Sensitive avidin-biotin amplified fluorogenic enzyme immunoassay using biotinylated monoclonal antibodies for the identification and quantitation of virus

A highly sensitive amplified fluorogenic enzyme-linked immunosorbent assay (FELISA), which utilizes the high affinity interaction of the vitamin biotin for the multiple binding sites on the glycoprotein avidin, was developed for the detection and identification of a model virus, Newcastle disease virus (NDV). Monoclonal antibodies (MCA) directed against the virus were purified and labelled with biotin. Biotinylated MCA was then used with avidin-labelled enzyme and a fluorogenic substrate to detect NDV adsorbed directly on nitrocellulose membranes. Reagents were standardized and, using purified virus, the theoretical lower limit of test sensitivity of the amplified FELISA was determined to be 1 fg/ml of test sample (50 ag/well). The specificity of the amplified FELISA was evaluated by challenging the assay system with homologous and heterologous strains of NDV, and with other serologically related and unrelated viruses. The test was simple to perform and multiple samples could be conveniently assayed with results obtainable in 3–4 h.