A screening assay for neuraminidase inhibitors using neuraminidases N1 and N3 from a baculovirus expression system.

CONTEXT

Development of inexpensive and safe enzymatic assays to screen for putative neuraminidase inhibitors.

OBJECTIVE

Validate the use of recombinant neuraminidase expressed in baculovirus located on the viral surface capsule to develop a neuraminidase inhibitor screening assay.

MATERIALS AND METHODS

Recombinant baculovirus particles displaying neuraminidase N1 and N3 were used as enzyme sources. The assay set-up required the use of 2'-(4-methylumbelliferyl)-α-D-acetyl neuraminic acid as substrate and oseltamivir carboxylate as benchmark inhibitor.

RESULTS

The assay was set up in a standard 96-well plate. The within- and between-assay coefficients of variation were, on average, less than 10%. The 50% inhibitory concentration values of the inhibitor were in good agreement with those determined by independent kinetic experiments.

DISCUSSION AND CONCLUSIONS

The assay showed satisfactory within- and between-assay repeatability. The obtained results suggest that recombinant baculovirus expressing neuraminidase located on the virus membrane capsule can be used to set up affordable and reliable neuraminidase inhibitors screening assays.