Research On The Development Of Broad Antibody And Rapid Detection Technology Towards Type Ⅰ Pyrethroids

Pyrethroids are a class of natural pyrethroid chemical structure developed on the basis of bionicdrugs. They have been widely used in pest control in agriculture, forestry, animal husbandry andhousehold. High pyrethroid exposure may cause hazardous to the human immune system, and evencause cancer. Many countries in the world have set up strict regulatories towards pyrethroids, resultinghuge economic loss of our export agro-products due to excess pyrethroid residues. Therefore, powerfuldetection techniques are in a great demand to keep harmful agro-products and food from consumers.Immunoassay has been widely used in small-weight-molecular detection, such as pesticides. Theimmunoassay towards pesticides mainly focuses on one pesticide, including antigen design andmonoclonal antibody development, which means one antibody, is only specific to one analyte. In realsample analysis, multi-components determination is always needed. In order to establish abroad-specificity immunoassay for determination of pyrethroid residues, the main contents andinnovation of this paper are as follows:1. Two positive hybridoma cell lines towards type I perythoid insecticides were achieved. Usingthe artificial antigen systhesized in the laboratory, two positive hybridoma cell lines secreting anti-typeI perythoid antibody, named Pyr1C5, Pyr3C8, were obtained by cell fusion, the improved semi-solidmedium, a gradient-step screening method.2. Two monoclonal antibodies which have broad-specificity towards etofenprox, permethrin andphenothrin were successfully developed for the first time. According to the results of the nature ofidentification, the two monoclonal antibodies were both lgG1subtype. The affinity constants ofmonoclonal antibody Pyr1C5and Pyr3C8were2.16×108L/moL and8.5×108L/moL, respecitively. Thesensitivity of antibody Pyr1C5(IC50) is0.090μg/mL, its cross-reactivity towards permethrin andphenothrin were82.2%and67.3%, respectively. The sensitivity of antibody Pyr3c8(IC50) is0.0552μg/mL, its cross-reactivity towards permethrin and phenothrin were84.3%and71.7%. Thecross-reactivities of the two antibodies to other type I and type II pyrethroids permethrin were less than1%.3. An indirect competitive enzyme-linked immunosorbent assay (ELISA) based on monoclonalantibody Pyr3C8was developed for the quantitative detection of etofenprox, permethrin and phenothrin.Under the optimized conditions, the sensitivity (IC50) of ELISA towards etofenprox was20.86μg/L, thelinear range is6.32-169.14μg/L and the detection limit of the method was3.16μg/L. The IC50of ELISAto permethrin was24.2μg/L, the linear range was assigned to a concentration6.19-478.0μg/L, and thedetection limit was3.72μg/L. The sensitivity towards phenothrin (IC50value) of the assay was29.4μg/L, the linear range was assigned to a concentration of6.37-404.0μg/L, the detection limit was4.27μg/L. The detection limit of this method is full able to meet the domestic and foreign etofenprox,permethrin and phenothrin’s requirements. The method was applied to etofenprox, permethrin andphenothrin in spiked recovery test in oilseed rape, cabbage and apple, the mean recovery rates variedfrom81%to95%, the relative standard deviation was between3.1%-7.2%.22samples of vegetablesand fruits were tested for the total concentration of permethrin, permethrin and phenothrin by both ELISA and GC-MS, the results have good correlation (R2=0.973).4. A highly sensitive time-resolved fluorescence immunochromatographic assay towardsethofenprox was developed for the first time. Its quantitative detection limit to ethofenprox is9.12ng/mL. the method was applied for determination of ethofenprox residues in15agricultural products.The results were compared with those detected by ELISA, which showed good correlation，thecorrelation coefficient reached0.986(R2).