title: Isolation of human monoclonal antibodies from peripheral blood B cells.
abstract: Isolation of monoclonal antibodies is an important technique for understanding the specificities and characteristics of antibodies that underlie the humoral immune response to a given antigen. Here we describe a technique for isolating monoclonal antibodies from human peripheral blood mononuclear cells. The protocol includes strategies for the isolation of switch-memory B cells from peripheral blood, the culture of B cells, the removal of the supernatant for screening and the lysis of B cells in preparation for immunoglobulin heavy-chain and light-chain amplification and cloning. We have observed that the addition of cytokines IL-2, IL-21 and irradiated 3T3-msCD40L feeder cells can successfully stimulate switch-memory B cells to produce high concentrations of IgG in the supernatant. The supernatant may then be screened by appropriate assays for binding or for other functions. This protocol can be completed in 2 weeks. It is adaptable to use in other species and enables the efficient isolation of antibodies with a desired functional characteristic without prior knowledge of specificity.
authors.affiliation: HIV-Specific Immunity Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases (NIAID), US National Institutes of Health (NIH), Bethesda, Maryland, USA.
authors.forename: Jinghe
authors.initials: J
authors.lastname: Huang
authors.forename: Nicole A
authors.initials: NA
authors.lastname: Doria-Rose
authors.forename: Nancy S
authors.initials: NS
authors.lastname: Longo
authors.forename: Leo
authors.initials: L
authors.lastname: Laub
authors.forename: Chien-Li
authors.initials: CL
authors.lastname: Lin
authors.forename: Ellen
authors.initials: E
authors.lastname: Turk
authors.forename: Byong H
authors.initials: BH
authors.lastname: Kang
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Migueles
authors.forename: Robert T
authors.initials: RT
authors.lastname: Bailer
authors.forename: John R
authors.initials: JR
authors.lastname: Mascola
authors.forename: Mark
authors.initials: M
authors.lastname: Connors
mesh_terms: D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D001402:B-Lymphocytes / Q000276:immunology*; D018929:Cell Culture Techniques; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal
references.citation: Nature. 1975 Aug 7;256(5517):495-7
references.pmid: 1172191
references.citation: J Immunol. 2005 Dec 15;175(12):7867-79
references.pmid: 16339522
references.citation: Science. 2009 Oct 9;326(5950):285-9
references.pmid: 19729618
references.citation: Nat Rev Immunol. 2012 Oct;12(10):709-19
references.pmid: 23007571
references.citation: Blood. 2009 Dec 10;114(25):5173-81
references.pmid: 19846886
references.citation: Cancer Res. 2001 Nov 1;61(21):7920-4
references.pmid: 11691813
references.citation: Science. 2010 Aug 13;329(5993):856-61
references.pmid: 20616233
references.citation: Res Virol. 1997 Mar-Apr;148(2):165-9
references.pmid: 9108620
references.citation: J Exp Med. 1984 Jan 1;159(1):208-20
references.pmid: 6319530
references.citation: Immunity. 2012 Sep 21;37(3):412-25
references.pmid: 22999947
references.citation: Nature. 2012 Nov 15;491(7424):406-12
references.pmid: 23151583
references.citation: Nature. 2009 Apr 2;458(7238):636-40
references.pmid: 19287373
references.citation: Nat Med. 2004 Aug;10(8):871-5
references.pmid: 15247913
references.citation: J Immunol Methods. 2008 Jan 1;329(1-2):112-24
references.pmid: 17996249
references.citation: Science. 2011 Aug 12;333(6044):850-6
references.pmid: 21798894
references.citation: Proc Natl Acad Sci U S A. 1999 Jun 8;96(12):6953-8
references.pmid: 10359820
references.citation: Science. 2011 Sep 16;333(6049):1633-7
references.pmid: 21764753
references.citation: J Virol. 2011 Oct;85(19):9998-10009
references.pmid: 21795340
references.citation: Nat Protoc. 2009;4(3):372-84
references.pmid: 19247287
references.citation: Proc Natl Acad Sci U S A. 2006 Mar 7;103(10):3557-62
references.pmid: 16505368
references.citation: Science. 2013 May 10;340(6133):751-6
references.pmid: 23661761