Generation Of Monoclonal Antibodies Against Copper And Its Application For Immunoassay Detection

Objectives:For rapid detection of heavy metals copper ion in food or en vironment, To produce the McAbs against Cu²⁺, and initially establish indirect-competitive enzyme-linked immunoassay and colloidal gold probe-based immuno chromatographic assay for the detection of the heavy metal, and make a basis for the ultimate establishment of immunological detection methods of Cu²⁺.Methods:In this study, the complete immune-antigen (Cu²⁺-DTPA-BSA) a nd detecting antigen (Cu²⁺-DTPA-OVA) were prepared by coupling Copper ion s and Bovin Serum Alburmin(BSA) and Ovalbmin(OVA) via bifunctional chelat ing agent p-NH2-Bn-DTPA. Six BALB/c mice were immunized with Cu²⁺-DTP A-BSA and three hybridoma cell strains secreting monoclonal antibodies against copper ions were established by the hybridoma technique. The best cell strain coded 4C2F10 was selected to produce ascites. The antibody titer of the ascit es is 1:1×106 and the subtype of the antibody is IgG1, and the specificity wer e identified in the way of indirect competetive enzyme-linked immunosorbent a ssay. Both indirect-competitive enzyme-linked immunoassay (ic-ELISA) and coll oidal gold immunochromatographic strip for Cu(II) were established primarily.Results:The complete antigen of Cu²⁺ were prepared successfully. After immunization and cell fusion, each of which has three hybridomaes which can stably secrete McAbs against ion copper or chromium were obtained. The best hybridoma of which can stably secrete McAbs against ion copper is 4C2F10, which secrete antibodies is IgG1, the relative affinity is 3.37±0.31×109 M-1, n o cross-reactivity exists between the antibodies and the carrier protein OVA. A lso not with DTPA, The cross-reactivity of the antibody with other metal ions are low except for Co²⁺,Zn²⁺ which are 17.5% and 8.75%. The IC50 of indi rect competitive enzyme-linked immunsorbent assay is 12.5 ng/mL respectively, the detectionlimit is 2ng/mL, the LOD of colloidal gold probe-based immunoc hromatographic assay is 80ng/mL. need 3 minutesConclusion:The indirect-competitive enzyme-linked immunoassay (ic-ELIS A) for Cu²⁺ detection was developed by optimizing the working concentration o f the antibody and antigen., Colloidal gold probe-based immunochromatographic assay for the detection of the ion copper. each of which was meet the nation al standard of water quality monitoring(GB/T 14848-93:1000ppb). It Can be us ed for actual testing.