کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1993656 1064696 2013 12 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Deep sequencing of phage display libraries to support antibody discovery
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Deep sequencing of phage display libraries to support antibody discovery
چکیده انگلیسی


• High throughput analysis of antibody sequences.
• Assess quality and diversity of large antibody libraries.
• Follow all sequences during phage display selection process.
• Identify rare antibody candidates by NGS.
• By-pass laborious screening steps.

The use of Next Generation Sequencing (NGS) for the analysis of antibody sequences both in phage display libraries and during in vitro selection processes has become increasingly popular in the last few years. Here, our methods developed for DNA preparation, sequencing and data analysis are presented. A key parameter has also been to develop new software designed for high throughput antibody sequence analysis that is used in combination with publicly available tools. As an example of our methods, we provide data from the extensive analysis of five scFv libraries generated using different heavy chain CDR3 diversification strategies. The results not only confirm that the library designs were correct but also reveal differences in quality not easily identified by standard DNA sequencing approaches. The very large number of reads permits extensive sequence coverage after the selection process. Furthermore, as samples can be multiplexed, costs decrease and more information is gained per NGS run. Using examples of results obtained post phage display selections against two antigens, frequency and clustering analysis identified novel antibody fragments that were then shown to be specific for the target antigen. In summary, the methods described here demonstrate how NGS analysis enhances quality control of complex antibody libraries as well as facilitates the antibody discovery process.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Methods - Volume 60, Issue 1, 15 March 2013, Pages 99–110
نویسندگان
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