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. 2021 Oct 11;16(10):e0252558. doi: 10.1371/journal.pone.0252558

Fig 3. Characterization of recombinant mAbs.

Fig 3

(a) Schematic of recombinant mAb constructs. VL sequences were cloned in frame with human kappa CL, while VH was cloned in frame with human CH1-CH3. The sortag sequence, LPETGG, was appended at the C-terminus, immediately after the CH3 domain. (b) SDS-PAGE of recombinant mAbs, 1 = non-boiled, non-reduced, 2 = boiled, reduced. (c) Size exclusion HPLC of SP2/SP4 and SP7/SP4 mAbs, showing a single peak on the A280 detector at the expected size for immunoglobulin (7.9 min). (d) Flow cytometry histogram showing binding of 20 nM SP2/SP4 recombinant mAb, but not 20nM SP7/SP4 mAb, to Cho-hCD63-eGFP cells. (e) Flow cytometry-based binding curve of Cho-hCD63-eGFP cells (neither mAb showed any binding to Cho WT cells). (f) The affinity of SP2/SP4 mAb was similar to that of H5C6 hybridoma-derived monoclonal antibody (n = 3).