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.

Nested RT-PCR BCR sequencing and analysis RT was performed using SuperScript IV (Thermo Fisher Scientific) as previously described (Tiller et al., 2009; Wu et al., 2010) . Briefly, 3 µl RT reaction mix consisting of 1.5 µl 50 µM random hexamers (Thermo Fisher Scientific), 0.4 µl 25 mM deoxyribonucleotide triphosphates (dNTPs; Thermo Fisher Scientific), 0.5 µl (10 U) SuperScript IV RT, and 0.6 µl water was added to each well containing a single sorted B cell in 10 µl lysis buffer and incubated at 50°C for 1 h. Following RT, 2 µl cDNA was added to 19 µl PCR reaction mix so that the final reaction contained 0.2 µl (0.5 U) HotStarTaq Polymerase (Qiagen), 0.075 µl 50 µM 39 reverse primers, 0.115 µl 50 µM 59 forward primers, 0.24 µl 25 mM dNTPs, 1.9 µl 10× buffer (Qiagen), and 16.5 µl water. The PCR program was 94°C 30 s, 57°C 30 s, 72°C 55 s, 50 cycles, 72°C 10 min for IgM/IgG/kappa light chains and 94°C 30 s, 60°C 30 s, 72°C 55 s, 50 cycles, 72°C 10 min for lambda light chains. After the first round of PCR, 2 µl of the PCR product was added to 19 µl of the second-round PCR reaction so that the final reaction contained 0.2 µl (0.5 U) HotStarTaq Polymerase, 0.075 µl 50 µM 39 reverse primers, 0.075 µl 50 µM 59 forward primers, 0.24 µl 25 mM dNTPs, 1.9 µl 10× buffer, and 16.5 µl water. PCR programs were the same as the first round of PCR. 4 μl of the PCR product was run on an agarose gel to confirm the presence of a ∼500-bp heavy chain band or 450-bp light chain band. 5 μl from PCR reactions showing the presence of heavy or light chain amplicons was mixed with 2 µl of ExoSAP-IT (Thermo Fisher Scientific) and incubated at 37°C 15 min, followed by 80°C for 15 min to hydrolyze excess primers and nucleotides. Hydrolyzed second-round PCR products were sequenced by Genewiz with the respective reverse primer, and sequences were analyzed using IMGT/V-Quest to identify V, D, and J gene segments.