results are shown in Table 2. b) Carrying of Peptide Each of the three peptides of SEQ ID NOs: 7 to 9 in the sequence listing was 10
A total of 300 μg of each 0 μg was added to 100 mM phosphate buffer (p
H7.4) 1 mL was added and dissolved. This solution 150
μL was added to 6 mL of the maleimide-introduced BSA of the above (1) a) and stirred at 25 ° C. for 1 hour. This solution was centrifuged at 18000 rpm at 10 ° C. for 30 minutes. To the obtained precipitate, 5 mL of 100 mM phosphate buffer (pH 7.4) containing 0.25% (w / v) of BSA was added to suspend the latex, and the suspension was mixed with a hepatitis C peptide antigen-sensitized latex solution. did.

[0029]

[Table 2]

By comparing Tables 1 and 2, it was found that, in the comparative example, false negatives occurred in 7 samples,
In the example, it can be seen that the number of specimens was reduced to two.

[0031]

According to the present invention, since it has the above-mentioned structure, C
In a screening test for hepatitis B, the occurrence of false negatives can be drastically reduced, and the reliability of the test results can be improved.

[0032]

[Sequence List] <110> SEKISUI CHEMICAL CO., LTD. <120> Peptide for hepatitis C antibody measurement and reagent for measurement <130> 99P01488 <160> 9 <170> PatentIn Ver. 2.0 <210 > 1 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Peptide <400> 1 Val Lys Phe Pro Gly Gly Gly Gly Asn Ile Val Gly Gly Leu Tyr Leu Leu 1 5 10 15 Pro Arg Arg Gly 20 <210> 2 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Peptide <400> 2 Val Lys Phe Pro Ala Gly Gly Gln Ile Leu Gly Gly Val Tyr Leu Val 1 5 10 15 Pro Arg Arg Gly 20 <210> 3 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Peptide <400> 3 Ser Arg Gly Asn His Leu Ser Pro Thr His Tyr Leu Pro Glu Ser Asp 1 5 10 15 Ala Ala Ala Arg 20 <210> 4 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Peptide <400> 4 Ser Arg Ala Asn His Val Ser Pro Ser His Tyr Val Pro Glu Se r Asp 1 5 10 15 Gly Ala Ala Arg 20 <210> 5 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Peptide <400> 5 Cys Glu Glu Met Glu Glu Phe Glu Gln Tyr Leu Ala Asp Arg Asp Pro 1 5 10 15 Ile Val <210> 6 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Peptide <400> 6 Cys Glu Glu Met Glu Asp Phe Asp Gln Tyr Leu Ala Glu Arg Glu Pro 1 5 10 15 Ile Val <210> 7 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Peptide <400> 7 Cys Glu Glu Met Glu Asp Phe Glu Gln Tyr Leu Ala Glu Arg Asp Pro 1 5 10 15 Ile Val <210> 8 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Peptide <400> 8 Cys Ser Arg Gly Asn His Val Ser Pro Thr His Tyr Val Pro Glu Ser 1 5 10 15 Asp Ala Ala Ala Arg 20 <210> 9 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Peptide <400> 9 Cys Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly Gly Val Tyr Leu 1 5 10 15 Leu Pro Arg Arg Gly 20

Claims (2)

[Claims] 1. A hepatitis C antibody measuring peptide comprising at least one peptide selected from the six peptides represented by SEQ ID NOs: 1 to 6. SEQ ID NO: 1: Val Lys Phe Pro Gly Gly Gly Gly Asn Ile V
al Gly Gly Leu Tyr LeuLeu Pro Arg Arg Gly SEQ ID NO: 2: Val Lys Phe Pro Ala Gly Gly Gly Gln Ile L
eu Gly Gly Val Tyr LeuVal Pro Arg Arg Gly SEQ ID NO: 3: Ser Arg Gly Asn His Leu Ser Pro Thr H
is Tyr Leu Pro Glu SerAsp Ala Ala Ala Arg SEQ ID NO: 4: Ser Arg Ala Asn His Val Ser Pro Ser H
is Tyr Val Pro Glu SerAsp Gly Ala Ala Arg SEQ ID NO: 5: Cys Glu Glu Met Glu Glu Phe Glu Gln T
yr Leu Ala Asp Arg AspPro Ile Val SEQ ID NO: 6: Cys Glu Glu Met Glu Asp Phe Asp Gln T
yr Leu Ala Glu Arg GluPro Ile Val 2. A reagent for measuring hepatitis C antibody, wherein the peptide of claim 1 is supported on an insoluble carrier.