Antibody discovery research has enabled the identification and development of many important biologic drugs, with >100 therapeutic antibody treatments already approved or under review in the US or EU [1,2]. Many researchers view antigen-specific B cells (AgSCs) as a particularly potent resource when mining for new immunotherapeutic antibodies. Unfortunately, focusing the discovery process solely on AgSCs from immunized individuals poses a huge challenge as they represent only 0.01%–0.1% of the total B cell population. In this article, Mahendra et al. tested a new method of purifying AgSCs from immunized mice. This improved workflow allowed them to quickly eliminate non-relevant cells from the antibody discovery process without damaging the AgSCs, enabling the generation of functional antibody clones from individual B cells. Using gBlocks Gene Fragments from IDT, they were able to express target-specific Fabs for further evaluation as part of a streamlined antibody discovery workflow.
The researchers used a multi-step enrichment workflow for bulk purification of AgSCs from immunized mice lymphoid tissues (spleen and lymph nodes). First, the total B cell population was isolated from other cell types by lysing red blood cells and selecting out unwanted cell types with antigen-coated beads. IgM+ and IgD+ B cells were also removed from the total B cell population, which was enriched for IgG+ B cells. AgSCs were selected out of the IgG+ B cell suspension using magnetic separation and biotinylated target antigen, then single-sorted for further use.
The researchers prepared cDNA from the individual B cells to amplify genes encoding IgG variable chains (VH and VK). They assessed sequence diversity of the recovered IgG genes using both Sanger and next generation sequencing (NGS). Recombinant antibody fragments (Fabs) from both AgSCs and non-antigen-selected B cells were cloned and expressed using their in-house method for antibody characterization. In brief, the variable regions of functional antibodies were synthesized as gBlocks Gene Fragments, cloned into an expression vector, then expressed and purified on an automated platform. Properties of the Fabs, including melting temperatures and antigen affinity, were measured using various assays.
Antigen-specific B cell (AgSC) bulk purification workflow produced viable AgSCs with minimal “escape” in flow-through population
The researchers used their multistep enrichment process to purify AgSCs from the initial suspension of dissociated cells from the harvested lymph node and spleen tissue. The collected AgSCs, which represented 0.19–1.12% of total B cells, were ~95% viable after the enrichment process. This represents a large improvement over another common method of antigen-specific B-cell enrichment, FACS, which sorts the fragile cells at such high speeds that it often causes significant damage and cell mortality. Importantly, IgG genes crucial for the antibody discovery process (VH and VK chains) were able to be efficiently recovered from single-sorted AgSCs for all 5 antigens (81%–99%), then expressed as recombinant IgGs (rIgGs) in a high-throughput system  for subsequent antigen binding tests.
The researchers investigated antigen binding of individual antibodies from three B-cell populations: the purified AgSCs, total B cell (TBC) population, and non-selected flow-through. 51–88% of rIgGs from the AgSC population bound to their target antigens, compared to only 1–8% of rIgGs from the TBC population and 0–2% from the flow-through population. The small percentage of target-specific rIgGs in the flow-through population suggests that few, if any, antigen-specific B cells escaped selection during the bulk purification process.