Standardization and application of immunofluorescence and immuno- peroxidase tests for detection of bluetongue virus antigen

Abstract

India is enzootic for bluetongue (BT), a predominant disease of small ruminants. The most important task in the control of disease is rapid and sensitive detection of virus. The present study was undertaken to standardize immunofluorescence (IFT) and immunoperoxidase tests (IPT) employing BTV serogroup specific VP7 monoclonal antibodies (MAbs), polyclonal homologous, and polyclonal heterologous antisera against specific serotypes of BTV for detection of BTV antigen. Serial tenfold dilutions of BTV-9 were tested for limit of detection (LoD) of IFT, IPT, and molecular assays by using MAbs against VP7, homologous anti-BTV-9 serum, and heterologous anti-BTV-16 serum. LoD of IFT was found to be 101 TCID50/mL using MAbs against VP7, anti BTV-9 serum, and anti BTV-16 serum. LoD of IPT was found to be 101 TCID50/mL, 102 TCID50/mL, and 102 TCID50/mL using MAbs against VP7, anti BTV-9 serum and anti BTV-16 serum, respectively. LoD of RT-PCR was 101 TCID50/mL and that of real time PCR was 100 TCID50/mL. This standardized assay was then applied for BTV detection in BTV suspected field samples collected from BT outbreaks followed by confirmation with virus isolation and NS3 group specific PCR. The current study shows that IFT and IPT are specific tests for group specific BTV identification. For IFT, monoclonal and polyclonal (homologous and heterologous) source of antibodies had similar sensitivity in the ability of BTV detection; whereas the most sensitive mode of detection by IPT was with MAbs.