Development of a Neutralization Assay for Influenza Virus Using an Endpoint Assessment Based on Quantitative Reverse-Transcription PCR
Figure 4
Influenza virus microneutralization assessed by qRT-PCR (qPCR-MN).
(A) An inoculum containing 1000 TCID50 of virus (Bris/07) was mixed with a dilution from a 2-fold dilution series of ferret antiserum in a well of a 96-well plate. After allowing the neutralization reaction to proceed for 1 hour at 37°C, trypsinized MDCK-London cells (30,000 per well) were added. TPCK-trypsin was present at 1 µg/mL. After 6 hours, experimental samples were prepared using SPR and subjected to qRT-PCR. The RNA copy numbers were normalized to the mean value obtained from infected wells in the absence of neutralizing serum (virus control wells). Each point represents the mean ± SEM (n = 3). The neutralization titer was defined as the reciprocal of the highest dilution factor of serum necessary to inhibit the PCR signal by 90%. (B) Same data as in (A); however, each experimental replicate was assessed independently. The mean of these curves would result in the curve depicted in (A).