| As thiobencarb pesticide residues in food is strictly limited in foreign countries,and is being determinated for plants and animal origin food in domestic.It is promising and has important practical significance to develope colloidal gold immunochromatographic strip for detection of herbicide thiobencarb for rapid test of field samples or rapid screening of mass samples.In this study,thiobencarb was choosed as a target,a kind of thiobencarb hapten was synthesized,polyclonal antibodies with specific identification were preparated through the mice immunization experiment,colloidal gold labled immunochromatographic test strip was established and was applied to the visual detection of herbicide thiobencarb in actual samples.The main contents and results were as follows:(1)2-(Ethylamino)ethanol,sublimed sulphur,carbon monoxide gas and p-chlorobenzyl chloride and succinic anhydride were used as raw materials to synthesize the hydroxyated thiobencarb through the chemical reaction.The hapten was conjugated respectively to bovine serum albumin BSA and OVA to prepare the artificial antigen and coating antigen of thiobencarb by the carbodiimide method.And the average coupling ratio of artificial antigen and coating antigen of thiobencarb were 6.59:1 and 5.15:1 under the condition of hosphate buffer solution(0.01 mol/L,pH=7.40)and room temperature was 26℃.The average concentration of artificial antigen and caoting antigen were 2.589 mg/mL and 1.928 mg/mL thought the coomassie brilliant blue method,respectively.(2)Polyclonal antibody of thiobencarb were preparated through the mice immunization experiment,and polyantiserum titer and its specificity were determined by indirect ELISA.During the immune cycle,the serum titers of five mice were around 1:4000.At the end of the immune cycle,the titers of the purified serum was determined between 1:8000 and 1:12500.The mice polyantiserum was highly sensitive to thiobencarb,with a cross-reaction rate of 3.89%to 12.29%for three thiocarbamate herbicides and less than 1%for three different herbicides.(3)Colloidal gold solution was prepared by trisodium citrate reduction method.The particle size of colloidal gold was about 20 nm.The optimal pH of colloidal gold labled antibody was about 8.0,adjusting with 40 μL 0.2 mol/L potassium carbonate solution,addiing antibody at the lowest concentration of 400 g/mL,and the antibody binding rate was 91.57%.The sample pad of GLb02,the gold standard pad of GL0194,the water absorption pad of H5072,the nitrocelluric acid membrane of Sartorious CN140 and the DB-6 base plate of QTY100 were used as the assembly material for the test strip.The gold labled antibody was diluted with 1.0 mL compound solution and sprayed on the gold standard pad,and the 0.964 mg/mL coated antigen of thiobencarb and 0.5 mg/mL sheep-anti-rat secondary antibody were scored at the T line and C line,respectively.The detection limit of the developed colloidal gold immunochromatographic strip was 100 ng/mL.The specificity and stability of the immunochromatographic strip were well.(4)Thiobencarb pesticide residues of three kinds of actual samples including radish,grass fish and chicken were determined,and the spike experiment was carried out,the sample extract was processed with purification adsorbent(composed of 0.30 g anhydrous magnesium sulfate,0.25 g Z-Sep+and 0.25 g C18),determination results of test strip was consistent with the UPLC-MS/MS results,indicating that the prepared colloidal gold immunochromatographic test strip can accurately detect sample of content up to 100 ng/mL.. |
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