CN1755365A - A method for detecting hepatitis C virus antibody - Google Patents

Abstract

The invention discloses a method for detecting hepatitis C virus antibody by using enzyme-linked immune technology, and also discloses the hepatitis C virus multi-antibody chimeric antigen and enzyme-labeled connecting arm adopted in the method. The detection method of the present invention comprises the preparation of hepatitis C virus multi-epitope chimeric antigen, the preparation of chimeric antigen and enzyme-labeled linker, the preparation of chimeric antigen-labeled horseradish peroxidase complex, and the coating of chimeric antigen Enzyme-linked plate detection plate, adding the antibody to be tested and enzyme-labeled antigen complex and other steps. The method of the invention has strong specificity, high sensitivity, good repeatability and early detection, can obtain satisfactory results when used for detecting hepatitis C virus antibody, and can be widely used for screening blood donors and clinical diagnosis.

Description

A method for detecting hepatitis C virus antibody

technical field

The invention relates to a method for detecting hepatitis C virus antibody, in particular to a method for detecting hepatitis C virus antibody by using enzyme-linked immunosorbent technology, and also relates to the multi-epitope embedding method of hepatitis C virus used in the method. Synthesize antigen and enzyme-labeled tether.

Background technique

Hepatitis C virus (HCV) is the main pathogen that causes viral non-A non-B hepatitis after blood transfusion. It is mainly transmitted through blood transfusion and intravenous drug use, and can also be transmitted through close contact and mother-to-child transmission. There are currently about 170 million HCV-infected people in the world. About half of those infected develop chronic hepatitis, and some develop cirrhosis and hepatocellular carcinoma. It is extremely harmful as there is currently no proven treatment and vaccine to prevent its further spread. According to recent reports, there are about 50,000 new cases of HCV infection in the United States each year, and about 251.2 million new liver cancer patients in the world every year. In addition to HBV infection, HCV is one of the main pathogenic factors that cause hepatocellular carcinoma.

Generally, about 15-20% of HCV-infected patients are self-limiting infections and can recover quickly. However, 80-85% of infected patients develop chronic hepatitis C, and 20% of them can develop liver fibrosis, and finally 4-5% of patients with liver fibrosis develop hepatocellular carcinoma, which is very serious. .

Currently, there is no other effective drug for the treatment of chronic hepatitis C except interferon, which has a definite curative effect. According to recent international reports, PEG-modified interferon plus ribavirin combination therapy can obtain good response in about 40-50% of patients, but it also has disadvantages such as long treatment course, high price, large toxic side effects, and easy rebound. In addition, the development of hepatitis C vaccine is also difficult due to the high mutation of HCV, and it is difficult to make breakthroughs in the short term. Therefore, it is imperative to detect HCV-infected patients as early as possible and block the spread of HCV.

Current status of HCV detection technology

HCV is generally transmitted through HCV-contaminated blood and blood products. There are two commonly used detection techniques for HCV: (1) indirect detection, which mainly detects anti-HCV antibodies and antigens; (2) direct detection, which detects HCV qualitatively or quantitatively. The composition of the virion determines the presence of the virus, eg HCV RNA. These two detection methods play a key role in the diagnosis of HCV infection, the selection of treatment options for HCV-infected patients, and the evaluation of the efficacy of anti-HCV treatment.

In the early 1990s, anti-HCV antibody detection technology was developed by using recombinant HCV antigen, that is, enzyme immunoassays (enzyme immunoassays, EIAs) technology, which has been developed to the third generation. This technology can detect mixed antibodies against different HCV antigens (such as C region, NS3 region, NS4 region and NS5 region, etc.) in the sample, and the specificity of detection has reached more than 99%.

The first generation of reagents: use HCV-C100 (511) as antigen, and apply the principle of indirect method to detect HCV antibody. Second-generation reagents: use genetic engineering to express recombinant or chemically synthesized polypeptides (HCV-C, NS3 NS4) HCV antigens, and use the principle of indirect ELISA to detect HCV antibodies, with higher specificity and sensitivity than the first-generation reagents. The third-generation reagents: use genetic engineering to express recombinant HCV antigens (HCV-C, NS3, NS4, NS5) and use the principle of indirect ELISA to detect HCV antibodies.

