Study Of Chemiluminescent Enzyme Immunoassay For The Determination Of Triazophos, Chloroamphenicol And Zearalenone

The residues of pesticide, veterinary drug and biotoxin may cause health implications for living organisms including humans. There has been a greater demand for developing analytical methods with very high sensitivity. Immunochemical techniques provide a simple and economical alternative to traditional methods in order to meet such demands for determination of pesticide, veterinary drug and biotoxin residues.Triazophos, chloroamphenicol and zearalenone were chosen to represent for the three kinds of compounds. The aim of this work was to develop an indirect competitive chemiluminescent enzyme immunoassay (ic-CLEIA) with an anti-mouse secondary antibody conjugated to peroxidase for the residual determination of triazophos, chloroamphenicol and zearalenone, thus exploring the similarities and differences between pesticide, veterinary drug and biotoxin, and providing the basis to future research.Luminol solution was used as the substrate of horseradish peroxidase. First, the optimal coating conjugate concentration and monoclonal antibodies dilution was studied. Then the chemiluminescence substrate solution was obtained after a variety of conditions were optimized, including the selection of buffer and enhancer, concentrations of luminol, p-iodophenol and H2O2. On the basis of it, the effects of several physicochemical parameters on ic-CLEIA performance were studied and optimized, such as the effects of ionic strength and pH, the comparison of different blocking substances and the tolerance to organic solvents (methanol, acetonitrile, acetone). After the condition optimization, the standard curves of CLEIA competitive reaction for triazophos, chloroamphenicol and zearalenone were established.Recoveries of the three compounds spiked into environmental samples, animal samples and food samples were determined by CLEIA after sample matrix effect testing. The optimized method was applied to the detection of triazophos residues in paddy water, soil, and rice grain samples; and detection of chloroamphenicol residues in shrimp, chicken and pork samples; and detection of zearalenone in corn, wheat and sorghum rice samples. The precision and accuracy were analyzed as well.Under the optimum conditions, the linear rang of the icCLEIA for triazophos was from0.097to12.5ng/mL. The limit of detection values (IC10) was0.14ng/mL, and the standard curve midpoint (IC50) was1.23ng/mL. The linear rang of the icCLEIA for chloroamphenicol was from1.95to125ng/mL, IC10was2.96ng/mL, and IC50was19.25ng/mL. The linear rang of the icCLEIA for zearalenone was from0.0625to2ng/mL, IC-10was0.031ng/mL, and IC50was0.19ng/mL.The assay performance indicated that the CLEIA could offer the possibility of improving the sensitivity of immunoassays by1-2orders of magnitude compared to conventional ELISA which is widely used to detect triazophos. The matrix effects of different samples were studied. According to the results, different dilution ratios was determined:the paddy water, soil, and rice grain samples were diluted with reacting buffer by10,100,50-fold respectively; the shrimp, chicken and pork samples were diluted with reacting buffer by2,1,2-fold respectively; the corn, wheat and sorghum rice samples were diluted with reacting buffer by14,14,80-fold respectively. The assay was then used to detect of triazophos, chloroamphenicol and zearalenone in the practical samples at above-mentioned dilution ratios. The results showed that when the spiked level was from5-800ng/g, the recoveries of triazophos from spiked samples ranged from67%to113%with coefficients of variation of0.08～24.53%; when the spiked level was from0.5-20ng/g, the recoveries of chloroamphenicol from spiked samples ranged from72%to95%with coefficients of variation of0.09～23.08%; when the spiked level was from7～400ng/g, the recoveries of zearalenone from spiked samples ranged from80%to111%with coefficients of variation of1.05～16.07%.Summarily, the established CLEIA was a simple, rapid, and economical tool which was suitable for the rapid screen of triazophos, chloroamphenicol and zearalenone residues in environmental samples, animal samples and grain samples with a high sensitivity. It could be the developing foundation of CLEIA test kits in the future, and it indicated that the CLEIA method had good prospects of application on pesticide, veterinary drug, and biotoxin residue detection.