Comparison between Phage-ELISA and Phage Dot-Blot Assay Methods for the Detection of Hepatitis B Surface Antigen and its Antibodies in Human Serum
DOI:
https://doi.org/10.6000/1927-3037.2014.03.04.3Keywords:
HBsAg, phage-ELISA, phage dot-blot assay, human sera.Abstract
A modified phage-enzyme link immunosorbent assay (phage-ELISA) and a phage dot-blot assay specific for hepatitis B surface antigen (HBsAg) and its antibody were developed by using phage display technology. The phage-ELISA and phage dot-blot assays enabled to detect HBsAg and anti-HBsAg in human sera, and compatible to commercial detection kit. The fusion phages were immobilized onto microtiter plate wells and nitrocellulose membrane sheets, then blocked with 10% milk diluent, and added with human serum at dilution of 1:5000. The absorbance at 405 nm was determined once the colour changes formed. The same human serum also applied on the commercial diagnostic kit for comparison. The statistical analysis was carried out using ANOVA and T Test (LSD) for variable comparison between phage-ELISA and phage dot-blot assays. Based on these studies, the phage-ELISA was found to be more sensitive compared to phage dot-blot assay as the detection of HBsAg in human sera was about 80% as compared to 51.7% by using phage dot-blot assay. Meanwhile, the sensitivity for detection of anti-HBsAg by using phage-ELISA was slightly higher which showed about 83.3%. However, the sensitivity of the assay was dropped almost half when using phage dot-blot assay. Therefore, they are practical to be used as a reliable alternative way for the detection HBsAg and its antibody in human sera.
References
Lee WM. Hepatitis B virus infection. New England J Med 1997; 337:1733-45. http://dx.doi.org/10.1056/NEJM199712113372406
Stevent CE, Toy PT, Tong MJ, Taylor PE, Vyas GN, Nair PV, Gudavalli M, Krugman S. Perinatal hepatitis B virus transmission in the United States. J Am Med Assoc 1985; 253: 1740-5. http://dx.doi.org/10.1001/jama.1985.03350360066020
Jung M-C, Pape GR. Immunology of hepatitis B infection. LANCET 2002; 2: 43-50. http://dx.doi.org/10.1016/S1473-3099(01)00172-4
Seitz, R. Hepatitis B virus. Infus Ther Transfus Med 2000; 27: 226-34. DOI: 10.1159/000025279.
Tsitsilonis OE, Thrasyvoulides A, Balafas A, Voutsas JF, Papamichail M, Lymberi P. Serological detection of hepatitis B viral infection by a panel of solid-phase enzyme-linked immunosorbent assays (ELISA). J Pharm Biomed Anal 2004; 34: 811-22. http://dx.doi.org/10.1016/S0731-7085(03)00563-6
Hollinger FB. Hepatitis B vaccines to switch or not to switch. J Am Med Assoc 1987; 257: 2634-6. http://dx.doi.org/10.1001/jama.1987.03390190112033
Tan WS, Tan GH, Yusoff K, Seow HF. A phage-displayed cyclic peptide that interacts tightly with the immunodominant region of hepatitis B surface antigen. Journal of Clinical Virology 2005; 34: 35-41. http://dx.doi.org/10.1016/j.jcv.2005.01.007
Tan GH, Yusof K, Seow HF, Tan WS. Antigenicity and Immunogenicity of the Immunodominant Region of Hepatitis B Surface Antigen Displayed on Bacteriophage T7. J Med Virol 2005; 77: 475-80. http://dx.doi.org/10.1002/jmv.20479
Hoofnagle TH, Dusheiko GM, Seeff LB, Jones EA, Waggoner JG, Bales ZB. Seroconversion from hepatitis B ‘e’ antigen to antibody in chronic type B hepatitis. Ann Intern Med 1981; 94: 744-8. http://dx.doi.org/10.7326/0003-4819-94-6-744
Rosenberg A, Griffin K, Studier W, McCormick M, Berg J, Novy R, Mierendorf R. T7 Select phage display system: a powerful new protein display system based on bacteriophage T7. Novations Novagen Newsletter 1996; 6:1-6. DOI: 10.3390/molecules19022481.
Guyatt GH, Oxman AD, Ali M, William A, Mcllroy W, Pattersow C. Laboratory diagnosis of iron deficiency anemia: an overview. J Gen Intern Med 1992. 7: 145-53. http://dx.doi.org/10.1007/BF02598003
Lowman HB, Bass SH, Simpson N, Wells JA. Selecting high-affinity binding proteins by monovalent phage display. Biochem 1991; 30: 10832-8. http://dx.doi.org/10.1021/bi00109a004
Soumillion P, Jespers L, Bouchet M, Marchand-Brynaert J, Winter G, Fastrez J. Selection of beta-lactamase on filamentous bacteriophage by catalytic activity. J Mol Biol 1994; 237: 415-22. http://dx.doi.org/10.1006/jmbi.1994.1244
Smith GP, Schultz DA, Ladbury JE. A ribonuclease S-peptide antagonist discovered with a bacteriophage display library. Gene 1993; 128: 37-42. http://dx.doi.org/10.1016/0378-1119(93)90150-2
Devlin JJ, Panganiban LC, Devlin PE. Random peptide libraries. A source of specific protein binding molecules. Science 1990; 249: 404-6. http://dx.doi.org/10.1126/science.2143033
Ramanujam P, Tan WS, Nathan S, Yusoff K. Selection of peptide inhibitors that inhibit the replication of Newcastle disease virus. Arch Virol 2002; 147: 981-93. http://dx.doi.org/10.1007/s00705-001-0778-y
Dyson MR, Germaschewski V, Murray K. Direct measurement via phage titer of the dissociation constants in solution of fusion phage-substrate complexes. Nucleic Acids Res 1995; 23:1531-5. http://dx.doi.org/10.1093/nar/23.9.1531
Ho KL, Yusoff K, Seow HF, Tan WS. Selection of high affinity binding phages on hepatitis B core particles with a disulfide constrained phage display library. J Med Virol 2003; 69:27-32. http://dx.doi.org/10.1002/jmv.10266
Barbas C, Kang A, Lerner R. and Benkovic S. Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc Nat Acad Sci USA 1991; 88: 7978-82. http://dx.doi.org/10.1073/pnas.88.18.7978
Clackson T, Hoogenboom HR, Griffiths AD, Winter G. Making antibody fragment using phage display libraries. Nature 1991; 352: 624-8. http://dx.doi.org/10.1038/352624a0
McCafferty J, Griffiths AD, Winter G, Chiswell DJ. Phage antibodies: filamentous phage displaying antibody variable domains. Nature 1990; 348: 552-4. http://dx.doi.org/10.1038/348552a0
Downloads
Published
How to Cite
Issue
Section
License
Policy for Journals/Articles with Open Access
Authors who publish with this journal agree to the following terms:
- Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution License that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.
- Authors are permitted and encouraged to post links to their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work
Policy for Journals / Manuscript with Paid Access
Authors who publish with this journal agree to the following terms:
- Publisher retain copyright .
- Authors are permitted and encouraged to post links to their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work .
