Detection and activation of HIV broadly neutralizing antibody precursor B cells using anti-idiotypes

Bancroft, Tara; DeBuysscher, Blair L.; Weidle, Connor; Schwartz, Allison; Wall, Abigail; Gray, Matthew D.; Feng, Junli; Steach, Holly R.; Fitzpatrick, Kristin S.; Gewe, Mesfin M.; Skog, Patrick D.; Doyle-Cooper, Colleen; Ota, Takayuki; Strong, Roland K.; Nemazee, David; Pancera, Marie; Stamatatos, Leonidas; McGuire, Andrew T.; Taylor, Justin J.

Kinetic analyses were performed at room temperature with shaking at 500 rpm. Anti-mouse Ig capture sensors were immersed in kinetics buffer containing 20 µg/ml purified antiidiotypic antibody for 240 s. After loading, the baseline signal was then recorded for 60 s in kinetics buffer. The sensors were next immersed into wells containing serial dilutions of purified iglb12 Fab in kinetics buffer for a 300-s association phase, followed by immersion in kinetics buffer for an additional 600-s dissociation phase. The background was signal measured from each analyte-containing well using sensors loaded with a negative control antibody and subtracted from the signal obtained with each corresponding ligand-coupled sensor at each time point. Kinetic analyses were performed at least twice with an independently prepared analyte dilution series. Curve fitting was performed using a 1:1 binding model and ForteBio data analysis software. The mean on rate and off rate values were determined by averaging all binding curves in each dilution series that matched the theoretical fit with an R 2 value of ≥0.95.