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Monoclonal antibodies isolated without screening by analyzing the variable-gene repertoire of plasma cells

Nature Biotechnology volume 28, pages 965–969 (2010)Cite this article

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Abstract

Isolation of antigen-specific monoclonal antibodies (mAbs) and antibody fragments relies on high-throughput screening of immortalized B cells1,2 or recombinant antibody libraries3,4,5,6. We bypassed the screening step by using high-throughput DNA sequencing and bioinformatic analysis to mine antibody variable region (V)-gene repertoires from bone marrow plasma cells (BMPC) of immunized mice. BMPCs, which cannot be immortalized, produce the vast majority of circulating antibodies. We found that the V-gene repertoire of BMPCs becomes highly polarized after immunization, with the most abundant sequences represented at frequencies between ∼1% and >10% of the total repertoire. We paired the most abundant variable heavy (VH) and variable light (VL) genes based on their relative frequencies, reconstructed them using automated gene synthesis, and expressed recombinant antibodies in bacteria or mammalian cells. Antibodies generated in this manner from six mice, each immunized with one of three antigens were overwhelmingly antigen specific (21/27 or 78%). Those generated from a mouse with high serum titers had nanomolar binding affinities.

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Figure 1: Isolation of monoclonal antibodies by mining the antibody variable (V)-gene repertoires of bone marrow plasma cells.
Figure 2: Comparison of high-frequency CDRH3s reveals unique VH genes in each of eight mice immunized with one of three antigens or an adjuvant control.

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Acknowledgements

We thank C. Das for her invaluable technical assistance on mammalian cell culture and transfection and M. Byrom for assistance with mouse experiments. This work was funded by grants from the Clayton Foundation, the Cockrell Chair in Engineering (to G.G.) and by a fellowship from Natural Sciences and Engineering Research Council of Canada (to X.G.).

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Authors and Affiliations

  1. Department of Chemical Engineering, University of Texas at Austin, Austin, Texas, USA

    Sai T Reddy, Xin Ge, Seung Hyun Kang, Constantine Chrysostomou & George Georgiou

  2. Department of Biomedical Engineering, University of Texas at Austin, Austin, Texas, USA

    Sai T Reddy, Kam Hon Hoi & George Georgiou

  3. Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, USA

    Aleksandr E Miklos, Randall A Hughes, Scott P Hunicke-Smith, Brent L Iverson, Philip W Tucker, Andrew D Ellington & George Georgiou

  4. Applied Research Laboratories, University of Texas at Austin, Austin, Texas, USA

    Aleksandr E Miklos, Randall A Hughes & Andrew D Ellington

  5. Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas, USA

    Brent L Iverson & Andrew D Ellington

  6. Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, Texas, USA

    Philip W Tucker & George Georgiou

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  1. Sai T Reddy
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Contributions

S.T.R. and G.G. developed the methodology, designed experiments, analyzed the data and wrote the manuscript; X.G., C.C., and K.H.H. carried out the bioinformatic analysis; S.T.R., A.E.M., R.A.H., S.H.K. and K.H.H. performed the experiments; S.P.H.-S. performed 454 DNA sequencing; B.L.I., P.W.T. and A.D.E. helped analyze the data.

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Correspondence to George Georgiou.

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The authors declare no competing financial interests.

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Supplementary Tables 1–13 and Supplementary Figs. 1–12 (PDF 4918 kb)

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Reddy, S., Ge, X., Miklos, A. et al. Monoclonal antibodies isolated without screening by analyzing the variable-gene repertoire of plasma cells. Nat Biotechnol 28, 965–969 (2010). https://doi.org/10.1038/nbt.1673

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