title: Molecular deconvolution of the monoclonal antibodies that comprise the polyclonal serum response.
abstract: We have developed and validated a methodology for determining the antibody composition of the polyclonal serum response after immunization. Pepsin-digested serum IgGs were subjected to standard antigen-affinity chromatography, and resulting elution, wash, and flow-through fractions were analyzed by bottom-up, liquid chromatography-high-resolution tandem mass spectrometry. Identification of individual monoclonal antibodies required the generation of a database of IgG variable gene (V-gene) sequences constructed by NextGen sequencing of mature B cells. Antibody V-gene sequences are characterized by short complementarity determining regions (CDRs) of high diversity adjacent to framework regions shared across thousands of IgGs, greatly complicating the identification of antigen-specific IgGs from proteomically observed peptides. By mapping peptides marking unique V(H) CDRH3 sequences, we identified a set of V-genes heavily enriched in the affinity chromatography elution, constituting the serum polyclonal response. After booster immunization in a rabbit, we find that the antigen-specific serum immune response is oligoclonal, comprising antibodies encoding 34 different CDRH3s that group into 30 distinct antibody V(H) clonotypes. Of these 34 CDRH3s, 12 account for ∼60% of the antigen-specific CDRH3 peptide mass spectral counts. For comparison, antibodies with 18 different CDRH3s (12 clonotypes) were represented in the antigen-specific IgG fraction from an unimmunized rabbit that fortuitously displayed a moderate titer for BSA. Proteomically identified antibodies were synthesized and shown to display subnanomolar affinities. The ability to deconvolute the polyclonal serum response is likely to be of key importance for analyzing antibody responses after vaccination and for more completely understanding adaptive immune responses in health and disease.
authors: Department of Chemical Engineering, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712-1062, USA.
authors: Yariv
authors: Y
authors: Wine
authors: Daniel R
authors: DR
authors: Boutz
authors: Jason J
authors: JJ
authors: Lavinder
authors: Aleksandr E
authors: AE
authors: Miklos
authors: Randall A
authors: RA
authors: Hughes
authors: Kam Hon
authors: KH
authors: Hoi
authors: Sang Taek
authors: ST
authors: Jung
authors: Andrew P
authors: AP
authors: Horton
authors: Ellen M
authors: EM
authors: Murrin
authors: Andrew D
authors: AD
authors: Ellington
authors: Edward M
authors: EM
authors: Marcotte
authors: George
authors: G
authors: Georgiou
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology; D002846:Chromatography, Affinity; D022801:Complementarity Determining Regions; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D040901:Proteomics; D011817:Rabbits
chemical_list: D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D007074:Immunoglobulin G
references: Nat Biotechnol. 2010 Jul;28(7):695-709
references: 20622844
references: J Immunol Methods. 2009 Apr 15;343(2):65-7
references: 19100741
references: Nat Biotechnol. 2008 Dec;26(12):1336-8
references: 19060866
references: Nature. 2008 May 29;453(7195):667-71
references: 18449194
references: J Am Soc Mass Spectrom. 1994 Nov;5(11):976-89
references: 24226387
references: Nat Biotechnol. 2008 Dec;26(12):1367-72
references: 19029910
references: Mol Cell Proteomics. 2010 Aug;9(8):1716-28
references: 20103567
references: J Biol Chem. 2000 May 5;275(18):13668-76
references: 10788485
references: J Chromatogr B Analyt Technol Biomed Life Sci. 2011 Apr 15;879(13-14):955-60
references: 21440514
references: Nat Biotechnol. 2012 Mar 25;30(5):447-52
references: 22446692
references: Nat Biotechnol. 2010 Sep;28(9):965-9
references: 20802495
references: Nat Biotechnol. 2012 Nov;30(11):1039-43
references: 23138294
references: Nat Rev Immunol. 2006 Oct;6(10):741-50
references: 16977339
references: Nature. 1955 Apr 2;175(4457):570-5; contd
references: 14370170
references: Science. 2011 Sep 16;333(6049):1593-602
references: 21835983
references: Science. 2012 Jan 20;335(6066):342-4
references: 22174128
references: J Proteome Res. 2010 Jun 4;9(6):2937-45
references: 20387908
references: Nat Methods. 2007 Nov;4(11):923-5
references: 17952086
references: Science. 2011 Aug 12;333(6044):850-6
references: 21798894
references: Keio J Med. 1991 Mar;40(1):35-9
references: 2046211
references: Proc Natl Acad Sci U S A. 1988 Dec;85(23):8998-9002
references: 2461560
references: Immunity. 1998 Mar;8(3):363-72
references: 9529153
references: Science. 2011 Sep 16;333(6049):1633-7
references: 21764753
references: PLoS One. 2011;6(10):e25797
references: 22039424
references: Arthritis Rheum. 2011 Nov;63(11):3477-86
references: 22038404
references: J Mol Biol. 2003 Jan 10;325(2):325-35
references: 12488098
references: Biotechnology (N Y). 1992 Jul;10(7):779-83
references: 1368267
references: Annu Rev Immunol. 1992;10:593-616
references: 1590997
references: Cell. 1990 Nov 30;63(5):987-97
references: 2124176
references: Proteins. 1993 May;16(1):1-7
references: 8497480
references: Anal Bioanal Chem. 2011 Jan;399(3):1081-91
references: 21107826
references: Cell. 1990 Mar 23;60(6):963-70
references: 2317867
references: J Immunol. 2011 Oct 15;187(8):4229-35
references: 21930965
references: Nature. 2011 Sep 22;477(7365):466-70
references: 21849977