Open Access

Expression of core antigen of HCV genotype 3a and its evaluation as screening agent for HCV infection in Pakistan

  • Muhammad Z Yousaf1Email author,
  • Muhammad Idrees1,
  • Zafar Saleem1,
  • Irshad U Rehman1 and
  • Muhammad Ali1
Virology Journal20118:364

https://doi.org/10.1186/1743-422X-8-364

Received: 23 May 2011

Accepted: 26 July 2011

Published: 26 July 2011

Abstract

Background

Pakistan is facing a threat from hepatitis C infection which is increasing at an alarming rate throughout the country. More specific and sensitive screening assays are needed to timely and correctly diagnose this infection.

Methods

After RNA extraction from specimen (HCV-3a), cDNA was synthesized that was used to amplify full length core gene of HCV 3a. After verification through PCR, DNA sequencing and BLAST, a properly oriented positive recombinant plasmid for core gene was digested with proper restriction enzymes to release the target gene which was then inserted downstream of GST encoding DNA in the same open reading frame at proper restriction sites in multiple cloning site of pGEX4t2 expression vector. Recombinant expression vector for each gene was transformed in E. coli BL21 (DE3) and induced with IPTG for recombinant fusion protein production that was then purified through affinity chromatography. Western blot and Enzyme Linked Immunosorbant Assay (ELISA) were used to detect immuno-reactivity of the recombinant protein.

Results

The HCV core antigen produced in prokaryotic expression system was reactive and used to develop a screening assay. After validating the positivity (100%) and negativity (100%) of in-house anti-HCV screening assay through a standardized panel of 200 HCV positive and 200 HCV negative sera, a group of 120 serum specimens of suspected HCV infection were subjected to comparative analysis of our method with commercially available assay. The comparison confirmed that our method is more specific than the commercially available assays for HCV strains circulating in this specific geographical region of the world and could thus be used for HCV screening in Pakistan.

Conclusion

In this study, we devised a screening assay after successful PCR amplification, isolation, sequencing, expression and purification of core antigen of HCV genotype 3a. Our developed screening assay is more sensitive, specific and reproducible than the commercially available screening assays in Pakistan.

Background

Hepatitis C is one of the most common liver diseases around the world. It is caused by hepatitis C virus (HCV) and a significant number of patients progress towards chronic hepatitis, hepatocellular carcinoma (HCC) and liver cirrhosis [1]. Viral infection is the major cause of liver cirrhosis in about 20% of patients that after 10 years lead to HCC in 3% of these patients per year [2]. The prevalence of HCV infection in various locations around the world ranges from 0.5 to 10% [3]. Currently, almost 200 million people of the world population are infected with HCV [4]. HCV genotypes and many subtypes have been identified and are generally studied for epidemiology, molecular diagnosis, development of vaccines, and clinical management of the infection [5]. Still no vaccine is available and the standard treatment is neither economical nor fully effective in all the patients [6].

HCV is a positive single stranded RNA virus (Flaviviridae family) [7, 8] that is nearly 9.6 Kb in length having a 5' non-coding region (5'NCR), a single open reading frame (ORF) encoding a polyprotein of about 3,000 amino acids and a non-coding region at 3' end (3'NCR). The HCV polyprotein is postranslationally cleaved into at least 3 structural (Core, E1 and E2) and 7 non-structural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins [9, 10] and these proteins play important roles in virus entry, replication, assembly, and pathogenesis through host peptidase and viral protease activities [11].

Core gene is one of the most conserved regions of HCV genome, involved in detection, quantitation [12] and genotyping [13, 14]. It also interact with the envelop protein (E1) and thus forms the HCV capsid [15]. The core antigen-based assays has been reported to be helpful for the measurement of HCV RNA among the patients undergoing dialysis [16] and shown to be useful indicator for HCV viremia in asymptomatic carriers [17]. It has also been reported that the HCV core antigen-based methods aree useful for the quantitative measurement of HCV with respect to rapidness, easiness and low cost [12]. Moreover, HCV core antigen-based assay can identify up to 94% of viraemic donations given during the seronegative window phase of infection. The performance of the assay appears to be suitable for large-scale screening of blood donations [18].

