CELLISA: Reporter Cell-Based Immunization and Screening of Hybridomas Specific for Cell Surface Antigens

Part of the Methods in Molecular Biology book series (MIMB, volume 748)


Monoclonal antibodies (mAbs) specific for cell surface antigens are an invaluable tool to study immune receptor expression and function. Here, we outline a generalized reporter cell-based approach to the generation and high-throughput screening of mAbs specific for cell surface antigens. Termed CELLISA, this technology hinges upon the capture of hybridoma supernatants in mAb arrays that facilitate ligation of an antigen of interest displayed on BWZ reporter cells in the form of a CD3ζ-fusion chimeric antigen receptor (zCAR); in turn, specific mAb-mediated cross-linking of zCAR on BWZ cells results in the production of β-galactosidase enzyme (β-gal), which can be assayed colorimetrically. Importantly, the BWZ reporter cells bearing the zCAR of interest may be used for immunization as well as screening. In addition, serial immunizations employing additional zCAR- or native antigen-bearing cell lines can be used to increase the frequency of the desired antigen-specific hybridomas. Finally, the use of a cohort of epitope-tagged zCAR (e.g., zCARFLAG) variants allows visualization of the cell surface antigen prior to immunization, and coimmunization using these variants can be used to enhance the immunogenicity of the target antigen. Employing the CELLISA strategy, we herein describe the generation of mAb directed against an uncharacterized natural killer cell receptor protein.

Key words

Monoclonal antibody Hybridoma technology High-throughput screening BWZ reporter cell assay CD3ζ-fusion chimeric antigen receptor β-Galactosidase (β-gal) 


  1. 1.
    Mesci, A., and Carlyle, J. R. (2007) A rapid and efficient method for the generation and screening of monoclonal antibodies specific for cell surface antigens. J Immunol Methods 323, 78–87.PubMedCrossRefGoogle Scholar
  2. 2.
    Karttunen, J., Sanderson, S., and Shastri, N. (1992) Detection of rare antigen-presenting cells by the lacZ T-cell activation assay suggests an expression cloning strategy for T-cell antigens. Proc Natl Acad Sci USA 89, 6020–4.PubMedCrossRefGoogle Scholar
  3. 3.
    Sanderson, S., and Shastri, N. (1994) Lacz Inducible, antigen/MHC-specific T-cell hybrids. Int Immunol 6, 369–76.PubMedCrossRefGoogle Scholar
  4. 4.
    Carlyle, J. R., Jamieson, A. M., Gasser, S., Clingan, C. S., Arase, H., and Raulet, D. H. (2004) Missing self recognition of Ocil/Clr-b by inhibitory NKR-P1 natural killer cell receptors. Proc Natl Acad Sci USA 101, 3527–32.PubMedCrossRefGoogle Scholar
  5. 5.
    Sancho, D., Mourao-Sa, D., Joffre, O. P., Schulz, O., Rogers, N. C., Pennington, D. J., Carlyle, J. R., and Reis e Sousa, C. (2008) Tumor therapy in mice via antigen targeting to a novel, DC-restricted C-type lectin. J Clin Invest 118, 2098–110.Google Scholar
  6. 6.
    Voigt, S., Mesci, A., Ettinger, J., Fine, J. H., Chen, P., Chou, W., and Carlyle, J. R. (2007) Cytomegalovirus evasion of innate immunity by subversion of the NKR-P1B:Clr-b missing-self axis. Immunity 26, 617–27.PubMedCrossRefGoogle Scholar
  7. 7.
    Carlyle, J. R., Mesci, A., Ljutic, B., Belanger, S., Tai, L. H., Rousselle, E., Troke, A. D., Proteau, M. F., and Makrigiannis, A. P. (2006) Molecular and genetic basis for strain-dependent NK1.1 alloreactivity of mouse NK cells. J Immunol 176, 7511–24.PubMedGoogle Scholar
  8. 8.
    Tai, L. H., Goulet, M. L., Belanger, S., Troke, A. D., St-Laurent, A. G., Mesci, A., Toyama-Sorimachi, N., Carlyle, J. R., and Makrigiannis, A. P. (2007) Recognition of H-2K(b) by Ly49Q suggests a role for class Ia MHC regulation of plasmacytoid dendritic cell function. Mol Immunol 44, 2638–46.PubMedCrossRefGoogle Scholar
  9. 9.
    Carlyle, J. R., Mesci, A., Fine, J. H., Chen, P., Belanger, S., Tai, L. H., and Makrigiannis, A. P. (2008) Evolution of the Ly49 and Nkrp1 recognition systems. Semin Immunol 20, 321–30.PubMedCrossRefGoogle Scholar
  10. 10.
    Mesci, A., Ljutic, B., Makrigiannis, A. P., and Carlyle, J. R. (2006) NKR-P1 biology: from prototype to missing self. Immunol Res 35, 13–26.PubMedCrossRefGoogle Scholar
  11. 11.
    Kearney, J. F., Radbruch, A., Liesegang, B., and Rajewsky, K. (1979) A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines. J Immunol 123, 1548–50.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of ImmunologyUniversity of Toronto and Sunnybrook Research InstituteTorontoCanada

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