Extended Data Fig. 1: On-cell binding assay to measure the binding affinity of wt-7D12, 7D12-32Bpa, 7D12-109Bpa, and 7D12-113Bpa to Epidermal Growth Factor Receptor (EGFR) expressed on the surface of A431 cells.
From: Site-specific encoding of photoactivity and photoreactivity into antibody fragments
(A) Results demonstrate that site-specially incorporated Bpa at position 109 in 7D12 has little effect on 7D12-EGFR binding affinity. To ensure reproducibility, experiments were performed in triplicates represented as REP 1, REP 2 and REP 3. (B) Quantitative assessment of binding between Bpa-containing 7D12 mutants and EGFR. Chemiluminescence intensities obtained from on-cell binding experiments for wt-7D12, 7D12-32Bpa, 7D12-109Bpa, and 7D12-113Bpa were quantified using CLARIOstar plate reader. For each plate the maximum value of intensity was normalized to 1 and the minimum normalized to zero. normalization is performed to ensure that data between replicates could be compared. The normalized intensity was plotted against concentration of 7D12. The data was fitted to sigmoidal nonlinear equation using GraphPad as shown in Fig. 2b. KD is the concentration of the 7D12, where chemiluminescence intensity is half of the maximum chemiluminescence intensity. IMAX is the normalized maximum chemiluminescence intensity at saturation. Values shown in brackets denote the standard deviation (s.d.) from the mean value of three replicates.
