| Abstract: | The ZstatFlu®‐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®‐II Test. The enzymatic reaction was carried out at 25°C and neutral pH, representing the optimum assay conditions for influenza types A and B viral neuraminidases. The results were outputted on a Polaroid? 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 25°C. In order to minimize false positives, the ZstatFlu®‐II Test was formatted with 0.3–0.4 Km substrate and freezing the test kit until use. The pH optimization of the ZstatFlu®‐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 © 2003 John Wiley & Sons, Ltd. |
