Differentiation between Acetylcholinesterase and the Organophosphate-inhibited Form Using Antibodies and the Correlation of Antibody Recognition with Reactivation Mechanism and Rate

Abstract

Two types of polyclonal antibodies were generated from (a) a decapeptide sequence that includes the active site serine of acetylcholinesterase (anti-AChE 10S ) and (b) the identical decapeptide sequence phosphorylated at the active site serine of acetylcholinesterase (antiAChE 10SP ). The anti-AChE 10S antiserum was found to specifically recognize native, control, and vehicletreated recombinant mouse AChE (rMoAChE) but did not recognize rMoAChE that was phosphorylated by the four organophosphate (OP) compounds tested. Conversely the anti-AChE 10SP antiserum recognized phosphoserine rMoAChE that resulted from reaction with phosphorous oxychloride (POCl 3 ) but did not recognize native or vehicle-treated rMoAChE. Anti-AChE 10SP also did not recognize OP-AChE conjugates that resulted from the reaction of rMoAChE with other OP compounds that afford neutral or monoanionic phosphoserine groups thereby indicating a high specificity for a precise OP conjugate. Antisera recognition correlated well with the rates of enzyme inhibition, aging, and oxime-induced reactivation indicating these antisera can both quantify the extent and type of inhibition and also differentiate between select mechanisms of inhibition. The ability to discern mechanistic differences between native AChE and OP-AChE conjugates suggests that these antisera can be used to identify biomarkers of OP exposure in a mechanism-based approach.

Immunodetection of Serum Albumin Adducts as Biomarkers for Organophosphorus Exposure

“…Previously, a report showed that antibodies could distinguish phosphorylated AChE and inhibited AChE using this strategy (George et al, 2003), but despite their utility, these polyclonal anti-phosphorylated AChE antibodies did not have the ability to distinguish different OP adducts. The results of that report did suggest that it was possible to raise antibodies to individually recognize specific modifications of the dianionic phosphorus of OP-adducted proteins (George et al, 2003).…”

“…Generally, the approach to detect protein-OP adducts has relied on MS (Thompson et al, 2010), although one report showed that antibodies could be raised against phosphorylated AChE (George et al, 2003). However, in that report, the antibodies reported to recognize the inhibited form of AChE did not distinguish OP-AChE adducts arising from different OPs (George et al, 2003).…”

“…Generally, the approach to detect protein-OP adducts has relied on MS (Thompson et al, 2010), although one report showed that antibodies could be raised against phosphorylated AChE (George et al, 2003). However, in that report, the antibodies reported to recognize the inhibited form of AChE did not distinguish OP-AChE adducts arising from different OPs (George et al, 2003). An attempt to develop antibodies against phosphonylated BChE showed that the chemical instability of phosphonylated decapeptide haptens designed to mimic phosphonylated BChE precluded using this approach (MacDonald et al, 2010, and unpublished observations).…”

Analysis and sequencing of the active-site peptide from native and organophosphate-inactivated acetylcholinesterase by electrospray ionization, quadrupole/time-of-flight (QTOF) mass spectrometry

“…1). A number of studies have been undertaken to understand the chemical nature of the OP-AChE modification [3,[8][9][10][11][12][13][14]. Importantly, OP-AChE conjugates differ in the phosphoester ligands, which defines a unique molecular weight identifier for analysis and potentially a method for discriminating between OP-AChE conjugates.…”

“…Therefore, MS methods developed for rMoAChE will be applicable to other mammalian AChEs, and other select esterases (e.g., butyrylcholinesterase) bearing homologous active-site sequences. To further aid the analysis, the active-site serine inclusive peptides TLFGESAGAA (AChE 10S ) [14] and GESAGAASVGMHIL (AChE 14S ; equivalent to the chymotryptic fragment, Fig. 1), were synthesized and used to develop methods for detection of the active-site peptide from rMoAChE.…”