A SOLID PHASE ASSAY FOR THE DETERMINATION OF HEPARAN SULFATE AND ITS APPLICATION TO NORMAL AND CANCEROUS HUMAN CARTILAGE SAMPLES

Abstract

A sensitive and accurate quantitative assay for the measurement of minor amounts of chondroitin/dermatan sulfate and heparan sulfate that does not require specific apparatus or reagents is described. The assay involves labeling of chondroitin sulfate A following reaction of carboxyl groups with biotin hydrazide in the presence of carbodiimide. ELISA plate wells were coated with glutaraldehyde and then spermine was coupled to it via a Schiff's base bond. In such activated wells, the biotinylated molecules were readily bound and detected after the interaction with avidin-peroxidase conjugates and the subsequent enzymic assay. Chondroitin/dermatan sulfate and heparan sulfate competed this interaction in a linear manner. Disaccharides derived from chondroitin sulfate A did not act as competitors, while heparan sulfate disaccharides showed significant competition. From the competition, before and after digestion with either chondroitinase ABC or heparitinases, the amounts of chondroitin sulfate and heparan sulfate in a sample could be calculated. The assay was applied for the determination of sulfated glycosaminoglycans in normal and cancerous human laryngeal cartilage samples. By using this procedure, the accurate determination, especially, of heparan sulfate in a mixture of glycosaminoglycans was achieved, which otherwise would require the use of very expensive technology.