Ultrasensitive Fluorogenic Reagents for Neuraminidase Titration.

Abstract

Influenza viral neuraminidase plays a crucial role during infections. It is a major target for the development of anti-influenza drugs and is also attracting increasing attention as a vaccine target as evidence accumulates that neuraminidase-neutralizing antibodies contribute to protection. However, no method currently exists to accurately and efficiently measure concentrations of active neuraminidase in virus samples or other crude mixtures, which hampers development on both fronts. In this report, we describe the development of a selective and sensitive active-site titration reagent for neuraminidase that can quantify viral neuraminidases down to sub-nanomolar levels in crude samples, with no background from non-viral neuraminidases. By using this reagent, we determined accurate kcat values for six influenza A and two influenza B neuraminidases for the first time. We also quantified the neuraminidase content in a commercial influenza vaccine, thus demonstrating that this titration reagent opens the possibility for better vaccine analysis.