Study Of Multi-residue Immunoassay For Pyrethroid Insecticides

Pyrethroid insecticides(SPs) had broad spectrum biological activity and low toxicity for non-target organisms. SPs were widely used for crop production,fruit tree treatment, household insects control and public health applications. Beneficially, the pesticides utilization was improved crop yield and quanlity, but there are contamination of foodstuffs associated with the remaining residues after its unreasonable application. The harm caused by food pesticides residues was a concerned issue. Therefore, people required a sensitive and rapid method for monitoring residue of pyrethroid insecticides.This study was to develop a multi-residue immunoassay for pyrethroid insecticides.The aritificial antigens of SPs was synthesized and identified in the study. Three generic haptens was designed and synthesized based on common structures of pyrethroid insecticides.Their structures were confirmed by 1H NHM and HRMS.The general haptens were conjugated with carrier proteins PLL by activated ester method to preparation immunogens.This method was also used to conjugatehaptens with BSA to preparation coating antigen. Using UV absorption spectrometry determined the conjugations were successful. The mouse immunized with the immunogens and titres of three antiserums(3.2×104,3.2×104,6.4×104)were determined by non-competitive indirect enzyme-linked assay procedure. These results show that these artificial antigens can be used to study an enzyme-linked immunosorbent assay for pyrethroid insecticides.On the basis of the work that has been done,the mouse with the highest antiserum titers was used to extract the total RNA from its spleen cells. After the RT-PCR, immunoglobulim variable heavy chain region(VH) genes and variable light chain region(VL) genes were first amplified. After being puried, equal mol VH and VL genes were jointed together by the use of Linker through SOE-PCR. The Sc Fv genes were applied by the primers that contain the slice sites of restriction endonuclease.The Sc Fv gene fragment were ligated into the phage vector p CANTAB5 e and were transformed into competent E.coli TG1 cells.With the help of helper phage M13KO7, the transformed bacterium were then reinfected. After overnight incubation in specific medium and sedimented by REG, the supernant was collected. That was the original phage display antibody library.Five rounds of selection were performed against haptens conjugated to Poly-L-Lysine(PLL) to enrich clones containing sc Fv specific to the haptens. A total of 6 clones were selected from round five, those Sc Fvs antibodies with the highest specificify for phosphorothioate hapten, termed S1-7, S1-13, S2-2, S2-13, S3-22 and S3-29 was produced via a enzyme-linked immunosorbent assay. The positive clone was express in fusion format in Escherichia coli TG1. To assess the binding affinity of positive Sc Fvs for haptens using the competitive inhibition enzyme-linked immunosorbent assay(CI-ELISA), the results showed that the IC50 values for the consanguinity hapten in assays constructed with sc Fvs were 0.329 μg/m L, 0.792 μg/m L, 0.834 μg/m L, 1.662 μg/m L,11.183 μg/m L, 0.993μg/m L.The result showed that sc Fv fragments antibodies of the positive clone had remarkable binding activity to the haptens.