Recombinant mac 1

Binding between Mac-1 and CD147 was evaluated using a modified enzyme-linked immunosorbent assay (ELISA). In a 96-well plate, wells were coated with recombinant Mac-1 (R&D Systems, Minneapolis, MN, USA) or bovine serum albumin (BSA) as the control. Recombinant CD147 was added in increasing concentrations (0–20 µg/mL) for 1 h. After removing CD147 and gentle washing, the wells were incubated with an anti-CD147 antibody (mouse-anti CD147, Abcam, Cambridge, UK) followed by a biotinylated secondary antibody (polyclonal rabbit anti mouse, Dako, Glostrup, Denmark) and a streptavidin/HRP complex (Life technologies, Carlsbad, CA, USA). The binding was detected using 3,3′,5,5′-tetramethylbenzidine (Serva, Heidelberg, Germany). The reaction was stopped using 1 M H₂SO₄. The absorption was measured using an ELISA plate reader at 450 nm with a reference value of 570 nm. Measurements from 6 independent experiments were analyzed.