Generation of monoclonal antibodies to cryptic collagen sites by using subtractive immunization

The extracellular matrix (ECM) plays a fundamental role in the regulation o f normal and pathological processes. The most abundantly expressed componen t found in the ECM is collagen. Triple helical collagen is known to be high ly resistant to proteolytic cleavage except by members of the matrix metall oproteinase (MMP) family of enzymes. To date little is known concerning the biochemical consequences of collagen metabolism on human diseases. This is due in part to the lack of specific reagents that can distinguish between proteolyzed and triple helical forms of collagen. Here we used the techniqu e of Subtractive Immunization (SI) to generate two unique monoclonal antibo dies (MAbs HUIV26 and HUI77) that react with denatured and proteolyzed form s of collagen, but show little if any reaction with triple helical collagen . Importantly, HUIV26 and HUI77 react with cryptic sites within the ECM of human melanoma tumors, demonstrating their utility for immunohistochemical analysis in vivo. Thus, the generation of these novel MAbs not only identif y specific cryptic epitopes within triple helical collagen, but also provid e important new reagents for studying the roles of collagen remodeling in n ormal as well as pathological processes.