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Generation of Polyclonal Antibodies Against Recombinant Human Glucocerebrosidase Produced in Escherichia coli

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Abstract

Deficiency of the lysosomal glucocerebrosidase (GCR) enzyme results in Gaucher’s disease, the most common inherited storage disorder. Treatment consists of enzyme replacement therapy by the administration of recombinant GCR produced in Chinese hamster ovary cells. The production of anti-GCR antibodies has already been described with placenta-derived human GCR that requires successive chromatographic procedures. Here, we report a practical and efficient method to obtain anti-GCR polyclonal antibodies against recombinant GCR produced in Escherichia coli and further purified by a single step through nickel affinity chromatography. The purified GCR was used to immunize BALB/c mice and the induction of anti-GCR antibodies was evaluated by enzyme-linked immunosorbent assay. The specificity of the antiserum was also evaluated by western blot analysis against recombinant GCR produced by COS-7 cells or against endogenous GCR of human cell lines. GCR was strongly recognized by the produced antibodies, either as cell-associated or as secreted forms. The detected molecular masses of 59–66 kDa are in accordance to the expected size for glycosylated GCR. The GCR produced in E. coli would facilitate the production of polyclonal (shown here) and monoclonal antibodies and their use in the characterization of new biosimilar recombinant GCRs coming in the near future.

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Acknowledgments

We are grateful to Dr. Gregory A. Grabowski (Cincinnati Children’s Hospital Medical Center, University of Cincinnati) for providing the rabbit anti-human GCR polyclonal antibody. This work was supported by grants from FAPESP, CNPq and Fundação Butantan.

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Authors and Affiliations

  1. Centro de Biotecnologia, Instituto Butantan, Av. Vital Brasil, 1500, São Paulo, SP, 05503-900, Brazil

    Juliana Branco Novo, Maria Leonor Sarno Oliveira, Isaías Raw & Paulo Lee Ho

  2. Interunidades em Biotecnologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brazil

    Juliana Branco Novo & Paulo Lee Ho

  3. Laboratório de Imunopatologia, Instituto Butantan, Av. Vital Brasil, 1500, 05503-900, São Paulo, SP, Brazil

    Geraldo Santana Magalhães

  4. Centro de Biotecnologia, Instituto de Pesquisas Energéticas e Nucleares, São Paulo, SP, Brazil

    Ligia Morganti

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  1. Juliana Branco Novo
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Correspondence to Paulo Lee Ho.

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Novo, J.B., Oliveira, M.L.S., Magalhães, G.S. et al. Generation of Polyclonal Antibodies Against Recombinant Human Glucocerebrosidase Produced in Escherichia coli . Mol Biotechnol 46, 279–286 (2010). https://doi.org/10.1007/s12033-010-9303-4

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  • DOI: https://doi.org/10.1007/s12033-010-9303-4

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