In March 1993, the Ministry of Health issued a document requiring the detection of HCV antibodies in blood donors. Subsequently, more than a dozen ELISA reagents for detecting HCV antibodies were launched and tested in batches. But all of them are indirect ELISA methods, but due to the defects of the indirect method technology itself and the quality problems of the products of various manufacturers, some misdiagnosis and missed detection have been caused, which has caused adverse social impact and certain harm to the society. For this reason, the quality problem of the reagent itself should be solved fundamentally.

At present, the double-antigen sandwich method has been successfully used to detect anti-HIV and Tp antibodies. The sensitivity and specificity of such reagents are better than the indirect method of labeled anti-human immunoglobulin. The detection of hepatitis C virus antibody still adopts indirect ELISA technology, mainly because peroxidase directly labels hepatitis C virus antigen, and its sensitivity cannot meet the requirements, especially after labeling the hepatitis C virus core antigen, the antigen activity is reduced. However, there are rapid gold standard reagents at home and abroad that use the principle of double-antigen sandwich method to detect anti-HCV antibodies by immunochromatography. However, the sensitivity is low, so it cannot be used for the screening of blood donors.

Contents of the invention

In order to solve the current problems of specificity and sensitivity in the detection of HCV antibodies, the invention discloses a method for detecting anti-HCV antibodies by using double-antigen sandwich ELISA technology. On the basis of the multi-protope HCV chimeric antigen, the present invention fuses four lysines (4K) in different connection modes, and is used to mark the tether of horseradish peroxidase (HRP); the present invention improves Antigen-labeled horseradish peroxidase (HRP) technology, and the establishment of double-antigen sandwich (ELISA) method for detection of hepatitis C virus antibody.

Method of the present invention comprises the steps:

1. Preparation of hepatitis C virus multi-antibody chimeric antigen and enzyme-labeled tether:

Firstly, the multi-epitope chimeric antigen of hepatitis C virus was prepared: the epitope of HCV antigen was analyzed by Goldkey computer-aided software. Determine the amino acid positions of the antigenic HCV different segment antigens (respectively HCV-C: 10-53aa; NS3: 1192-1457aa; NS4: 1916-1947aa;), synthesize genes respectively, construct pBVIL-1 vector, and The antigens were linked into multi-epitope chimeric antigens, and HCV multi-epitope antigens (HCV-C, NS3, NS4) were expressed in the form of inclusion bodies in E.coli, purified by ion exchange chromatography, and the purity was identified by SDS-PAGE.

Then, on the basis of the HCV fusion antigen expressed by the above clone, enzyme-labeled tethers, namely four lysines (4K), were introduced at the 5' end, 3' end and the middle of the antigen, respectively.

Firstly, primers are synthesized, and the primer sequences are shown in sequences 1-6 in the sequence listing. Then, the pILla-NS ₄ CNS ₃ plasmid was used as a template for PCR amplification to obtain gene fragments, which were digested and inserted into vectors respectively to obtain expression plasmids.

Using the pIL1-HCV/C plasmid as a template, PCR amplification was performed with F3/R3 primers to obtain gene fragments, which were then digested with EcoR I and BamH I and inserted into the pIL1 vector to obtain the expression plasmid pIL1-4K+HCV /C, using pIL1’s ability to connect multiple target genes conveniently, connect pIL1-HCV/NS4, pIL1-4K+HCV/C, and pIL1-HCV/NS3 to construct pIL1-NS ₄ -4KC-NS ₃ expression plasmid.

Transform the three expression plasmids obtained above into E. coli recipient bacteria for high-efficiency expression, extract inclusion bodies, and use Q-Sepharose-FF anion transfer column, S-Sepharose-FF cation transfer column, gel filtration column, etc. Perform chromatography, SDS-PAGE identification, Lowy method to determine protein content, freeze-dry, and store at -25°C.

2. Preparation of horseradish peroxidase labeled with multi-antibody chimeric antigen of hepatitis C virus:

(7) HRP activation: add horseradish peroxidase (HRP) and SMPB with slight stirring.

