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Lectin Affinity Chromatography: An Efficient Method to Purify Horse IgG3

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Protein Downstream Processing

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2178))

Abstract

Affinity chromatography is a separation method based on a specific binding interaction between an immobilized ligand and its binding partner. An important class of ligands for the effective separation and purification of biotechnologically important substances is lectins, a group of naturally occurring molecules widely found in plants that display a range of specificities to bind different sugars. As sugars are often added to proteins through the process of glycosylation, ∼1/3 of all genetically encoded proteins are glycosylated, numerous cognate pairs of lectins with glycosylation groups have been discovered. Their specific binding interactions have not only allowed the development of numerous methodological strategies involving immobilized lectins to isolate molecules of interests but also for understanding the intermolecular interactions and alterations in glycosylation during a diverse set of biological phenomena, including tumor cell metastasis, intracellular communication, and inflammation. In this chapter, we describe a basic procedure for the separation of horse antibody classes by affinity chromatography based on differences in their glycosylation patterns. This procedure has been utilized for the purification of horse IgG3 (hoIgG3) from other six Ig from equine sera in a single step by using an Artocarpus integrifolia Jacalin column. This class of antibody comprises the therapeutic fraction generated in equine for passive antibody therapy and can serve as a biomarker for patient hypersensitivity. During the course of developing the protocol, the affinity interaction constant between the huIgE-hypersensitive immunoglobulin and the purified hoIgG3 was also determined.

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Abbreviations

EDTA:

ethylenediaminetetraacetic acid

hoIgG3:

horse immunoglobulin G3

HPLC:

high-performance liquid chromatography

huIgE:

human immunoglobulin E

Ig:

immunoglobulin

IgE:

immunoglobulin E

IgG:

immunoglobulin G

PAGE:

polyacrylamide gel electrophoresis

PBS-NP40:

phosphate buffer of pH 7.3 containing 150 mM NaCl and 0.1% Nonidet P-40

SDS:

sodium dodecyl sulfate

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Acknowledgments

We thank Dr. Daniel Maturana for assistance in the experiments with thermophoresis along with V.G. Mendes and K. Felisbino for support with technical laboratory analysis.

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Authors and Affiliations

  1. FIOCRUZ, Center for Technological Development in Health (CDTS)/National Institute of Science and Technology for Innovation on Neglected of Population Diseases (INCT-INDP), Rio de Janeiro, RJ, Brazil

    Salvatore G. De-Simone & David W. Provance Jr.

  2. FIOCRUZ, Oswaldo Cruz Institute, Laboratory of Experimental and Computational Biochemistry of Pharmaceuticals, Rio de Janeiro, RJ, Brazil

    Salvatore G. De-Simone

  3. Department of Cellular and Molecular Biology, Biology Institute, Federal Fluminense University, Niterói, RJ, Brazil

    Salvatore G. De-Simone

  4. FIOCRUZ, Oswaldo Cruz Institute, Interdisciplinar Laboratory of Medical Research, Rio de Janeiro, RJ, Brazil

    David W. Provance Jr.

Authors
  1. Salvatore G. De-Simone
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  2. David W. Provance Jr.
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Corresponding author

Correspondence to Salvatore G. De-Simone .

Editor information

Editors and Affiliations

  1. Laboratory of Enzyme Technology, Department of Biotechnology, School of Applied Biology and Biotechnology, Agricultural University of Athens, Athens, Greece

    Nikolaos E. Labrou

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De-Simone, S.G., Provance, D.W. (2021). Lectin Affinity Chromatography: An Efficient Method to Purify Horse IgG3. In: Labrou, N.E. (eds) Protein Downstream Processing. Methods in Molecular Biology, vol 2178. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0775-6_20

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  • DOI: https://doi.org/10.1007/978-1-0716-0775-6_20

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0774-9

  • Online ISBN: 978-1-0716-0775-6

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