Hrp conjugated antibody specific for monkey igg

Nunc MaxiSorp Elisa plates were coated with 1 μg/well of PR8 in sodium carbonate/bicarbonate coating buffer (pH 9.5) overnight at 4 °C. Plates were blocked with 1X Blocking Buffer (10X Casein Blocking Buffer, Sigma-Aldrich, St. Louis, MO, USA) plus 2% goat serum (Lampire Biologicals, Pipersville, PA, USA) for 1 h and then washed. The wash buffer contained PBS with 0.1% Tween 20. Plasma samples were serially diluted in 1X blocking buffer. Wells that contained no virus served as a negative control. Horseradish peroxidase (HRP)-conjugated antibody specific for monkey IgG (Fitzgerald, Acton, CA, USA) or IgM (LifeSpan Biosciences, Seattle, WA, USA) was used to detect bound antibodies. Plates were developed using 3,3′,5,5′-Tetramethylbenzidine dihydrochloride (TMB)(Sigma-Aldrich, St. Louis, MO, USA) and read at 450 nm on a Elx800 Absorbance Microplate Reader (BioTek, Winooski, VT, USA). For each dilution, the OD from the non-virus-coated wells was subtracted from the virus-coated wells. The threshold titer was defined as the value that reached 3X the assay background, i.e., wells that only received 1X blocking buffer + virus (no sample). Fold change was determined by dividing the threshold titer at each given time point by the d0 threshold titer.

Avidity assays were performed as the ELISAs described above with the addition of a NaSCN dissociation step following sample incubation. To normalize the total amount of antibody in the assay, the plasma dilution used was determined for each animal based on the dilution that yielded 50% of the MAX OD450 in the ELISA binding curve. Following incubation with plasma, 2-fold dilutions of NaSCN starting at 5M were added to each plate for 15 min. Plates were then washed, and the HRP-conjugated antibody specific for monkey IgG (Fitzgerald) was used to detect bound antibodies and developed like the ELISA assay. The 50% maximum inhibitory concentration (IC₅₀) was calculated using GraphPad Prism software.