Preparation and characterization of diethoxy- and monoethoxy phosphylated (‘aged’) serine haptens and use in the production of monoclonal antibodies

Highlights

• Phosphonate analogs of phosphoserine were prepared.

• Diethoxyphosphoserine and monoethoxyphosphoserine antibodies were prepared.

• A new mass spectrometry method was devised to assess hapten density.

• Antibodies recognize paraoxon-modified rat blood.

Abstract

In this study, the first mechanism-based monoclonal antibodies have been produced that recognize and differentiate diethoxy- and monoethoxyphosphorylated serine residues. Haptens were synthesized as the stable phosphonate form of phosphoserine esters to improve the immunoresponse. Following condensation with a glutaric anhydride to link the phosphoserine moieties to carrier protein, the hapten densities attached to bovine serum albumin and keyhole limpet henocyanin were determined by partial trypsin digestion and MALDI mass spectrometry, and confirmed using a fluorescent assay (FITC) to quantify unmodified lysine residues. The conjugation reactions were pH optimized to improve hapten density. Screening of subclones led to the identification of two monoclonal antibodies: (a) N257/25.11 that specifically recognizes (EtO)₂P(O)-Ser as the phosphylated or inhibited form, and (b) N262/16 that recognizes (EtO)(HO)P(O)-Ser as the ‘aged’ form. Analysis of blood samples treated with paraoxon (EtO)₂P(O)-OPhNO₂ showed a concentration dependent recognition of the phosphylated form.

Introduction

Chronic, low-level exposure to organophosphate (OP) insecticides has been associated with a number of minor conditions, including fatigue, headache, and common cold or flu-like symptoms, but also other, more concerning morbidities such as acute cholinergic poisoning, ataxia, delayed neuropathy, intermediate syndrome, pulmonary toxicity, genotoxicity, Parkinson’s disease and vision loss [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13]. The diversity of conditions and illnesses suggests that OPs react with numerous protein targets [14], [15] other than acetylcholinesterase (AChE) and early detection of a wider range of OP-modified targets could indicate courses of treatment and aid in disease prevention.

Most commercial OP insecticides share a common structural motif: a diethoxyphosphorothionate 1 that vary in the leaving group Z (Scheme 1). However, it is widely known that the myriad of cholinergic and non-cholinergic effects following OP exposure are due to the highly reactive oxon form 2 of the insecticide from oxidative desulfurization that acts as an indiscriminate phosphylating agent with chemical properties similar to nerve agents. For example, diethoxy OP oxons react readily with the target enzyme AChE to form DEP-AChE adducts (Scheme 1) that trigger cholinergic toxicity.

Formation of the OP-AChE conjugate can be reversed by water or oxime antidotes to partially restore the enzymatic activity [16], [17], [18]. Subsequent to the inhibition, a process known as ‘aging’ can also occur that results in the loss of a phosphoester group and formation of the oxyanion, or monoethoxyphosphoryl (MEP) AChE conjugate (Scheme 1).

Oxons also react to afford other OP-modified proteins [14], [15], [19], [20]. However, OP oxons are too reactive to quantify in vivo, and researchers have focused their attention on OP-modified proteins or OP-biomarkers since they offer a more reliable alternative for confirming low-level exposures. Identification of insecticide-based biomarkers should be straightforward since a majority of OP insecticides form the aforementioned diethoxy- and monoethoxyphosphoryl conjugates at serine (OP-tyrosine adducts are also known, see: [21], [22], [23], [24]) thereby reducing the biomarker panel to the DEP-serine and MEP-serine subtypes. Detection of DEP- and/or MEP-serine conjugates would provide accurate, mechanistically precise indicators of OP insecticide exposure. Of key importance is that those two general biomarkers of OP insecticide exposure are chemically distinct as neutral (DEP) or charged (MEP) conjugates that can be differentiated by antibodies (Scheme 2) [25]. Moreover, a dual-analyte approach introduces the possibility of identifying each conjugate, which may distinguish between acute and chronic exposure and thus may prove important for directing proper therapeutic intervention.

Although antibodies for detecting organophosphorus compounds in vitro are known [26], [27], [28], [29], [30], antibodies to OP-adducted proteins have not been widely reported [30], [31], [32]. Indirectly, immunoprecipitation of OP-protein targets using antibodies to butyrylcholinesterase followed by digestion and mass spectral characterization of the OP-modified peptide has been applied to address the problem [20], [33], [34], [35], [36], [37].

As noted, insecticide oxons are similar to chemical nerve gas agents in their reactivity and selectivity toward protein residues such as serine. As a result, DEP- or MEP-modified serines 3 and 4 (Scheme 2) represent chemically precise, small molecule representations of insecticide oxon biomarkers. Antibodies thus derived from DEP-serine and MEP-serine would be expected to selectively recognize proteins modified at serine by insecticide oxons. Therefore, this study seeks to prepare and characterize DEP- and MEP-serine moieties as haptens (Scheme 2) and produce antibodies that selectively recognize those structures.