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A proteomics approach for the identification and cloning of monoclonal antibodies from serum

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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.

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Figure 1: Overview of proteomics approach for identifying functionally relevant monoclonal antibodies from an immunized animal.
Figure 2: Affinity purification of progesterone receptor–specific polyclonal rabbit IgG.
Figure 3: Identification and characterization of functional monoclonal antibodies against progesterone receptor A/B.

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  • 28 March 2012

    In the version of this supplementary file originally posted online, the Figures and Tables were corrupted. The error has been corrected in this file as of 28 March 2012.


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We would like to dedicate this work to the memories of César Milstein and George Kohler. We thank A. Singh and S. Kane for polyclonal antibody development, and W. Colpoys, L. Cunningham, J. Simendinger, K. Crosby and G. Innocenti for help with western blot analysis, immunohistochemistry, immunofluorescence and flow cytometry. We thank K. Smith for help with polyclonal purification and M. Lewis for help with animal immunization and spleen isolation. We thank C. Reeves for help with DNA sequencing and J. Knott for peptide antigen synthesis. Finally, we thank P. Hornbeck, C. Hoffman, S. Chow and T. Singleton for reading the manuscript and providing useful discussions.

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Authors and Affiliations



W.C.C., S.A.B. and R.D.P. developed the methodology, designed experiments, analyzed the data and wrote the manuscript. W.C.C. and S.A.B. performed experiments and did the bioinformatic analysis. S.S. designed experiments, analyzed data and wrote the manuscript. X.Z., S.M.S., J.S.W., J.G.B., R.K.R. and L.P. performed experiments. M.J.C. and J.R. helped analyze the data and write the manuscript.

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Correspondence to Roberto D Polakiewicz.

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All authors are employees of Cell Signaling Technology.

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Cheung, W., Beausoleil, S., Zhang, X. et al. A proteomics approach for the identification and cloning of monoclonal antibodies from serum. Nat Biotechnol 30, 447–452 (2012).

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