(8) Antigen activation: Dithiothreitol (DTT) activates the hepatitis C virus multi-epitope chimeric antigen (HCVAg) with gentle stirring.

(9) Desalting: desalting the above-mentioned activated antigen and HRP.

(10) Binding: the desalted antigen is bound to HRP.

(11) Termination: add iodoacetamide and stir gently.

(12) Storage: subpackage and store at -20°C.

3. Detection of HCV antibody by double-antigen sandwich method

1. The basic principle of double-antigen sandwich detection of hepatitis C virus antibody:

Enzyme-linked detection reagent for hepatitis C virus antibody (double antigen sandwich): coat the enzyme-linked plate with hepatitis C disease epitope chimeric antigen, add the sample to be tested and the enzyme-labeled hepatitis C disease epitope chimeric antigen, such as Antibodies exist in the sample to form an antigen-antibody complex. After washing, the enzyme substrate develops color, and the optical density is measured, and the result is judged according to the optical density.

2. The research procedure of the method for detecting hepatitis C virus antibody: see accompanying drawing 1.

3. The composition of the detection kit for hepatitis C virus antibody: including HCV antigen-coated enzyme-linked plate; HCV antibody positive control serum; HCV antibody negative control serum; enzyme conjugate; chromogenic reagent A; chromogenic reagent B; stop solution; Washing fluid, etc.

4. Operating procedures:

(1) Take out the dry package enzyme-linked plate, add the sample to be tested in each well; add the enzyme-labeled antigen conjugate, add double wells for negative and positive controls, add no reagent to the blank well, mix well and then water bath.

(2) Wash the plate with washing solution and pat dry for the last time.

(3) Wash the plate with 2.

(4) Add color developer A liquid first, then add color developer B liquid, avoid light and develop color.

(5) Add stop solution to each well.

5. Result judgment:

(1) Instrument measurement: Measure the OD value of each well with an enzyme-linked analyzer at 450 nm (calculated by subtracting the blank control). The positive control OD > 0.8, and the negative control OD < 0.10. The kit is valid, and the OD value is measured within 10 minutes after termination.

(2) Critical value: the average OD value of the negative control +0.05. If the OD value of the negative control is <0.05, calculate as 0.05; if >0.05, calculate as the actual OD value.

Divided into four groups according to the connection mode, of which

A: 5'-KKKK-NS3-C-NS4; B: NS3-C-NS4-KKKK-3'; C: 5'-KKKK-NS3-C-NS4-KKKK-3'; D: NS3-KKKK- C-NS4

The results of the detection of hepatitis C virus antibody activity by multi-epitope chimeric antigen-labeled horseradish peroxidase double-antigen sandwich in four different connection methods showed that the four groups of different connection methods were significantly different from the HCV serum reactivity. HCV serum reactivity was better than that of B, C and D groups. Use group A to label HPR for double-antigen sandwich detection of hepatitis C virus antibodies.

The detection results of HRP-labeled modified antigen double-antigen sandwich method on the national third-generation standard serum showed that the detection of anti-HCV antibody by double-antigen sandwich ELISA technology can greatly improve the specificity and sensitivity of detection reagents.

The development of double-antigen sandwich ELISA to detect anti-HCV antibodies has the following advantages over indirect methods:

(1) The antigen sandwich method can simultaneously detect all types of IgG subtypes and IgM in HCV-infected patients, and can be used for early diagnosis. The indirect method can only capture IgG, not IgM. And the secondary antibody uses anti-human IgG enzyme-labeled antibody, only one antigen-antibody specific reaction occurs, and some IgG subtypes or IgM often cause missed detection; (2) The double-antigen sandwich performs secondary specific reaction on the antibody, reducing the indirect False positives caused by enzyme-labeled antibodies with only one specific reaction.

The method of the present invention has strong specificity, high sensitivity, good repeatability, early detection, can detect IgG subtype and IgM subtype, can obtain satisfactory results when used for detecting hepatitis C virus antibody, and can be widely used in blood donor screening and clinical practice diagnosis.

Description of drawings

Figure 1 is a schematic diagram of the research procedure of the method for detecting antibodies to hepatitis C virus.

