A solid-phase assay for the quantitative analysis of hyaluronic acid at the nanogram level

A sensitive and accurate solid-phase assay for the quantitative determination of hyaluronic acid (HA) is described. The wells of the polystyrene microplates used were coated with glutaraldehyde followed, via a Schiff's base bond, with spermine to introduce amino groups. HA was added to the activated microwells in the presence of carbodiimide and left to bind via a peptide bond to the amino groups. Then aggrecan solution was added to the wells of the microtiter plates to interact with its G1 domain with hyaluronic acid, and the amounts of aggrecan bound were measured immunochemically. The inhibition of the binding between aggrecan and immobilized HA due to soluble HA present in reference solutions showed linearity in the range of concentrations 0.1 to 0.7 microg/ml. The reaction is specific and rapid and can be widely used for the calculation of HA in body fluids directly and in tissue samples after a brief digestion with a proteolytic enzyme.