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Production of Metallothionein Polyclonal Antibodies Using Chickens as Model

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Abstract

The production of polyclonal antibodies (pAbs) against metallothioneins (MT) has been done in mammals. In this work, we describe a model where pAbs against rat liver MT were produced in chickens. Liver MT-1 and MT-2 isoforms isolated from rats were used as immunogens. MT was purified by exclusion chromatography and MT isoforms isolated by ionic exchange chromatography. Chickens were immunized with each isoform emulsified with Freund adjuvant over 6 weeks. MT-pAbs obtained from egg yolk were purified by ammonium sulfate precipitation followed by thiophilic interaction chromatography. MT-pAbs were characterized by ELISA, SDS-PAGE electrophoresis, and Western blot assays. Results showed significant titers (1:1,000) of MT-1 and MT-2 IgY in the eggs collected 30 days after the first immunization as determined by a direct ELISA assay; results also show a cross-reaction between MT-1 and MT-2 isoforms: however, the Abs obtained did not react with other non-MT proteins in hepatic homogenates. Sensitivity assays showed that MT-pAbs detected MT-1 and MT-2 at nanogram levels. These data suggest that chickens are an alternative model for producing pAbs against mammal high-homology proteins such as MT.

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Acknowledgments

This work was partially supported by the grant II 108 G04 from VIEP, BUAP. We thank Dr. Carlos Escamilla W. for his assistance and the donation of the animals used in this work.

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Authors and Affiliations

  1. Laboratorio de Investigaciones Químico Clínicas, Facultad de Ciencias Químicas, Benemérita Universidad Autónoma de Puebla, Puebla, Puebla, México

    Angélica María Ortiz-Bueno, Bertha Alicia León-Chávez, Alejandro Ruiz-Tagle, Leopoldo Castro-Caballero & Eduardo Brambila

  2. Centro de Investigaciones en Ciencias Microbiológicas. Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla, Puebla, Puebla, México

    Patricia Lozano-Zarain

  3. Department of Toxicology, Bristol-Myers Squibb Pharmaceutical Research Institute, New Brunswick, NJ, USA

    William E. Achanzar

  4. Facultad de Ciencias Químicas, BUAP. Gral. Pedro Hinojosa 17, Lomas de Loreto, Puebla, Puebla, 72260, México

    Eduardo Brambila

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  1. Angélica María Ortiz-Bueno
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  2. Bertha Alicia León-Chávez
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  3. Alejandro Ruiz-Tagle
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  4. Patricia Lozano-Zarain
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  5. Leopoldo Castro-Caballero
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  6. William E. Achanzar
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  7. Eduardo Brambila
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Correspondence to Eduardo Brambila.

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Ortiz-Bueno, A.M., León-Chávez, B.A., Ruiz-Tagle, A. et al. Production of Metallothionein Polyclonal Antibodies Using Chickens as Model. Appl Biochem Biotechnol 158, 502–511 (2009). https://doi.org/10.1007/s12010-008-8349-2

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  • DOI: https://doi.org/10.1007/s12010-008-8349-2

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