Figure 1
Development, production and purification of a pSn-specific recombinant antibody, rec41D3. (A) Vector map of p41D3 (B) Non-reducing Western blot analysis of hybridoma supernatant of mAb 41D3 compared to p41D3-transfected HEK293T cell supernatant using polyclonal antibodies specific for mouse immunoglobulins (C) SDS-PAGE of protein G purified transfected cell supernatants to evaluate rec41D3 purity (D) Western blot analysis of purified rec41D3 using polyclonal antibodies specific for mouse immunoglobulins (E) Confocal microscopical analysis of CHO cells and recombinant pSn expressing CHO cells (CHO-pSn) incubated with mAb 41D3, rec41D3 or isotype matched controls mAb 13D12 and rec13D12 at 4°C for 1 hour. Cells were fixed and stained with FITC-labelled goat-anti-mouse IgG. Images represent a single confocal z-section through the middle of the cell. Scale bar: 5μm (F) Kinetic profile of the pSn-41D3/pSn-rec41D3 interaction by Surface Plasmon Resonance measurements. Immobilized pSn4D-Fc was flowed with rising concentrations of mAb 41D3 or rec41D3, after which injection was halted and dissociation was monitored during 5 minutes. Sensorgrams were fitted to a bivalent model and an approximation of the equilibrium dissociation constant was calculated. M, protein marker; mAb, mAb 41D3; rec, rec41D3; KD, equilibrium dissociation constant.