Optimization of the concentrations of MAb (A), PAb (B), and the third antibody, GAR-AP (C) over a range of concentration of purified Cel7A. (A). Two different concentrations of Cel7A MAb 10 μg/ml (◊) and 50 μg/ml (□) were added to the well. PAb and GAR-AP concentrations were kept constant at 1/400 and 1/1750 dilutions, respectively (B). Using 10 μg/ml Cel7A MAb, the Cel7A PAb was diluted to different degrees: 14(◊), 7(□), 3.5(∆), and 1.75(X) μg/ml. The GAR-AP was diluted at 0.3 μg/ml. (C). The concentration of GAR-AP was varied by diluting it to 1 (◊) or 0.3 (□)μg/ml in PBS. Cel7A MAb concentration was kept at 10 μg/ml, and Cel7A PAb was diluted to 14 μg/ml in PBS.