Application of enzyme-linked immunosorbent assay for the diagnosis of Infectious Bovine Rhinotracheitis and Bovine Viral Diarrhea

Abstract

Enzyme-linked immunosrobent assays (ELISA) were investigated for the quantitation of immunoglobulin G (IgG) and immunoglobulin M (IgM) to Infectious Bovine Rhinotracheitis (IBR) and Bovine Viral Diarrhea (BVD) viruses, and for the detection of IBR and BVD viruses. Sera from calves experimentally inoculated with IBR virus (IBRV) or BVD virus (BVDV) and 100 field-collected sera were assayed by ELISA, complement fixation, (CF), and virus neutralization (VN) tests for antibodies against IBRV and BVDV. The time-kinetics study of ELISA for the quantitation of IgG to IBRV and BVDV showed that the absorbance values kept increasing for the first 100 minutes until they reached their maximum and remained stable at 105 to 120 minutes. The specificity of the ELISA was demonstrated by blocking IBRV positive control calf serum with IBRV-specific antiserm produced in a rabbit. In the ELISA for quantitation of IgG to BVDV, the specificity could not be demonstrated by the blocking test. Formulations for calculating results in terms of signal to noise (S/N) ratio were determined. The optimal concentration for quantitation of IgG to IBRV by ELISA in this study was 3.0 ug/ml by using purified IBRV antigen or a 1:32 dilution of crude IBRV antigen. The optimal reading was accomplished at 30 minutes after the substrate was added. By using crude IBRV antigen, the correlation, sensitivity, and specificity values in relation to the VN test were 94, 96, and 88%, respectively. For practical and economical reasons, using crude IBRV antigen is probably preferable in most instances. The optimal concentration for quantitation of IgG to BVDV by ELISA in this study was 2.0 ug/ml of purified BVDV viral antigen. The optimal reading was accomplished at 2 hours after the substrate was added. Its correlation, sensitivity, and specificity values in relation to VN test were 98, 99, and 83%, respectively. ELISA for quantitation of IgM to IBRV or BVDV was hampered by nonspecific bindings between the bovine serum tested and rabbit antiserum to IBRV or BVDV, respectively. ELISA for the detection of IBRV was found to be equally sensitive to electron microscopy, but less sensitive than virus isolation method. ELISA checkerboard test for the detection of BVDV indicated that this technique was much less sensitive than the conventional cell culture virus isolation method.