A new competitive binding enzyme-linked immunosorbent assay for glycocalicin in plasma and platelet concentrate supernatants.

A new competitive binding enzyme-linked immunosorbent assay (CB ELISA) for glycocalicin (GC) was developed using GC coated wells and a monoclonal antibody (MAb) to glycoprotein Ib (AN51). The principal stages of the CB ELISA consisted of coating the plate with GC extract overnight, blocking with 3% BSA, incubating the wells with test or standard sample dilution and AN51, and a final incubation with horseradish peroxidase-conjugated goat anti-mouse IgG. Serial dilutions of purified GC, starting in 2% BSA, yielded standard curves which were linear between 10 and 0.4 micrograms/ml. Parallel curves were obtained for platelet concentrate supernatants and for citrated plasma. We used the ELISA to measure GC levels in platelet concentrates during storage. The results indicated that soluble GC increased progressively during storage from 3.3 to 6.7 micrograms/ml, while GC levels in platelet-poor plasma remained at 1.9-2.2 micrograms/ml. These results show that the new CB ELISA is a simple and short assay for the direct measurement of GC in plasma solutions, and may be of use in clinical studies.