Endoglycosidase assay

Abstract

A solid phase substrate which yields soluble labeled products upon hydrolysis by a glycosaminoglycan endoglycosidase and methods of producing said substrate are comprised in the present invention. The solid phase substrate comprises glycosaminoglycan bearing labeled substances bound to amino groups. The labeled glycosaminoglycan substrate is reductively aminated at its reducing terminal end to produce an amine-terminus. The substrate is further coupled to an amino-reactive solid matrix through its amine-terminus. A method of producing the solid phase substrate comprises the steps of: at least partially N-desulfating or N-deacylating a glycosaminoglycan; labeling at least partially N-deacylated or N-desulfated glycosaminoglycan with a substance yielding detectable signals to produce labeled glycosaminoglycan; completely N-acylating the labeled glycosaminoglycan with acyl anhydride or acyl halide; reductively aminating a reducing terminal end of said labeled glycosaminoglycan to produce labeled amine-terminal glycosaminoglycan; and coupling, through its terminal amine, the labeled amine-terminal glycosaminoglycan to an amino-reactive solid phase support to produce the solid phase substrate. The solid phase substrate is usable to detect metastatic tumors by measurement of serum heparanase levels. The potential metastases of a tumor may also be determined by its heparanase levels.