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.

BLI assays were performed on the Octet.Red instrument (For-teBio). For anti-idiotype binding screens, anti-mouse IgG capture sensors (ForteBio) were immersed in kinetics buffer (1× PBS, 0.01% BSA, 0.02% Tween 20, and 0.005% NaN 3 , pH 7.4) containing 5 µg/ml purified anti-idiotypic antibody for 100 s. After loading, the baseline signal was then recorded for 60 s in kinetics buffer. The sensors were then immersed in kinetics buffer containing 20 µg/ml purified human antibody for a 100 s association step. The maximum response was determined by averaging the nanometer shift over the last 5 s of the association step after subtracting the background signal from each analytecontaining well using empty anti-mouse IgG Fc capture sensors at each time point.