Molecular Biotechnology

, Volume 29, Issue 2, pp 101–109 | Cite as

In Situ isolation of immunoglobulin sequences expressed by single tumor-infiltrating B cells using laser-assisted microdissection

  • Philippa M. O'Brien
  • David W. M. Millan
  • Jonathon A. Davis
  • M. Saveria Campo


The isolation of fully human monoclonal antibodies (MAb) against tumor targets has to date relied largely on combinatorial library-based antibody display techniques, which generally require lengthy antigen selection procedures due to a low frequency of clones expressing compatible heavy (VH) and light chain (VL) variable genes. Here we describe a method to directly isolate immunoglobulin sequences in situ from antibody-producing cells infiltrating human tumor tissue. Single B cells and plasma cells infiltrating cervical cancer were microdissected from tissue sections using laser-assisted microscopy, and VH and VL expressed by each individual cell amplified using nested reverse transcriptase-polymerase chain reaction (RT-PCR), thus retaining the native VH and VL pairing. Sequencing analysis determined that the isolated cells expressed functional immunoglobulin variable genes, consistent with an antitumor antibody response. The immunoglobulin sequences can be reassembled as Fab or scFv fragments using conventional recombinant antibody expression plasmids. This method will allow a more direct assessment of the humoral immune response to cancer, and the potential identification of novel human therapeutic cancer antibodies.

Index Entries

Antibody cancer B cell immunoglobulin phage display 


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  1. 1.
    Kohler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495–497.PubMedCrossRefGoogle Scholar
  2. 2.
    Carter, P. (2001) Improving the efficacy of antibody-based cancer therapies. Nature Rev. Cancer 1, 118–129.CrossRefGoogle Scholar
  3. 3.
    Hudson, P. J. and Souriau, C. (2003) Engineered antibodies. Nature Med. 9, 129–134.PubMedCrossRefGoogle Scholar
  4. 4.
    Kipriyanov, S. M. and Le Gall, F. (2004) Generation and production of engineered antibodies. Mol. Biotechnol. 26, 39–60.PubMedCrossRefGoogle Scholar
  5. 5.
    Kretzschmar, T. and von Ruden, T. (2002) Antibody discovery: phage display. Curr. Opin. Biotech. 13, 598–602.PubMedCrossRefGoogle Scholar
  6. 6.
    Holt, L. J., Enever, C., de Wildt, R. M. T., and Tomlinson, I. M. (2000) The use of recombinant antibodies in proteomics. Curr. Opin. Biotech. 11, 445–449.PubMedCrossRefGoogle Scholar
  7. 7.
    Lagerkvist, A. C., Furebring, C., and Borrebaeck, C. A. (1995) Single, antigen-specific B cells used to generate Fab fragments using CD40-mediated amplification or direct PCR cloning. Biotechniques 18, 862–869.PubMedGoogle Scholar
  8. 8.
    Zhang, H., Lake, D. F., Barbuto, J. A. M., Bernstein, R. M., Grimes, W. J., and Hersh, E. M. (1995) A human monoclonal antimelanoma single-chain Fv antibody derived from tumor-infiltrating lymphocytes. Cancer Res. 55, 3584–3591.PubMedGoogle Scholar
  9. 9.
    De Wildt, R. M. T., Steenbakkers, P. G., Pennnings, A. H. M., van den Hoogen, F. H. J., van Venrooij, W. J., and Hoet, R. M. A. (1997) A new method for the analysis and production of monoclonal antibody fragments originating from single human B cells. J. Immunol. Meth. 207, 61–67.CrossRefGoogle Scholar
  10. 10.
    Coronella, J. A., Telleman, P., Kingsbury, G. A., Truong, T. D., Hays, S., and Junghans, R. P. (2001) Evidence for an antigen-driven humoral immune response in medullary ductal breast cancer. Cancer Res. 61, 7889–7899.PubMedGoogle Scholar
  11. 11.
    De Wildt, R. M. T. and Hoet, R. M. A. (2002) The recovery of immunoglobulin sequences from single human B cells by clonal expansion. Meths Mol. Biol. 178, 121–131.Google Scholar
  12. 12.
    Fend, F., Emmert-Buck, M.R., Chuaqui, R., Cole, K., Lee, J., Liotta, L.A., and Raffeld, M. (1999) Immuno-LCM: laser capture microdissection of immunostained frozen sections for mRNA analysis. Am. J. Pathol. 154, 61–66.PubMedGoogle Scholar
  13. 13.
    OBrien, P.M., Tsirimonaki, E., Coomber, D.W., Millan, D.W., Davis, J.A. and Campo, M.S. (2001) Immunoglobulin genes expressed by B-lymphocytes infiltrating cervical carcinomas shown evidence of antigen-driven selection. Cancer Immunol. Immunother. 50, 523–532.CrossRefGoogle Scholar
  14. 14.
    Coronella, J.A., Telleman, P., Truong, T.D., Ylera, F., and Junghans, R.P. (2000) Amplification of IgG VH and VL (Fab) from single human plasma cells and B cells. Nucleic Acids Res. 28, E85.Google Scholar
  15. 15.
    Wang, X., and Stollar, B.D. (2000) Human immunoglobulin variable region gene analysis by single cell RT-PCR. J. Immunol. Meth. 244, 217–225.CrossRefGoogle Scholar
  16. 16.
    Preuss, K-D., Zwick, C., Bormann, C., Neumann, F., and Pfreundschuh, M. (2002) Analysis of the B-cell repertoire against antigens expressed by human neoplasms. Immunol. Rev. 188, 43–50.PubMedCrossRefGoogle Scholar
  17. 17.
    Burton, D.R., and Barbas, C.F.3rd (1994) Human antibodies from combinatorial libraries. Adv. Immunol. 57, 191–280.PubMedCrossRefGoogle Scholar
  18. 18.
    Lefranc, M.-P., and Lefranc, G. (2001) The Immunoglobulin Facts Book. Academic Press.Google Scholar

Copyright information

© Humana Press Inc 2005

Authors and Affiliations

  • Philippa M. O'Brien
    • 1
  • David W. M. Millan
    • 2
  • Jonathon A. Davis
    • 2
  • M. Saveria Campo
    • 1
  1. 1.Institute of Comparative MedicineUniversity of Glasgow Veterinary SchoolGlasgow
  2. 2.North Glasgow NHS TrustGlasgowUK

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