Chapter title |
Immunosenecence
|
---|---|
Chapter number | 16 |
Book title |
Immunosenescence
|
Published in |
Methods in molecular biology, January 2015
|
DOI | 10.1007/978-1-4939-2963-4_16 |
Pubmed ID | |
Book ISBNs |
978-1-4939-2962-7, 978-1-4939-2963-4
|
Authors |
Wu, Yu-Chang, Kipling, David, Dunn-Walters, Deborah, Yu-Chang Wu, David Kipling, Deborah Dunn-Walters |
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. |
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