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-keratan sulfate (KS) monoclonal antibody 5-D-4 (5D4). Undegraded aggrecan was recognized with an IC₅₀ of 0.27 μg/ml. When aggrecan was treated with human stromelysin (SLN), human leukocyte elastase (HLE), or papain, the degradation fragments had different hydrodynamic sizes. Treatment 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 the ELISA (IC₅₀-0.5 μg/ml), degradation by both HLE and papain significantly decreased the sensitivity for detection of KS epitope (IC₅₀-170 and 215 μg/ml, respectively). In addition, 5D4 detected single chain costal and corneal KS with much less sensitivity (IC₅₀-21 and 469 μg/ml, respectively) than undegraded aggrecan (IC₅₀-0.27 μg/ml).