An antigen-capture ELISA detects pestivirus antigens in blood and tissues of immunotolerant carrier cattle.

An antigen-capture ELISA was developed for the detection of pestivirus-specific antigens in peripheral blood leucocytes (PBLs), blood clots and tissue samples of immunotolerant cattle persistently infected with virus. The ELISA demonstrated complete agreement with conventional virus isolation procedures undertaken on specimens from a total of 58 carrier animals and 360 uninfected animals. The technique is based on capturing antigen with a high-titred goat polyclonal antiserum and detecting the bound antigen with a combination of 3 broadly-reactive monoclonal antibodies. Increased sensitivity was obtained with the use of an avidin-biotin complex (ABC) amplification method. On average, ELISA optical densities (ODs) for PBL and blood clot samples derived from carrier animals were 1.53 and 0.95, respectively, while uninfected animals had corresponding values of less than 0.15 for all blood samples. Tissue samples from carrier cattle had OD values ranging from an average of 0.95 for liver to 1.77 for spleen, with negative values for all tissues again averaging less than 0.20. Signal-to-noise (S/N) ratios calculated from the ELISA OD readings for carrier cattle showed an average of 15.6 for blood samples and 16.4 for tissues. In contrast, all samples from negative cattle had S/N ratios less than 2.0. The antigen-capture ELISA has been validated on field samples and is suitable for routine diagnostic and certification testing.