a, Amplified heavy chains were paired with universal cow light chain or with NC-Cow1 light chain and tested for expression (anti-Fc ELISA), antigen binding (BG505 SOSIP), autologous neutralization (BG505 pseudovirus), and heterologous neutralization (Q23 pseudovirus). Sequence alignments of recovered heavy chains are listed underneath. As ultralong HCDR3 antibodies have been reported to pair with a single germline light chain (V30), amplified heavy chain genes were first paired with the universal cow light chain and screened for expression, binding to BG505 SOSIP, neutralization of BG505 virus, and neutralization of a clade A heterologous virus, Q23. From this dataset, three antibodies (NC-Cow1, NC-Cow2, and NC-Cow3) were selected that showed autologous neutralization (all from day 238) and corresponding native light chains were amplified, with success for only NC-Cow1. These three antibodies were expressed and purified at a larger scale for additional characterization by maintaining native pairing for NC-Cow1 and pairing with germline V30 for NC-Cow2 and NC-Cow3. For the day 70 time point, three heavy chains were selected that showed binding to BG505 SOSIP, but no neutralization, and these antibodies were produced with their native light chain pairs (NC-Cow4 to NC-Cow6). Finally, as NC-Cow1 could neutralize isolate Q23 in the neutralization screen, an additional sort with PBMCs from day 381 was performed, but used BG505 SOSIP liganded with and without NC-Cow1 to enrich for epitope-specific antibodies. The enrichment yielded an additional five hits by neutralization screen, and four out of these five antibodies were produced at larger scale with their native light chain pairs (NC-Cow7 to NC-Cow10). Small-scale screening was also performed with all heavy chains paired with NC-Cow1 light chain and no significant increase in neutralization breadth was observed, although there were slight improvements in BG505 SOSIP affinity and autologous potency. b, Nucleotide alignment of heavy chain sequences of isolated monoclonal antibodies36.