Detailed ways

Example 1 Preparation of Hepatitis C Virus Multi-epitope Chimeric Antigen and Enzyme-labeled Linker

1. Materials:

pIL1 vector and pIL1a-NS ₄ CNS ₃ plasmid: Chinese patent has been applied for and authorized, patent number: ZL00100695.9, patent name: "expression vector PBVIL1 and its construction method and use", and has been preserved, deposit number: CGMCC No: 0437.

pIL1-HCV/C plasmid:

E. coli HB101:

Q-Sepharose-FF anion exchange column:

S-Sepharose-FF cation exchange column:

Horseradish Peroxidase (HRP):

SMPB:

Dithiothreitol (DTT):

HCV-Ag:

Iodoacetamide:

Enzyme Linker:

EcoR I, BamH I enzymes:

2. Method results:

1. Development of chimeric antigens with multiple epitopes of hepatitis C virus:

The HCV antigen epitopes were analyzed with Goldkey computer-aided software. Determined the amino acid positions of the antigens of different segments of HCV with strong antigenicity (respectively HCV-C: 10-53aa; NS3: 1192-1457aa; NS4: 1916-1947aa;), synthesized genes respectively, constructed pBVIL-1 vector, and The antigens were linked into multi-epitope chimeric antigens, and HCV multi-epitope antigens (HCV-C, NS3, NS4) were expressed in the form of inclusion bodies in E.coli, purified by ion exchange chromatography, and the purity was identified by SDS-PAGE. For the specific operation process, please refer to literature (1). Song Xiaoguo, Ling Shigan, Zhang Heqiu, Chen Kun. Research on Recombinant Hepatitis C Virus Antigen. Journal of Academy of Military Medical Sciences; 2001; 25(2): 91-95. (2). Song Xiaoguo, Ling Shigan , Zhang Heqiu, Chen Kun. Construction of high-efficiency prokaryotic fusion expression vector (pBVIL1) and its application in HCV expression. Journal of Cellular and Molecular Immunology; 2001; 17(3): 231-233.).

On the basis of the HCV fusion antigen expressed by the above clones, enzyme-labeled tethers, i.e. 4 lysines (4K), were introduced into the 5' end, 3' end and the middle of the antigen respectively, and the following primers were first synthesized:

1. F ₁ GC GAATTC ATG AAAAAAAAAAAA GCACCTGTACGATCACTG

EcoR I 4K IL1 5’ end

2. R ₁ GC GGATCC TTA ACACGTATTGCAGTCTAT

BamH I only NS3 3' end

3. F ₂ 5'GC GAA TTC ATG GCA CCT GTA CGA TC

EcoR I IL1 5'

4. R ₂ GC GGATCC TTA CTTCTTCTTCTT ACACGTATTGCAGTCTAT

BamH I Only 4K NS3

5. F ₃ 5'GC ACT AGT AAA AAA AAA AAA GAG GGT GGA TCT

                                          

6. R3: 5'CGC GGA TCC TTA GGA AGA CAC AAA

BamH I IL1

Using the pIL1a-NS ₄ CNS ₃ plasmid as a template (constructed and preserved by the Vaccine Engineering Research Office of the Institute of Basic Medical Sciences, Academy of Military Medical Sciences), PCR amplification was performed with F1/R1, F2/R2, and F3/R3 primers respectively, and three The gene fragment was digested with EcoR I and BamH I, and inserted into the pIL1 vector respectively to obtain three expression plasmids pIL1a/4K-NS ₄ -CNS ₃ , pIL1a/NS ₄ -C-NS ₃ -4K, pIL1a /4K -NS ₄ -C -NS ₃ -4K.

Using the pIL1-HCV/C plasmid as a template, PCR amplification was performed with F3/R3 primers to obtain gene fragments, which were then digested with EcoR I and BamH I and inserted into the pIL1 vector to obtain the expression plasmid pIL1-4K+HCV /C, using pIL1’s ability to connect multiple target genes conveniently, connect pIL1-HCV/NS4, pIL1-4K+HCV/C, and pIL1-HCV/NS3 to construct pIL1-NS ₄ -4KC-NS ₃ expression plasmid,

Transform the three expression plasmids obtained above into E.coli HB101 recipient bacteria for high-efficiency expression, extract inclusion bodies, use Q-Sepharose-FF anion exchange column, S-Sepharose-FF cation exchange column, and gel filtration Column chromatography, SDS-PAGE identification, Lowy method to determine protein content, freeze-dried, and stored at -25°C.

