Generation of Recombinant Monoclonal Antibodies from Immunised Mice and Rabbits via Flow Cytometry and Sorting of Antigen-Specific IgG+ Memory B Cells

Abstract

Single B cell screening strategies, which avoid both hybridoma fusion and combinatorial display, have emerged as important technologies for efficiently sampling the natural antibody repertoire of immunized animals and humans. Having access to a range of methods to interrogate different B cell subsets provides an attractive option to ensure large and diverse panels of high quality antibody are produced. The generation of multiple antibodies and having the ability to find rare B cell clones producing IgG with unique and desirable characteristics facilitates the identification of fit-for-purpose molecules that can be developed into therapeutic agents or research reagents. Here, we describe a multi-parameter flow cytometry single-cell sorting technique for the generation of antigen-specific recombinant monoclonal antibodies from single IgG+ memory B cells. Both mouse splenocytes and rabbit PBMC from immunised animals were used as a source of B cells. Reagents staining both B cells and other unwanted cell types enabled efficient identification of class-switched IgG+ memory B cells. Concurrent staining with antigen labelled separately with two spectrally-distinct fluorophores enabled antigen-specific B cells to be identified, i.e. those which bind to both antigen conjugates (double-positive). These cells were then typically sorted at one cell per well using FACS directly into a 96-well plate containing reverse transcriptase reaction mix. Following production of cDNA, PCR was performed to amplify cognate heavy and light chain variable region genes and generate transcriptionally-active PCR (TAP) fragments. These linear expression cassettes were then used directly in a mammalian cell transfection to generate recombinant antibody for further testing. We were able to successfully generate antigen-specific recombinant antibodies from both the rabbit and mouse IgG+ memory B cell subset within one week. This included the generation of an anti-TNFR2 blocking antibody from mice with an affinity of 90 pM.

The PRRSV-Specific Memory B Cell Response Is Long-Lived in Blood and Is Boosted During Live Virus Re-exposure

“…Previous studies of the porcine memory B cell, as well as that of the mouse, have shown that memory B cells develop and are maintained in several lymphoid structures (20,33). However, studies from other species would suggest that at least some component of memory is maintained in circulation (30,34,35). In this study, there was no evidence of nsp7-specific cells within key secondary lymphoid tissues after day 56, including day 118, 5 days after challenge.…”

“…The reason for the decline of these antigen specific populations in the lymph node can be attributed to the involution of germinal centers. The large increase in nsp7-specific B cells in the blood is likely due to the migration of nsp7-specific memory B cells out of the lymph nodes and into blood where these cells would be able to more actively search for specific antigen, as has been observed in other species with several pathogens (28)(29)(30). The delayed expansion of nsp7-specific B cells in the spleen is less clear.…”

Rapid single B cell antibody discovery using nanopens and structured light

“…Quantitative ranking of the binding of antibodies is frequently used to characterize candidate molecules and can be accomplished using ELISA, FACS, surface plasmon resonance systems (e.g., Biacore), and other methodologies. 7,8,11,15,26,[35][36][37][38][39][40] As of early 2019, the Beacon software was unable to generate quantitative values for fluorescent blooms. Despite the high-content imaging capabilities, this limited the built-in analysis to a binary (yes/no) result.…”

“…Moreover, we feel the overall workflow timeline can be executed even more rapidly. Successful VH and VL recovery and recombinant expression in 5-7 days have been demonstrated, 35,37,38 as have rapid immunization strategies. 42,43 Combining approaches such as these with the 1-day screening process of the NanOBlast method described here, complete antibody discovery workflows using antigen-experienced, affinity-matured ASCs could be executed in as little as 3-4 weeks.…”

From rabbit antibody repertoires to rabbit monoclonal antibodies

“…Single B-cell isolation based on antigen capture by fluorescence-activated cell sorting, magnetic beads, solid-phase panning or hemolytic plaques followed by light- and heavy-chain cloning has also been applied to the generation of rabbit mAbs from peripheral B cells, 172, 173, 174, 175, 176 which does not require killing of the animal and allows for multiple sampling points from primary and secondary lymphoid tissues. 177 Antigen capture-driven bulk spleen B-cell isolation from immunized rabbits followed by light- and heavy-chain-encoding DNA amplification and their combinatorial assembly and expression in scFv–Fc or IgG format in mammalian cells has also been reported. 178 In the fluorescent foci method, plasma cells from rabbit bone marrow are identified by fluorescent microscopy, isolated and subjected to light- and heavy-chain-encoding DNA amplification and sequencing.…”