Goat anti mouse igg h l secondary antibody conjugated to hrp

Cellular ELISA [30 (link)] was performed to measure the binding of the anti-ADAM10 human mAb 1H5, relative to the binding of the commercial anti-ADAM10 mAb1427 (monoclonal mouse IgG2B Clone # 163003, R&D), to ADAM10 expressed on the cell surface of colon cancer cell lines LIM1215 and COLO205 as well as to HEK293 cells and HEK293 cells transfected with full-length human ADAM10. Briefly, 5 × 10⁴ cells/well were immobilized on 96-well ELISA plates (Greiner bio-one) with 1 % paraformaldehyde for 2hrs at 37° C. The plate was washed thrice with PBS and blocked for 2 hrs at room temperature with 4 % non-fat dry milk. The anti-ADAM10 mAbs were then added in varying concentrations. Mouse mAb conjugated to HRP and recognizing human IgG was used as a secondary antibody (Abcam) to detect 1H5 binding to ADAM10 expressed on the cells. For the commercial mAb1427 recognizing human ADAM10, we used goat anti-mouse IgG (H+L) secondary antibody conjugated to HRP (Thermofischer Scientific). Color was developed using the TMB substrate kit (Thermo Scientific). The data was recorded at 450 nm.