title: Preparation of specific monoclonal antibodies against chelated copper ions.
abstract: Copper ions are too small to elicit an immune response. Therefore, copper was conjugated to carrier proteins using S-2-(4-isothiocyanatobenzyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid, a bifunctional chelator, to make artificial antigens for copper. Several mice were immunized, and monoclonal antibodies (MAbs) against chelated copper were produced. Spleen cells of immunized mice were fused with myeloma cells. The resulting hybridomas were screened using protein conjugates which were covalently bound to metal-free 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA) or Cu-DOTA. Two hybridoma cell lines (F4 and B2) that produced MAbs with high selectivity and sensitivity were expanded for further study. Cross-reactivities with other metals were below 1%. These antibodies were used to construct competitive ELISAs for copper ions. The IC(50) for F4 and B2 were 0.39 and 1.66 mg/l, respectively. The detection range and the lowest detection limit for copper using the antibody F4 was 0.019-8.22 and 0.003 mg/l, respectively. Spike recovery studies in tap water showed that the most sensitive antibody could be used for copper detection in drinking water.
authors.affiliation: College of Animal Science and Veterinary Medicine, Henan University of Science and Technology, 70 Tianjin Road, Luoyang, Henan 471003, People's Republic of China.
authors.forename: T
authors.initials: T
authors.lastname: Kong
authors.forename: X B
authors.initials: XB
authors.lastname: Li
authors.forename: G W
authors.initials: GW
authors.lastname: Liu
authors.forename: G H
authors.initials: GH
authors.lastname: Xie
authors.forename: Z
authors.initials: Z
authors.lastname: Wang
authors.forename: Z G
authors.initials: ZG
authors.lastname: Zhang
authors.forename: Y
authors.initials: Y
authors.lastname: Zhang
authors.forename: J
authors.initials: J
authors.lastname: Sun
authors.forename: J
authors.initials: J
authors.lastname: Tang
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002460:Cell Line; D002614:Chelating Agents / Q000737:chemistry*; D003300:Copper / Q000737:chemistry / Q000276:immunology*; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D057230:Limit of Detection; D051379:Mice; D008807:Mice, Inbred BALB C; D013054:Spectrophotometry, Atomic
chemical_list: D000911:Antibodies, Monoclonal; D002614:Chelating Agents; D003300:Copper