Suppr超能文献

一种新型亲和标签ABTAG及其在噬菌体展示筛选的单域抗体亲和筛选中的应用。

A Novel Affinity Tag, ABTAG, and Its Application to the Affinity Screening of Single-Domain Antibodies Selected by Phage Display.

作者信息

Hussack Greg, Baral Toya Nath, Baardsnes Jason, van Faassen Henk, Raphael Shalini, Henry Kevin A, Zhang Jianbing, MacKenzie C Roger

机构信息

Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON, Canada.

Human Health Therapeutics Research Centre, National Research Council Canada, Montréal, QC, Canada.

出版信息

Front Immunol. 2017 Oct 30;8:1406. doi: 10.3389/fimmu.2017.01406. eCollection 2017.

Abstract

ABTAG is a camelid single-domain antibody (sdAb) that binds to bovine serum albumin (BSA) with low picomolar affinity. In surface plasmon resonance (SPR) analyses using BSA surfaces, bound ABTAG can be completely dissociated from the BSA surfaces at low pH, over multiple cycles, without any reduction in the capacity of the BSA surfaces to bind ABTAG. A moderate throughput, SPR-based, antibody screening assay exploiting the unique features of ABTAG is described. Anti-carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) sdAbs were isolated from a phage-displayed sdAb library derived from the heavy chain antibody repertoire of a llama immunized with CEACAM6. Following one or two rounds of panning, enriched clones were expressed as ABTAG fusions in microtiter plate cultures. The sdAb-ABTAG fusions from culture supernatants were captured on BSA surfaces and CEACAM6 antigen was then bound to the captured molecules. The SPR screening method gives a read-out of relative expression levels of the fusion proteins and kinetic and affinity constants for CEACAM6 binding by the captured molecules. The library was also panned and screened by conventional methods and positive clones were subcloned and expressed for SPR analysis. Compared to conventional panning and screening, the SPR-based ABTAG method yielded a considerably higher diversity of binders, some with affinities that were three orders of magnitude higher affinity than those identified by conventional panning.

摘要

ABTAG是一种骆驼科单域抗体(sdAb),它以低皮摩尔亲和力与牛血清白蛋白(BSA)结合。在使用BSA表面的表面等离子体共振(SPR)分析中,结合的ABTAG在低pH下可在多个循环中从BSA表面完全解离,而BSA表面结合ABTAG的能力不会有任何降低。本文描述了一种基于SPR的中等通量抗体筛选测定法,该方法利用了ABTAG的独特特性。从用癌胚抗原相关细胞粘附分子6(CEACAM6)免疫的羊驼重链抗体库衍生的噬菌体展示sdAb文库中分离出抗CEACAM6 sdAb。经过一轮或两轮淘选后,富集的克隆在微量滴定板培养物中表达为ABTAG融合蛋白。培养上清液中的sdAb-ABTAG融合蛋白被捕获在BSA表面,然后将CEACAM6抗原与捕获的分子结合。SPR筛选方法可读出融合蛋白的相对表达水平以及捕获分子与CEACAM6结合的动力学和亲和力常数。该文库也通过传统方法进行淘选和筛选,阳性克隆被亚克隆并表达用于SPR分析。与传统的淘选和筛选相比,基于SPR的ABTAG方法产生的结合物多样性要高得多,其中一些结合物的亲和力比通过传统淘选鉴定的结合物高三个数量级。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4b70/5674936/efd82ae9c00f/fimmu-08-01406-g001.jpg

