Monoclonal Antibodies That Recognize Various Folding States of Pure Human Butyrylcholinesterase Can Immunopurify Butyrylcholinesterase from Human Plasma Stored at Elevated Temperatures

Abstract

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 capture completely denatured, boiled HuBChE. The highest quantity of HuBChE protein was extracted from 45 °C heat-denatured human plasma when HuBChE was immunopurified with a combination of monoclonals B2 18-5 and C191. Using a mixture of these two antibodies in future emergency response assays may increase the capability to confirm exposure to cholinesterase inhibitors.

Development of immunosorbents coupled on-line to immobilized pepsin reactor and micro liquid chromatography–tandem mass spectrometry for analysis of butyrylcholinesterase in human plasma

“…At the end of the grafting step, the supernatant containing unbound antibodies was analyzed by BCA assay to quantify the amount of antibodies grafted on sepharose. Monoclonal B2 18-5 antibodies (100 µg, 1 mg.mL -1 in PBS with 0.1% sodium azide) were easily bound to sepharose (35 mg) with 95% grafting yields, close to the 99% yields described by Peng et al for the immobilization of 5 mg of B2 18-5 antibodies on 1 g of CNBr activated sepharose, for 20 h [38]. Polyclonal antibodies N-15 and C-18 (100 µg, 200 µg.mL -1 in PBS with 0.1% sodium azide and 0.1% gelatin) were grafted on sepharose and the grafting yield was estimated to be 35% for N-15 and 38% for C-18 antibodies.…”

“…According to the results found in the literature in terms of affinity for HuBuChE, three antibodies directed against three epitopes were chosen for the preparation of immunosorbents: two polyclonal antibodies whose epitopes where respectively located near the N-terminal (N-15) [24] and C-terminal (C-18) amino acid sequence of HuBuChE [39] and one monoclonal antibody B2 18-5 whose epitope was not mentioned but whose affinity for HuBuChE was demonstrated by Peng et al [20,38,40]. The choice of the immobilization support is a key parameter for antibody grafting since it conditioned the chemical and mechanical properties of the immunosorbent.…”

“…epoxy activated polymethacrylate resin [36] and CNBr activated sepharose [19,37] were chosen. Sepharose is an agarose gel that has been widely used for antibody immobilization [19,29,37,38]. It displays several advantages such as good chemical stability covering a wide range of pH (2-9) and a relative hydrophilicity that minimizes non specific interactions between sepharose and hydrophobic proteins.…”

“…Its 700 Å pore size provides high capacity and relatively good accessibility of target molecules to the pores. Polymethacrylate resin was also chosen as grafting support for its high pore size (> 1000 Å), which allows proteins with high molecular weight and hydrodynamic diameter like HuBuChE (200 Å [38]) to easily access the antibodies bound inside the pores. It also has low cost and good stability at low pH, which is crucial regarding the acidic condition applied on IS during the elution step.…”

Online coupling of immunoextraction, digestion, and microliquid chromatography-tandem mass spectrometry for the analysis of sarin and soman-butyrylcholinesterase adducts in human plasma

“…Antibodies 3E8 are commercially available, and were mainly used for the extraction of HuBuChE from plasma, using the immunomagnetic approach [7,18,23,24,[38][39][40]43]. The use of mAb2 and B2 18-5 antibodies for HuBuChE purification is more recent and was developed by Oksana Lockridge, using different immobilization supports like Pansorbin cells [43], Dynabeads-Protein G [39,43,44] or CNBr-activated sepharose [45,46]. In these works [39,43], the potential of the three antibodies to extract unadducted HuBuChE from plasma was compared, but only in a semi-quantitative way.…”