Following the selection of anti-IDs for the second phase, the anti-IDs were both biotinylated and Sulfotagged to be used in a pairwise sandwich ECL assay with the therapeutic mAb. This screening was done at three concentrations of mAb across at least 2 orders of magnitude in the presence and absence of the soluble target. In addition, six different PK assay formats and two concentrations of anti-IDs were used to simultaneously identify the most robust PK assay format and optimal reagent combination and concentration. A second scoring system measuring ten different parameters was used to determine the optimal anti-ID pairing and PK assay format (Figure -F; Supplementary Tables ). The ten parameters include, 1) background signal, 2) and 3) signal to noise with and without soluble ligand, 4) and 5) upper level of quantitation with and without soluble ligand, 6) and 7) lower level of quantitation with and without soluble ligand, and 8), 9) and 10) soluble ligand interference at three different drug levels. This multifactorial scoring system identified the top two performing pairs of ant-IDs in the PK assay format that would yield the most robust PK assay (Figure and). These results were further confirmed by the results the recombinant anti-ID pairings (Figure and 3B). To enable this complex approach, all assay steps were automated on a liquid handling system to ensure consistency of the method given the range of pipetting steps required on each sample plate and the number of total plates created.