CN101477126A - Hepatitis C virus antigen-antibody combined detection method - Google Patents

Abstract

The invention discloses a method for jointly detecting hepatitis C virus (HCV) antigen-antibody. A monoclonal antibody of an anti-HCV core antigen and a chimeric antigen are coated with enzyme-linked plate to be detected simultaneously, so that the method can shorten the 'window period' of HCV virus detection. Moreover, HCV antibodies in serum and the compatibility with the coated monoclonal antibody can be detected as possible so as to avoid the combination of the chimeric antigen and the monoclonal antibody by modifying HCV overall length core antigen, telescoping non-structural areas and realizing the expression of the chimeric antigen, and the positive detection rate is close to the PCR positive detection rate. The HCV detection method also has the advantages of simple operation, low price, so the method is suitable for promotion and application.

Description

A kind of method of hepatitis C virus antigen-antibody combined detection

Technical field

The present invention relates to the virus detection techniques field, be specifically related to the method for hepatitis C virus antigen-antibody combined detection.

Background technology

Hepatitis C is that (Hepatitis C virus HCV) infects and a kind of global infectious disease of causing by hepatitis C virus.There are 1.7 hundred million hepatitis c virus infection persons in the whole world at present according to estimates, and the third liver infection rate of China is approximately 3.0%, has 4000～6,000 ten thousand third hepatopaths at present at least.Wherein there is the infected of 80～85% will develop into chronic hepatitis C, wherein has 20% can develop into liver fibrosis again, 4～5% patients with liver fibrosis generation hepatocellular carcinoma is finally arranged, endanger very serious.

At present, still needleless is to the specific treatment medicine of hepatitis C virus, and also variation is difficult fast because of HCV in the development of hcv vaccine, and difficulty has the progress of breakthrough in a short time.Therefore, the early diagnosis of HCV is of great importance for the examination HCV infection sources, guiding clinical treatment and prognosis judgement.

Existing HCV detection mode commonly used mainly contains: (1) indirect detection: be used for HCV antigen/antibody combination in the detection bodies, because HCV infects have one about 40～70 day " window phase " of back to HCV antigen/antibody combination, so anti-HCV can not be as the early diagnosis index of HCV infection; (2) directly detect: by constituent the existing of qualitative or detection by quantitative HCV virion with definite virus.Because HCV infects the back promptly had HCV-RNA in 6～15 days in the infected's blood appearance, and before seropositive conversion, reach a higher level, therefore adopt this detection technique that the blood donor is carried out conventional screening and can reduce the danger that " window phase " infects greatly, though method qualitative or detection by quantitative HCV virion has in early days, characteristics such as responsive and special, but because the expensive accurate instrument of this Technology Need, higher experiment skill, expensive reagent, and cause cross pollution to cause false positive higher easily, cause it to be difficult to be generalized to single blood donor, also be subjected to limiting significantly in the application of developing country.

In view of the limitation of above HCV method for detecting virus, develop quick, accurate, cheap as early as possible and necessitate in the HCV detection method that various big hospital can be popularized.The joint-detection of pair HCV antibody and HCV cAg is not arranged at present as yet, thus the relevant report that " window phase " that HCV is infected shortens greatly.

Summary of the invention

The object of the present invention is to provide a kind of method to HCV cAg and the antibody combined detection of HCV, " window phase " that HCV is infected foreshortened to for 2 weeks, thus reach accurately, fast, testing goal easily.

For achieving the above object, method of the present invention comprises the steps:

1) the cAg sequence by Computer Analysis hepatitis C virus different subtype, determine 2 high conservatives, the amino acid sequence that antigenicity is strong: QDVKFPGGGQIVGGVY, PRGSRPSWGPTDPRRR, by the complete sequence antigen of technique for gene engineering clonal expression HCV, purification of Recombinant HCV-cAg also carries out activity identification again;

2) utilize monoclonal antibody technique to prepare the mouse source hybridoma cell strain that 2 plant heights are imitated the anti-HCV cAg of secretion specificity, be preserved in Chinese typical culture collection center, deposit number is respectively CCTCCNO:C200823, CCTCC NO:C200824, carries out antibody purification and obtains the anti-HCV core antibody that height ratio is lived;

3) enzyme conjugates preparation: behind the horseradish peroxidase purifying with freeze-drying, mixes to make it form enzyme conjugates with the monoclonal antibody of preparation, again enzyme conjugates is carried out dual purifying and concentrated ,-20 ℃ of preservations are standby;

4) chimeric antigen of reconstruct HCV cAg and other non-structural protein: described chimeric antigen prepares in the following manner: according to synthetic 8 primers of HCV complete genome sequence design, divide the fragment clone NS-3 with PCR, the corresponding gene fragment of NS-4, NS-5 and Core core, make up recombinant vector and transformed into escherichia coli abduction delivering albumen, expression product carries out purifying with affinity chromatography and adopts Western blot method identified activity again.

