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.

Complexes of IB2 Fab + iglb12 c/scFv and IB3 Fab + iglb12 Fab were formed by mixing both components at a 1:1 molar ratio and incubating overnight at 4°C. Complexes were purified by size exclusion chromatography using Superdex 200 in 5 mM Hepes and 150 mM NaCl at pH 7.4. Crystals of IB2 Fab + iglb12 c/scFv and IB3 Fab + iglb12 Fab were obtained using a mosquito and an NT8 dispensing robots, and screening was done using commercially available screens (Rigaku Wizard Precipitant Synergy block #2, Molecular Dimensions Proplex screen HT-96, Hampton Research Crystal Screen HT) by mixing 0.1 µl/0.1 µl (protein/reservoir) by the vapor diffusion method. Crystals used for diffraction data were grown in the following conditions: IB3 Fab + iglb12 Fab crystals were grown in solution containing 0.2 M ammonium phosphate monobasic, 0.1 M Tris, pH 8.5, and 50%(+/−) 2-methyl-2,4-pentanediol. IB2 Fab + iglb12 c/scFv crystals were grown in solution containing 13.4% polyethylene glycol 8000, 13.4 PEG 400, 0.1 M MgCl 2 , and 0.1 M Tris, pH 8.5, using the vapor diffusion method. Crystals were cryoprotected in solutions containing 30% molar excess of their original reagents and 20% glycerol for IB3 Fab + iglb12 Fab and 20% 2R-3R Butanediol for IB2 Fab + iglb12 c/scFv. Crystal diffracted to 3Å for IB3 Fab + iglb12 Fab and 2.6Å for IB2 Fab + iglb12 c/scFv. Data were collected with advanced light source 5.1 and 5.2 and processed using HKL2000 (Otwinowski and Minor, 1997) .