Indian Journal of Pathology and Microbiology
Home About us Instructions Submission Subscribe Advertise Contact e-Alerts Ahead Of Print Login 
Users Online: 281
Print this page  Email this page Bookmark this page Small font sizeDefault font sizeIncrease font size


 
  Table of Contents    
ORIGINAL ARTICLE  
Year : 2019  |  Volume : 62  |  Issue : 1  |  Page : 43-48
Expression and immune recognition of polypeptides derived from Hepatitis C virus structural proteins


Department of Biochemistry, Faculty of Science, Ain Shams University, Cairo, Egypt

Click here for correspondence address and email

Date of Web Publication31-Jan-2019
 

   Abstract 


Background: Hepatitis C virus (HCV) is characterized by a high degree of nucleotide sequence variability between genotypes. This variability extends to functional and immunological determinants. Serological tests using antigenic segments derived from the HCV polyprotein have been used for the diagnosis of HCV infection. However, available diagnostic Kits do not necessarily take type variability into consideration and are not optimized for HCV genotype 4a (HCV4a), the predominant genotype in Egypt. Aim: The aim of this study was to express some HCV4a-derived polypeptides in order to identify those with immunodiagnostic utility. Materials and Methods: Six sequential/overlapping genomic segments encoding 100–266 amino acid peptides from the core (peptide 1), envelope 1 (E1; peptide 2), envelope 2 (E2; peptides 4, 5, and 6), and E1/E2 (peptide 3) regions of the HCV4apolyprotein were selected for in vitro expression as glutathione S-transferase-fusion proteins. The immunoreactivity of the expressed peptides was evaluated against sera from HCV-infected/uninfected individuals using dot blot, western blot, and enzyme-linked immunosorbent assay. Results: The expressed polypeptides were recognized by HCV-infected sera from 20 patients, while showing no immunoreactivity toward uninfected serum. Peptide 1 derived from the core protein was found to be the most immunoreactive. Conclusion: Expressed polypeptides hold good potential for use in the development of improved HCV immunodiagnostics.

Keywords: Diagnostic reagents, hepatitis C virus, immune recognition, polypeptide expression

How to cite this article:
Halim AS, Mohamed MR, Hamid FF, Karim AM. Expression and immune recognition of polypeptides derived from Hepatitis C virus structural proteins. Indian J Pathol Microbiol 2019;62:43-8

How to cite this URL:
Halim AS, Mohamed MR, Hamid FF, Karim AM. Expression and immune recognition of polypeptides derived from Hepatitis C virus structural proteins. Indian J Pathol Microbiol [serial online] 2019 [cited 2019 Feb 21];62:43-8. Available from: http://www.ijpmonline.org/text.asp?2019/62/1/43/251258





   Introduction Top


Hepatitis C virus (HCV) represents a major worldwide public health problem. There are about 150 million people chronically infected with HCV, corresponding to 2%–2.5% of the world's population according to the world health organization (WHO). The WHO estimated prevalence of HCV in Egypt, the largest reservoir of HCV in the world, is >10%.[1]

Comparisons of HCV nucleotide sequences derived from individuals from different geographical regions revealed the presence of six major HCV genotypes (labeled 1–6), later on, a seventh genotype was characterized.[2] A recent study confirmed the presence of at least 7 different HCV genotypes and 67 subtypes.[3]

HCV genotype 4 is most prevalent in the Middle East and Egypt where it accounts for >80% of all HCV infections,[4] with the 4a subtype responsible for most infections [5] HCV genotype 4 is a very heterogeneous genotype showing significant genetic divergence and more subtypes compared with other genotypes.[6]

HCV infection usually appears as an asymptomatic disease and is often diagnosed accidentally and, unfortunately, remains heavily underdiagnosed.[7] It is estimated that only 30%–50% of individuals infected with HCV are aware of their disease and can take advantage of treatment options.[8]

Serologic assays that detect specific antibodies to HCV (anti-HCV antibodies) are being utilized for the diagnosis of HCV infection. In current clinical practice, antibodies against multiple HCV epitopes are detected by commercially available second and third generation enzyme-linked immunosorbent assay (ELISA).[9]

It has been reported that HCV infection and its complications are among the leading public health challenges in Egypt.[10] Improved immunodiagnostics requires a deeper understanding of the immunogenicity of the different segments of the viral polyprotein. As a part of studies to construct an epitope map for HCV4a that may be of value in developing improved diagnostics and vaccines, we envisioned an approach for expressing viral polypeptides covering the whole genome. In this study, we demonstrate the utility of this approach through expressing a number of polypeptides from the core, envelope 1 (E1), envelope 2 (E2), and E1/E2 regions of the polyprotein and testing their recognition by sera from infected persons.


