BUXTON R, EDWARDS B, JUO RR, VOYTA JC, TISDALE M, BETHELL R; Interscience Conference on Antimicrobial Agents and Chemotherapy.

Abstr Intersci Conf Antimicrob Agents Chemother Intersci Conf Antimicrob Agents Chemother. 1999 Sep 26-29; 39: 421 (abstract no. 286).

Glaxo Wellcome, Stevenage, UNITED KINGDOM

BACKGROUND: Determination of the sensitivity of influenza viruses to neuraminidase (NA) inhibitors is based presently on assays of NA function because, unlike available cell culture methods, the results of such assays are predictive of susceptibility in vivo. A rapid assay with improved sensitivity is required because a proportion of clinical isolates have insufficient NA to be detectable in the current fluorogenic assay, and because some mutations associated with resistance to NA inhibitors reduce the activity of the enzyme.METHODS: A chemiluminescence based assay of neuraminidase activity has been developed that uses a 1,2-dioxetane derivative of sialic acid (NA-STAR) as the substrate. The limit of detection of influenza virus neuraminidase with NA-STAR as the substrate has been compared with that for the fluorogenic substrate using purified neuraminidase from influenza B/Beijing/1/87. A panel of isolates from phase 2 clinical studies of zanamivir, which were undetectable in the fluorogenic assay, were tested for activity using the NA-STAR substrate. A comparison of sensitivity to zanamivir of influenza A and B virus NAs using the two assay methods has been performed using a panel of recent isolates.RESULTS: When compared with the fluorogenic assay, use of the NA-STAR substrate results in a 67-fold reduction in the limit of detection of the neuraminidase assay, from 200pM (11 femtomoles) to 3pM (0.16 femtomoles). Of the 12 isolates with undetectable neuraminidase activity from NAIA/B 2008, 10 (83%) were found to have detectable NA activity using the NA-STAR substrate. IC[50] values for zanamivir using the NA-STAR were in the range 1.0-7.5nM and those for the fluorogenic assay in the range 1.0-5.7nM (n=6).CONCLUSION: The NA-STAR assay is a highly sensitive, rapid assay of influenza virus neuramini dase, which is applicable to the monitoring of the susceptibility to NA inhibitors of clinical isolates of influenza viruses.

Publication Types:

• Meeting Abstracts

Keywords:

• Biological Assay
• Chemiluminescent Measurements
• Influenza Vaccines
• N-Acetylneuraminic Acid
• Neuraminidase
• Orthomyxoviridae
• Sialic Acids
• Zanamivir
• analysis
• antagonists & inhibitors

Other ID:

• GWAIDS0008116

UI: 102245613

From Meeting Abstracts