To combat and timely diagnose HCV, community based serologic screening is of extreme significance due to dodgy trend of asymptomatic nature of the HCV infection [18]. For this purpose rapid, economical, sensitive and more specific assays are needed. The present work involved an effort to design such an assay using purified HCV core antigen from local isolates and to check out the opportunity of these cloned HCV core gene to be further employed in the possibility of vaccine development. We also describe the application of recombinant HCV core antigen from local isolates to formulate more specific screening assay for Pakistani population where HCV is becoming a big health problem.

Methods

PCR amplification, TA cloning and characterization of HCV Core gene

The viral RNA was first reverse transcribed and then used as template for polymerase chain reaction (PCR) [19]. Full-length HCV Core gene (573-bp) [GenBank: EU435145] was amplified with the following primers: the forward primer (Core-F) 5'-GGATCC TGCAACATG AGCACACTTCC -3' containing the BamHI restriction site and the reversed primer (Core-R) 5'-CTCGAG AGACGTGCCCGCCACTCT -3' containing the XhoI restriction site. The gel purified PCR product was then ligated with T4 DNA ligase to yield the constructs. The constructs were transformed into E. coli, and the selected bacterial transformants were verified by restriction enzyme analysis, colony PCR and sequencing.

Homology search

The DNA sequences of the core gene of HCV 3a obtained were searched for homology with other sequences in GenBank data base using blastn, at http://www.ncbi.nlm.nih.gov/BLAST/. Different clones constructed in present study were aligned with the representative HCV core gene sequences in the GenBank database using Multalign software package. Pair wise comparisons were performed to determine percent nucleotide homology.

Sub-cloning and construction of recombinant expression vector

Recombinant TA vector containing core cDNA of HCV genotype 3a and expression vector pGEX4t2 (Invitrogen) were digested with same restriction enzymes and ligated by T4 DNA ligase (Fermentas-Life Sciences, USA) overnight at 14°C, and stored at -20°C. The ligation product was routinely transformed into Escherichia coli (E. coli ) DH5α by heat shock method and selected on LB broth containing ampicilline (100 ug/ml). Then the recombinant expression vector carrying target gene (pGEX4t2-C) was transformed into E. coli BL21 (DE3) resulting in the production of GST-C fusion protein.

Expression and purification

The positive individual clone was cultured in 5 mL Luria bertani (LB) medium containing 100 μg/mL ampicilline and then induced for 4 hours at 37°C, adding isopropyl-β-D-thiogalactoside (IPTG) at concentration of 1 mM. For IPTG dose optimization, the bacterial culture was induced with different concentrations of IPTG [0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 (mM)] and allowed to grow for 4 h at 37°C. For temperature optimization, the bacterial culture was induced with IPTG [1.0 (mM)] and allowed to grow for 4 h at three different temperatures (25, 30 and 37°C). For time optimization, the bacterial culture was induced with IPTG [1.0 (mM)] and allowed to grow for 3 h, 4 h, 5 h and overnight (~16 h) at 37°C. Total cell proteins from each optimization experiment were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, small-scale expression was done by optimized conditions as described above to prepare for purification [20]. The protein amount was determined by Bradford assay using bovine serum albumin (BSA) as previously described [21].

The supernatant was then poured on to a purification column and allowed to bind for 1 h with gentle shaking. Finally, the proteins were collected and analyzed by SDS-PAGE to assess the level of homogeneity. A 500 ml induced bacterial culture (E. coli BL21 (DE3) was harvested after 4 h, centrifuged at 6000 × g for 10 min and the cell pellet was suspended in 20 mM Tris buffer (pH 8.0). Different conditions like suitable liquid growth media for E. coli, suitable growth temperature and pH, IPTG final concentration for induction, post-induction time for maximum recombinant production, were optimized. The cells were later lysed using lysozyme (0.1 mg/mL) at 4°C for 1 h and sonicated on ice for 5 min at an amplitude of 30% with a 30 s pulse frequency. The lysate was centrifuged at 10,000 × g for 20 min at 4°C and the supernatant was collected as soluble fraction. The resulting pellet was washed twice with 10 mL 2M urea containing 50 mM Tris buffer (pH = 8.0), 1 mM EDTA, 150 mM NaCl and 0.1% Triton X-100. The suspension was centrifuged at 10,000 × g for 20 min at 4°C and then the resulting subsidence was resuspended in regeneration buffer containing 6 M urea, 0.5 M NaCl, 20 mM Tris-HCl (pH 7.9) and incubated at room temperature for 30 min. The incubated mixtures were then centrifuged at 10,000 × g for 20 min, and the supernatant was submitted to further purification.

