A method for the quantitation of hyaluronan (hyaluronic acid) in biological fluids using a labeled avidin-biotin technique

Abstract

An improved method for the detection and quantitation of hyaluronan (hyaluronic acid) (HA) in biological fluids is described. The principle on which the method is based is that HA binds strongly to a biotinylated HA-binding protein (B-HABP) which was prepared from cartilage proteoglycans. HA was immobilized on polyvinyl chloride plates which had been precoated with poly-L-lysine. The unknown sample or HA standards together with excess B-HABP are then added. The B-HABP that binds to the immobilized HA is then incubated with the enzyme-conjugated avidin (e.g., alkaline phosphatase), and the color which develops on addition of enzyme substrate (e.g., p-nitrophenyl phosphate) is determined by light absorption using a microtitration plate reader. The assay is not only convenient and reliable but is capable of measuring HA in solution at the picogram level. The assay was used to determine HA levels in human sera and synovial fluid taken from volunteers and patients with rheumatoid arthritis and osteoarthritis.