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

Abstract

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 < Lipoprotein (a) < low density lipoprotein; rat chondrosarcoma proteoglycan < bovine nasal cartilage proteoglycan < human aorta proteoglycan; chondroitin 4-sulfate < chondroitin 6-sulfate < dermatan sulfate for lipoproteins, proteoglycans, and glycosaminoglycans, respectively.