Sensitive fluorogenic enzyme immunoassay on nitrocellulose membranes for quantitation of virus

Abstract

A highly sensitive fluorogenic enzyme-linked immunosorbent assay (FELISA), which utilizes nitrocellulose membranes as solid phase support, has been developed for the detection and identification of virus in clinical samples. Reagents were standardized and, using purified Newcastle disease virus (NDV) as a model, the theoretical lower limits of test sensitivity of the FELISA were compared, in both “sandwich” and “indirect” formats, to those of a comparable chromogenic enzyme-linked immunosorbent assay (CELISA). Of the systems evaluated, the “sandwich” FELISA exhibited maximum sensitivity and detected 10 fg of purified virus protein per milliliter of test sample (500 ag per test volume). Specificity of the “sandwich” FELISA was evaluated by challenging the system with heterologous strains of NDV and with other serologically related and unrelated viruses. In a clinical trial in which fecal materials from chickens undergoing vaccination with NDV were assayed directly by FELISA, the virus was detected from the first to approximately the tenth day post-vaccination. The test is simple to perform and results can be obtained in approximately 4 h.