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Preparation of Novel Monoclonal Antibodies Against Chelated Cadmium Ions

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Abstract

The detection of cadmium ions using enzyme-linked immunosorbent assays (ELISA) has been reported by several research groups. Because cadmium ions are too small to stimulate the immune system, high molecular weight immunogens of cadmium are constructed using bifunctional chelators. At present, the most commonly used bifunctional chelator for the preparation of antigens for heavy metal ions is 1-(4-isothiocyanobenzyl) ethylenediamine N,N,N′,N′-tetraacetic acid (ITCBE). However, the price of ITCBE is high. So we are interested in a cheaper bifunctional chelator, 1-(4-aminobenzyl) ethylenediamine N,N,N′,N′-tetraacetic acid (aminobenzyl-EDTA). Here, cadmium ions were conjugated to carrier proteins using aminobenzyl-EDTA to make artificial antigens. Then, several mice were immunized with the antigen. And monoclonal antibodies (MAbs) against cadmium 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 EDTA or cadmium. Three hybridoma cell lines (A3, E4 and B5) 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. The IC50 for A3 was 8.4 μg/l. The detection range and the lowest detection limit using the antibody A3 was 0.394–64.39 and 0.051 μg/l, respectively. Spike–recovery studies in tap water showed that the antibody A3 could be used for cadmium detection in drinking water.

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Abbreviations

Aminobenzyl-EDTA:

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

EDTA:

N,N,N′,N′-Ethylenediamine-tetraaceticacid

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

Ka:

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, the National Natural Science Foundation of China (nos. 30600441, 30230260, and 30871897), and The PhD Start-up Foundation of Henan University of Science and Technology (09001514).

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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

    Tao Kong, Xue-Qin Hao & Zhi-Jun Yang

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

    Xiao-Bing Li, Guo-Wen Liu, Zhe Wang, Jia Tang & Wei Yang

  3. College of Veterinary Medicine, Northeast Agriculture University, Harbin, 150030, People’s Republic of China

    Zhi-Gang Zhang

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

    Jia Sun

Authors
  1. Tao Kong
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  2. Xue-Qin Hao
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  3. Xiao-Bing Li
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  4. Guo-Wen Liu
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  5. Zhi-Gang Zhang
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  6. Zhi-Jun Yang
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  7. Zhe Wang
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  8. Jia Tang
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  9. Wei Yang
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  10. Jia Sun
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Corresponding authors

Correspondence to Xiao-Bing Li or Guo-Wen Liu.

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Authors Tao Kong, Xue-Qin Hao, and Xiao-Bing Li of Jilin University and Henan University of Science and Technology contributed equally to this work.

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Kong, T., Hao, XQ., Li, XB. et al. Preparation of Novel Monoclonal Antibodies Against Chelated Cadmium Ions. Biol Trace Elem Res 152, 117–124 (2013). https://doi.org/10.1007/s12011-013-9599-6

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  • DOI: https://doi.org/10.1007/s12011-013-9599-6

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