13] Immunological characterization of cartilage proteoglycans

Abstract

Immunological and some affinity methods offer possibilities for recognizing and quantitating the protein core, and specific regions of the protein core, and are potentially of great importance for probing proteoglycan structure. This chapter presents procedures for isolating and purifying cartilage proteoglycan antigens and methods for raising antibodies and testing their specificities. Isolation of proteoglycan antigens from cartilage include: extraction with 4M guanidine HCl and protease inhibitor filter, dialyzation, addition of CsCl centrifuge, and addition of guanidine hydrochloride. The mixture is then stirred overnight before centrifuging under similar conditions. Tubes are sliced to give an A1D1and an A1D4 fraction. Fractions are dialyzed successively against water, NaC1, and water. Each dialysis step is for 24 hr at 4 °. Fractions are recovered by lyophilization. Yields are approximately 3 g of A1D1 and 100 mg of A1D4. Several animal species have been used by various research groups to raise antisera against the proteoglycan aggregate components, proteoglycan monomer, and link proteins.