Development, characterization, and diagnostic applications of monoclonal antibodies against bovine rotavirus

Abstract

Hybridomas secreting monoclonal antibodies (MAbs) against the Nebraska calf diarrhea strain of bovine rotavirus (BRV) were characterized. Indirect fluorescent-antibody assay, immunodot assay, and immunoprecipitation were used to select hybridomas that produced anti-BRV MAbs. Seven of the MAbs were shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot assay to be reactive with the BRV outer capsid protein, VP7, which has a molecular mass of 37.5 kDa. None of the seven MAbs were reactive with canine rotavirus, bovine coronavirus, or uninfected Madin-Darby bovine kidney cells. Two clones, 8B4 (immunoglobulin G2a [IgG2a]) and 2B11 (IgG1), were found suitable for use in an antigen capture enzyme-linked immunosorbent assay for detecting BRV in bovine fecal samples. Both were subtype A specific (G6 subtype) but did not react with all isolates of BRV group A.

FIG. 1

Immunoprecipitation of BRV-infected cytoplasmic cell lysate. MDBK cells were infected with BRV, and infected cytoplasmic extracts were immunoprecipitated with MAbs. The lysates were immunoprecipitated with MAbs 2B11 (lane 6), 8B4 (lane 5), 5F9 (lane 4), and 5H8 (lane 3). Noninfected cell lysate was used as a negative control (lane 2), and polyclonal serum against BRV was used as a positive control (lane 1).