We have developed and optimized a new technology, termed Nanobody MiSeq/MS, which combines deep sequencing with mass spectrometry to rapidly generate large numbers of bacterially-expressed nanobodies with extremely high affinity and specificity. In our nanobody development pipeline, we first immunize llamas with the antigen target. After a strong immune response is elicited, we collect lymphocytes from bone marrow, highly enriched for active B cells. We then purify cDNA from these cells, and perform high-throughput sequencing on the PCR-amplified variable region (VHH) of heavy-chain-only IgG variants (HCAbs). In parallel, we collect animal sera, and affinity purify HCAbs with strong affinity and specificity to each antigen. The purified HCAbs are then analyzed by MS, and by correlating peptides to the DNA database of full-length sequences, we can identify candidate nanobody clones.
We first demonstrated the effectiveness of this approach by generating more than 30 high affinity nanobodies against GFP and mCherry. We freely provide these clones to the public, and have shared ~400 plasmids with more than 120 outside groups. Since this initial proof of principle, we have applied our nanobody generation pipeline to over 30 new antigens, generating a total of more than 150 unique nanobodies. Among our current interests, we are developing new nanobodies for use as therapeutics against cancer targets.
If you would like to learn more about our nanobody production pipeline, read it here! Also, please contact us if you are interested in a particular reagent/software or collaborating with us.