Heise, N and Klein, U orcid.org/0000-0002-4789-967X (2017) Somatic hypermutation and affinity maturation analysis using the 4-hydroxy-3-nitrophenyl-acetyl (NP) system. In: Calado, DP, (ed.) Germinal Centers. Methods in Molecular Biology, 1623 . Springer (Humana) , pp. 191-208. ISBN 978-1-4939-7094-0
Abstract
Somatic hypermutation of immunoglobulin variable region (IgV) genes and affinity maturation of the antibody response are the hallmarks of the germinal center (GC) reaction in T cell-dependent immune responses. Determining the consequences of the experimental manipulation of the GC response on somatic hypermutation and affinity maturation requires the availability of a system that allows measuring these parameters. Immunization of mice of the C57/Bl6 genetic background with the hapten 4-hydroxy-3-nitrophenyl-acetyl (NP) coupled to a carrier protein leads to the predominant usage of one particular IgV heavy chain gene segment, V186.2, among the responding B cells. Moreover, a specific somatic mutation in codon 33 of V186.2 that leads to a tryptophan to leucine amino acid exchange increases the affinity of the corresponding antibody by ~10-fold, thus representing a molecular marker for affinity maturation. In addition, due to the simplicity of the antigen and the virtual absence of NP-specific plasma cells prior to immunization, NP-based immunizations represent ideal tools to quantify the plasma cell response by measuring NP-specific antisera by ELISA and the generation of NP-specific plasma cells by ELISPOT analysis. We here describe approaches to (1) measure the anti-NP plasma cell response by ELISA and ELISPOT analysis, and to (2) amplify and sequence V186.2 rearrangements from GC B cells and plasma cells to determine the level of somatic hypermutation and the extent of affinity maturation in the anti-NP response.
Metadata
Item Type: | Book Section |
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Authors/Creators: |
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Editors: |
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Copyright, Publisher and Additional Information: | (c) 2017, Springer Science+Business Media LLC. This is an author produced version of a chapter published in Methods in Molecular Biology volume 1623. Uploaded in accordance with the publisher's self-archiving policy. |
Keywords: | B cell; plasma cell; Ig variable region gene; germinal center; somatic hypermutation; affinity maturation; T cell-dependent; T cell-independent |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Medicine and Health (Leeds) > Institute of Molecular Medicine (LIMM) (Leeds) > Section of Experimental Haematology (Leeds) |
Depositing User: | Symplectic Publications |
Date Deposited: | 06 Nov 2018 12:57 |
Last Modified: | 06 Nov 2018 12:57 |
Status: | Published |
Publisher: | Springer (Humana) |
Series Name: | Methods in Molecular Biology |
Identification Number: | 10.1007/978-1-4939-7095-7_16 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:138157 |