After washing the column twice with 1X PBS, the clear cell free lysate was loaded directly on GST-Fast Flow Sepharose column (Amersham) with speed of 0.5 ml/min. Elution of the GST fusion protein complex was done with 10 mM reduced glutathione prepared in 15 mM Tris-Cl (pH 8). Eluted fusion protein was dialyzed in buffer containing 50 mM Tris-Cl pH 8 and 1 mM EDTA for 4 hours. Peak fractions were analyzed by western blot and ELISA.

Western blot

Western blot analysis of samples was carried out as described previously [22]. In short, the proteins were separated on a 10-12% separating gel and transferred to nitrocellulose membrane electrophoretically. To prevent non-specific binding, the membrane was blocked in PBST (containing 0.5% Tween 20 and 2% BSA) blocking buffer. Recombinant fusion protein was identified by using rabbit polyclonal anti-GST antibody (Sigma) as primary antibody and anti-rabbit horse radish peroxidase conjugated antibody (Sigma) as secondary antibody.

Indirect ELISA

Enzyme linked immunosorbant assay (ELISA) was performed by modifying the methods described previously [23, 24] (Engvall and Perlman, 1971; Lin et al. 2008). All the values were represented by mean and standard deviation (SD) as performance of the serological assays is reported to be improved by adding 3 to 4 times the SD to the mean OD value. For the comparison and analysis of different ELISA assays SD and coefficient of variation (CV%) were used where needed.

HCV RNA PCR

HCV RNA qualitative PCR was done using SmartCyclerII Real-time PCR with kits from Sacace Biotechnologies, Italy as per procedure given in kit protocol.

Results

Expression and purification of recombinant core HCV 3a antigen

The cloning strategy adapted for constructing the recombinant plasmids is revealed in Figure 1. The N-terminally GST-fused recombinant core protein was produced in E. coli strain BL21 (DE3) with molecular weight of about 46 kDa (Figure 2). The highest level of expression was achieved in 2 × YT growth media (pH 7.5) at 25°C, with a 3 hours post-induction time, using 0.5 mM IPTG as an inducer. The strategy of our research showed more promising results than other eukaryotic systems or insect cell system because of its less time consuming, high-level yield, convenience and cost-effective. Finally, after the optimization of all conditions for expression, we obtained 8.25 mg/500 ml of protein from GST + Core construct.
Figure 1

Strategy of vector construction. (A) Scheme of the arrangement of HCV core gene in the expression vector pGEX4t2 that leads to the construction of recombinant vector pGEX4t2C.

Figure 2

Western blot analysis of recombinant GST+C proteins fusion complex. Lane M refers to the resolved pre-stained protein marker, lanes 1- 2 to cell lysate of non-induced E. coli BL21 (DE3) cells while lanes 3-6 refer to fusion complex of GST+E1 recombinant proteins with a specific band of 46 kDa.

Validation of recombinant core antigen as screening agent

A standardized panel, established by the Division of Molecular Virology, Center of Excellence in Molecular Biology, consisting of 200 HCV negative and 200 HCV positive specimens were tested by ELISA using the recombinant structural core HCV 3a fusion protein and the anti-human IgG conjugate. The results obtained after calculating the means, standard deviations and co-efficient of variation (CV %). Positive and negative results were differentiated on the basis of cut-off value, which was calculated from the arithmetic mean of sera negative for HCV infection plus four randomly chosen standard deviations. Based on this cut-off value (= 0.21), the results of anti-HCV screening assay were calculated as described previously [25]. Based on this value, all positive samples were found positive and all negative samples were found negative. Validating in-house anti-HCV screening assay via known sera, 100% positivity and negativity respectively were achieved.

In this connection, the present assay was analyzed by classifying the HCV-positive and HCV-negative samples into three groups each, and having them assayed three times by the same person or by three different persons. The coefficient of variations obtained from the intra-person reproducibility for HCV positive and negative sera were 4.88% and 5.88% respectively as shown in Table 1. After validating and establishing the reproducibility (Table 2) of present in-house anti-HCV screening assay with known positive and negative sera, a total of 120 serum specimens of unknown results for HCV infection were randomly collected representing almost all geographical regions of Pakistan.
Table 1

Results of in-house anti-HCV screening assay

 

Mean

Minimum

Maximum

S.D.

