Antibody Cloning from Env-Sorted B Cells

The antibody cloning of Env-sorted single B cells was conducted as follows. A mix containing 3 μl of random hexamers (GeneLink), 2 μl of deoxynucleoside triphosphates (dNTPs), and 1 μl of SuperScript IV enzyme (Thermo Fisher) was added to each well of a single-cell-sorted 96-well plate that underwent thermocycling according to the program outlined in the SuperScript IV protocol, resulting in 25 μl of cDNA for each single cell. cDNA (5 μl) was then added to a PCR mix containing 12.5 μl of 2× multiplex PCR mix (Qiagen), 9 μl of H₂O, 0.5 μl of forward primer mix, and 0.5 μl of reverse primer mix (mouse [105 (link)] and rabbit [55 (link)]) for HCs and KCs within each well. A second PCR was then performed using 5 μl of the first PCR as the template and respective primers (mouse [105 (link)] and rabbit [55 (link)]) utilizing the same recipe as the first PCR. The PCR products were run on a 1% agarose gel, and those with correct HC and KC bands were then used for Gibson ligation (New England Biolabs), cloning into IgG expression vectors, and transformation into competent cells. Mouse and rabbit MAbs were expressed by the transient transfection of ExpiCHO cells (Thermo Fisher) with equal amounst of paired HC and KC plasmids and purified from the culture supernatant after 12 to 14 days using protein A bead columns (Thermo Fisher).