The Studies On The Immunoassay For Rapid Detection And Diagnosis Of Exposure To Organophosphorous Pesticides As The Nerve Agent

A sandwich ELISA (sELISA) has been developed for detection of organophosphorylated butyrylcholinesterase (OP-BChE, paraoxon-BChE as a model), a potential biomarker for human exposure to organophophate insecticides and nerve agents. A pair of antibodies specific to OP-BChE adduct were identified through systematic screening of several anti BChE antibodies (anti-BChE) and anti-phosphoserine antibodies (anti-Pser) from different sources. The selected anti-BChE (set as capture antibody) recognized both phosphorylated and nonphosphorylated BChE. The anti-Pser (set as detecting antibody) was used to recognize the OP moiety of OP-BChE adducts. With the combination of the selected antibody pair, several key parameters (such as the concentration of anti-BChE and anti-Pser, and the blocking agent) were optimized to enhance the sensitivity and selectivity of the sELISA. Under the optimal conditions, the sELISA has shown a wide linear range from0.03nM to30nM, with a detection limit of0.03nM. Furthermore, the sELISA was successfully applied to detect OP-BChE from in vitro biological samples such as rat plasma spiked with OP-BChE with excellent adduct recovery (z>99%). These results demonstrate that this novel approach holds great promise to develop an ELISA kit and offers a simple and cost-effective tool for screening/evaluating exposure to organophosphate insecticides and nerve agents.We present a novel disposable electrochemical immunosensor for highly selective and sensitive detection of organophosphorylated butyrylcholinesterase (OP-BChE), a specific biomarker for exposure to organophosphorus nerve agents. In our new approach, the zirconia nanoparticles (ZrO2) were employed to selectively capture the OP moiety of OP-BChE adducts, and followed by quantum dot (QD)-tagged anti-BChE antibodies for amplified quantification. The captured CdSe-QD tags can be sensitively detected by stripping voltammetry. The nerve agent, diisopropylfluorophosphate (DFP), was selected to prepare OP-BChE adducts in various matrices. The developed electrochemical immunosensor demonstrates a highly linear voltammetric response over the range of0.1to30nM OP-BChE, with a detection limit of0.03nM (based on S/N=3) coupled with a good reproducibility (R.S.D=4.5%). Moreover, the immunosensor can be successfully applied for diagnosis of in-vitro OP nerve agent exposure through biomonitoring of OP-BChE adducts in the plasma samples. This novel nanoparticle-based electrochemical immunosensor thus provides an alternative way for designing simple, fast, sensitive, and cost-effective sensing platform for on-site screening/evaluating exposure to a variety of OP nerve agents.In this study, we also present a novel potable immunochromatographic electrochemical biosensor (IEB) for simple, rapid, and sensitive biomonitoring of trichloropyridinol (TCP), a metabolite biomarker of exposure to organophosphorus insecticides. Our new approach takes the advantage of immunochromatographic test strip for a rapid competitive immunoreaction and a disposable screen-printed carbon electrode for a rapid and sensitive electrochemical analysis of captured HRP labeling. Several key experimental parameters (e.g. immunoreaction time, the amount of HRP labeled TCP, concentration of the substrate for electrochemical measurements, and the blocking agents for the nitrocellulose membrane) were optimized to achieve a high sensitivity, selectivity and stability. Under optimal conditions, the IEB has been demonstrated a wide linear range (0.1-100ng/ml) with a detection limit as low as0.1ng/ml TCP. Furthermore, the IEB has been successfully applied for biomonitoring of TCP in the rat plasma samples with in vivo exposure to organophosphorous insecticides like Chlorpyrifos-oxon (CPF-oxon). The IEB thus opens up new pathways for designing a simple, rapid, clinically accurate and quantitative tool for TCP detection, as well as holds a great promise for in-field screening of metabolite biomarkers, e.g., TCP, for humans exposed to organophosphorous insecticides.In this study, we also reported a new approach for electrochemical quantification of enzymatic (BChE) inhibition for biomonitoring of exposure to organophosphorous (OP) pesticides and nerve agents based on magnetic beads (MB) immunosensing platform. The principle of this approach is based on the combination of MB immunocapture-based enzyme activity assay and competitive immunoassay of the total amount of BChE for simultaneous detection of enzyme inhibition and phosphorylation in biological fluids. In competitive immunoassay, the target BChE in a sample competes with the BChE immobilized on the MBs to bind to limited sites of anti-BChE antibody labeled with quantum dots (QD-anti-BChE), followed by stripping voltammetric analysis of the bound QD conjugate on the MBs. This assay shows a linear response over the total BChE concentration range of0.1-20nM. Simultaneous real time BChE activity was measured on an electrochemical carbon nanotube-based sensor coupled with a microflow injection system after immunocapture by the MB-anti-BChE conjugate. Therefore, he formed phosphorylated BChE adduct (OP-BChE) can be estimated by the difference values of the total amount of BChE (including active and OP-inhibited) and active BChE from established calibration curves. Under the optimal conditions, it is sensitive enough to detect0.5nM OP-BChE, which is less than2%BChE inhibition.A new specific monoclonal antibody (McAb) against the organophosphorous pesticide fenthion was generated based on5synthesized haptens (H1-H5), and a sensitive and specific Enzyme-Linked Immunosorbent Assay (ELISA) for detection of fenthion based on the new immunizing/coating hapten combination was developed. In this study, the H3(6-(methoxy(4-(methylthio) phenoxy) phosphorothioylamino) hexanoic acid) which attempts to expose the aromatic ring group was conjugated with Bovine Serum Albumin (BSA) used as immuogen; and H1(4-(2-(dimethoxyphosphorothioyloxy)-4-methyl-5-(methylthio)phenylamino)-4-oxobutanoic acid, H2(4-(4-(dimethoxyphosphorothioyloxy)-2-methylphenylamino)-4-oxobutanoic acid, H4(4-(dimethoxyphosphorothioylamino)butanoic acid) and H5(4-(3-methyl-4-(methylthio) phenoxy) butanoic acid) were conjugated with ovalbumin (OVA) for the coating antigen.The BLAB/C mice were immunized by the BSA conjugation (H3-BSA, immunogen) with the intraperitoneal injection. The hybridoma cell lines which can stable secrete McAb with the high specificity to fenthion were established by hybridoma technology. All of the5OVA conjugations were used as coating antigen. The efficient antibody/coating antigen was selected from the5combinations. Furthermore, the optimal competitive indirect ELISA was developed, with the detection limit (IC20) of0.028ng/ml. The cross reactivity of the antibody with the organophophorous pesticide Parathion was0.5%, even lower than0.2%with the any other tested pesticides. Also, the average recovery of the optimal ELISA from real samples such as soil, water, rice and Chinese cabbages was80%-124%.