Application of Elisa in the diagnosis of rotavirus infections in buffalo calves

Abstract

The conditions for diagnosis of group A rotavirus infection in buffalo calves by enzyme linked immunosorbent assay (ELISA) were optimised in terms of type of microtitre plates, all reagents and the cut off points for positivity. Irradiated polystyrene plates were the plates of choice. The optimal dilution for the clinical samples (faecal extracts), capture and detector antibodies and the enzyme conjugate were I : 10, I : 5,000, I : 10,000 and I : 300, respectively. Further, we found that the cut off point for positivity by the screening ELISA was an optical density (OD) of ≥ 0.170 at 450 nm wave length, and for confirmation when blocking activity was ≥ 30%. 'The positivity of a faecal sample was graded as strongly positive if the PIN value was ≥2.7 by screening ELISA and ≥ 50% blocking activity in the confirmatory blocking ELISA. Samples having PIN value < 2.7 but ≥ 2. I and < 50% but ≥ 30% blocking activity were regarded as weakly positive. in addition, pre and post colostral buffalo sel'd as negative and positive control sera respectively, were selected and used for detection of antirotavlral antibodies by the blocking ELISA. 'This study establishes that the ELISA technique can be profitably used (once required parameters are defined), in the diagnosis of rotavirus infection in buffalo calves.