Example 2 Preparation of Hepatitis C Virus Multi-epitope Chimeric Antigen Labeled Horseradish Peroxidase

One, material: with embodiment one.

2. Method results:

(1) HRP activation: 1ml (10mg HRP+1mlPBS PH7.6) horseradish peroxidase (HRP), add 100μl SMPB and stir gently at 18-20°C for 20 minutes.

(2) Antigen activation: Activation of hepatitis C virus multi-epitope chimeric antigen (HCVAg) by DTT: 5 mg HCVAg plus 115 μl (30.9 mg/ml) dithiothreitol (DTT) at 18-20° C. and gently stirred for 20 minutes.

(3) Desalting: Use PD-10 desalting column to desalt the above-mentioned activated antigen and HRP respectively, and collect 1.5ml of each volume.

(4) Binding: the desalted antigen is combined with HRP and gently stirred at 18-20°C for 45 minutes.

(5) Termination: add 40 μl of 0.1M iodoacetamide and stir gently at 37°C for 30 minutes.

(6) Storage: aliquot 100 μl/tube, and store at -20°C.

Example 3 Double Antigen Sandwich Method for Detecting Hepatitis C Virus Antibody

One, material: with embodiment one.

2. Method results:

1. Dilute the purified multi-epitope fusion antigen of hepatitis C virus to 0.33μg/ml with 0.05M pH9.6 carbonate buffer, coat the enzyme-linked assay plate with 100μl/well, and wash the plate with buffer 2 The second time, 3% BSA was blocked for 2 hours, and dried at room temperature for later use. Enzyme-labeled HCV multi-epitope fusion antigens of 4 different tethers were used, and the labeled antigens were diluted to 1:2000 times with a special diluent for enzyme-labeled antigens at 4°C for later use. The 4 HCV antibody positive and 4 negative sera were tested separately.

2. The research procedure of the method for detecting hepatitis C virus antibody: see accompanying drawing 1.

3. Kit composition:

(1) 1 HCV antigen-coated enzyme-linked plate

(2) HCV antibody positive control serum 2ml 1 bottle

(3) HCV antibody negative control serum 2ml 1 bottle

(4) 1 bottle of enzyme conjugate 12ml

(5) Color developer A 6ml 1 bottle

(6) Color developer B 6ml 1 bottle

(7) Stop solution 6ml 1 bottle

(8) One bottle of 50ml washing liquid (20×concentration)

4. Operating procedure:

(1) Take out the dry package enzyme-linked plate, add 50 μl of the sample to be tested in each well; add 100 μl of enzyme-labeled antigen conjugate, add 100 μl to each hole for the negative and positive control, add no reagent to the blank well, mix well and bathe in 37°C water 60 minutes.

(2) Wash the plate 5 times with 1:20 diluted washing solution, and pat dry for the last time.

(3) Wash the plate with 2.

(4) First add 50 μl of color developer A solution, then add 50 μl of color developer B solution, and develop color for 20+2 minutes in a water bath at 37° C. in the dark.

(5) Add 50 μl of stop solution to each well.

5. Result judgment:

(1) Instrument measurement: Measure the OD value of each well with an enzyme-linked analyzer at 450 nm (calculated by subtracting the blank control). The positive control OD > 0.8, and the negative control OD < 0.10. The kit is valid, and the OD value is measured within 10 minutes after termination.

(2) Critical value: the average OD value of the negative control +0.05. If the OD value of the negative control is <0.05, calculate as 0.05; if >0.05, calculate as the actual OD value.