Described 8 primers are respectively:

①HCV-core-F：TTG?CTC?GAG?AGG?CGA?CAA?CCT?ATC?C；

②HCV-core-R：GGC?GGA?GAC?GGG?CAG?TCG?GGG?TGA?CAG?GAG；

③HCV-NS3-F：CTC?CTG?TCA?CCC?CGA?CTG?CCC?GTC?TCC?GCC；

④HCV-NS3-R：GCT?ATC?AGC?CGG?TTC?ATG?TCT?ACA?TTG?GTG?TAC；

⑤HCV-NS4-F：GTA?CAC?CAA?TGT?AGA?CAT?GAA?CCG?GCT?GAT?AGC；

⑥HCV-NS4-R：GGA?GTA?CGA?CTC?AAC?CTG?GGT?AAC?ACG?CGC；

⑦HCV-NS5-F：GCG?CGT?GTT?ACC?CAG?GTT?GAG?TCG?TAC?TCC；

⑧HCV-NS5-R：CAC?TCG?GGC?ACA?GGG?AAG?TTT?GGC?CTT?GAC。

5) bag is by elisa plate and detect: monoclonal antibody and the chimeric antigen of the anti-HCV cAg of above preparation are wrapped simultaneously by elisa plate, add testing sample and detect.

Described mouse source hybridoma cell strain 140-1 in August 14 in 2008 be preserved in Chinese typical culture collection center (be called for short: CCTCC, the address is: Chinese Wuhan Lopa Nationality an ancient woman's ornament mountain Wuhan University), deposit number is CCTCC NO:C200823; Mouse source hybridoma cell strain 140-2 is preserved in Chinese typical culture collection center August 14 in 2008, and (be called for short: CCTCC, the address is: Chinese Wuhan Lopa Nationality an ancient woman's ornament mountain Wuhan University), deposit number is CCTCC NO:C200824.

Technical scheme of the present invention can produce following technique effect:

1, will resist the monoclonal antibody of HCV cAg and chimeric antigen to wrap by elisa plate simultaneously and detect, and when shortening HCV " window phase " greatly, can effectively reduce viral loss;

2, the total length cAg is changed structure, chimeric non-structural area is also realized the expression of chimeric antigen, can detect as far as possible in the serum HCV antibody and with the bag quilt monoclonal antibody compatible, avoided chimeric antigen and monoclonal antibody to mutually combine, its positive rate and PCR positive rate are approaching;

3, the antigen-antibody joint-detection is simple to operate, compatible, cheap with present enzyme linked immunological equipment is easy to promote.

Description of drawings

Fig. 1: the pcr amplification result of HCV core fragment

M, molecular weight standard.1 negative serum; 2, positive serum sample

Fig. 2: the SDS-PAGE electrophoretogram of the IgG behind the purifying

1, monoclonal antibody strain NO:C200823; 2, monoclonal antibody strain NO:C200824

Fig. 3: the purification result of fusion

M, molecular weight of albumen standard; 1, inclusion body; 2, chimeric protein

Embodiment

Following examples are intended to illustrate the present invention rather than limitation of the invention further, and the present invention can implement by the described arbitrary mode of summary of the invention.In addition, those of ordinary skills will be understood that HCV antigen, detection of antibodies can separate or carry out simultaneously.

Embodiment one: the antibody combined detection of hepatitis C antigen

One, the preparation of HCV core recombinant antigen

1, selects HCV cAg determinant epi-position

Different subtype HCV cAg amino acid sequence is analyzed, and to the Macvector process analysis of the antigenicity of HCV-cAg, water wettability, epitope, select the antigen position that sequence is conservative, antigenicity is strong, determined 2 different antigenic determinant amino acid (AA) sequence, as shown in table 1:

Four different HCV hypotype antigenic determinant sequences of table 1

2, the clonal expression of HCV core recombinant antigen

Select 2-160 amino acids sequence to carry out recombinant clone and express, as shown in table 2 with the gene source that pBVIL1 expresses:

Table 2 pBVIL1 plasmid expression gene source

All contain restriction enzyme site (being XhoI in the forward primer, is XbaI in the reverse primer) at the primer that is used for amplification gene, amplified production inserts the carrier pBVIL1 with same double digestion behind double digestion.The HCV-c antigen gene fragment expression plasmid of above-mentioned structure, transform E.coli bacterium HB101 with conventional Calcium Chloride Method sensitization, flat board through containing ammonia benzyl antibiotic is cultivated, after choosing the bacterium colony amplification cultivation, with alkaline denaturation extracting plasmid, with PCR method screening positive clone, amplified production is identified (Fig. 1) with agarose gel electrophoresis.