CV%

Positive (n = 200)

0.82

0.58

0.91

0.051

6.21

Negative (n = 200)

0.17

0.09

0.18

0.012

6.66

Number of HCV positive and negative sera was 200 each. Cut-off value was calculated 0.21. Samples exhibiting O.D. greater than 0.21 were considered positive while those exhibiting less than 0.21 considered negative. Lower the value of Coefficient of Variation (CV%) more authentic the results. Statistically, CV% of maximum 10 is acceptable for the validation of the results. Coefficient of Variation percentage is mostly affected by the extreme values in the data.

Table 2

Intra-person and inter-person reproducibility of in-house anti-HCV screening assay

 

Intra-person reproducibility

Inter-person reproducibility

Anti-HCV

Mean OD ± S.D

CV (%)

Mean OD ± S.D.

CV (%)

Positive (n = 200)

0.860 ± 0.042

4.88

0.859 ± 0.051

5.93

Negative (n = 200)

0.17 ± 0.010

5.88

0.160 ± 0.011

6.87

Mean optical densities (OD), standard deviations (SD) and co-efficient of variations (CV) of an established panel of HCV positive and negative sera are shown depicting high reproducibity.

To detect HCV infection, samples were subjected to a commercially available ELISA assay (following the manufacturer's protocol), ELISA assay validated in this study and reverse transcriptase PCR (as a 'Gold standard'). Comparative analysis of unknown sera by these three assays is summarized in Table 3. Out of total 120 sera, 36 sera were positive by both our assay (sensitivity 100%) and PCR but one sample out of these 36 was found negative by the commercial assay (sensitivity 97.2%).
Table 3

Comparative analysis of in-house screening ELISA assay, commercially available ELISA assay and HCV RNA PCR as reference standard

 

Core antigen assay

Commercial assay

PCR

'Gold Standard'

HCV true positive

36 (100%)

35 (97.2%)

36

HCV true negative

83 (98.8%)

80 (95.2%)

84

HCV false positive

0 (0%)

1 (2.88%)

0

HCV false negative

1 (1.2%)

4 (4.76%)

0

Total

120

120

120

Sensitivity of our assay and commercial assay was 100 and 97.2%, while specificity was 98.8 and 95.2% respectively. The HCV RNA PCR was used as a standard reference in the comparison.

Remaining 84 sera were negative by PCR, out of these 84 negative samples, our assay confirmed 83 (98.8%) as negative but commercial assay gave false negative results for 4 samples. It is demonstrated that in-house anti-HCV screening assay has a high sensitivity, specificity and reproducibility for detection of anti-HCV antibodies.

Discussion

Screening and diagnosing HCV is a key factor to treat this infection as early as possible and to ensure the recovery before it gets lethal. HCV infection is spreading at an alarming rate in Pakistan and 10% of the population is already HCV infected and the rate is still ascending [26]. Imported kits are used for the screening of HCV in Pakistan, however, these assays are associated with two main concerns. First, the kits are not prepared from the antigens representing local HCV strain owing to this a considerable percentage of the suspected individuals are being given false results especially false negative. Secondly, Pakistan is poor country and cannot afford precious foreign exchange that is being utilized as a result of importing these HCV screening kits. Moreover, in routine healthy blood donors screening at blood collection centers of developing countries like Pakistan, rapid assay kit to screen HCV is erroneous because of misleading results [27]. The asymptomatic nature of HCV 'The Silent Killer' [28] is posing a serious threat to Pakistani society.

In this study, we did isolate, cloned, characterized, expressed and purified the full length core antigen of Pakistani hepatitis C virus genotype 3a. The in vitro antigenic activity of purified recombinant proteins paved the way to develop an in-house anti-HCV screening assay for the most prevalent HCV genotype (3a) in Pakistan. The main aim of our study was to evaluate core antigen as anti-HCV screening agent and for that purpose E. coli expression system was convenient. Heterogeneously expressed proteins in E. coli are widely used for the development of screening assays for a number of diseases including HCV infection. Our research findings shows more promised results than other eukaryotic systems and insect cell system as it is more effective, produces relatively high-level yield and convenient. Further studies are needed, including elucidation of more characteristics of the recombinant structural fusion proteins and detection of anti-HCV antibodies in human sera on large scale.

