NCM 2022: 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. Sanger sequencing has been ‘the gold standard’ for antibody gene sequencing but relies on the availability of species-specific degenerate primer sets and is lengthy and expensive. We developed Nanopore Antibody Sequencing (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 using Oxford Nanopore sequencing, benefiting from the long reads produced.

When compared with Sanger sequencing of two hybridoma cell lines, NAb-seq was highly accurate, reliable, amenable to high-throughput, and identified the presence of multiple productive heavy and light chains within a single cell. 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 the identification and validation of hybridoma and single B-cell-derived antibodies used in research, diagnostics, and therapeutics.