The Preparation Of Antibodies And Development Of Immunoassays For Clothianidin

Immunoassay is simple,fast,sensitivity,economic and high-throughput detection technique,which have emerged as research hotspot and development trend for testing the quality and safety of agricultural products.This study was designed to prepare antibodies and develop immunoassays for detecting clothianidin residue in agricultural products and environmental samples.The results might be provided reliable theoretical basis and date support for the development of immunoassay kits and test strips for detecting clothianidin,and provided new research ideas for rapid detection of pesticide and other small molecules.The main research contents were as follows:1.The preparation of antibodies for clothianidinClothianidin hapten was synthesized by combining with 3-mercaptopropionic acid,and then conjugated to bovine serum albumin(BSA)and ovalbumin(OVA)to produce immunogen and coating antigen.The polyclonal antibody(PcAb)was obtained from immunized New Zealand white rabbit.The titre and affinity for PcAbs were 4.096×10~6 and 1.15×109 L/mol,respectively.The spleen cells of immunized BALB/c female mouse were fused with SP2/0 murine myeloma cells.After several times of screening and subcloning,hybridoma cell line(2B5C9G3)that stably produced monoclonal antibody(McAb)against clothianidin was selected.The McAb was IgG3 and kappa type,and the titre and affinity were 2.56×10~5 and 9.95×10~7 L/mol,respectively.The results showed that the PcAb and McAb had high titre and affinity,which were good materials for development immunoassays to detect clothianidin.The heavy chain variable region gene(V_H)and light variable region gene(V_L)of antibody were obtained from hybridoma cell line(2B5C9G3).The single variable region gene(ScFv)was formed by connecting the V_H and V_L using a Linker gene segment,and then inserted into phagemid vector(pComb3XSS).After the recombination phagemid was transformed into host bacterium(E.coli K12 ER2738),the anti-clothianidin phage-ScFv was produced under the condition of help phage(M13K07).The phage-ScFv retained the properties of McAb and could be used as an alternative antibody for detecting clothianidin.The preparation and study of phage-ScFv have unified the genotype and phenotype of clothianidin antibody,and might be provided the important information and materials for further study the mechanism of antigen-antibody,antibody evolution,quantitative structure-activity relationship,and so on.2.The development of enzyme-linked immunosorbent assays for clothianidinUnder optimal conditions,the enzyme-linked immunosorbent assays(ELISAs)based on three-type of antibodies were developed for detecting clothianidin.The half-maximal inhibition concentration(IC50)and the limit of detection(LOD,IC10)were 46.0 and 1.08 ng/mL for PcAb-ELISA,25.6 and 3.84 ng/mL for McAb-ELISA,and 62.3 and 8.43 ng/mL for Phage-ScFv-ELISA,respectively.There were no obvious cross-reactivities(CRs)of the antibodies with its analogues except for dinotefuran(11.8%,46.5%,and 51.9%,respectively).In addition,the accuracy,precision and correlation of the ELISAs have been studied.The results indicated that the established ELISAs would be rapid,simple,specific,and accurate analytical methods for detecting clothianidin residue in agricultural and environmental samples.3.The development of chemiluminescent enzyme immunoassay for clothianidinUsing pattern of chemiluminescent assay,the chemiluminescent enzyme immunoassay(CLEIA)based on PcAb for detecting clothianidin was developed under optimal conditions,and the IC50 and LOD were 14.8 and 0.66 ng/mL.There were no obvious CRs with clothianidin analogues except for dinotefuran(9.4%).The recoveries of 76.4-10~7.4%and relative standard deviations(RSDs)of 2.9-9.4%for CLEIA were achieved from the spiked river water,soil,rice,cabbage,and tomato samples.The results of CLEIA for the authentic samples were largely consistent with GC(R2=0.9899).The CLEIA provided 3 times higher sensitivity than the ELISA using the same PcAb,and could be a satisfactory analytical tool for the monitoring of clothianidin in agricultural products.4.The development of fluorescence immunoassays for clothianidinIn the development of fluorescence immunoassays,the time-resolved fluoroimmunoassay(TRFIA)for determination of clothianidin,fluorescence-linked immunosorbent assay(FLISA)based on quantum dots for the detection of clothianidin and thiacloprid simultaneously,and fluorescence polarization immunoassay(FPIA)for detecting clothianidin were developed.Under optimal conditions,the standard curves were established;the IC50 and LOD were 2.07 and 0.022 ng/mL for TRFIA;the IC50 and LOD of the FLISA were 12.5 and 0.301 ng/mL for clothianidin,and 9.27 and 0.447 ng/mL for thiacloprid;the IC50 and LOD were 87.3 and 5.53 ng/mL for FPIA,respectively.The specificity,accuracy and precision of the developed fluorescence immunoassays were evaluated by the CRs,recoveries and correlation with instrument-based detection methods.The results indicated that the sensitivity of TRFIA has a significantly improvement,which might become a highly sensitive detection method;the FLISA is a satisfactory tool for detecting clothianidin and thiacloprid residue simultaneously;using the FPIA,the detection could be achieved without the steps of separation and washing,which showed significant advantages in detection time,detection step,and stability of tracer;The proposed fluorescence immunoassays could be ideally suitable for simple,rapid,sensitive,specific,accurate and high-throughput screening for clothianidin residue in agricultural and environmental samples.5.The development of gold immunochromatographic assay for clothianidinBased on the preparation of McAb and colloidal gold,a simple and rapid gold immunochromatographic assay(GICA)for detection of clothianidin was developed.The results can be judged by the naked eye within 10 min,and had a visual detection limit of 8 ng/mL.The CR of GICA was only 50%with dinotefuran.The matrix effects for different samples,sensitivity,accuracy and convenience of GICA were evaluated.Compared with ELISA,the GICA has advantages in detection time,detection step,result show,and test site.With respect to its simplicity,rapidity,sensitivity,and lower expenses,the clothianidin residue in agricultural and environmental samples might achieve semiquantitative determination by GICA.Furthermore,it was important for developing the commercial test strip and testing on-site of clothianidin.