Isolating Of Mimic Epitope And Phage-enzyme Linked Immunosorbent Assay For Thiacloprid

Thiacloprid hapten was synthesized and conjugated to ovalbumin via mixed anhydride method to produce a coating antigen and bovine serum albumin by active ester method to produce an immunogen for immunizing BALB/c mice.Spleen cells of immunized mouse were fused with well-growing SP2/0 murine myeloma cells to obtain the hybridoma cell.Finally,one hybridoma cell line?3A5?that stablely produced anti-thiacloprid monoclonal antibody was obtained on the basis of several screening and subcloning.The ascites were obtained by intraperitoneally injection of the 3A5 hybridoma cell line,and purified by caprylic acid-ammonium sulfate method to acquire the monoclone antibody.A monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay?ic-ELISA?was developed to detect thiacloprid.And the concentrations of coating antigen and antibody and working solution?concentration of methanol,iron strength and pH values?were optimized.Under the optimal conditions?0.14M Na+,pH 7.4 PBS containing 5%methanol?,the half maximal inhibition concentration(IC₅₀)and the limit of detection?IC10?of the ic-ELISA were 26.3 and 1.4 ug/L,respectively.The cross-reactivities?CR?were less than 0.01%for the tested structural analogues of thiacloprid and regarded as negligible.A line 8-amino-acid random peptide library was constructed by using the Ph.D.Peptide Display Cloning System.The library was made by ligating asynthetic 33-bp degenerate sequence into the M13KE vector and transfecting E.coli cells with the ligationproduct by electroporation.The transducing unit?TU?and titer were 8.7×108 pfu/?g and 1.6×1012 pfu/mL,respectively.Six phage-displayed peptides were isolated from the linear 8-residue peptide phage library and a cyclic 8-residue peptide phage library.Phage ELISAs were developed to detect thiacloprid by using the phage-displayed peptides.Under the optimal conditions?0.14M Na+,pH 8.5 PBS containing 5%methanol?,the half maximal inhibition concentration(IC₅₀)and the limit of detection?IC10?of the developed phage ELISA were 8.3 and 0.7?g/L,respectively.Compared to the conventional ELISAs,the sensitivity was improved more than 3-fold.The cross-reactivity?CR?was less than 0.01%for the tested structural analogues and regarded as negligible.The recoveries of thiacloprid ranged from 80.3 to 116.3%in environmental and agricultural samples,and the relative standard deviations?RSDs?werel.98-9.76%,which conformed to the requirements for residue detection.The amount of thiacloprid detected by phage ELISA in the samples was significantly?R2=0.9894,y=1.0867x+0.0318?correlated with that detected by high-performance liquid chromatography?HPLC?.The current study indicates that isolating phage-displayed peptides from phage display libraries is an alternative method for the development of a sensitive immunoassay and that the developed assay is a potentially useful tool for detecting thiacloprid in environmental and agricultural samples.