Refined protocol for generating monoclonal antibodies from single human and murine B cells

被引：49

作者：

Ho, Irvin Y. ^{[1
]}

Bunker, Jeffrey J. ^{[2
,3
]}

Erickson, Steven A. ^{[2
,3
]}

Neu, Karlynn E. ^{[1
,3
]}

Huang, Min ^{[1
]}

Cortese, Mario ^{[4
]}

Pulendran, Bali ^{[5
]}

Wilson, Patrick C. ^{[1
,3
]}

机构：

[1] Univ Chicago, Rheumatol Sect, Dept Med, Gwen Knapp Ctr Lupus & Immunol Res, 924 E 57th St R422, Chicago, IL 60637 USA

[2] Univ Chicago, Dept Pathol, 929 E 57th St W503S, Chicago, IL 60637 USA

[3] Univ Chicago, Comm Immunol, Chicago, IL 60637 USA

[4] Emory Vaccine Ctr, Yerkes Natl Primate Res Ctr, Atlanta, GA 30329 USA

[5] Emory Univ, Sch Med, Dept Pathol, Atlanta, GA 30322 USA

来源：

JOURNAL OF IMMUNOLOGICAL METHODS | 2016年 / 438卷

基金：

美国国家卫生研究院;

关键词：

Monoclonal antibodies; Single-cell PCR; Human antibodies; Gibson assembly; Antibody expression; NEUTRALIZING ANTIBODIES;

D O I：

10.1016/j.jim.2016.09.001

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

Generating monoclonal antibodies from single B cells is a valuable tool for characterizing the specificity and functional properties of humoral responses. We and others developed protocols that have facilitated major advances in our understanding of B cell development, tolerance, and effector responses to HIV and influenza. Here, we demonstrate various refinements and dramatically reduce the time required to produce recombinant antibodies. Further, we present new methods for cloning and isolating antibodies from cells with lower immunoglobulin mRNA levels that may be resistant to traditional techniques. Together, these refinements significantly increase single-cell antibody expression efficiency and are easily integrated into established and novel pipelines. (C) 2016 Elsevier B.V. All rights reserved.

引用

页码：67 / 70

页数：4

共 9 条

[1] A Neutralizing Antibody Selected from Plasma Cells That Binds to Group 1 and Group 2 Influenza A Hemagglutinins [J].

Corti, Davide ;

Voss, Jarrod ;

Gamblin, Steven J. ;

Codoni, Giosiana ;

Macagno, Annalisa ;

Jarrossay, David ;

Vachieri, Sebastien G. ;

Pinna, Debora ;

Minola, Andrea ;

Vanzetta, Fabrizia ;

Silacci, Chiara ;

Fernandez-Rodriguez, Blanca M. ;

Agatic, Gloria ;

Bianchi, Siro ;

Giacchetto-Sasselli, Isabella ;

Calder, Lesley ;

Sallusto, Federica ;

Collins, Patrick ;

Haire, Lesley F. ;

Temperton, Nigel ;

Langedijk, Johannes P. M. ;

Skehel, John J. ;

Lanzavecchia, Antonio .

SCIENCE, 2011, 333 (6044) :850-856

[2] Both Neutralizing and Non-Neutralizing Human H7N9 Influenza Vaccine-Induced Monoclonal Antibodies Confer Protection [J].

Dunand, Carole J. Henry ;

Leon, Paul E. ;

Huang, Min ;

Choi, Angela ;

Chromikova, Veronika ;

Ho, Irvin Y. ;

Tan, Gene S. ;

Cruz, John ;

Hirsh, Ariana ;

Zheng, Nai-Ying ;

Mullarkey, Caitlin E. ;

Ennis, Francis A. ;

Terajima, Masanori ;

Treanor, John J. ;

Topham, David J. ;

Subbarao, Kanta ;

Palese, Peter ;

Krammer, Florian ;

Wilson, Patrick C. .

CELL HOST & MICROBE, 2016, 19 (06) :800-813

[3]

Gibson DG, 2009, NAT METHODS, V6, P343, DOI [10.1038/nmeth.1318, 10.1038/NMETH.1318]

[4] Atypical and classical memory B cells produce Plasmodium falciparum neutralizing antibodies [J].

Muellenbeck, Matthias F. ;

Ueberheide, Beatrix ;

Amulic, Borko ;

Epp, Alexandra ;

Fenyo, David ;

Busse, Christian E. ;

Esen, Meral ;

Theisen, Michael ;

Mordmueller, Benjamin ;

Wardemann, Hedda .

JOURNAL OF EXPERIMENTAL MEDICINE, 2013, 210 (02) :389-399

[5] Broad diversity of neutralizing antibodies isolated from memory B cells in HIV-infected individuals [J].

Scheid, Johannes F. ;

Mouquet, Hugo ;

Feldhahn, Niklas ;

Seaman, Michael S. ;

Velinzon, Klara ;

Pietzsch, John ;

Ott, Rene G. ;

Anthony, Robert M. ;

Zebroski, Henry ;

Hurley, Arlene ;

Phogat, Adhuna ;

Chakrabarti, Bimal ;

Li, Yuxing ;

Connors, Mark ;

Pereyra, Florencia ;

Walker, Bruce D. ;

Wardemann, Hedda ;

Ho, David ;

Wyatt, Richard T. ;

Mascola, John R. ;

Ravetch, Jeffrey V. ;

Nussenzweig, Michel C. .

NATURE, 2009, 458 (7238) :636-640

[6] Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen [J].

Smith, Kenneth ;

Garman, Lori ;

Wrammert, Jens ;

Zheng, Nai-Ying ;

Capra, J. Donald ;

Ahmed, Rafi ;

Wilson, Patrick C. .

NATURE PROTOCOLS, 2009, 4 (03) :372-384

[7] Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning [J].

Tiller, Thomas ;

Meffre, Eric ;

Yurasov, Sergey ;

Tsuiji, Makoto ;

Nussenzweig, Michel C. ;

Wardemann, Hedda .

JOURNAL OF IMMUNOLOGICAL METHODS, 2008, 329 (1-2) :112-124

[8]

Trombetta J.J., 2014, CURRENT PROTOCOLS MO

[9] Predominant autoantibody production by early human B cell precursors [J].

Wardemann, H ;

Yurasov, S ;

Schaefer, A ;

Young, JW ;

Meffre, E ;

Nussenzweig, MC .

SCIENCE, 2003, 301 (5638) :1374-1377

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