Studies On Immunological Determination Of Heavy Metal Copper And Cadmium

Heavy metals cannot be rendered harmless by chemical or biological remediation processes and can persist in body of human and animals for long periods. Through the food chain heavy metals may deposit into human and animals body and even a little quantity can cause serious health problems which could transfer to the next generation on the genetic level. So in environmental and agricultural food monitoring it is very important to quantitatively analyze heavy metals.The aim of this study is to establish hybridoma cell lines that can secrete high affinity and specificity monoclonal antibodies that are against copper and cadmium respectively and develop enzyme linked immunosorbent assay (ELISA) for copper and cadmium. Copper and cadmium was coupled to carrier proteins such as keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) via bifunctional chelator. Heavy metal-chelator-KLH had taken as immunizing antigen and metal-chelator-BSA as detection antigen. The hybridoma cell lines were established by hybridoma technique after immunization. The titer and affinity of monoclonal antibodies were determined by indirect ELISA and the specificity of monoclonal antibodies was determined by competitive inhibition ELISA. The working concentration of antigen and antibody, blocking agent and blocking time so on were studied to determine the optimal working parameter of ELISA. The results showed that 4 anti-copper monoclonal antibodies were prepared and 2 of them, designed B2 and F4, were high secrete high affinity and specificity monoclonal antibodies; 3 anti-cadmium monoclonal antibodies were prepared and 1 of them, designed A3, was high secrete high affinity and specificity monoclonal antibodies. The subclass of B2, F4 and A3 was IgG1. The affinity constant of anti-copper monoclonal antibodies B2, F4 was 4.69×109, 9.63×109 L/mol, of anti-cadmium monoclonal antibody A3 was 3.27×109 L/mol. Competitive ELISA for determination of copper and cadmium was developed. The working concentration of antigen for copper and cadmium was 0.5μg/mL and 1.0μg/mL, respectively. The optimal working parameter of ELISA was coating overnight at 4℃, blocking with rabbit serum at 37℃for 1.5 h, reacting with HRP secondary antibodies at 37℃for 1 h, reacting with substrate at 37℃for 10 min。The IC50 of anti-copper monoclonal antibody F4 was 0.39 mg/L showing the lowest detection limit of 0.003 mg/L and of anti-cadmium monoclonal antibody A3 was 8.4μg/L showing the lowest detection limit of 0.051μg/L. The recovery from copper ions-fortified was in the range of 89.9%-102.6%, and cadmium ions-fortified was in the range of 90.5%-104.4%. The results indicated that these monoclonal antibodies appeared to be very promising analytical tools for the rapid and sensitive determination of copper and cadmium in the environment.