NAb-seq: an accurate, rapid and cost-effective method for antibody long-read sequencing in hybridoma cell lines and single B cells

Despite their common use in research, monoclonal antibodies are currently not systematically sequenced. This can lead to issues with reproducibility and the occasional loss of antibodies with loss of cell lines. Hybridoma cell lines have been the primary means of generating monoclonal antibodies from immunized animals including mice, rats, rabbits and alpacas. Excluding therapeutic antibodies, few hybridoma-derived antibody sequences are known. Sanger sequencing has been “the gold standard” for antibody gene sequencing but relies on the availability of species-specific degenerate primer sets for amplification of light and heavy antibody genes, in addition to lengthy and expensive cDNA preparation. Here we leveraged recent improvements in long-read Oxford Nanopore Technologies (ONT) sequencing to develop NAb-seq: a three-day, species-independent, and cost-effective workflow to characterize paired full-length immunoglobulin light and heavy chain genes from hybridoma cell lines. When compared to Sanger sequencing of two hybridoma cell lines, long-read ONT sequencing was highly accurate, reliable, and amenable to high throughput. We further show that the method is applicable to single cells, allowing efficient antibody discovery in rare populations such as memory B cells. In summary, NAb-seq promises to accelerate identification and validation of hybridoma antibodies as well as antibodies from single B cells used in research, diagnostics and therapeutics. ### Competing Interest Statement The authors have declared no competing interest. * APC : allophycocyanin BAX : Bcl-2-associated X protein BLAST : basic local alignment search tool BV : brilliant violet DPBS : Dulbecco’s phosphate-buffered saline FBS : fetal bovine serum Ig : immunoglobulin IMGT® : international ImMunoGeneTics information system® KLH : keyhole limpet hemocyanin NAb-seq : nanopore antibody sequencing ONT : Oxford Nanopore Technologies PE : phycoerythrin RAGE-seq : Repertoire and Gene Expression by Sequencing VH : variable heavy chain VL : variable light chain V-QUEST : V-QUEry and Standardization