3, the purifying of recombinant HCV-cAg and activity identification

HCV-C antigen is collected 0.15M NaCl eluting peak through S-Sepharose post stage gradient wash-out, through the desalination of Sephadex G50 post, collects first eluting peak again.Identify that with the SDS-PAGE method antigen purity, protein applied sample amount are only to deposit a band, scanning purity 〉=95% under the condition of 10 μ g.With purifying HCV-c antigen indoor quality controlled serum is carried out the ELISA test to measure activity.Antigen behind the purifying is in-20 ℃ of cryopreservation.Purifying HCV-c antigen carries out the ELISA test to measure activity to indoor quality controlled serum (10 parts of HCV antibody positive serum, 10 parts of HCV negative antibody serum).

Two, the Monoclonal Antibody of anti-hepatitis c virus cAg

1, the Monoclonal Antibody of different epi-position anti-hepatitis c virus cAgs

With recombinant HCV-cAg immunity Balb/c mouse (mouse 6-8 in age week), extracting spleen cell mixes in the 9:1 ratio with myeloma cell's (SP2/0 cell), and PEG urgees fusion, (is fused into hybridoma on 20% hyclone/DMEM1 * HAT) in the HAT nutrient solution.

When the hybridoma clonal growth reaches total area 15%-20%, can begin its supernatant is detected, utilize nutrient culture media to combine, detect hybridoma with the ELISA method and whether produce antibody, thereby filter out target clone system with the porous culture plate of embedding target antigen.

With hybridoma by preparing ascites in the subcutaneous implantation mouse body.Get ascites and filter out the high secretion of antibody sexual cell, be transferred to conventional 1640 nutrient culture media clonings and cultivate, be collected into 10-20L cell line nutrient solution with the ELISA method.In the culture tank of transferring to after the 1640 conventional nutrient culture media dilutions, antibody specificity monoclonal cell system is turned out in cloning.Cellular incubation is observed material balance in the culture tank, and the supernatant collection rate is 99%, and the residual cells collection rate is 1%.

2, monoclonal antibody purifying and evaluation

Adopt sad-ammonium sulfate precipitation method precipitation odd contradictive hydroperitoneum or collect liquid, dialysis separation and purification monoclonal antibody composition carries out ion-exchange chromatography with dislysate supernatant Sephadex G-50 chromatographic column, uses the pH7.2/0.01MPBS wash-out, collect first eluting peak, concentrate with PEG2000 again and collect.To obtain 10 times of concentrate dilutions at last, survey 280nm OD value, calculate the concentration of antibody purification.This technology makes the antibody concentration standard for being not less than 3ug/ml.

Identify purity with the SDS-PAGE method: applied sample amount 10ug, the anti--IgG behind the purifying are Er Tiao district band (see figure 2) on SDS-PAGE.Packing behind the assay approval, freeze-drying ,-20 ℃ of preservations are standby.

Polypeptide amino acid position with synthetic different core antigen of C type hepatitis virus: 20-35aa, 35-50aa, 100-115aa, 115-130aa wrap the screening of being carried out monoclonal antibody by elisa plate respectively, screening positive clone carries out subclone, picks out the monoclonal of different epi-positions.Thereby to the monoclonal antibody screening of the anti-hepatitis c virus of different epi-positions with identify.

Three, enzyme conjugates preparation

The HRP that gets 5mg is dissolved in 1.0ml NaHCO ₃In the solution, add the NaIO of an amount of 0.06M ₄The room temperature lucifuge stirred 30 minutes, moved into bag filter, with 0.01M pH9.6 carbonate buffer solution dialysed overnight.Get purifying enzyme mark monoclonal antibody 5mg, use 0.01M NaHCO ₃Solution fully dissolves the back to be mixed with enzyme liquid, the bag filter of packing into, and with 4 ℃ of lucifuge dialysis of 0.01M pH9.6 carbonate buffer solution 20 hours, liquid was changed 3 times in the centre.Get the dialysis back and add 0.4%NaBH in conjunction with liquid ₄0.2ml, stir after 30 minutes, placed 4 hours for 4 ℃.

