Fig. 1. Flowchart of the study depicting antigen-specific B-cell bulk selection and their characterization by single cell cloning, and phage library construction.

Cell suspension was prepared from spleen and lymph nodes and total B-cell was enriched by RBC lysis followed by immuno-depletion of non-B cells by negative selection. IgG⁺ B cells were enriched by depleting IgM⁺ and IgD⁺ B cells. Antigen-specific B cells were isolated by using biotinylated target antigen in solution. Input, selected and flow-through B-cell populations were single-sorted by FACS in 96-well plate. From single B cells, cDNA was prepared, and variable regions of individual IgG genes were amplified. Heavy and kappa chain expression cloning were performed for each sorted B-cell and co-expressed in Expi293F cells to generate recombinant antibodies for antibody identification and method validation.