ZstatFlu®-II test:: a chemiluminescent neuraminidase assay for influenza viral diagnostics

The ZstatFlu(R)-II test is a highly sensitive, specific, rapid, point-of-care chemiluminescent diagnostic test for influenza infection. Influenza viral neuraminidase-specific substrate, spiroadamantyl-1,2-dioxetane-4,7-dimethoxy-N-acetyl-neuraminic acid, is at the core of the ZstatFlu(R)-II Test. The enzymatic reaction was carried out at 25degreesC and neutral pH, representing the optimum assay conditions for influenza types A and B viral neuraminidases. The results were outputted on a Polaroid(TM) High Speed Detector Film. Positive results appeared as a '+'-shaped white film image; negative results produced no image. The 'glow' kinetics, facilitated by a unique combination of light enhancers, also 'tuned' the wavelength of emission to match the spectral properties of the film. The substrate hydrolysed non-enzymatically at acid pH or at temperatures above 25degreesC. In order to minimize false positives, the ZstatFlu(R)-II Test was formatted with 0.3-0.4 K-m substrate and freezing the test kit until use. The pH optimization of the ZstatFlu(R)-II test is discussed with reference to model compounds of sialyl-glycosides. A nucleophilic attack or an electrostatic stabilization of a developing carbonium ion under the influence of the adjacent carboxyl group was probably responsible for non-enzymatic hydrolysis of the substrate. Intramolecular general acid catalysis is proposed as a mechanism for the lability of the O-glycosidic linkage of the substrate. Copyright (C) 2003 John Wiley Sons, Ltd.