With the enzyme conjugates bag filter of packing into, with pH 7.2 0.01M PBS dialysis 48 hours.The SephadexG-200 gel filtration: with Sephadex G-200 chromatographic column on the dislysate, with the PBS wash-out of pH 7.2,0.01M, collect first eluting peak, concentrate with PEG2000 ,-20 ℃ of preservations are standby.Anti-human IgG enzyme labeling thing adopts the square formation titration, measures with indirect elisa method and tires.Be 1:5000 times.

Four, the chimeric antigen of reconstruct HCV cAg and other non-structural protein

With reference to the gene order of Hepatitis C virus subtype 1b (AY460204), design 8 overlap extension PCR primers, it is synthetic that primer is given birth to the worker by Shanghai:

1)HCV-core-F：TTG?CTC?GAG?AGG?CGA?CAA?CCT?ATC?C；

2)HCV-core-R：GGC?GGA?GAC?GGG?CAG?TCG?GGG?TGA?CAG?GAG；

3)HCV-NS3-F：CTC?CTG?TCA?CCC?CGA?CTG?CCC?GTC?TCC?GCC；

4)HCV-NS3-R：GCT?ATC?AGC?CGG?TTC?ATG?TCT?ACA?TTG?GTG?TAC；

5)HCV-NS4-F：GTA?CAC?CAA?TGT?AGA?CAT?GAA?CCG?GCT?GAT?AGC；

6)HCV-NS4-R：GGA?GTA?CGA?CTC?AAC?CTG?GGT?AAC?ACG?CGC；

7)HCV-NS5-F：GCG?CGT?GTT?ACC?CAG?GTT?GAG?TCG?TAC?TCC；

8)HCV-NS5-R：CAC?TCG?GGC?ACA?GGG?AAG?TTT?GGC?CTT?GAC。

Divide the fragment clone corresponding gene fragment of NS-3, NS-4, NS-5 and Core core with PCR, with overlap extension PCR these four fragments are connected into mosaic gene then, be inserted into expression vector pBV-IL2 again and be built into recombinant vector, with its transformed into escherichia coli (JM109) abduction delivering albumen, expression product carries out purifying (Fig. 3) with Q type ion exchange chromatography and molecular sieve.

Purified product adopts Western blot method identified activity: at first carry out SDS-PAGE (gel strength is 100g/L), transfer on the nitrocellulose membrane again, add people HCV antibody positive serum and 1: 20 000HRP2 mouse-anti human IgG of 100g/L skimmed milk power, dilution in 1: 1000 successively, develop the color with diaminobenzidine (DAB) at last.

Five, HCV antigen-antibody joint-detection

1, material

1) the blood positive sample of HCV antigen-antibody:

From HCV antigen or the antibody or the antigen-antibody while positive sample of Yueyang Jun Mountain blood-collecting station of Hunan Jynda Bioengineering Co., Lts. and the collection of in the wrong former blood-collecting station, the detectable that the affirmation of these HCV antibody or antigen positive is used is from Roche Holding Ag's kit (COBAS AMPLICOR HCV MONITORKIT; HCV ELISA TEST KIT (SP-NANBASE C-96 3.0)) and Shenzhen basic HCV of company nucleic acid quantitative determination reagent kit.

2) monoclonal antibody:

Monoclonal antibody c20-35 is by mouse source hybridoma cell strain CCTCC NO:C200823 secretion, at the N-end portion (amino acid 20-35) of HCV core protein; Monoclonal antibody c100-115 is by mouse source hybridoma cell strain CCTCC NO:C200824 secretion, at the C-end portion (amino acid/11 00-115) of HCV core protein.C100-115 antibody and horseradish peroxidase (HRP) are used the standard method coupling.

3) chimeric antigen:

Chimeric antigen contains 399 amino acid residues, wherein merged the fragment of core (amino acid 60-100), NS3 (1000-1105), NS4 (1917-1947) and NS5 (2383-2453), and obtain at the expression in escherichia coli purifying with interleukin (IL2, contain 150 amino acid based) fused polypeptide.

