Dual-enzyme cascade-magnetic separation immunoassay for respiratory syncytial virus

A new immunoassay developed for the detection of respiratory syncytial virus (RSV) makes use of magnetic separation and amplification by a dual-enzyme cascade for signal generation. Magnetic particles are conjugated to monoclonal anti-RSV antibodies through the heterobifunctional crosslinker sulfosuccinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate, yielding particles of high specific activity and low background. The dual-enzyme cascade is initiated by activation of a masked inhibitor for the enzyme rabbit liver esterase (RLE) by an alkaline phosphatase-antibody conjugate. The esterase activity is then measured to determine the degree of inhibition and, hence, the amount of specifically labeled antigen. These methods yield an immunoassay which is rapid and sensitive. Formation of the immunometric complex requires only 7 min and the total assay time is 40 min. The limit of detection for RSV fusion protein was found to be 1 ng or 10(-14) mol per test. Pre-clinical evaluation of the assay with 52 clinical specimens (nasal washes or aspirates) gave 96% sensitivity (25/26) and 96% specificity (25/26) with respect to a microtiter ELISA procedure and blocking antibody assay.