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.