Development of spot enzyme immunoassay for Bluetongue Virus (BTV) antibodies

Spot enzyme immunoassay (SEI) is a highly versatile solid phase immunoassay for antibody or antigen detection of Bluetongue (BT). This assay uses a minute amount of reagent dotted onto solid surface such as nitrocellulose membrane (NCM) or other membrane bound to plastic stick known as dipstick. After incubation with antigen specific antibody, enzyme conjugate followed by addition of a precipitable chromogenic substrate, the formation of coloured dot on the solid phase is visually read. Bluetongue is an infectious, non-contagious, insect borne viral emerging disease of ruminants and has been reported from most of the tropical and subtropical regions of the world. Bluetongue virus (BTV) belongs to genus Orbivirus of the family Reoviridae, consisting of 10ds RNA viral genome segments. The work was planned to monitor BTV infection and to develop diagnostic assay for easy, rapid, reliable and economic detection of BTV in bovines. The spot enzyme immunoassay (SEI) is the doorstep method. A total of 395 serum samples collected from seven districts of Madhya Pradesh (MP) were tested for detection of group-specific BTV antibodies using cELISA. Out of 395 samples, 100 bovine sera samples categorized into strong positive, weak positive and negative based on cELISA were used for the development and assessment of SEI. Out of 100 bovine sere samples, 79 samples were found positive and 21 samples were found negative for BTV by cELISA and 63 samples were found positive and 37 samples were found negative for BTV by SEI. The best results were found with BTV VP7 recombinant antigen, skimmed milk 2.5% and gelatin 1% as blocking solution, sera dilution of 1:80 and 1:800 conjugate dilution.