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J Immunoassay Immunochem. 2016 Feb 1. [Epub ahead of print]
EXPRESSION AND PURIFICATION OF RECOMBINANT H5HA1 PROTEIN OF H5N1 AVIAN INFLUENZA VIRUS IN E. coli AND ITS APPLICATION IN INDIRECT ELISA.
Redda YT1, Venkatesh G1, Kalaiyarasu S1, Bhatia S1, Senthil Kumar D1, Nagarajan S1, Pillai A1, Tripathi S1, Kulkarni DD1, Dubey SC1.
Author information
Abstract
The PCR amplified HA1 fragment of H5N1 (H5HA1) avian influenza virus (AIV) haemagglutinin gene was cloned into pET28a (+) expression vector and expressed in Rosetta Blue (DE3) pLysS cells. The recombinant H5HA1 (rH5HA1) protein purified by passive gel elution after SDS-PAGE of the inclusion bodies reacted specifically with H5N1 serum in Western blot analysis. A subtype specific indirect enzyme linked immunosorbent assay (iELISA) using the rH5HA1 protein as the coating antigen was developed for detecting antibodies to H5 subtype of AIV. The assay had 89.04% sensitivity and 95.95% specificity when compared with haemagglutination inhibition test. The Kappa value of 0.842 indicated a perfect agreement between the tests. The iELISA developed can be used for serosurveillance of avian influenza in chickens.
KEYWORDS:
AIV H5N1; characterization; expression; haemagglutinin; indirect ELISA
PMID: 26829111 [PubMed - as supplied by publisher]
EXPRESSION AND PURIFICATION OF RECOMBINANT H5HA1 PROTEIN OF H5N1 AVIAN INFLUENZA VIRUS IN E. coli AND ITS APPLICATION IN INDIRECT ELISA.
Redda YT1, Venkatesh G1, Kalaiyarasu S1, Bhatia S1, Senthil Kumar D1, Nagarajan S1, Pillai A1, Tripathi S1, Kulkarni DD1, Dubey SC1.
Author information
Abstract
The PCR amplified HA1 fragment of H5N1 (H5HA1) avian influenza virus (AIV) haemagglutinin gene was cloned into pET28a (+) expression vector and expressed in Rosetta Blue (DE3) pLysS cells. The recombinant H5HA1 (rH5HA1) protein purified by passive gel elution after SDS-PAGE of the inclusion bodies reacted specifically with H5N1 serum in Western blot analysis. A subtype specific indirect enzyme linked immunosorbent assay (iELISA) using the rH5HA1 protein as the coating antigen was developed for detecting antibodies to H5 subtype of AIV. The assay had 89.04% sensitivity and 95.95% specificity when compared with haemagglutination inhibition test. The Kappa value of 0.842 indicated a perfect agreement between the tests. The iELISA developed can be used for serosurveillance of avian influenza in chickens.
KEYWORDS:
AIV H5N1; characterization; expression; haemagglutinin; indirect ELISA
PMID: 26829111 [PubMed - as supplied by publisher]