Advertisement

Antibodies and Selection of Monoclonal Antibodies

Chapter
First Online:
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 917)

Abstract

Monoclonal antibodies are universal binding molecules with a high specificity for their target and are indispensable tools in research, diagnostics and therapy. The biotechnological generation of monoclonal antibodies was enabled by the hybridoma technology published in 1975 by Köhler and Milstein. Today monoclonal antibodies are used in a variety of applications as flow cytometry, magnetic cell sorting, immunoassays or therapeutic approaches. First step of the generation process is the immunization of the organism with appropriate antigen. After a positive immune response the spleen cells are isolated and fused with myeloma cells in order to generate stable, long-living antibody-producing cell lines – hybridoma cells. In the subsequent identification step the culture supernatants of all hybridoma cells are screened weekly for the production of the antibody of interest. Hybridoma cells producing the antibody of interest are cloned by limited dilution till a monoclonal hybridoma is found. This is a very time-consuming and laborious process and therefore different selection strategies were developed since 1975 in order to facilitate the generation of monoclonal antibodies. Apart from common automation of pipetting processes and ELISA testing there are some promising approaches to select the right monoclonal antibody very early in the process to reduce time and effort of the generation. In this chapter different selection strategies for antibody-producing hybridoma cells are presented and analysed regarding to their benefits compared to conventional limited dilution technology.

Keywords

B lymphocytes Monoclonal antibodies Hybridoma technology Selection of antibody producing cells ELISA 
This is a preview of subscription content, log in to check access.

References

  1. 1.
    Hufford MM, Richardson G, Zhou H, Manicassamy B, García-Sastre A, Enelow RI, Braciale TJ (2012) Influenza-infected neutrophils act within the infected lungs act as antigen presenting cells. PLoS ONE 7(10):e46581. Epub 2012 Oct 8CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Segura E, Villadangos JA (2011) A modular and combinatorial view of the antigen cross-presentation pathway in dendritic cells. Traffic 12:1677–1685CrossRefPubMedGoogle Scholar
  3. 3.
    Bergtold A, Desai DD, Gavhane A, Clynes R (2005) Cell surface recycling of internalized antigens permits dendritic cell priming of B cells. Immunity 23:503–514CrossRefPubMedGoogle Scholar
  4. 4.
    Cerutti A, Chen K, Chorny A (2011) Immunoglobulin responses at the mucosal interface. Annu Rev Immunol 29:273–293CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Chen K, Xu W, Wilson M, He B, Miller NW, Bengten E, Edholm E-S, Santini PA, Rath P, Chiu A, Cattalini M, Litzman J, Bussel JB, Huang B, Meini A, Riesbeck K, Cunningham-Rundles C, Plebani A, Cerutti A (2009) Immunoglobulin D enhances immune surveillance by activating antimicrobial, proinflammatory and B cell-stimulating programs in basophils. Nat Immunol 10(8):889–898CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Köhler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495–497CrossRefPubMedGoogle Scholar
  7. 7.
    Spieker-Polet H, Sethupathi P, Yam PC, Knight KL (1995) Rabbit monoclonal antibodies: generating a fusion partner to produce rabbit-rabbit hybridomas. PNAS USA 92:9348–9352CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Kilmartin JV, Wright B, Milstein C (1982) Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line. J Cell Biol 93:576–582CrossRefPubMedGoogle Scholar
  9. 9.
    Osborne J, Harrison P, Butcher R, Ebsworth N, Tan K (1999) Novel super-high affinity sheep monoclonal antibodies against CEA bind colon and lung adenocarcinoma. Hybridoma 18:183–191CrossRefPubMedGoogle Scholar
  10. 10.
    Messerschmidt K, Hempel S, Holzlöhner P, Ulrich RG, Wagner D, Heilmann K (2012) IgA antibody production by intrarectal immunization of mice using recombinant major capsid protein of hamster polyomavirus. Eur J Microbiol Immunol 2:231–238. doi: 10.1556/EuJMI.2.2012.3.9 CrossRefGoogle Scholar
  11. 11.
    Puck TT, Marcus PI (1995) A rapid method for viable cell titration and clone production with HeLa cells in tissue culture: the use of x-irradiated cells to supply conditioning factors. PNAS USA 41:432–437CrossRefGoogle Scholar
  12. 12.
    Browne SM, Al-Rubeai M (2007) Selection methods for high-producing mammalian cell lines. Trends Biotechnol 25:425–432CrossRefPubMedGoogle Scholar
  13. 13.
    Underwood PA, Bean PA (1988) Hazards of the limiting-dilution method of cloning hybridomas. J Immunol Methods 107:119–128CrossRefPubMedGoogle Scholar
  14. 14.
    Coller HA, Coller BS (1986) Poisson statistical analysis of repetitive subcloning by the limiting dilution technique as a way of assessing hybridoma monoclonality. Methods Enzymol 121:412–417CrossRefPubMedGoogle Scholar
  15. 15.
    Caroll S, Al-Rubeai M (2004) The selection of high-producing cell lines using flow cytometry and cell sorting. Expert Opin Biol Ther 4:1821–1829CrossRefGoogle Scholar
  16. 16.
    Dharshanan S, Hung CS (2014) Screening and subcloning of high producer transfectomas using semisolid media and automated colony picker. Methods Mol Biol 1131:105–112CrossRefPubMedGoogle Scholar
  17. 17.
    Breitling F, Poustka A, Moldenhauer G (2000) Selection of monoclonal antibodies. PCT application WO 00/42176Google Scholar
  18. 18.
    Holmes P, Al-Rubeai M (1999) Improved cell line development by a high throughput affinity capture surface display technique to select for high secretors. J Immunol Methods 230:141–147CrossRefPubMedGoogle Scholar
  19. 19.
    Messerschmidt K, Heilmann K (2013) Toxin-antigen conjugates as selection tools for antibody producing cells. J Immunol Methods 387:167–172CrossRefPubMedGoogle Scholar
  20. 20.
    Borth N, Zeyda M, Kunert R, Katinger H (2000) Efficient selection of high-producing subclones during gene amplification of recombinant Chinese hamster ovary cells by flow cytometry and cell sorting. Biotechnol Bioeng 71:266–273CrossRefPubMedGoogle Scholar
  21. 21.
    Weaver JC, McGrath P, Adams S (1997) Gel microdrop technology for rapid isolation of rare and high producer cells. Nat Med 3:583–585CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2016

Authors and Affiliations

  1. 1.Research Group “Antibody Technologies”University of PotsdamPotsdam – GolmGermany

Personalised recommendations

Cite chapter

Buy options