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Unbiased RACE-Based Massive Parallel Surveys of Human IgA Antibody Repertoires

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Natural Antibodies

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1643))

Abstract

For investigations of human B-cell receptor (BCR) repertoires, we have developed a protocol for large-scale surveys of human antibody heavy chain (VH) rearrangements. Here we study IgA repertoires, as more IgA antibodies are synthesized in the human body on a daily level than all other isotypes combined. In fact, IgA is secreted at all mucosal surfaces, and it is also secreted in the perspiration that coats our cutaneous surfaces. In these studies we can characterize the IgA clonal diversity of B-cell populations obtained from any donor. To recover representative repertoire libraries, we make our libraries from antibody gene transcript templates (i.e., cDNA), as these are closer reflections of the immune repertoire expressed at the antibody protein level. To avoid biases potentially introduced by upstream oligonucleotide primers that hybridize to variable region framework regions, our approach also uses rapid amplification of cDNA ends (RACE) of antibody transcripts. For exploration of human IgA responses, we have designed a duplexing antisense constant region primer that efficiently amplifies, side-by-side, heavy chain transcripts of both the IgA1 and IgA2 subclasses. By these methods we have begun to define the molecular differences in the IgA1 and IgA2 responses occurring simultaneously in different donors. These methods will be used to investigate the effects of microbial virulence factors on host defenses, during autoimmune responses, and in B-cell malignancies.

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Acknowledgments

Our work has been supported by the National Institutes of Health, R0141090118, R01A1068063, American Recovery and Reinvestment Act supplement; the Lupus Research Institute; the Alliance for Lupus Research; and the P. Robert Majumder Charitable Trust. This protocol was developed through an NIAID contract for B Cell Epitope Discovery and Mechanisms of Antibody Protection, HHSN272201400019C and a gift from the Judith and Stewart Colton Foundation. For NGS support we thank the NYUMC Genome Technology Center, under the supervision of Dr. Adriana Heguy, which is a shared resource partially supported by the Cancer Center Support Grant, P30CA016087, at the Laura and Isaac Perlmutter Cancer Center. We thank Adam Pelzek for editorial assistance.

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Authors and Affiliations

  1. Department of Medicine, NYU School of Medicine, 450 E. 29th Street, New York, NY, 10016, USA

    Hanane El Bannoudi, Céline Anquetil & Marc J. Braunstein

  2. Temple University, Philadelphia, PA, 19122, USA

    Sergei L. Kosakovsky Pond

  3. Department of Medicine, NYU School of Medicine, Alexandria Center for Life Science, 8th Floor, Rm 804, 450 E. 29th Street, New York, NY, 10016, USA

    Gregg J. Silverman

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  1. Hanane El Bannoudi
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  2. Céline Anquetil
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  3. Marc J. Braunstein
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  4. Sergei L. Kosakovsky Pond
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  5. Gregg J. Silverman
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Correspondence to Gregg J. Silverman .

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Editors and Affiliations

  1. Centre de Recherche des Cordeliers, INSERM, PARIS, Paris, France

    Srinivas V. Kaveri

  2. Centre de Recherche des Cordeliers, INSERM, PARIS, Paris, France

    Jagadeesh Bayry

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El Bannoudi, H., Anquetil, C., Braunstein, M.J., Pond, S.L.K., Silverman, G.J. (2017). Unbiased RACE-Based Massive Parallel Surveys of Human IgA Antibody Repertoires. In: Kaveri, S., Bayry, J. (eds) Natural Antibodies. Methods in Molecular Biology, vol 1643. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7180-0_5

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  • DOI: https://doi.org/10.1007/978-1-4939-7180-0_5

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7179-4

  • Online ISBN: 978-1-4939-7180-0

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