2, method

With monoclonal antibody C20-35 and the 1 * PBS (pH7.4) of the anti-HCV cAg of 2mg/ml purifying, pH7.5 also fully mixes.In same damping fluid, add 2mg/ml reorganization chimeric protein (comprising core fragment C-NS3-NS4-NS5), mixed solution 20 minutes, the every hole of coated elisa plate 100 μ L/ is placed dull and stereotyped in 4 ℃ of refrigerator 16-24 hours.Then with dull and stereotyped three times of 1 x PBS washing. every hole adds 300 μ L sealing damping fluid (1% BSA in the microwell plate of antigen-antibody bag quilt then, 1 * PBS (pH7.4), sweet mellow wine, polyglycol, gelatin) one hour, purge is dull and stereotyped and on 4 ℃ of lyophilizers dry 24 hours, dull and stereotypedly preserves having under the condition of drying agent.

In flat board, add 100mLPBS solution with dilution 100mL testing sample, the sample that dilute is moved in the micropore of HCV antigen/antibody bag quilt,, use the PBS solution washing 5 times that contains 0.5%Tween20 then then 37 ℃ of cultivations 60 minutes with micro tube.Add the anti-human IgG in mouse source of 200 μ L horseradish peroxidase (HRP) marks and the anti-HCV core monoclonal antibody (AntiC100-115) of HRP mark in the micropore, cultivate the PBS solution washing 5 times that usefulness after 30 minutes contains Tween20.Add o-phenylenediamine and hydrogen peroxide again in micropore, lucifuge colour developing 10 minutes adds 2mol/L sulfuric acid 50 μ L cessation reactions at last.Detect microwell plate with the survey of BIO-RAD microplate reader under the light of A450nm, calculate the ratio (P/N) of testing sample hole A450nm and negative control hole average A 450nm, P/N 〉=2.1 are judged to be the positive.Manifest orange-colored light in any one micropore and just show that tested sample is infected by HCV.

3, result

The result of test is as shown in table 3, and the blood sample of HCV infection is detected.The antibody/antigen joint-detection can detect HCV virion and the antibody in the blood simultaneously.Improve the recall rate that HCV infects.

Table 3:HCV positive ELISA testing result

Claims (3)

1, a kind of method of hepatitis C virus antigen-antibody combined detection is characterized in that realizing by following steps:

1) by the cAg sequence of computer software analysis hepatitis C virus different subtype, determines 2 high conservatives, the amino acid sequence that antigenicity is strong: QDVKFPGGGQIVGGVY, PRGSRPSWGPTDPRRR;

2) utilize monoclonal antibody technique to prepare the mouse source hybridoma cell strain that 2 plant heights are imitated the anti-HCV cAg of secretion specificity, be preserved in Chinese typical culture collection center, deposit number is respectively CCTCCNO:C200823, CCTCC NO:C200824, carries out antibody purification and obtains the anti-HCV core antibody that height ratio is lived;

3) behind the horseradish peroxidase purifying with freeze-drying, mix the formation enzyme conjugates with the monoclonal antibody of preparation;

4) chimeric antigen of reconstruct HCV cAg and other non-structural protein;

5) monoclonal antibody and the chimeric antigen with the anti-HCV cAg of above preparation wraps simultaneously by elisa plate, adds testing sample and detects.

2, the method for a kind of hepatitis C virus antigen-antibody combined detection according to claim 1, it is characterized in that, described chimeric antigen prepares in the following manner: according to synthetic 8 primers of HCV complete genome sequence design, divide the fragment clone NS-3 with PCR, the corresponding gene fragment of NS-4, NS-5 and Core core, make up recombinant vector and transformed into escherichia coli abduction delivering albumen, expression product carries out purifying with affinity chromatography and adopts Western blot method identified activity again.

3, the method for a kind of hepatitis C virus antigen-antibody combined detection according to claim 2 is characterized in that, described 8 primers are respectively:

1)HCV-core-F：TTG?CTC?GAG?AGG?CGA?CAA?CCT?ATC?C；

2)HCV-core-R：GGC?GGA?GAC?GGG?CAG?TCG?GGG?TGA?CAG?GAG；

3)HCV-NS3-F：CTC?CTG?TCA?CCC?CGA?CTG?CCC?GTC?TCC?GCC；

4)HCV-NS3-R：GCT?ATC?AGC?CGG?TTC?ATG?TCT?ACA?TTG?GTG?TAC；

5)HCV-NS4-F：GTA?CAC?CAA?TGT?AGA?CAT?GAA?CCG?GCT?GAT?AGC；

6)HCV-NS4-R：GGA?GTA?CGA?CTC?AAC?CTG?GGT?AAC?ACG?CGC；

7)HCV-NS5-F：GCG?CGT?GTT?ACC?CAG?GTT?GAG?TCG?TAC?TCC；

8)HCV-NS5-R：CAC?TCG?GGC?ACA?GGG?AAG?TTT?GGC?CTT?GAC。

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