Preparation of Specific Monoclonal Antibodies Against Chelated Copper Ions

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...

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Bibliographic Details
Published in:Biological trace element research 2012-03, Vol.145 (3), p.388-395
Main Authors: Kong, T, Li, X. B, Liu, G. W, Xie, G. H, Wang, Z, Zhang, Z. G, Zhang, Y, Sun, J, Tang, J
Format: Article
Language:English
Subjects:
Animals
Antibodies, Monoclonal - immunology
Antigens
Biochemistry
Biomedical and Life Sciences
Biotechnology
Cell Line
chelating agents
Chelating Agents - chemistry
Copper
Copper - chemistry
Copper - immunology
Cross Reactions
detection limit
Drinking water
Enzyme-Linked Immunosorbent Assay
Female
hybridomas
Immune response
Immunization
Immunology
inhibitory concentration 50
Ions
Life Sciences
Limit of Detection
Mice
Mice, Inbred BALB C
Monoclonal antibodies
myeloma
Nutrition
Oncology
Proteins
Spectrophotometry, Atomic
splenocytes
tap water
transport proteins
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Summary: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 IC50 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.
ISSN:0163-4984
1559-0720
DOI:10.1007/s12011-011-9206-7