Chapter title |
Primer Design and Inverse PCR on Yeast Display Antibody Selection Outputs
|
---|---|
Chapter number | 4 |
Book title |
Schizosaccharomyces pombe
|
Published in |
Methods in molecular biology, January 2018
|
DOI | 10.1007/978-1-4939-7546-4_4 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7545-7, 978-1-4939-7546-4
|
Authors |
Fortunato Ferrara, Andrew R. M. Bradbury, Sara D’Angelo, Ferrara, Fortunato, Bradbury, Andrew R. M., D’Angelo, Sara |
Abstract |
The display of antibodies on the surface of Saccharomyces cerevisiae cells enables the high-throughput and precise selection of specific binders for the target antigen. The recent implementation of next-generation sequencing (NGS) to antibody display screening provides a complete picture of the entire selected polyclonal population. As such, NGS overcomes the limitations of random clones screening, but it comes with two main limitations: (1) depending upon the platform, the sequencing is usually restricted to the variable heavy chain domain complementary determining region 3 (HCDR3), or VH gene, and does not provide additional information on the rest of the antibody gene, including the VL; and (2) the sequence-identified clones are not physically available for validation. Here, we describe a rapid and effective protocol based on an inverse-PCR method to recover specific antibody clones based on their HCDR3 sequence from a yeast display selection output. |
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