James E. Christner

A competitive assay of lipoprotein: Proteoglycan interaction using a 96-well microtitration plate

A method for the microassay in vitro of lipoprotein: proteoglycan interactions is described. The wells of a plastic 96-well microtitration plate are coated with low density lipoprotein. A limiting quantity of biotin-conjugated proteoglycan is allowed to bind to each coated well, and the amount of the latter retained in wells is estimated spectrophotometrically through subsequent binding of alkaline phosphatase-conjugated avidin. Many of the incubation parameters (e.g., time, pH, salt concentration, divalent cations), which influence the extent of binding of biotin-conjugated proteoglycan, have been studied and optimized. The effect upon binding of introducing different levels of proteoglycans or lipoproteins at the interaction step can be measured readily. Thus, the orders of increasing relative binding affinities were found to be high density lipoprotein less than Lipoprotein (a) less than low density lipoprotein; rat chondrosarcoma proteoglycan less than bovine nasal cartilage proteoglycan less than human aorta proteoglycan; chondroitin 4-sulfate less than chondroitin 6-sulfate less than dermatan sulfate for lipoproteins, proteoglycans, and glycosaminoglycans, respectively.

[13] Immunological characterization of cartilage proteoglycans

Publisher Summary 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.

The specificity of a mouse monoclonal antibody to human aorta proteoglycans

Mouse monoclonal antibodies to human aorta proteoglycans have been raised. A sensitive radioimmunoassay which can be employed to measure binding of proteoglycans to these antibodies is described. It offers advantages of simplicity and speed over other immunoassays currently employed for proteoglycan determination. One of the monoclonal antibodies, 6D2/B5, recognizes keratan sulfate-bound antigens (e.g. cartilage proteoglycans). Further work has indicated that this antibody is not specific for keratan sulfate, as brain sulfatide and the seaweed polysaccharide, fucoidan, also possess its epitope. The 6D2/B5 antibody can be of value in localizing and estimating keratan sulfate if additional biochemical criteria for keratan sulfate recognition are utilized.