In several studies HCV core antigen based ELISA has been used as a screening method for the identification of HCV infection in human sera [2931]. No screening method has been developed so far in Pakistan based on HCV types and isolates exist in this region and the sensitivity and specificity of the commercially available methods may be low for HCV strains common in Pakistan. Keeping in view these limitations of the available assays, anti-HCV screening assay based on local HCV isolates was developed and validated using recombinant core antigen purified in the present study.

The HCV core antigen was first tested using 200 serum samples with established HCV infection cases and 200 serum samples from anti-HCV and HCV RNA negative sera to evaluate the sensitivity and specificity respectively of the developed method. Detection of HCV RNA through RealTime PCR was used as a reference to compare and validate the results. The HCV core antigen and the reference tests detected all 200 HCV positive samples as positive and all 200 negative samples as negative. The results are in accordance with the previous reports. A report from Iran [32] described the expression of HCV core antigen in E. coli but instead of ELISA, dot blot assay was preferred to capture the antibodies in HCV infected human sera. Important factors for a commercial assay are its specificity and reproducibility. An assay must yield concordant results when tests are repeated [33]. In the present study, the assay was analyzed by classifying the HCV-positive and negative sera and concluded that the reproducibility was sufficiently high.

To avoid false results, we used core antigen of HCV for the most prevalent genotype (3a) in Pakistan. As compared to ELISA, the rapid tests have not shown any promising results and hence should not be recommended in transfusion centers for screening blood donors. Moreover, the failure of the rapid kits to detect HCV reactive samples may be due to inadequate coating of the antigens, heterogeneity of the virus nature of the antigens used [34]. These evaluations focused on the working characteristics of the present in house anti-HCV screening assay, such as ease of handling, specificity and sensitivity on a group of well-characterized samples obtained from geographically diverse regions of Pakistan, and we report their suitability for manipulation in small laboratories, i.e. blood collection centers. A possible drawback of the present HCV core antigen based ELISA is that it may not detect HCV infection in the sample taken prior to the production of anti-HCV antibodies in the patients i.e. the period between actual infection and production of antibodies.

Conclusions

In the present study, we were able to obtain a high-level expression of the recombinant HCV core antigen. In this study, we devised a screening assay by using core antigen of local HCV genotype 3a. This is the first report of its own kind in which the core antigen of HCV from a local strain was successfully used as screening agent. The sensitivity, specificity and reproducibility of the developed assay is high than the commercially available ELISA assays.

Sources of support

This work was partially supported by the Higher Education Commission (HEC) of Pakistan.

Declarations

Acknowledgements

We thank all the hepatologists, gastroenterologists, clinicians and patients for their cooperation in the study.

Authors’ Affiliations

(1)
Division of Molecular Virology & Molecular Diagnostics,National Centre of Excellence in Molecular Biology, University of the Punjab

