Assessment of B Cell Repertoire in Humans

Wu, Yu-Chang, Kipling, David and Dunn-Walters, Deborah 2015. Assessment of B Cell Repertoire in Humans. Shaw, Albert C., ed. Immunosenescence: Methods and Protocols, Vol. 1343. Methods in Molecular Biology, New York: Humana Press, pp. 199-218. (10.1007/978-1-4939-2963-4_16)

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Abstract

The B cell receptor (BCR) repertoire is highly diverse. Repertoire diversity is achieved centrally by somatic recombination of immunoglobulin (Ig) genes and peripherally by somatic hypermutation and Ig heavy chain class-switching. Throughout these processes, there is selection for functional gene rearrangements, selection against gene combinations resulting in self-reactive BCRs, and selection for BCRs with high affinity for exogenous antigens after challenge. Hence, investigation of BCR repertoires from different groups of B cells can provide information on stages of B cell development and shed light on the etiology of B cell pathologies. In most instances, the third complementarity determining region of the Ig heavy chain (CDR-H3) contributes the majority of amino acids to the antibody/antigen binding interface. Although CDR-H3 spectratype analysis provides information on the overall diversity of BCR repertoires, this fairly simple technique analyzes the relative quantities of CDR-H3 regions of each size, within a range of approximately 10-80 bp, without sequence detail and thus is limited in scope. High-throughput sequencing (HTS) techniques on the Roche 454 GS FLX Titanium system, however, can generate a wide coverage of Ig sequences to provide more qualitative data such as V, D, and J usage as well as detailed CDR3 sequence information. Here we present protocols in detail for CDR-H3 spectratype analysis and HTS of human BCR repertoires.

Item Type:	Book Section
Date Type:	Publication
Status:	Published
Schools:	Medicine
Uncontrolled Keywords:	Antibody; B cell; B cell receptor; High-throughput sequencing; Immunoglobulin; Next generation sequencing; Repertoire; Spectratyping
Publisher:	Humana Press
ISBN:	978-1-4939-2962-7
ISSN:	1064-3745
Last Modified:	14 Oct 2021 13:45
URI:	https://orca.cardiff.ac.uk/id/eprint/95979

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