1 step abts

Immunogenicity of the vaccines was determined by enzyme-linked immunosorbent assay (ELISA) of sera samples collected on Day 35. HA1 and HA0 influenza B HA recombinant antigens were applied to 96-well plates and incubated overnight at 4°C (1.25 μg/mL). Plates were washed (405 TS ELISA Plate Washer, Agilent Technologies) with PBS plus 0.1% Tween 20 between all steps. After blocking (1% Omniblok, AmericanBio, Inc., and 0.1% Tween 20 in PBS), serum samples from vaccinated mice were plated at a 1:125 dilution followed by two-fold dilutions. Plates were then incubated at 37°C with a HRP conjugated secondary antibody (goat anti-mouse IgG (H+L), Thermo Fisher Scientific). Colorimetric detection occurred at room temperature and measured for absorbance at 405 nm (1-Step ABTS, Thermo Fisher Scientific). Endpoint titers levels were statistical defined per plate (31 (link)) and analysis of variance (ANOVA) tests were conducted with Tukey’s multiple comparisons test to determine group differences.

Antigen was applied to 96-well plates and incubated overnight at 4°C (1.25 μg/mL), following PBS washes and blocking (1% Omniblok, AmericanBio, Inc. and 0.1% Tween 20 in PBS), primary antibody was incubated for 2 hr at room temperature (serum samples were initially diluted 1:1000). Primary monoclonal antibodies were stocks at 1mg/ml and for ELISAs primary mAbs C179, FI6v3, and CR6261 was used at 0.5 μg/ml while discovered antibodies mAb-11, 49, 63, 67, 85 were tested at both 0.5 and 5 μg/ml as primary antibodies. Plates were washed and placed at 37°C for 1 hr with the addition of an HRP conjugated secondary antibody (goat anti-mouse IgG (H+L), Thermo Fisher Scientific). Colorimetric detection occurred for 15 min at room temperature and absorbance was read at 405 nm (1-Step ABTS, Thermo Fisher Scientific). Samples were run in quadruplicate. Endpoint titers levels were statistical defined per plate using saline sera to determine thresholds [95 (link)]. Statistical calculations were carried out with the software Prism (GraphPad). Statistical analysis was conducted for analysis of variance (ANOVA) using the F-test. The F statistic is reported [F() =] with the parenthesis being the degrees of freedom within groups separated by a comma followed by the significance level (p) at the end.

CHO cells were transfected with 0.1 μg/mL wildtype or single alanine mutant xtMC2R and 0.1 μg/mL cMRAP1 using jetPRIME transfection reagent (Polyplus transfection SA, Illkirch, France). Cells were grown for 48 h at 37 °C and then incubated on ice. Cells were surface-labeled with anti-V5 antibody (1:1000, Rockland Immunochemicals, Limerick, PA, USA) and then fixed in 4% paraformaldehyde, pH 7.2 (Sigma, St. Louis, MO, USA). Fixed cells were washed in 1x phosphate-buffered saline and incubated at room temperature with HRP-conjugated goat-anti-rabbit secondary antibody (Sigma, St. Louis, MO, USA). Cells were then washed and treated with 1-step ABTS (Thermo-Fisher Scientific, Watham, MA, USA). Absorbance was measured at 405 nm using a Bio-Tek Synergy HTX plate reader (Winooski, VT, USA). The analyses were done in triplicate. For each analysis the negative control was xtMC2R transfected alone, and the positive control xtMC2R co-transfected with cMRAP1.