References

  1. Hoofnagle JH: Course and outcome of hepatitis C. Hepatology 2002,36(5):S21-29. 10.1053/jhep.2002.36227View ArticlePubMedGoogle Scholar
  2. Zein N: Clinical significance of hepatitis C virus genotypes. Clin Microbiol Rev 2000, 13: 223-235. 10.1128/CMR.13.2.223-235.2000PubMed CentralView ArticlePubMedGoogle Scholar
  3. Brinster C, Inchauspe G: DNA vaccine for hepatitis-C virus. Immunology 2001, 44: 143-153.Google Scholar
  4. Butt S, Muhammad Idrees M, Akbar H, Rehman I, Awan Z, Afzal S, Hussain A, Shahid M, Manzoor S, Rafique S: The changing epidemiology pattern and frequency distribution of hepatitis C virus in Pakistan. Infection, Genetics and Evolution 2010, 10: 595-600. 10.1016/j.meegid.2010.04.012View ArticlePubMedGoogle Scholar
  5. Ali A, Ahmed H, Idrees M: Molecular epidemiology of Hepatitis C virus genotypes in Khyber Pakhtoonkhaw of Pakistan. Virol J 2010, 203: 1-7.Google Scholar
  6. Arichi T, Saito T, Major ME, Belaykov IM, Shirai M, Engelhard VH: Prophylactic DNA vaccine for hepatitis C virus infection and protection from HCV recombinant vaccinia infection in an HLA-A2.1 transgenic mouse model. Proc Nat Acad Sci USA 2000, 97: 297-302. 10.1073/pnas.97.1.297PubMed CentralView ArticlePubMedGoogle Scholar
  7. James MC, The Liver and Biliary Tract, Kumar V, Abbas AK, Fausto N: Pathologic Basis of Disease. Volume 894. 7th edition. Edited by: Robbins, Cotran. Elsevier Saunders Philadelphia, Pennsylvania 19106; 2005.Google Scholar
  8. Rho J, Ryu JS, Hur W, Kim CW, Jang JW, Bae SH, Choi JY, Jang SK, Yoon SK: Hepatitis C virus (HCV) genotyping by annealing reverse transcription-PCR products with genotype-specific capture probes. J Microbiol 2008,46(1):81-87. 10.1007/s12275-007-0121-8View ArticlePubMedGoogle Scholar
  9. Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M: (1989) Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 1989, 244: 359-362. 10.1126/science.2523562View ArticlePubMedGoogle Scholar
  10. Reed KE, Rice CM: Overview of hepatitis C virus genomestructure, polyproteinprocessing, and protein properties. Curr Top Microbiol Immumol 2000, 242: 55-84.Google Scholar
  11. Luo G: Molecular virology of hepatitis C virus. Birkhauser, Basel, Switzerland; 2004.View ArticleGoogle Scholar
  12. Okazaki K, Nishiyama Y, Saitou T: Fundamental evaluation of HCV core antigen method comparison with Cobas Amplicor HCV monitor v2.0 (high range method). Rinsho Byori 2008,56(2):95-100.PubMedGoogle Scholar
  13. Idrees M, Riazuddin S: A study of best positive predictors for sustained virological response to interferon alpha plus ribavirin therapy in naive hepatitis C patients. BMC Gastretrol 2009, 9-5.Google Scholar
  14. Ohno TM, Mizokami RR, Wu M: New hepatitis C virus (HCV) genotyping system that allows for identification of HCV genotypes 1a, 1b,2a, 2b, 3a, 3b, 4, 5a, and 6a. J Clin Microbiol 1997,35(1):201-207.PubMed CentralPubMedGoogle Scholar
  15. Lo SY, Selby M, Tong M, Ou JH: Comparative studies of the core gene products of two different hepatitis C virus isolates: two alternative forms determined by a single amino acid substitution. Virology 1994, 199: 124-131. 10.1006/viro.1994.1104View ArticlePubMedGoogle Scholar
  16. Fabrizi F, Lunghi G, Aucella F, Mangano S, Barbisoni F: Novel assay using total hepatitis C virus (HCV) core antigen quantification for diagnosis of HCV infection in dialysis patients. J Clin Microbiol 2005,43(1):414-420. 10.1128/JCM.43.1.414-420.2005PubMed CentralView ArticlePubMedGoogle Scholar
  17. Agha S, Tanaka Y, Saudy N, Kurbanov F, Abo-Zeid M: Reliability of hepatitis C virus core antigen assay for detection of viremia in HCV genotypes 1, 2, 3, and 4 infected blood donors: a collaborative study between Japan, Egypt, and Uzbekistan. J Med Virol 2004,73(2):216-222. 10.1002/jmv.20078View ArticlePubMedGoogle Scholar
