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

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.

Towards the fabrication of a label-free amperometric immunosensor using SWNTs for direct detection of paraoxon.

A label-free immunosensor based on SWNTs modified GC electrodes has been developed for the direct detection of paraoxon. Based on aryldiazonium salt chemistry, forest of SWNTs can be vertically aligned on mixed monolayers of aryldiazonium salt modified GC electrodes by C-C bonding, which provides an interface showing efficient electron transfer between biomolecules. PEG molecules were introduced to the interface to resist non-specific protein adsorption. Ferrocenedimethylamine (FDMA) was subsequently attached to the ends of SWNTs through the amide bonding followed by the attachment of epitope i.e., paraoxon hapten to which a paraoxon antibody would bind. This immunosensor shows good selectivity and high specificity to paraoxon, and is functional for the detection of paraoxon in both laboratory and field by a displacement assay. There is a linear relationship between electrochemical signal of FDMA and the concentration of paraoxon over the range of 2-2500 ppb with a lowest detected limit of 2 ppb in 0.1 M phosphate buffer at pH 7.0. The SWNTs based amperometric immunosensor provides an opportunity to develop the sensing system for on-site sensitive detection of a spectrum of insecticides.

Lateral flow immunochromatographic assay for sensitive pesticide detection by using Fe3O4 nanoparticle aggregates as color reagents.

Magnetic Fe(3)O(4) particle aggregates were prepared by cross-linking Fe(3)O(4) nanoparticles bearing surface carbonyl groups with poly-L-lysine. Upon further coupling with antiparaoxon methyl polyclonal antibody, the resultant particle aggregate-based probes were used in a lateral flow immunochromatographic assay (LFIA) of pesticide residue of paraoxon methyl. The results were compared with that achieved by using the mother Fe(3)O(4) nanoparticles. More quantitative results on the signal amplification effect endowed by the controlled aggregation of Fe(3)O(4) nanoparticles were extracted by relative optical density analysis. Under optimized conditions, a detection limit of 1.7 ng/mL for paraoxon methyl was achieved by using the particle aggregates, which is almost 40-fold lower than that based on the mother Fe(3)O(4) nanoparticles.

Squaric monoamide monoester as a new class of reactive immunization hapten for catalytic antibodies.

A squaric monoester monoamide motif was employed as an effective reactive immunogen for the discovery of monoclonal antibodies with reactive residue(s) in their combining sites. Two antibodies, 2D4 and 3C8, were uncovered that enhance paraoxon hydrolysis over background. Kinetic analysis of these antibodies was performed and interestingly both undergo a single turnover event due to covalent modification within the antibody combining site. Because antibodies 2D4 and 3C8 result in covalent attachment and thus inactivation of paraoxon, they could be useful probes for investigating paraoxon intoxication.

[Production and identification of anti-paraoxon monoclonal antibodies].

Paraoxon (E600) was conjugated to bovine serum albumin(BSA) or tachypleus tridentatus hemocyanin (TTH) by diazotization. Two hybridoma cell lines secreting monoclonal antibodies(McAb) against paraoxon have been established by fusing mouse myeloma cells and splenocytes from Balb/c mice immunized with E600-BSA. The chromosomes of the hybridoma cell 2B10 were analyzed. The immunoglobulin of the McAb was classified. The affinity and specificity of this antibody were determined. The hybridoma cells have fairly reserved the antibody-producing capacity after continuously growing or stored in liquid nitrogen for 10 weeks.

A novel immunoassay with direct relevance to protection against organophosphate poisoning.

Antiparaoxon immune sera were employed in a new immunoassay based on competition between acetylcholinesterase and antibodies for the binding of paraoxon. Unlike radioimmunoassay, the new assay described herein can be extended to predict the feasibility of antibodies to confer in vivo protection of acetylcholinesterase against organophosphate poisoning. The toxicity of paraoxon was reduced in mice which were preinjected with the immune sera.

[Changes in the cholinesterase activity and glucose concentration in the blood induced by paraoxon in rabbits immunized against paraoxon]. / Veränderungen der Cholinesteraseaktivitäten und der Glukosekonzentration im Blut durch Paraoxon bei gegen Paraoxon immunisierten Kaninchen.

The immunization of rabbits with a paraoxon-HSA-conjugate resulted in an antibody response with titres of 1:25000 to 1:100000 of antisera dilution and average affinity constants K0 of 10(6) M-1 and heterogeneity indices of 0.8 to 0.9 calculated by means of the Sips equation. The serum cholinesterases and the erythrocyte acetylcholinesterase in immunized rabbits were protected against a stronger inhibition by parenteral application of paraoxon. An increase of blood glucose after paraoxon application to immunized rabbits could not be observed but was detectable in unimmunized animals.

[Production and characterization of antisera against O,O-diethyl-O-(p-nitrophenyl)-phosphate (paraoxon)]. / Erzeugung und Charakterisierung von Antiseren gegen O,O-Diäthyl-O-(p-nitrophenyl)-phosphat (Paraoxon).

Methods for the production of antibodies against Paraoxon in rabbits are described. The highest-titre antisera were produced with conjugates containing a succinyl spacer group with a degree of derivatisation between 8 and 12 haptens per albumin molecule. The antibody response was tested by immunoprecipitation in agar gel with a hapten-rabbit-albumin-complex and by a radioimmunoassay. The synthesis of 125I labelled tracers is reported. The most potent antisera bound 50% of the radioactive tracer after 1:10(5) dilution and showed apparent affinity constants of 10(6) to 10(7) M-1 for Paraoxon. Compared to Paraoxon these antisera possessed cross reactivities with Parathion of 8.8 to 13.8%, with Methylparathion of 0.35 to 0.82%, and with Dimethoat of 0.0006 to 0.0012%.