Production of guinea pig monoclonal antibodies (mAbs) against human insulin. (A) Representative fluorescence-activated cell sorting (FACS) graph of human insulin-immunized guinea pig lymphocytes stained with anti-guinea pig IgG, ER-tracker and insulin-Cy3. Gates for lymphocytes (R1), plasma/plasmablast cells (PCs) (IgGlow endoplasmic reticulum (ER)high, R2) and antigen-specific plasma/plasmablast cells (ASPCs) (IgGlow ERhigh insulin+, R3) were established. (B) Representative agarose gel electrophoresis of cognate pairs of V genes amplified from single-cell-sorted R3-gated cells. (C) polymerase chain reaction (PCR) success ratio for the V genes. (D) Antigen specificity of mAbs produced from the R3-gated cells. (E) Binding affinity and binding epitopes of the highly binding clones in (D). (F) Immunohistochemical staining of insulin with c08 mAb in (E) (red) and glucagon (green) in a mouse pancreatic section.