H.K. Pradhan

Development of a capsid based competitive inhibition enzyme-linked immunosorbent assay for detection of bovine immunodeficiency virus antibodies in cattle and buffalo serum.

The aim of this study was to develop a more specific and sensitive competitive inhibition ELISA (CI-ELISA) than the currently used indirect ELISA for detection of antibodies to bovine immunodeficiency virus (BIV) in cattle and buffaloes. Murine monoclonal antibodies (MAbs) were generated against a recombinant capsid (CA) protein of bovine immunodeficiency virus. Of the 13 anti-CA MAbs developed, MAb-9G10 was selected for CI-ELISA based on the maximum inhibition (98%) obtained with reference BIV antibody positive serum. Based on the distribution of percent inhibition of known negative sera (n=50), a cut-off value was set at 40% inhibition. The MAb-based CI-ELISA showed much higher agreement (concordance: 95.4%) than the indirect ELISA (concordance: 77.8%) with Western blot. Out of 672 sera of cattle and buffaloes tested by CI-ELISA from four states of India, 22% (113/516) of cattle and 19% (30/156) of buffalo were sero-positive for BIV with an overall seroprevalence of 21% (143/672) in India.

Single-chain fragment variable antibody against the capsid protein of bovine immunodeficiency virus and its use in ELISA.

Recombinant antibody specific for the capsid (CA) protein of bovine immunodeficiency virus (BIV) was generated in the form of single-chain fragment variable (ScFv) using the phage display technique for affinity selection. The variable heavy (V(H)) and variable light (V(L)) chain gene fragments were recovered from cells of CA-specific hybridoma (9G10) described previously. The V(H) and V(L) DNA fragments were assembled through a flexible linker DNA to generate ScFv fragment which was cloned in a phagemid expression vector to express ScFv protein. The specific reactivity of the expressed ScFv to the CA antigen was confirmed by Western blot, and the ScFv fragment was used to develop a competitive inhibition ELISA for detection of antibodies to BIV in cattle and buffalo. The recombinant antibody was shown to be more than four times sensitive than its parent monoclonal antibody (MAb, 9G10) by testing of spiked samples of reference positive sera. The improved sensitivity of the recombinant antibody-based ELISA was confirmed by the detection of a larger proportion of animals with BIV antibody by it than by the MAb-based ELISA.

Development of single-chain Fv against the nucleoprotein of type A influenza virus and its use in ELISA

Single chain fragment variable (ScFv) antibodies specific to the nucleoprotein (NP) of avian influenza virus (AIV) were developed using a phage display system. The variable heavy (VH) and the variable light (VL) chain gene fragments were derived from spleen cells of Balb/c mouse immunized with a recombinant NP (rNP) antigen (∼63 kDa) of H5N1 influenza virus. The VH and the VL DNA fragments were assembled through a flexible linker DNA to generate ScFv DNA that was cloned subsequently in a phagemid to express ScFv protein in Escherichia coli cells. The specific reactivity of the ScFv with the rNP antigen and viral antigen (H5N1) was confirmed by Western blot and ELISA. A competitive inhibition ELISA (CI-ELISA) was developed using the rNP and the anti-NP ScFv for detection of type-specific antibodies to AIV in chicken sera. The ScFv based CI-ELISA was compared with hemagglutination inhibition (HI) test and agar gel immunodiffusion (AGID) test over 850 sera. Sensitivity of the CI-ELISA was 100% with HI and AGID and specificity was 98.7% with HI and 100% with AGID.

Genetic Analysis and Expression of NS3 Gene of Bovine Viral Diarrhoea Virus 1 from India for Detection of Antibodies in Cattle

Abstract Mishra, N., Pitale, S.S. and Pradhan, H.K. 2008. Genetic analysis and expression of NS3 gene of bovine viral diarrhoea virus 1 from India for detection of antibodies in cattle. J. Appl. Anim. Res., 33: 99–103. Considering the importance of NS3 antigen in diagnosis of bovine viral diarrhea virus (BVDV) infection, we analysed genetically selected Indian isolates in NS3 gene region and generated recombinant NS3 protein in Escherichia coli for its use as ELISA antigen. High degree of conservation was observed when the nucleotide and deduced amino acid sequence of Indian and the reference BVDV 1 isolates were analysed. The helicase domain of NS3 gene of an Indian isolate was cloned into pTriEx-2-Neo expression vector and a recombinant protein of 50 KDa was expressed as cytoplasmic inclusion bodies. The affinity purified protein showed antigenic properties in western blot with BVDV infected cattle serum. When used in ELISA, it could detect and BVDV antibodies in bovine sera. The expression of NS3 recombinant protein showing immunological and diagnostic significance is first such study in India.