Establishment Of ELISA And CLEIA With The Prepared Heavy Metal Lead Complete Antigen

Lead is a widely-existing heavy metal.Due to its serious neurotoxic effects,various organizations worldwide have set limits on the lead contents of the food and environment.Therefore,it is necessary to set a quick and easy method to monitor the lead content in food and environment.In this study,a bifunctional chelating agent was used to conjugate lead with keyhole limpet hemocyanin（KLH）to synthesize a whole antigen-immunized mouse,a lead monoclonal antibody was prepared to establish an enzyme-linked immunosorbent assay（ELISA）and an enzyme-free chemiluminescence method（CLEIA）.The two methods were compared with the conventional graphite furnace atomic absorption spectrometry（GFAAS）in order to establish an efficient,fast and convenient lead detection method.This study include the follow three parts:1.Heavy metal lead complete antigen preparation.In this experiment,a bifunctional chelating agent isothiocyanobenzyl-EDTA（ITCBE）was used to prepare a heavy metal lead total antigen.The chelating agent is linked to the lead ion,and the synthesized metal ion-chelator is chelated with the carrier protein to obtain a whole antigen,then the compounds-of unique structures and larger relative molecular mass-Pb²⁺-ITCBE-KLH and Pb²⁺-ITCBE-BSA are formed,they could reduce the toxic effects and enhance the immunogenicity.2.The establishment of ELISA assay.On the basic operation procedure of ELISA,confirm the reaction concentration of the antigen-antibody,then select the experimental conditions such as buffer type,blocking time,incubation time,determine that the antigen is on 1:1000 dilution,antibody on 1:1600 dilution,and use CBS coat overnight,block BSA for2 hours,incubate the primary antibody for 60 min and the secondary antibody with 1:8000dilution for 45 min,then develop color for 8 min.On this condition,the IC₅₀ of the lead was9.43 ng/m L（ELISA）.The detection limit（LOD）was 0.68 ng/m L,and an indirect competitive ELISA method（ic-ELISA）was established for lead detection.Use this method detect other metal ions and the cross-reactivity is of less than 0.83%.Through acid leaching method enrich the lead ions in water,food and feed samples and the recovery was 82.10%-108.25%（ic-ELISA）.3.The establishment of CLEIA assay.On the basic operation procedure of CLEIA,determine the antigen-antibody reaction concentration,select the experimental conditions such as buffer type,blocking time,incubation time,determine that the antigen is on 1:1000 dilution,antibody on 1:1500 dilution,and use CBS was coat overnight,block BSA for 2 hours,incubate the primary antibody for 45 min and the secondary antibody with 1:8000 dilution for 45 min,and then develop color for 5 min to establish a fast,convenient and efficient ic-CLEIA for lead detection.Under the optimal condition,lead IC₅₀ was 7.93 ng/m L（CLEIA）,detection limit（LOD）was 0.67 ng/m L（ic-CLEIA）,acid leaching is used to concentrate lead ions in water,food and feed samples.The recovery rate was 80.13%-98.80%（ic-CLEIA）.In this study,lead complete antigen was successfully synthesized,ELISA and CLEIA detection methods were established.With reference to GFAAS,ELISA and CLEIA all showed the consistency of test results.An efficient,fast and convenient lead detection method has been established.