SENSITIVITY OF MONOCLONAL-ANTIBODY, 5-D-4, FOR THE DETECTION OF AGGRECAN, AGGRECAN FRAGMENTS, AND KERATAN SULFATE

Bovine nasal septum aggrecan and selected proteinase-digested products of aggrecan were evaluated in an inhibition ELISA using the anti-kera tan sulfate (KS) monoclonal antibody 5-D-4 (5D4). Undegraded aggrecan was recognized with an IC50 of 0.27 mug/ml. When aggrecan was treated with human stromelysin (SLN), human leukocyte elastase (HLE), or papai n, the degradation fragments had different hydrodynamic sizes. Treatme nt with SLN produced the largest fragments, HLE generated intermediate fragments, and papain the smallest fragments. Whereas degradation of aggrecan by SLN had little effect on recognition of proteoglycan in th e ELISA (IC50 - 0.5 mug/ml), degradation by both HLE and papain signif icantly decreased the sensitivity for detection of KS epitope (IC50 - 170 and 215 mug/ml, respectively). In addition, 5D4 detected single ch ain costal and corneal KS with much less sensitivity (IC50 - 21 and 46 9 mug/ml, respectively) than undegraded aggrecan (IC50 - 0.27 mug/ml).