   Materials and Methods Top


Basic molecular biology techniques were conducted according to previously described standard procedures,[11] unless otherwise specified. Diagnosis of chronic infection with HCV was based on biochemical, serological, and molecular findings. The study protocol was reviewed and approved by the scientific ethical committee of our institution. All procedures followed were in accordance with the standards of the scientific ethical committee of our institution on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. Informed consent was obtained from all patients for being included in the study.

Amplification and cloning of HCV-cDNA segments into a TA vector

DNA segments were amplified from HCV type 4a genome isolate ED43[12] cloned in pUC119 plasmid kindly provided by Dr. Richard Elliot. Six overlapping or sequential segments within the recombinant pUC119 plasmid were amplified by the polymerase chain reaction (PCR) using primer pairs flanked with unique restriction enzyme cloning sites [Table 1].
Table 1: Amplification primers and expressed peptides

Click here to view


Fresh PCR products were cloned into the pCR2.1vector and transferred into competent cells using the TA Cloning Kit and One Shot TOP10 Chemically Competent Escherichia coli cells (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The presence and orientation of the PCR products were analyzed by restriction enzyme (Bam HI, Bgl II, and Sal I; Promega, Madison, WI, USA) digestion of the isolated plasmids.

Subcloning and expression in pGEX

The pGEX-4T-1 expression vector (Pharmacia LKB) was used for subcloning the inserts from the TA vector and their subsequent expression as fusion proteins with Schistosoma japonicum 26-kDa glutathione S-transferase (Sj26 GST). Insert 1 cloned in the TA vector was excised by digestion with Bam HI, whereas inserts 2 through 6 were excised by double digestion with either Bgl II and Sal I (inserts 2 and 3) or Bam HI and Sal I (inserts 4, 5, and 6), respectively. The pGEX-4T-1 vector was digested with either Bam HI or BamHI and Sal I. Both the excised inserts and the linearized pGEX-4T-1 vector were purified on 1% agarose gel (Applied Biosystems/Ambion, Austin, TX, USA) and recovered from the gel using the freeze-squeeze method.[13]

Inserts were subsequently ligated into the corresponding BamHI or Bam HI and Sal I cloning sites of the pGEX-4T-1 expression vector. The ligation reactions were then used to transform E. coli Top10 competent cells. Positive recombinants were identified using cracking gel analyses. To verify insert size, small-scale plasmid DNA preparations and digestion with restriction enzymes were performed for pGEX-4T-1 plasmids harboring inserts 1, 4, 5, and 6. As inserts 2 and 3 were excised from the respective TA vectors using a combination of Bgl II and Sal I restriction enzymes and subsequently cloned in Bam HI and Sal I digested pGEX-4T-1, these inserts could not be excised with the same restriction enzymes. Therefore, the presence and size of inserts 2 and 3 were verified by PCR using the flanking primers shown in [Table 1].

Bacterial colonies harboring the pGEX-4T-1 vector and colonies harboring recombinant pGEX-4T-1 with each of the six inserts were inoculated individually, in LB-ampicillin medium and incubated at 37°C overnight. Expression of Sj26 GST and the fusion proteins was induced by Isopropyl β-D-1-thiogalactopyranoside. The proteins were purified by affinity chromatography where the filtered supernatant was loaded onto a polypropylene column (Poly-Prep Chromatography Columns, Bio-Rad Laboratories, Hercules, CA, USA) packed with glutathione cellulose resin (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) according to the manufacturer's instructions. The protein concentration was determined using the Pierce bicinchoninic acid [14] Protein Assay Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). The size and integrity of the purified fusion protein were then verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 50 μg of the purified fusion protein.

Analysis of the immunoreactivity of the expressed fusion proteins

The immunoreactivity of the expressed fusion proteins was tested against sera from both HCV-infected and uninfected individuals in Egypt as well as serum from a rabbit immunized with Sj26 GST (anti-GST serum) using Dot and western blot analysis as well as ELISA. An alkaline phosphatase secondary antibody conjugate together with a p-nitrophenyl phosphate substrate (pNPP; Sigma-Aldrich, Saint Louis, Missouri, USA) was used for detection.

