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. 2002 Nov 12;73(1):177–188. doi: 10.1016/0022-1759(84)90043-7

Western and dot immunoblotting analysis of viral antigens and antibodies: Application to murine hepatitis virus

Pierre J Talbot 1,1, Robert L Knobler 1, Michael J Buchmeier 1,2
PMCID: PMC7131576  PMID: 6208281

Abstract

Viral proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred quantitatively to nitrocellulose by electroblotting in SDS-containing buffer. Monoclonal antibodies directed against previously defined epitopes on the viral proteins were used as probes to detect viral protein synthesis and processing, as well as expression in animal tissues. Circulating polyclonal antibodies were also probed and characterized for their polypeptide specificities. Under appropriate conditions, this Western immunoblotting technique was quantitative. Finally, a highly sensitive dot immunoblotting assay was used to analyze the sensitivity to denaturation of various epitopes on the viral proteins. This assay detected picogram quantities of viral antigens and antibodies.

Keywords: immunoblotting, dot blot, nitrocellulose replica, antigenic analysis, antibodies, virus

Abbreviations: SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; MHV-4, JHM strain of murine hepatitis virus-4; DBM, diazobenzyloxymethyl; N, nucleocapsid; Mr, relative molecular weight; CPE, cytopathic effect; PBS, Dulbecco's phosphate-buffered saline, pH 7.4; MAb, monoclonal antibodies; PFU, plaque-forming units; LD50, lethal dose for 50% of animals; BSA, bovine serum albumin; m.o.i., multiplicity of infection; Ig, immunoglobulin; cpm, counts per min; NGS, normal goat serum; NP-40, Nonidet P40; EDTA, ethylene diamine tetraacetic acid; PMSF, phenyl methyl sulfonyl fluoride

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