Figure 2.

Tissue‐based assay protocol diagram. An illustration of the experimental procedure to measure the binding affinity in human colon tissue VRH. Matching the test sample and reference sample RI by splitting it into two aliquots, it is possible to perform RI measurements at <10⁻⁶ RIU sensitivity in the presence of significant matrix complexity. 1) Tissue VRH samples are prepared as described in methods. 2) A set of samples with varying concentrations of PF‐00547659 are prepared in PBS. 3) A set of samples with varying concentrations of an isotype control mAb, using the same concentrations as PF‐00547659 series, are prepared in PBS. Either samples from 2) or 3) are mixed with tissue VRH producing 4) and 6) test samples or 5) reference samples, and the samples were allowed to reach equilibrium. Samples were measured in the same manner sequentially starting with the reference and then the binding samples with increasing concentrations of mAb and fixed target protein, up to the highest concentration of mAb (1 nM). This step‐wise procedure constitutes a single binding assay run producing a saturation isotherm. 7) The binding signal was calculated as the difference between the sample and reference signals at the same mAb concentration and represents the amount of specific binding between mAb and target protein occurring within the sample because all other binding events are cancelled by the reference sample. 8) The binding signal (δ RI in milliradians) was then plotted versus concentration of mAb.