For dot blots, 5 μL (25 μg) of each purified fusion protein or purified Sj26 GST fusion partner was pipetted onto 0.45-μm pore nitrocellulose membrane (Bio-Rad Laboratories) and allowed to dry, the dot blot was then probed with serum diluted 1:100.[11] For western blots, proteins separated on SDS-PAGE as described above were transferred onto a nitrocellulose membrane in a Trans-Blot Semi-Dry Electrophoretic Transfer Cell (Bio-Rad Laboratories) and probed with serum diluted1:100.[15]

For ELISA, polystyrene microtiter plate wells were coated with the expressed protein antigens diluted to 5 μg/mL in 0.05 M bicarbonate buffer pH 9.5 (100 μL per well) for 2 h at room temperature and then transferred to 4°C overnight. Next day, the antigen solution was discarded and the plate was washed three times with phosphate-buffered saline (PBS). The empty sites in the wells were then blocked with PBS containing 2% bovine serum albumin (215 μL per well) for 30 min at room temperature and washed three times with PBS-0.05% Tween-20. The primary antibody diluted in PBS-0.05% Tween-20 at 1:10 was added (100 μL per well) and the plate was incubated at 37°C for 2 h. The excess unbound primary antibody was washed four times with PBS-0.05% Tween-20. Secondary antibody conjugate diluted 1:5,000 in PBS-0.05% Tween-20 was added (100 μL per well) and incubated for 1 h at room temperature. The excess secondary antibody was washed 4 times with PBS-0.05% Tween-20 followed by an additional wash with PBS. Substrate (pNPP) was added (100 μL per well) and the plate was incubated in the dark for 15 min before O.D. measurement at 405 nm.[16]


   Results Top


Expression of fusion peptides

SDS-PAGE was used to determine the size of six HCV polypeptides expressed as fusion proteins with Sj26 GST. the observed molecular weight for fusion proteins 1, 2, 3, and 5 of ~48 kDa, whereas the observed molecular weights for fusion proteins 4 and 6 were ~55 and 33 kDa, respectively. Taking into consideration the size of the fusion partner, those molecular weights reflect the relative polypeptide lengths shown in [Table 1].

Analysis of the immunoreactivity of the expressed peptides

Dot blot

The immunoreactivity of Sj26 GST as well as the six HCV fusion proteins was tested by dot blots using sera from HCV-infected and uninfected individuals. As shown in [Figure 1]a, both Sj26 GST and the six HCV fusion proteins reacted with anti-GST serum. On the other hand, fusion proteins 1-6 but not Sj26 GST reacted with sera from 20 HCV-infected individuals, with fusion protein 1 showing highest reactivity with all sera and fusion protein 6 least reactive with most sera. Among the infected sera, serum 7 was the most reactive with most but not all fusion proteins [Figure 1]b. Neither Sj26 GST nor the six HCV fusion proteins showed immunoreactivity toward normal uninfected serum [Figure 1]c.
Figure 1: Dot blot of Sj26 GST and Sj26 GST-HCV fusion proteins. B: Blank (no antigen); GST: purified Sj26 GST; Ag1-6: purified Sj26 GST-HCV fusion proteins 1–6. Blots were probed with (a) anti-GST serum, (b) sera from HCV-infected patients 1-20, and (c) uninfected serum

Click here to view


Western blot

Western blots for the reactivity of the six fusion proteins and their Sj26 GST fusion partner with anti-GST serum, HCV-infected serum 7, and normal uninfected serum are shown in [Figure 2]. Both Sj26 GST and the six HCV fusion proteins were recognized by anti-GST [Figure 2]a, whereas infected serum reacted with the six fusion proteins but not with Sj26 GST [Figure 2]b. Of the six fusion proteins probed with serum 7, the strongest signal was observed with fusion protein 1 as was the case in the dot blot.
Figure 2: Western blot of Sj26 GST and Sj26 GST-HCV fusion proteins. M = molecular weight marker, GST = purified Sj26 GST, Lanes 1-6 = purified Sj26 GST-HCV fusion proteins 1–6. Blots were probed with (a) anti-GST serum and (b) HCV-infected serum from patient 7

