Rapid detection of bovine herpesvirus type 1 antigens in nasal swab specimens with an antigen capture enzyme-linked immunosorbent assay

Abstract

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of bovine herpesvirus type 1 (BHV-1) antigens in nasal swab specimens collected from infected animals. Development of the ELISA involved screening and selection of BHV-1-specific monoclonal antibodies for their ability to capture BHV-1 antigens and for their stability and activity after conjugation to horseradish peroxidase. Forty combinations of capture-conjugate monoclonal antibody pairs were screened for detection of nanogram amounts of purified BHV-1 by using a double-antibody-sandwich ELISA in which antigen and conjugated antibody were simultaneously added to antibody-coated wells. Of the 40 monoclonal antibody pairs, 4 were analyzed further and 1 was selected for routine application to clinical specimens. Of 129 nasal swab specimens collected during the first 10 days after experimental infection with BHV-1, 66 were found to be positive by both virus isolation and ELISA and 34 were positive for infectious virus but negative by ELISA. One specimen was positive by ELISA but negative by virus isolation, and the remaining 28 specimens were negative by both tests. Quantitation of the virus-containing specimens showed that the ELISA had a lower detection limit of 10(3.5) median tissue culture infective doses. The ELISA was judged to be highly useful for diagnosis of BHV-1 infections, since all of the nasal swab specimens that were collected from 12 animals during the first 5 days of the infection, when the clinical signs were the most apparent, were positive.