Preparation Of Monoclonal Antibodies And Establishment Of ELISA For Detection Of Heavy Metal Copper Ions In Animal Food

The heavy metal copper is an important element to be used in industry, agriculture, husbandry and our life, but the excess copper ions will bring a big harm to our bodies. The methods of physical and chemical detection for heavy metal have much shortcomings, such as the high cost, complicated sample handling, hardly rapid testing and the like. Immunological technique was used to prepare anti-Cu²⁺-EDTA monoclonal antibody and establishment the blocking ELISA method of anti-Cu²⁺-EDTA in this paper.Copper ion [Cu（II）] was chemical modified with chelator Isothiocyanobenzyl-EDTA（ITCBE）to obtain the hapten. The hapten was coupled to carrier proteins bovine serum albumin（BSA） and ovalbumin（OVA） to obtain the artificial immunogen Cu²⁺-ITCBE-BSA and obtain the coating antigen Cu²⁺-ITCBE-OVA which were identified by UV, ICP-AES, SDS-PAGE, and ELISA of polyclonal antibody（pAb）. The results showed that the concentrations of Cu²⁺-ITCBE-BSA and Cu²⁺-EDTA-OVA were 9.21 mg/mL and 6.25 mg/mL. The concentrations of Cu²⁺ were 16.83 μg/mL and 6.42 μg/mL. The Cu²⁺-EDTA pAb of Balb/C mice was obtained and had the good imunological properties with titers of 1∶（5.12×104） by indirect ELISA, with the 50% inhibitive concentration（IC50） of Cu²⁺-EDTA 29.92 ng/mL by blocking ELISA, with 27.01% cross-reactivity（CR） to Zn2+-EDTA and little or no CR with other compounds.The hybridomas secreted monoclonal antibody against Cu²⁺-EDTA were chosen by indirect ELISA and blocking ELISA through cell fusion technology. The titers, sensitivity and specificity of anti-Cu²⁺-EDTA mAb were identified with indirect ELISA and blocking ELISA. The results showed that four cell lines were chosen and named 1F7, 2B8, 3G5 and 3D10. The 2B8 was the best with indirect ELISA titers of 1∶（1.28×103） in supernatant and 1∶（5.12×105） in ascites. Its isotype was Ig G1/κ and its Ka was 7.87×1011 L/mol. It had good sensitivity with IC50 of Cu²⁺-EDTA 29.78 μg/L and it had good specificity with little or no CR with other compounds except 26% cross-reactivity to Zn2+-EDTA.The blocking ELISA kit for determination of Cu²⁺ was established with anti-Cu²⁺-EDTA mAb of 2B8, and its performances were measured. The results showed that it had good sensitivity with IC50 of Cu²⁺-EDTA 11.91 ng/mL, it had good precision and accuracy with the coefficient variation（CV%） were below 15% by inter-assay and intra-assay. The kit had 26% CR to Zn2+-EDTA and little or no CR with other compounds. The validity of Cu²⁺ kit in 4 was above six months.℃The artificial antigen of anti-Cu²⁺-EDTA was synthetised successfully. The monoclonal antibodies of high titers, sensitivity and specificity were prepared. The blocking ELISA was establishmented and it would be used to determinate Cu²⁺ in animal food.