Click here to view


Enzyme-linked immunosorbent assay

The immunoreactivity of fusion proteins 1, 2, 3, and 4 was quantitatively tested by ELISA against uninfected human serum and HCV-infected patient sera (1-20). The immunoreactivity of the fusion proteins was expressed as optical density (OD) at a wavelength of 405 nm [Figure 3]. The quantitative results support the dot blot findings showing a pronounced OD reading for all four fusion proteins with fusion protein 1 consistently producing highest OD and serum 7 showing best overall reactivity with all fusion proteins.
Figure 3: ELISA reactivity of Sj26 GST-HCV fusion proteins 1-4. Ag 1-4: fusion proteins 1–4 reacted with sera from HCV infected patients (1–20) and uninfected serum. Wells were coated with antigens diluted to 5 μg/mL in 0.05 M bicarbonate buffer (100 μL per well)

Click here to view


To investigate potential additive, synergistic or antagonistic immunoreactivities of the expressed HCV fusion proteins against the various sera, we prepared fusion protein cocktails (a cocktail of fusion proteins 1 + 2 and a cocktail of fusion proteins 1 + 2 + 3 + 4). Comparison of the ELISA results of both cocktails with each of the 20 HCV-infected patient sera [Figure 4] does not show consistent higher recognition for either one. Moreover, [Table 2] shows that the addition of fusion protein 1 to fusion protein 2 slightly diminished immunoreactivity as compared with fusion protein 1 alone. Furthermore, mixing fusion proteins 1, 2, 3, and 4 was not in favor of improved immunoreactivity in comparison to fusion proteins 1 and 2 alone or combined.
Figure 4: ELISA reactivity of Sj26 GST-HCV fusion protein cocktails. Ag 1 + Ag2: Wells were coated with 100-μL solution of fusion proteins 1 and 2 at a concentration of 5 μg/mL for each protein; Ag1 + Ag2 + Ag3 + Ag4: Wells were coated with 100-μL solution of fusion proteins 1 + 2 + 3 + 4 at a concentration of 5 μg/mL for each protein. Antigens were reacted with sera from HCV-infected patients 1–20 and uninfected serum

Click here to view
Table 2: Immunoreactivity of the Sj26 GST HCV fusion proteins

Click here to view



   Discussion Top


An understanding of the immunological properties of the expressed hepatitis C genome is important for the development of sensitive reagents for immunodiagnosis, genotyping, and protective vaccination and studies aimed at understanding the variabilities that help the virus evade the host's immune response. The availability of a repertoire of overlapping polypeptides covering the viral genome provides important reagents toward achieving this goal. In this study, we report on the cloning and expression of six sequential or overlapping segments of the HCV4a genome isolate ED43,[12] encoding parts of the structural proteins: core, E1, and E2.

The core is one of the most conserved regions of the HCV genome. The core molecule has a structural function since it interacts with the envelope 1 protein (E1) and thus forms the viral capsid that contains the HCV genome.[17] In addition, the HCV core protein is highly antigenic, induces specific cellular and humoral responses, and most probably plays a key role in the pathogenesis of HCV infection.[18] It is one of the main components of the kits used for detecting HCV infection in clinical settings.[19]

The E1 and E2 envelope proteins harbor 6 and 11 putative N-glycosylation sites, respectively, those provide targets for immune recognition. Moreover, the envelope proteins are the first antigens to be exposed to the host cells, and formation of antibodies to the envelope region of a virus begins as early as 2 h after viral infection. The envelope proteins were found to induce only a humoral immune response in the host with E2 being more immunogenic than E1.[20]

Anti-E2 antibodies were detected in > 90% of HCV RNA-positive individuals using E2 antigen [21] and the E2 protein was suggested as useful for monitoring HCV infection.[22] Zaaijer et al.[23] demonstrated that HCV antibodies could be detected using E2-derived peptides, and therefore, the E2 protein was recommended as a promising antigen for the detection of HCV antibodies.

In our study, the polypeptides [Table 1] were expressed as 100–200 amino acid segments of the viral polyprotein fused to Sj26 GST, their recognition by the human immune system was studied by dot blots, western blots and ELISA. Dot blots probed by sera from 20 different patients show that all polypeptides are recognized by the sera; however, signal strength varied differentially between the peptides [Figure 1]. Peptide 1 was the best recognized by all sera. Interestingly, it is a 200-amino acid peptide derived from the core region incorporated in many commercial ELISA kits.[19],[20] Reactivity of the remaining polypeptides with the 20 sera was variable, a peptide poorly recognized by one serum may be better recognized by another, and conversely, a peptide well recognized by the second serum may be less well recognized by the first, e.g., compare peptide 2(E1) and peptide 5(E2) recognition by serum 7 and 9.

