For each sample, normalized pps were diluted 1∶1 in NA-Star Assay buffer (the same concentration as in the infection assay) then incubated with 10 µL of NA-Star substrate for 30 min at room temperature.
Twenty-five µl of each drug dilution was combined with 25 µl diluted viruses (PR8, NC, NY, WSN, H274Y and B) or recombinant NA proteins [recombinant N1 protein (influenza A/Beijing/262/95) and N4 protein (influenza A/Gray Teal/Aus/2/79) (Protein Sciences Corporation, Meriden, CT, USA)] in NA-Star assay buffer.
Briefly, serial dilutions of ATA, AH and NAA ranging from 2000 µg/ml to 0.002 µg/ml were prepared in NA-Star assay buffer, i.e. 26 mM MES (2-[N-Morpholino] ethanesulfonic acid), pH 6.0, 4 mM CaCl2, such that dilutions were double the final concentration.
For each sample, normalized pseudoparticles were diluted 1∶1 in NA-Star Assay buffer, incubated with 10 µL of NA-Star chemiluminescent substrate for 30 min at room temperature, and analyzed using a luminometer (2103 Envision Multilabel Reader; Perkin-Elmer, Waltham, MA).
The NA-Star assay was applied to determine the IC50 of NA enzymatic activity of SH5190 and SH5190 R292K in the presence of three clinically available NAIs: oseltamivir carboxylate, zanamivir and peramivir.
Briefly, 25 µL of half-log dilutions of neuraminidase inhibitors (0.03 1000 nM) in NA-Star Assay buffer were added to each well of a white 96-well microplate plate, 25 µL of virus dilution was added and plates were pre-incubated at 37 °C for 20 min.
Study Design: The luteinizing-granulosa cells of 6 patients were isolated after follicular aspiration and assayed for StAR expression.
In vitro susceptibility of virus isolates to OC were tested by a chemiluminiscent neuraminidase inhibition assay (NA-Star, Applied Biosystems).
After 48 h of infection, β-gal activity was measured with the Tropix Galacto-Star chemiluminescent reporter gene assay (Applied Biosystems Inc, Bedford, MA) according to the manufacturer's guidelines.
ß-galactosidase reporter enzyme activity in the two-hybrid strains was measured using the Galacto-Star™ chemiluminescent reporter assay system according to the manufacturer's instructions (Applied Biosystems, Foster City, California), except that cells were lysed by glass bead disruption.
In vitro susceptibility of virus isolates to oseltamivir carboxylate was determined by a chemiluminiscent neuraminidase inhibition assay (NA-Star, Applied Biosystems) and to zanamivir by fluorescent neuraminidase inhibition assay, as described previously [12], [13].
