Table 3. Summary of recovery of recombinant anti-human TNF antibodies from antigen-specific rat plasma cells.

Number of CD138+ bone marrow cells analyzed	Number of h TNF -specific plasma cells isolated	Number of VH and VL cognate gene pairs recovered	Number of hTNF-specific recombinant antibodies (via TAP)
2.5 × 106	287	226	147

CD138⁺ bone marrow cells from 5 immunized Sprague Dawley rats were analyzed via the fluorescent foci method using biotinylated human TNF as the target antigen. Following RT-PCR, recoveries of heavy (VH) and light (VL) chain variable region gene pairs were determined by agarose gel electrophoresis of the secondary PCR products. Binding of recombinant IgG to TNF was determined using supernatants from HEK-293F cells which had been transiently co-transfected with heavy and light chain TAP products. The assay was performed using a fluorescence-based homogeneous assay with biotinylated human TNF immobilized on Superavidin beads and a goat anti-rat Fcγ-specific-Cy5 secondary reagent.