Regarding the three E2 polypeptides, peptide 4 (266 amino acids) encompasses the sequence of peptide 5 (166 amino acids), which, in turn, encompasses the sequence of peptide 6 (100 amino acids). Signal intensity in the dot blot for the three peptides generally appears to decrease with decreased peptide size for most sera probably reflecting fewer epitopes. Surprisingly, signal intensity for four infected sera (13, 14, 15, and 19) is strongest for peptide 5 followed by peptide 6 (the smallest), while peptide 4, the largest peptide, displayed the weakest signal. Possibly those sera due to viral or host genetic factors do not recognize predominant epitope(s), which involve sequences within the N-terminal amino acids of peptide 4; removal of those amino acids in peptides 5 and 6 may expose other epitope(s), which are masked by those N-terminal amino acids and are better recognized by the 4 variant sera.

Western blots [Figure 2] probed with anti-GST and patient serum confirmed fusion protein size and integrity. In contrast to dot blots and ELISA, which recognize conformational epitopes, western blots due to the use of SDS gels in their preparation react primarily but probably not exclusively with linear epitopes.[24] Despite this difference, peptide 1, the antigen best recognized using dot blots, was also the best recognized antigen in western blots.

A quantitative assessment of the affinity of peptides 1–4 to antibodies in the 20 patients' sera using ELISA [Figure 3] reflected the affinities observed in dot blots; furthermore, combining peptides in ELISA [Figure 4] did not appear to enhance signal output above that observed for the most reactive peptide but produced a signal reflecting the relative antibody binding affinity of the component antigens. This is evident on comparing ODs reflecting the average immunoreactivity of patients 1–20 with the four peptides alone and in combinations in [Table 2].

In conclusion, the development of sensitive immunodiagnostic reagents for HCV using recombinant 100- to 200-amino acid peptides is a feasible approach. For best results, the genome needs to be screened for the best reactive peptides as detected by ELISA assays using infected human sera. Combining a number of high affinity peptides is probably necessary in order to compensate for decreased sensitivity resulting from HCV type or patient variability in antigen–antibody recognition.

Financial support and sponsorship

This research has been funded by a research grant from Ain Shams University to Amr M. Karim.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
World Health Organization (WHO) Hepatitis C Fact Sheet, No. 164. July 2013. Available from: http://www.who.int/mediacentre/factsheets/fs164/en/. [Last accessed on 2016 May 22].  Back to cited text no. 1
    
2.
Murphy DG, Sablon E, Chamberland J, Fournier E, Dandavino R, Tremblay CL. Hepatitis C virus genotype 7, a new genotype originating from central Africa. J Clin Microbiol 2015;53:967-72.  Back to cited text no. 2
    
3.
Smith DB, Bukh J, Kuiken C, Muerhoff AS, Rice CM, Stapleton JT, et al. Expanded classification of hepatitis C virus into 7 genotypes and 67 subtypes: Updated criteria and genotype assignment web resource. Hepatology 2014;59:318-27.  Back to cited text no. 3
    
4.
Khattab MA, Ferenci P, Hadziyannis SJ, Colombo M, Manns MP, Almasio PL, et al. Management of hepatitis C virus genotype 4: Recommendations of an international expert panel. J Hepatol 2011;54:1250-62.  Back to cited text no. 4
    
5.
World Health Organization. Hepatitis C. Geneva, Switzerland: World Health Organization, 2000 WHO fact sheet 164. Available from: http://www.who.int/mediacentre/factsheets/fs164/en/print.html/. [Last accessed on 2016 May 22].  Back to cited text no. 5
    
6.
Simmonds P, Bukh J, Combet C, Deléage G, Enomoto N, Feinstone S, et al. Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes. Hepatology 2005;42:962-73.  Back to cited text no. 6
    
7.
Villano SA, Vlahov D, Nelson KE, Cohn S, Thomas DL. Persistence of viremia and the importance of long-term follow-up after acute hepatitis C infection. Hepatology 1999;29:908-14.  Back to cited text no. 7
    
