Preparation Of Artificial Antigens For Heavy Metals Lead And Cadmium And Establishment Of Immune Detection System

Pollution of heavy metals lead and cadmium in feed not only affects animal health,but also affects food safety through accumulation in animals and residues in animal derived foods.Therefore,it is necessary to strictly monitor the residues of lead and cadmium in feed and food.Compared with flame atomic absorption spectrometry,graphite furnace atomic absorption spectrometry and other chemical detection methods,the immunological detection method of heavy metals has the advantages of rapid detection,high sensitivity and low cost.The preparation of high specificity and high sensitivity artificial antigens and antibodies is the key to study of heavy metal immunological detection technology.In this study,lead and cadmium artificial antigens were prepared,purified and identified,and Balb/c mice were immunized to explore the relationship between the production of serum polyclonal antibodies and the immune cycle and dosage of mice,monoclonal antibodies against lead and cadmium were prepared.1.Preparation and identification of lead artificial antigensTwo lead artificial antigens of BSA-EDTA-Pb²⁺and BSA-DTPA-Pb²⁺were prepared by carbon diimine method,and identified by ultraviolet full wavelength scanning,SDS-PAGE,BCA and flame atomic absorption method.The coupling rates of BSA-EDTA-Pb²⁺and BSA-DTPA-Pb²⁺were 8.5:1 and 11.9:1,respectively.Balb/c mice were immunized with BSA-DTPA-Pb²⁺with better coupling rate.After the fifth immunization,the serum titer of polyclonal antibody reached 1:16000.After optimizing the detection conditions of indirect ELISA,IC₅₀was 46.55 ng/m L,and there was no cross-reaction with Ba²⁺,Fe³⁺,Zn²⁺and Cd²⁺.2.Preparation and identification of cadmium artificial antigensTwo kinds of artificial antigens of BSA-EDTA-Cd²⁺and BSA-DTPA-Cd²⁺were prepared by physical method and identified by ultraviolet full wavelength scanning,SDS-PAGE,BCA and flame atomic absorption method.The coupling rates of BSA-EDTA-Cd²⁺and BSA-DTPA-Cd²⁺were 14.2:1 and 21.6:1,respectively.Balb/c mice were immunized with two artificial antigens,and the serum titer of multiantibody reached1:25600 after four immunizations,the overall serum titer of BSA-EDTA-Cd²⁺group was higher than that of BSA-DTPA-Cd²⁺group,and the IC₅₀of BSA-EDTA-Cd²⁺group and BSA-DTPA-Cd²⁺group were 208.88 ng/m L and 83.91 ng/m L,respectively.There were no cross-reactions between serum and other metal ions in the two groups.3.Establishment of Cd²⁺artificial antigen immune modelThe artificial antigen KLH-EDTA-Cd²⁺was prepared by physical method.The artificial antigen was identified by ultraviolet full wavelength scanning,Bradford method and ICP-MS,and the successful preparation of the artificial antigen was confirmed.Mice were immunized with KLH-EDTA-Cd²⁺.The serum indexes of mice with different immune cycles（2、3 and 4 weeks）and different immune doses（25,50 and 100μg/rat）were detected and compared,the serum titer of mice with immune cycle of 4 weeks and injection dose of 50μg group was the highest,and the serum sensitivity was 41.17 ng/m L.There were no cross-reactions with Ba²⁺,Fe³⁺,Zn²⁺and Pb²⁺.4.Preparation of monoclonal antibodies against lead and cadmiumCell fusion test was performed on mice immunized with the highest titer of BSA-DTPA-Pb²⁺and KLH-EDTA-Cd²⁺,and the cell fusion rate reached 86.46%.Cell lines1G1 and 1H12 which could secrete monoclonal antibodies against lead and cadmium were successfully screened.The titers of lead and cadmium monoclonal antibodies were more than 1:6.4×10⁴and the IC₅₀values were 123.67 ng/m L and 91.68 ng/m L,respectively.There was no cross-reaction with other ions.