ZstatFlu-II test: a chemiluminescent neuraminidase assay for influenza viral diagnostics.
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 degrees C and neutral pH, representing the optimum assay conditions for influenza types A and B viral neuraminidases. The results were outputted on a Polaroid trade mark 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 degrees C. In order to minimize false positives, the ZstatFlu-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-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.
Achyuthan
Komandoor E
KE
QTL Biosystems, Santa Fe, NM 87507, USA. achyuthan@qtlbio.com
Pence
Lisa M
LM
Appleman
James R
JR
Shimasaki
Craig D
CD
D006571:Heterocyclic Compounds / Q000737:chemistry; D006573:Heterocyclic Compounds, 1-Ring; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D009980:Influenza A virus / Q000201:enzymology* / Q000302:isolation & purification; D009981:Influenza B virus / Q000201:enzymology* / Q000302:isolation & purification; D007251:Influenza, Human / Q000175:diagnosis* / Q000821:virology; D007700:Kinetics; D008163:Luminescent Measurements; D009297:Nasal Mucosa / Q000821:virology; D009439:Neuraminidase / Q000032:analysis*; D010614:Pharynx / Q000821:virology; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity; D012972:Sodium Hydroxide; D013379:Substrate Specificity; D013696:Temperature; D014773:Virology / Q000295:instrumentation / Q000379:methods
D006571:Heterocyclic Compounds; D006573:Heterocyclic Compounds, 1-Ring; D011933:Reagent Kits, Diagnostic; C079142:1,2-dioxetane; D012972:Sodium Hydroxide; D009439:Neuraminidase