Soluble CAP256 Env Binding ELISA for Antibody Titers

To assess Env binding antibody titers in rabbit sera, a matching soluble CAP256 Env binding ELISA was performed as described earlier (80 (link)). In short, Nunc MaxiSorp flat-bottom 96-well plates (Sigma) were coated with 10 ng/well of soluble, trimeric Env. Rabbit sera were used in the primary incubation in a serial dilution range starting at 1:10. Anti-rabbit IgG-horseradish peroxidase (HRP) (1:5,000, Roche) was used for detection with TMB ELISA substrate (Abcam). The reaction was stopped after 10 min with 1 N H₂SO₄. The ELISA signal was analyzed using a VersaMax ELISA microplate reader (Molecular Devices), which subtracted absorbance values at 540 nm from values at 450 nm. ELISAs for the whole time course and each group were performed at the same time on duplicate plates. Duplicate data points were averaged and fitted to a four-parameter logistic regression curve (4PL curve) in GraphPad Prism 5.0. Antibody endpoint titers were calculated from 4PL curves with the threshold set as 4PL curve minimum + standard error of minimum for each time point. Data were plotted as the mean ± SEM for the whole group. ELISAs for binding to CAP256 SU V1V2 loop scaffold for week 0 and 2 weeks after the final MVA inoculation or protein boost were performed in a similar fashion, but wells were coated with 500 ng/well of protein.