B cells play an important role in combating infections. The humoral immune response is initiated when a B cell encounters its cognate antigen. Following antigen-dependent activation, the B cell differentiates into plasmablasts and plasma cells that secrete copious amounts of antibodies (Nutt et al., 2015; Cyster and Allen, 2019). The antibodies help in clearing the invading pathogen from the host via multiple mechanisms, that include neutralization, and opsonization followed by uptake and killing by phagocytic cells such as neutrophils and macrophages. Monoclonal antibodies (mAbs) are being used as therapeutics to treat a wide range of disease conditions such as cancers, infectious diseases, transplantation and metabolic disorders (Carter and Rajpal, 2022). Due to their high specificity, mAbs are finding application as probes in diagnostics and research. Monoclonal antibodies are being covalently coupled with cytotoxic substances to generate antibody-drug conjugates and make them more effective (Beck et al., 2017).

The huge antibody diversity in humans is achieved by a process called somatic V(D)J recombination which occurs during B cell ontogeny (Schatz and Ji, 2011). During this process, one of the large numbers of variable (V) recombines with one of the several diversity (D) and joining (J) gene segments in the IgH locus. In the case of the lgκ and Igλ light chain loci the V gene segment recombines with the J gene segment as the light chain loci lacks D gene segments. The V gene segments exhibit sequence similarity and are categorized into subgroups by IMGT (https://www.imgt.org). In humans, V gene segments in the H, κ and λ chains are classified into 8, 7 and 17 subgroups, respectively. Some of the gene segments are non-functional. In secondary lymphoid organs such as the spleen and lymph nodes, the clonally expanding antigen-reactive B cells undergo somatic hypermutation which results in alteration in the sequence of the rearranged V genes (Peled et al., 2008).

Multiple methodologies have been developed to study B cell responses, each with its unique advantages and disadvantages. These include hybridoma technology, phage and yeast antibody display libraries, and antigen-specific single B cell based technologies for generating mAbs (Frenzel et al., 2013; Pedrioli and Oxenius, 2021). In the latter case, the expressed IgH and IgL chain is amplified from sorted single antigen-specific B cells and cloned into a suitable vector system for the expression of recombinant mAb. For groups interested in developing therapeutics or validating antigen-specific mAbs, generating human recombinant mAbs derived from single B cells is the method of choice (Weitkamp et al., 2003; Tiller et al., 2008; Smith et al., 2009; Zhou et al., 2020). This approach allows expression of naturally occurring IgH and IgL pairs, which is not possible with current phage display techniques. The single B cell based technologies methodology not only allows rapid development of recombinant mAbs but also is amenable to antibody engineering. While in vivo Ig pairing is maintained in hybridomas and B cell immortalization approaches but the success rate of generating mAbs is quite low. In recent years, artificial intelligence-enabled approaches have been used for optimizing antibody affinity and producing new antibody candidates (Banach et al., 2023; Cheng et al., 2024; Hie et al., 2024).

The success rate of generating recombinant mAbs from single-sorted B cells is critically dependent on the coverage of the primer set. The currently available primer sets do not cover all the functional human Ig gene rearrangements (Coronella et al., 2000; Weitkamp et al., 2003; Johansson et al., 2007). Incomplete coverage of the primer set shows up as gaps in the B cell repertoire. Here, we describe the design of a primer set that can theoretically amplify all possible V(D)J rearrangements. Furthermore, the primers can be used to clone paired VH-VL and express as recombinant mAbs from single B cells for further functional validation. We utilized the germline human IgH and IgL chain genes from the NCBI (https://www.ncbi.nlm.nih.gov) and IMGT databases for designing the primers. We adopted a nested RT-PCR based strategy for amplifying the variable region of the expressed IgH and IgL transcripts from sorted single human B cells. We experimentally validated the primer set using multiple strategies. VH-VL pairs amplified from sorted single B cells were cloned, expressed and purified from HEK293T cells as full-length recombinant antibodies. Our primer set can facilitate the analysis of the B cell pre-immune, and immune repertoires elicited following vaccination and infection in its full diversity, better than has been previously possible.