Refined protocol for generating monoclonal antibodies from single human and murine B cells.
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.
Department of Medicine, Section of Rheumatology, Gwen Knapp Center for Lupus and Immunology Research, University of Chicago, 924 E 57th Street R422, Chicago, IL 60637, USA. Electronic address: irvin@uchicago.edu.
Irvin Y
IY
Ho
Department of Pathology, University of Chicago, 929 E 57th Street W503S, Chicago, IL 60637, USA; Committee on Immunology, University of Chicago, USA. Electronic address: jeffbunker@uchicago.edu.
Jeffrey J
JJ
Bunker
Department of Pathology, University of Chicago, 929 E 57th Street W503S, Chicago, IL 60637, USA; Committee on Immunology, University of Chicago, USA. Electronic address: sae@uchicago.edu.
Steven A
SA
Erickson
Department of Medicine, Section of Rheumatology, Gwen Knapp Center for Lupus and Immunology Research, University of Chicago, 924 E 57th Street R422, Chicago, IL 60637, USA; Committee on Immunology, University of Chicago, USA. Electronic address: karlynn@uchicago.edu.
Karlynn E
KE
Neu
Department of Medicine, Section of Rheumatology, Gwen Knapp Center for Lupus and Immunology Research, University of Chicago, 924 E 57th Street R422, Chicago, IL 60637, USA. Electronic address: huang2@uchicago.edu.
Min
M
Huang
Emory Vaccine Center, Yerkes National Primate Research Center, Atlanta, GA 30329, USA. Electronic address: mario.cortese@emory.edu.
Mario
M
Cortese
Emory Vaccine Center, Yerkes National Primate Research Center, Atlanta, GA 30329, USA; Department of Pathology, Emory University School of Medicine, Atlanta, GA 30322, USA. Electronic address: bpulend@emory.edu.
Bali
B
Pulendran
Department of Medicine, Section of Rheumatology, Gwen Knapp Center for Lupus and Immunology Research, University of Chicago, 924 E 57th Street R422, Chicago, IL 60637, USA; Committee on Immunology, University of Chicago, USA. Electronic address: wilsonp@uchicago.edu.
Patrick C
PC
Wilson
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000918:Antibody Specificity / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology*; D003001:Cloning, Molecular / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D011994:Recombinant Proteins / Q000096:biosynthesis*
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins
Antibody expression; Gibson assembly; Human antibodies; Monoclonal antibodies; Single-cell PCR