                                                                              

The results of the activity determination of double-antigen sandwich detection of hepatitis C virus antibody Types of labeled antigens The measurement results P1 P2 P3 P4 N1 N2 N3 N4 ABCD 1.2380.6181.4590.402 1.0960.6561.2340.294 2.2591.5072.3300.583 0.2750.0890.19l0.066 0.0470.0420.0520.042 0.0510.0580.0520.054 0.0790.0660.0810.065 0.0460.0520.0540.047

A: 5'-KKKK-NS3-C-NS4;

B: NS3-C-NS4-KKKK-3';

C: 5'-KKKK-NS3-C-NS4-KKKK-3';

D: NS3-KKKK-C-NS4

The results confirmed that there were obvious differences in the reactivity between different connection methods and HCV serum in the four groups, and the connection method and HCV serum reactivity in group A were better than those in groups B, C, and D. Use group A to label HPR for double-antigen sandwich detection of hepatitis C virus antibodies.

Table 2 The results of HRP-labeled modified antigen double-antigen sandwich against the national third-generation standard serum 1 2 3 4 5 6 7 8 9 10 11 ABCDEFGH 0.0050.0050.0540.0530.0050.0052.0082.015 0.0760.0760.0910.0800.0720.0680.0770.082 0.0710.0770.0800.0890.0830.0780.0730.076 0.0830.0800.0770.0880.0880.0770.0780.076 0.0930.0740.0800.0750.0700.0940.0710.105 0.0810.0960.0980.1010.0760.0720.0770.076 0.2743.4140.3230.7360.1770.1190.7640.274 1.2010.2170.3330.1160.3910.1060.2740.183 0.1290.1300.1590.4811.7110.1550.1910.186 0.1490.2090.1620.1810.4473.3170.1350.176 0.9700.1640.1400.1440.1150.5060.3130.101

A1, B1, E1, F1 are blank wells, C1, D1 are negative controls, G1, H1 are positive controls;

●A2 and B2-H6 are negative standard serums from 1 to 40; A7 and B7-H11 are positive standard serums from 1 to 40. Cutoff = 0.102

Sequence Listing

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Claims (7)

1. a method that detects antibody of HCV comprises the steps:

(1) preparation of hepatitis C virus multi-epitope chimeric antigen;

(2) preparation of hepatitis C virus multi-epitope chimeric antigen and enzyme labeling linking arm;

(3) the anti-position of hepatitis C virus multilist chimeric antigen mark horseradish peroxidase;

(4) with hepatitis C virus multi-epitope inosculating antibody primordial covering enzyme linked immunosorbent detection plate;

(5) add antibody to be measured and (3) prepared enzyme-labelled antigen;

(6) add developer, chromogenic enzyme substrate;

(7) color development stopping reaction is measured optical density, according to the optical density judged result.

2. according to the described method of claim 1, it is characterized in that the preparation of described hepatitis C virus multi-epitope chimeric antigen comprises the steps:

(1) determines the amino acid position of the different section antigens of the strong HCV of antigenicity;

(2) the difference synthetic gene makes up the pBVIL-1 carrier, and antigen is connected into the multi-epitope chimeric antigen;

(3) in E.coli with inclusion body formal representation HCV multi-epitope antigen;

(4) by the ion-exchange chromatography purifying, SDS-PAGE identifies purity.

3. method according to claim 2, the amino acid position of the different section antigens of the HCV that wherein antigenicity is strong is HCV-C:10-53aa; NS3:1192-1457aa; NS4:1916-1947aa.

4. according to the described method of claim 1, it is characterized in that the preparation of hepatitis C virus multi-epitope chimeric antigen and enzyme labeling linking arm comprises the steps:

(1) synthetic primer carries out pcr amplification, obtains genetic fragment;

(2) enzyme is cut genetic fragment, is inserted in the carrier preparation expression plasmid;

(3) transformed into escherichia coli is expressed, and extracts inclusion body, carries out purifying.

5. according to the described method of claim 4, wherein the nucleotide sequence of the primer that is synthesized is shown in sequence 1-6 in the sequence table.

6. according to the described method of claim 4, carrier wherein is pIL1.

7. according to the described method of claim 1, wherein the anti-position of hepatitis C virus multilist chimeric antigen mark horseradish peroxidase comprises the steps:

(1) horseradish peroxidase (HRP) activation;

(2) hepatitis C virus multilist anti-position chimeric antigen (HCVAg) activation;

(3) desalination;

(4) combination;

(5) stop;

(6) preserve.

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