The Study Of Chlorothalonil Enzyme Immunoassay

Chlorothalonil is a efficiency broad-spectrum protective fungicide which was developed by Diamond Alkali Company of United States in1963. At home and abroad, chlorothalonil is widely used for agricultural production. It is mainly applied in vegetables, crops, fruit trees, tea, tobacco and golf, which prevents and treats the fungal diseases. Chlorothalonil and its metabolites have great residues in fruits, vegetables, and other environmental samples, because it has been extensively used and has a good adhesion on the plant. Some researches have shown that chlorothalonil can be detected out in the Arctic. Chlorothalonil has low toxicity to humans and livestock, but it high to fish, shell and other aquatic organisms. Chlorothalonil is classified in the B2group, which is considered as a probable human carcinogen by the researches of US EPA.4-hydroxy-chlorothalonil is the major degradation products of chlorothalonil in soil and water, which has higher toxic, better adsorption capacity and longer half-life than chlorothalonil, at the same time it also has a good water-soluble. Therefore,4-hydroxy chlorothalonil will cause more serious effects on the environment and human health. In2009, the US EPA revised the maximum residue limits of chlorothalonil, which was not only focus on chlorothalonil, but also on4-hydroxy chlorothalonil.Currently, in China, the commonly detection methods of chlorothalonil are gas chromatography, high performance liquid chromatography and other instrumental analysis methods, the detection of4-hydroxy chlorothalonil is rarely reported. There are some problems of instrumental methods, such as pre-treatment complex, time-consuming and high cost, which are not suitable for rapid detection for a large number of samples. Thence, the establishment of a simple, fast and flux detection method is significant. Immunosorbent assay is a rapid detection method commonly used for pesticide residue analysis, whose principle is that the immune responses between the antigen and antibody combine with the amplification of the catalytic effect of enzym. This method maintains the sensitivity of enzymatic reaction, while gets the specificity of antigen-antibody reaction, thus greatly improving the sensitivity.Since the widely used of chlorothalonil had harmed on human health and ecological environment, in this paper, a variety of haptens and polyclonal antibodies of chlorothalonil were prepared to establish an indirect competitive ELISA (icELISA) and colloidal gold chromatography methods. Simultaneously, the icELISA was set up to detect the total residues of chlorothalonil and4-hydroxy-chlorothalonil. The mainly results were as follows:1. Preparation and identification of chlorothalonil artificial antigen.In this work, the6chlorothalonil derivatives (i.e. hapten) which had specific groups or different structures of coupling arm were synthesized starting from its parent drug through one or two-step chemical reactions. The12chlorothalonil artificial antigens were prepared via the hapten conjugated with carrier protein (bovine serum albumin, BSA and ovalbumin, OVA) by glutaraldehyde, carbonyl imidazole, activated-ester and mixed-anhydride method, respectively. The coomassie brilliant blue method was applied to estimate the coupling ratio of artificial antigens, which extent were from2:1to40:1.2. Preparation and purification of chlorothalonil polyclonal antibodies.The male New Zealand white rabbits were immunized with immunogen to obtain antiserum, which titer range was from1.6×103:1to4.0×104:1with the non-competitive ELISA. DEAE column chromatography and caprylic acid-saturated ammonium sulfate were used for purifying antiserum to receive IgG, whose affinity constant was from109to1010and titer range was from1.6×103:1to5.0×104:1.3. Establishing of chlorothalonil indirect competitive ELISA.The icELISA method of chlorothalonil was established through the optimization of reaction and detection conditions.3good quality antibodies were applied, which IC50were ranged from2.275ng/mL to8.34ng/mL, IC20from0.9ng/mL to3.35ng/mL. All antibodies are highly specific. The detection results shown that chlorothalonil recovery was from84.5%to90.1%and the relative standard deviation was from2.9%to7.4%. HPLC method was used for verification, which results illustrated that the consistency of precision and accuracy between ELISA and HPLC was satisfactory.4. ELISA kit of Chlorothalonil.The recoveries of chlorothalonil by indirect competitive ELISA kit, was from83.60%to106.00%, the relative standard deviation of within-batch was from0.57%to3.26%, between-batch was from3.07%to9.01%, and the detection limit (LOD) was25ng/mL. The study of ELISA kit storage stability shown that the Bo value was still under control at4℃for6months and the correlation coefficient remained about0.99.5. The development of chlorothalonil colloidal gold immunochromatographic test strip.Sodium citrate method was used for reducing gold chloride acid to colloidal gold particles with uniform particle size. The diameter of them was about18nm, analyzed by UV spectrum and electron microscopy. Colloidal gold were labeled by polyclonal antibody against chlorothalonil, and then the immunochromatographic test strips were assembled. The whole detection time was about30min, and the LOD was60ng/mL.6. The detection methods of chlorothalonil and its metabolite residues.The ELISA method was applied to analyze chlorothalonil and its major multi-residue, named4-hydroxy-chlorothalonil. By ELISA, the recoveries of chlorothalonil was from 86.60%to91.60%,4-hydroxy-chlorothalonil was from105.20%to111.20%and the total of chlorothalonil and4-hydroxy-chlorothalonil equal added was from85.40%to106.80%; the relative standard deviation of chlorothalonil,4-hydroxy-chlorothalonil and total were from4.50%to7.60%,4.40%to5.30%and4.90%to6.30%, respectively.