Fig. 7From: Antigen-specific single B cell sorting and expression-cloning from immunoglobulin humanized rats: a rapid and versatile method for the generation of high affinity and discriminative human monoclonal antibodiesCD22 specificity analysis of antibodies by ELISA and flow cytometry. Supernatants from HEK 293 cells transfected with light and heavy chain coding constructs from single anti-human CD22 B cells were analysed for reactivity against human CD22. a Human CD22 or β-Gal were coated at a concentration of 5 μg/mL in 96-well plates. 50 μL of IgG expression supernatants collected 5 days after transfection were used pure or diluted at 1:10 in PBS and used as the primary antibody. Biotin conjugated anti human IgG H + L antibody was used as the secondary antibody. Streptavidin HRP was used for detection. OD450 read from blank control was deducted from all wells. Cell culture supernatants from human anti-β-Gal H + L (clone 44A8, β-Gal Control) and CD22 H + L (clone 5E1, CD22 H + L Control) transfected cells were used as positive controls in β-Gal and CD22 ELISA, respectively. Non-transfected cell culture supernatant was used as the negative control. b Representative flow cytometry analysis of heavy and light chain co-transfected HEK cell culture supernatants. Supernatants from the indicated heavy + light chain positive transfections in ELISA (the first 5 from left) or negative (the following ones) and were used as primary antibodies on Ramos CD22+ and NK92 CD22− cell lines. Cell culture supernatants from human anti-CD22 H + L (clone 5E1, CD22 H + L) transfected cells and a commercial mouse mAb anti-human CD22 (Ms anti-hu CD22) were used as positive controls. Biotin conjugated anti human IgG H + L antibody was used as a secondary antibody and streptavidin Alexa405 was used for cytometry detection. Histograms for transfected cell supernatant shown in black non-transfected cell supernatant in filled grey. Control Ramos and NK92 cell lines were labelled with a commercial mouse anti human CD22 FITC (black line) or with an isotype control (filled grey)Back to article page