相似文献

3
Llama-derived single-domain antibodies for the detection of botulinum A neurotoxin.
Anal Bioanal Chem. 2010 Sep;398(1):339-48. doi: 10.1007/s00216-010-3905-3. Epub 2010 Jun 28.
5
Isolation of functional single domain antibody by whole cell immunization: implications for cancer treatment.
J Immunol Methods. 2011 Aug 31;371(1-2):70-80. doi: 10.1016/j.jim.2011.06.017. Epub 2011 Jun 30.
6
Facile Affinity Maturation of Single-Domain Antibodies Using Next-Generation DNA Sequencing.
Methods Mol Biol. 2022;2446:245-268. doi: 10.1007/978-1-0716-2075-5_12.
7
Isolation of monoclonal antibody fragments from phage display libraries.
Methods Mol Biol. 2009;502:341-64. doi: 10.1007/978-1-60327-565-1_20.
8
Generation of llama single-domain antibodies against methotrexate, a prototypical hapten.
Mol Immunol. 2007 Mar;44(7):1680-90. doi: 10.1016/j.molimm.2006.08.007. Epub 2006 Sep 29.

引用本文的文献

1
Protein Engineering: Advances in Phage Display for Basic Science and Medical Research.
Biochemistry (Mosc). 2022 Jan;87(Suppl 1):S146-S110. doi: 10.1134/S0006297922140127.
2
3
Molecular basis for protein-protein interactions.
Beilstein J Org Chem. 2021 Jan 4;17:1-10. doi: 10.3762/bjoc.17.1. eCollection 2021.
4
Recombinant expression of nanobodies and nanobody-derived immunoreagents.
Protein Expr Purif. 2020 Aug;172:105645. doi: 10.1016/j.pep.2020.105645. Epub 2020 Apr 11.

本文引用的文献

1
Exploring sensitivity & throughput of a parallel flow SPRi biosensor for characterization of antibody-antigen interaction.
Anal Biochem. 2017 May 15;525:8-22. doi: 10.1016/j.ab.2017.02.007. Epub 2017 Feb 20.
2
Isolation of TGF-β-neutralizing single-domain antibodies of predetermined epitope specificity using next-generation DNA sequencing.
Protein Eng Des Sel. 2016 Oct;29(10):439-443. doi: 10.1093/protein/gzw043. Epub 2016 Sep 8.
3
Identification of cross-reactive single-domain antibodies against serum albumin using next-generation DNA sequencing.
Protein Eng Des Sel. 2015 Oct;28(10):379-83. doi: 10.1093/protein/gzv039. Epub 2015 Aug 27.
4
Shark variable new antigen receptor biologics - a novel technology platform for therapeutic drug development.
Expert Opin Biol Ther. 2014 Oct;14(10):1527-39. doi: 10.1517/14712598.2014.937701. Epub 2014 Aug 4.
5
Disulfide linkage engineering for improving biophysical properties of human VH domains.
Protein Eng Des Sel. 2012 Oct;25(10):581-9. doi: 10.1093/protein/gzs055. Epub 2012 Aug 30.
6
A V(L) single-domain antibody library shows a high-propensity to yield non-aggregating binders.
Protein Eng Des Sel. 2012 Jun;25(6):313-8. doi: 10.1093/protein/gzs014. Epub 2012 Apr 6.
7
Multivalent anchoring and oriented display of single-domain antibodies on cellulose.
Sensors (Basel). 2009;9(7):5351-67. doi: 10.3390/s90705351. Epub 2009 Jul 7.
8
Beyond natural antibodies: the power of in vitro display technologies.
Nat Biotechnol. 2011 Mar;29(3):245-54. doi: 10.1038/nbt.1791.
9
Introduction to current and future protein therapeutics: a protein engineering perspective.
Exp Cell Res. 2011 May 15;317(9):1261-9. doi: 10.1016/j.yexcr.2011.02.013. Epub 2011 Mar 1.
10
Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain.
J Biol Chem. 2011 Mar 18;286(11):8961-76. doi: 10.1074/jbc.M110.198754. Epub 2011 Jan 7.

文献AI研究员

20分钟写一篇综述,助力文献阅读效率提升50倍。

立即体验

用中文搜PubMed

大模型驱动的PubMed中文搜索引擎

马上搜索

文档翻译

学术文献翻译模型,支持多种主流文档格式。

立即体验