Monoclonal Antibodies ThatRecognize Various Folding States of Pure Human ButyrylcholinesteraseCan Immunopurify Butyrylcholinesterase from Human Plasma Stored atElevated Temperatures

摘要

Human plasma to be analyzed for exposure to cholinesterase inhibitors is stored at 4 °C or lower to prevent denaturation of human butyrylcholinesterase (HuBChE), the biomarker of exposure. Currently published protocols immunopurify HuBChE using antibodies that bind native HuBChE before analysis by mass spectrometry. It is anticipated that the plasma collected from human casualties may be stored nonideally at elevated temperatures of up to 45 °C for days or maybe weeks. At 45 °C, the plasma loses 50% of its HuBChE activity in 8 days and 95% in 40 days. Our goal was to identify a set of monoclonal antibodies that could be used to immunopurify HuBChE from plasma stored at 45 °C. The folding states of pure human HuBChE stored at 4 and 45 °C and boiled at 100 °C were visualized on nondenaturing gels stained with Coomassie blue. Fully active pure HuBChE tetramers had a single band, but pure HuBChE stored at 45 °C had four bands, representing native, partly unfolded, aggregated, and completely denatured, boiled tetramers. The previously described monoclonal B2 18-5 captured native,partly unfolded, and aggregated HuBChE tetramers, whereas a new monoclonal,C191 developed in our laboratory, was found to selectively capturecompletely denatured, boiled HuBChE. The highest quantity of HuBChEprotein was extracted from 45 °C heat-denatured human plasmawhen HuBChE was immunopurified with a combination of monoclonals B218-5 and C191. Using a mixture of these two antibodies in future emergencyresponse assays may increase the capability to confirm exposure tocholinesterase inhibitors.