  18. Lee SR, Peterson J, Niven P: Efficacy of a hepatitis C virus core antigen enzyme-linked immunosorbent assay for the identification of 'window-phase' blood donations. Vox Sang 2001,80(1):19-23. 10.1046/j.1423-0410.2001.00008.xView ArticlePubMedGoogle Scholar
  19. Guo YF, Cheng AC, Wang MS, Shen CJ, Jia RY, Chen S, Zhang N: Development of TaqMan ® MGB fluorescent real-time PCR assay for the detection of anatid herpesvirus 1. Virology Journal 2009, 6: 71. 10.1186/1743-422X-6-71PubMed CentralView ArticlePubMedGoogle Scholar
  20. Zhang SC, Ma GP, Xiang J, Cheng AC, Wang MS, Zhu DK, Jia RY, Luo QH, Chen ZL, Chen XY: Expressing gK gene of duck enteritis virus guided by bioinformatics and its applied prospect in diagnosis. Virology Journal 2010, 7: 168. 10.1186/1743-422X-7-168PubMed CentralView ArticlePubMedGoogle Scholar
  21. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976, 72: 248-254. 10.1016/0003-2697(76)90527-3View ArticlePubMedGoogle Scholar
  22. Towbin H, Staehelin T, Gordon J: Proc Natl Acad Sci USA. 1979, 76: 4350-4354. 10.1073/pnas.76.9.4350PubMed CentralView ArticlePubMedGoogle Scholar
  23. Engvall E, Perlman P: Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 1971,8(9):871-4. 10.1016/0019-2791(71)90454-XView ArticlePubMedGoogle Scholar
  24. Lin G, Qiu C, Zheng F, Zhou J, Cao X: Secretory expression of E2 main antigen domain of CSFV C strain and the establishment of indirect ELISA assay. Virologica Sinica 2008,23(5):363-368. 10.1007/s12250-008-2970-7View ArticleGoogle Scholar
  25. Griner RJ, Mayewski AI, Mushlin P: Greenland, Selection and interpretation of diagnostic tests and procedures. Principles and applications. Ann Intern Med 1981, 94: 557-592.PubMedGoogle Scholar
  26. Farhana M, Hussain I, Haroon TS: Hepatitis C: the dermatologic profile. J Pak Assoc Derm 2009, 18: 171-181.Google Scholar
  27. Torane VP, Shastri JS: Comparison of ELISA and rapid screening tests for the diagnosis of HIV, hepatitis B and hepatitis C among healthy blood donors in a tertiary care hospital in Mumbai. Indian J Med Microbiol 2008, 26: 284-285. 10.4103/0255-0857.42071View ArticlePubMedGoogle Scholar
  28. Vito R, Barbara R: Hepatitis C virus infection: when silence is deception. Trends Immunol 2003,24(8):456-464. 10.1016/S1471-4906(03)00178-9View ArticleGoogle Scholar
  29. Lee DS, Lesniewski RR, Sung YC: Significance of anti-E2 in the diagnosis of HCV infection in patients on maintenance hemodialysis: Anti-E2 is frequently detected among anti-HCV antibody-negative patients. J Am Soc Nephrol 1996, 7: 2409-2413.PubMedGoogle Scholar
  30. Leon P, Lopez JA, Elola C, Quan S, Echevarria JM: Typing of hepatitis C virus antibody with specific peptides in seropositive blood donors and comparison with genotyping of viral RNA. Vox Sang 1997, 72: 71-75. 10.1046/j.1423-0410.1997.7220071.xView ArticlePubMedGoogle Scholar
  31. da-Silva-Cardoso M, Sturm D, Koerner K: Anti-HCV envelope prevalence in blood donors from Baden-Wurttemberg. Ann Hematol 1997, 74: 135-137. 10.1007/s002770050271View ArticlePubMedGoogle Scholar
  32. Kazemi B, Bandehpour M, Seyed N, Roozbehi M, Mosaff N: Cloning and Expression of Hepatitis C Virus Core Protein in pGemex-1 Expression Vector. Archives of Iranian Medicine 2008.,11(2):Google Scholar
  33. Yeom J, Jun G, Chang Y, Sohn M, Yoo S, Kim E, Ryu S, Kang H, Kim Y, Ahn S, Cha J, Youn S, Park J: Evaluation of a new fourth generation enzyme-linked immunosorbent assay, the LG HIV Ag-Ab Plus, with a combined HIV p24 antigen and anti-HIV-1/2/O screening test. J Virol Meth 2006,137(2):292-297. 10.1016/j.jviromet.2006.07.002View ArticleGoogle Scholar
  34. Torane V, Shastri J: Comparison of ELISA and rapid screening tests for the diagnosis of HIV, hepatitis B and hepatitis C among healthy blood donors in a tertiary care hospital in Mumbai. Indian J Med Microbiol 2008,26(3):284-85. 10.4103/0255-0857.42071View ArticlePubMedGoogle Scholar

Copyright

© Yousaf et al; licensee BioMed Central Ltd. 2011

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.