Characterization of anti-OPN MAb. All Mab were screened based on their ELISA detection of full-length human OPN (fl-OPN). A) Recombinant human or mouse fl-OPN were used for immunoblot with the anti-OPN MAb indicated. Each lane was loaded with 20 ng OPN protein for detection. While the recognition of human OPN was greater by all MAb, they all cross-reacted with murine OPN. B) An ELISA was performed using the human N-terminal (aa-1-167) or C-terminal (aa167-314) human recombinant fragments. Each lane was loaded with 250 ng protein. MAb 1E3 is an irrelevant antibody used as a negative control. 2C5, 2H9, 2F10, and 2E11 recognize OPN epitopes on the N-terminal fragment, while 1F11 recognizes an epitope on the C-terminal fragment. C) The same OPN fragments were tested by immunoblot, and yielded consistent results with the ELISA.