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Preparation of Specific Monoclonal Antibodies Against Chelated Copper Ions

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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 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.

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Abbreviations

p-SCN-Bn-DOTA:

S-2-(4-Isothiocyanatobenzyl)-1 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid

DOTA:

1, 4, 7, 10-Tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid

MAb:

Monoclonal antibodies

BSA:

Bovine serum albumin

KLH:

Keyhole limpet hemocyanin

HAT:

Hypoxanthine aminopterin, and thymidine

HT:

Hypoxanthine/thymidine

FBS:

Fetal bovine serum

OPD:

o-Phenylenediamine

ELISA:

Enzyme-linked immunosorbent assay

PEG:

Polyethylene glycol

K a :

Affinity constants

CR:

Cross-reactivity

CI-ELISA:

Indirect competitive enzyme-linked immunosorbent assay

ITCBE:

1-(4-Isothiocyanobenzyl) ethylenediamine N, N, N′, N′-tetraacetic acid

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Acknowledgments

This work was supported by the National “863” Project (2007AA10Z440 and 2007AA10Z426) of China and the National Natural Science Foundation of China (Nos. 30600441, 30230260, and 30871897).

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Authors and Affiliations

  1. College of Animal Science and Veterinary Medicine, Henan University of Science and Technology, 70 Tianjin Road, Luoyang, Henan, 471003, People’s Republic of China

    T. Kong

  2. Laboratory of Animal Nutritional and Metabolic Diseases and Toxicopathy, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xian Road, Changchun, Jilin, 130062, People’s Republic of China

    X. B. Li, G. W. Liu, G. H. Xie, Z. Wang, Y. Zhang & J. Tang

  3. College of Veterinary Medicine, Northeast Agriculture University, Harbin, 150030, China

    Z. G. Zhang

  4. Karolinska Institutet Huddinge, Novum, plan 8 Baxter Novum, SE 141 86, Stockholm, Sweden

    J. Sun

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  1. T. Kong
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  2. X. B. Li
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  3. G. W. Liu
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  4. G. H. Xie
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  5. Z. Wang
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  6. Z. G. Zhang
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  7. Y. Zhang
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  8. J. Sun
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  9. J. Tang
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Corresponding authors

Correspondence to G. W. Liu or Z. Wang.

Additional information

T. Kong X.B. Li, G.W. Liu, and G.H. XIE (Henan University of Science and Technology and Jilin University, respectively) contributed equally to this work.

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Kong, T., Li, X.B., Liu, G.W. et al. Preparation of Specific Monoclonal Antibodies Against Chelated Copper Ions. Biol Trace Elem Res 145, 388–395 (2012). https://doi.org/10.1007/s12011-011-9206-7

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  • DOI: https://doi.org/10.1007/s12011-011-9206-7

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