8.
Thimme R, Oldach D, Chang KM, Steiger C, Ray SC, Chisari FV. Determinants of viral clearance and persistence during acute hepatitis C virus infection. J Exp Med 2001;194:395-1406.  Back to cited text no. 8
    
9.
Scott JD, Gretch DR. Molecular diagnostics of hepatitis C virus infection: A systematic review. JAMA 2007;297:724-32.  Back to cited text no. 9
    
10.
Miller FD, Abu-Raddad LJ. Evidence of intense ongoing endemic transmission of hepatitis C virus in Egypt. Proc Natl Acad Sci USA 2010;107:14757-62.  Back to cited text no. 10
    
11.
Green MR, Sambrook J. Molecular Cloning: A Laboratory Manual. 4th ed. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2012.  Back to cited text no. 11
    
12.
Chamberlain RW, Adams N, Saeed AA, Simmonds P, Elliott RM. Complete nucleotide sequence of a type 4 hepatitis C virus variant, the predominant genotype in the Middle East. J Gen Virol 1997;78:1341-7.  Back to cited text no. 12
    
13.
Tautz D, Renz M. An optimized freeze-squeeze method for the recovery of DNA fragments from agarose gels. Anal Biochem 1983;132:14-9.  Back to cited text no. 13
    
14.
Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, et al. Measurement of protein using bicinchoninic acid. Anal Biochem 1985;150:76-85.  Back to cited text no. 14
    
15.
Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 1979;76:4350-4.  Back to cited text no. 15
    
16.
Parker SP, Cubitt WD, Ades AE. A method for the detection and confirmation of antibodies to hepatitis C virus in dried blood spots. J Virol Methods 1997;68:199-205.  Back to cited text no. 16
    
17.
Ait-Goughoulte M, Hourioux C, Patient R, Trassard S, Brand D, Roingeard P. Core protein cleavage by signal peptide peptidase is required for hepatitis C virus-like particle assembly. J Gen Virol 2006;87:855-60.  Back to cited text no. 17
    
18.
Nelson DR, Marousis CG, Davis GL, Rice CM, Wong J, Houghton M, et al. The role of hepatitis C virus-specific cytotoxic T lymphocytes in chronic hepatitis C. J Immunol 1997;158:1473-81.  Back to cited text no. 18
    
19.
Pawlotsky JM. Use and interpretation of hepatitis C virus diagnostic assays. Clin Liver Dis 2003;7:127-37.  Back to cited text no. 19
    
20.
Bräutigam J, Scheidig AJ, Egge-Jacobsen W. Mass spectrometric analysis of hepatitis C viral envelope protein E2 reveals extended microheterogeneity of mucin-type O-linked glycosylation. Glycobiology 2013;23:453-74.  Back to cited text no. 20
    
21.
Lesniewski R, Okasinski G, Carrick R, Van Sant C, Desai S, Johnson R, et al. Antibody to hepatitis C virus second envelope (HCV-E2) glycoprotein: A new marker of HCV infection closely associated with viremia. J Med Virol 1995;45:415-22.  Back to cited text no. 21
    
22.
Hüssy P, Faust H, Wagner JC, Schmid G, Mous J, Jacobsen H. Evaluation of hepatitis C virus envelope proteins expressed in insect cells for use as tools for antibody screening. J Hepatol 1991;26:1179-86.  Back to cited text no. 22
    
23.
Zaaijer HL, Vallari DS, Cunningham M, Lesniewski R, Reesink HW, van der Poel CL, et al. E2 and NS5: New antigens for detection of hepatitis C virus antibodies. J Med Virol 1994;44:395-7.  Back to cited text no. 23
    
24.
Zhou YH, Chen Z, Purcell RH, Emerson SU. Positive reactions on Western blots do not necessarily indicate the epitopes on antigens are continuous: Immunol Cell Biol 2007;85:3-8.  Back to cited text no. 24
    

Top
Correspondence Address:
Alyaa S. Abdel Halim
Department of Biochemistry, Faculty of Science, Ain Shams University, Abbassia, Cairo - 11566
Egypt
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJPM.IJPM_604_18

Rights and Permissions


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2]



 

Top
 
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Email Alert *
    Add to My List *
* Registration required (free)  


    Abstract
   Introduction
    Materials and Me...
   Results
   Discussion
    References
    Article Figures
    Article Tables

 Article Access Statistics
    Viewed87    
    Printed1    
    Emailed0    
    PDF Downloaded2    
    Comments [Add]    

Recommend this journal