Enzyme-linked immunosorbent assay for proteolytically inactivated antithrombin-III: use of sodium dodecyl sulfate to eliminate signal due to intact antithrombin-III.
Antithrombin III (AT-III) is a serine protease inhibitor (serpin) that can be catalytically inactivated by human neutrophil elastase (HNE) without inhibiting HNE activity. As with catalytic inactivation of most serpins, the cleaved form of the inhibitor is difficult to measure in the presence of active inhibitor. One major difference between the cleaved and intact forms of AT-III is that the cleaved form adopts a more stable conformation. Using sodium dodecyl sulfate (SDS), we were able to devise an enzyme-linked immunosorbent assay (ELISA) capable of detecting cleaved AT-III in the presence of intact AT-III. It seems likely that the SDS alters the intact AT-III so that it is not detected in the ELISA. As little as 5 micrograms/ml HNE-cleaved AT-III could be detected when spiked into human plasma; HNE-cleaved AT-III spiked into human plasma at different levels was recovered as expected. Thrombin-cleaved AT-III was also detected using this ELISA. The generation of cleaved AT-III in human plasma by HNE in the presence of heparin could be monitored as well. The cleaved AT-III ELISA is a novel, yet simple way to measure proteolytically inactivated AT-III in the presence of intact AT-III and should be useful for studying the role of proteolytic inactivation of serpins such as AT-III in vivo.
Esmon
P C
PC
Department of Experimental Therapeutics, Cutter Biological, Miles Inc., Berkeley, California 94701.
Yee
E
E
D000990:Antithrombin III / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008722:Methods; D009504:Neutrophils / Q000201:enzymology; D010196:Pancreatic Elastase / Q000494:pharmacology*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry
D012967:Sodium Dodecyl Sulfate; D000990:Antithrombin III; D010196:Pancreatic Elastase