title: A proteomics approach for the identification and cloning of monoclonal antibodies from serum.
abstract: We describe a proteomics approach that identifies antigen-specific antibody sequences directly from circulating polyclonal antibodies in the serum of an immunized animal. The approach involves affinity purification of antibodies with high specific activity and then analyzing digested antibody fractions by nano-flow liquid chromatography coupled to tandem mass spectrometry. High-confidence peptide spectral matches of antibody variable regions are obtained by searching a reference database created by next-generation DNA sequencing of the B-cell immunoglobulin repertoire of the immunized animal. Finally, heavy and light chain sequences are paired and expressed as recombinant monoclonal antibodies. Using this technology, we isolated monoclonal antibodies for five antigens from the sera of immunized rabbits and mice. The antigen-specific activities of the monoclonal antibodies recapitulate or surpass those of the original affinity-purified polyclonal antibodies. This technology may aid the discovery and development of vaccines and antibody therapeutics, and help us gain a deeper understanding of the humoral response.
authors.affiliation: Cell Signaling Technology, Inc., Danvers, Massachusetts, USA.
authors.forename: Wan Cheung
authors.initials: WC
authors.lastname: Cheung
authors.forename: Sean A
authors.initials: SA
authors.lastname: Beausoleil
authors.forename: Xiaowu
authors.initials: X
authors.lastname: Zhang
authors.forename: Shuji
authors.initials: S
authors.lastname: Sato
authors.forename: Sandra M
authors.initials: SM
authors.lastname: Schieferl
authors.forename: James S
authors.initials: JS
authors.lastname: Wieler
authors.forename: Jason G
authors.initials: JG
authors.lastname: Beaudet
authors.forename: Ravi K
authors.initials: RK
authors.lastname: Ramenani
authors.forename: Lana
authors.initials: L
authors.lastname: Popova
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Comb
authors.forename: John
authors.initials: J
authors.lastname: Rush
authors.forename: Roberto D
authors.initials: RD
authors.lastname: Polakiewicz
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000918:Antibody Specificity / Q000235:genetics; D001402:B-Lymphocytes / Q000166:cytology; D001798:Blood Proteins / Q000737:chemistry*; D002853:Chromatography, Liquid / Q000379:methods; D007074:Immunoglobulin G / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D051379:Mice; D040901:Proteomics / Q000379:methods*; D011817:Rabbits; D011980:Receptors, Progesterone / Q000737:chemistry; D044967:Serum / Q000276:immunology; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D000906:Antibodies; D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D011980:Receptors, Progesterone