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Generation and application of anti-ouabain IgY antibodies

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Abstract

Ouabain is a bioactive hapten and is very difficult to be accurately quantified because of the lack of useful reagents. Furthermore, where ouabain is produced in the adrenal glands has not been identified. In this study, ouabain–BSA was generated for immunizing the laying hens to generate ouabain-specific IgY antibodies in chicken eggs. The anti-ouabain IgY antibodies were detected in eggs 1 week after the last immunization and their concentrations increased with time. The highest concentrations of anti-ouabain IgY antibodies reached at 1:10,240 for ELISA 5 weeks after immunization and maintained for 4 weeks in chicken eggs. Following PEG precipitation, an average of 8.5 mg of anti-ouabain IgY antibodies with a purity of 87.6% was achieved from a single egg. Further analysis revealed that the anti-ouabain IgY antibodies had little immunoreactivity to hydrocortisone, dexamethasone, cedilanid, and digoxin, indicating their high specificity, and the purified IgY antibodies effectively detected endogenous ouabain in the cytoplasm of cells predominately in the zona reticularis of rat and human adrenal glands, indicating their high immunoreactivity. Given that IgY has an unique structure and bioactive features, the generated anti-ouabain IgY antibodies may be used as a new reagent for accurately quantifying ouabain in biological studies.

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Acknowledgments

This study was supported by the National Natural Science Foundation of China (NSFC, No. 30971222), and the Shanxi Province Science and Technology Program (No: 2005K11-G3(1)). The authors thank Medjaden Bioscience Limited for assisting in the preparation of this manuscript.

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Authors and Affiliations

  1. Department of Cardiology, The Second Affiliated Hospital, School of Medicine, Xi’an Jiao Tong University, Xi’an, 710004, Peoples’ Republic of China

    Ming Juan Zhang, Can Zhan Zhu, Zong Ming Duan & Xiao Lin Niu

  2. Department of Pathology, The Second Affiliated Hospital, School of Medicine, Xi’an Jiao Tong University, Xi’an, 710004, Peoples’ Republic of China

    Jun Yang

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  1. Ming Juan Zhang
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  2. Jun Yang
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  3. Can Zhan Zhu
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Correspondence to Ming Juan Zhang.

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Zhang, M.J., Yang, J., Zhu, C.Z. et al. Generation and application of anti-ouabain IgY antibodies. Mol Cell Biochem 358, 241–247 (2011). https://doi.org/10.1007/s11010-011-0974-3

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