Improved production of monoclonal antibodies against the LcrV antigen of Yersinia pestis using FACS-aided hybridoma selection
For about four decades, hybridoma technologies have been the “work horse” of monoclonal antibody production. These techniques proved to be robust and reliable, albeit laborious. Over the years, several major improvements have been introduced into the field, but yet, antibody production still requires many hours of labor and considerable resources. In this work, we present a leap forward in the advancement of hybridoma-based monoclonal antibody production, which saves labor and time and increases yield, by combining hybridoma technology, fluorescent particles and fluorescence-activated cell sorting (FACS). By taking advantage of the hybridomas’ cell-surface associated antibodies, we can differentiate between antigen-specific and non-specific cells, based on their ability to bind the particles. The speed and efficiency of antibody discovery, and subsequent cell cloning, are of high importance in the field of infectious diseases. Therefore, as a model system, we chose the protein LcrV, a major virulence factor of the plague pathogen Yersinia pestis, an important re-emerging pathogen and a possible bioterror agent.
1. Milstein, C., The hybridoma revolution: an offshoot of basic research. Bioessays, 1999. 21(11): p. 966-73.
2. de StGroth, S.F. and D. Scheidegger, Production of monoclonal antibodies: strategy and tactics. J Immunol Methods, 1980. 35(1-2): p. 1-21.
3. Samra, H.S. and F. He, Advancements in high throughput biophysical technologies: applications for characterization and screening during early formulation development of monoclonal antibodies. Mol Pharm, 2012. 9(4): p. 696-707.
4. Valihrach, L., P. Androvic, and M. Kubista, Platforms for Single-Cell Collection and Analysis. Int J Mol Sci, 2018. 19(3).
5. Dippong, M., et al., Hapten-Specific Single-Cell Selection of Hybridoma Clones by Fluorescence-Activated Cell Sorting for the Generation of Monoclonal Antibodies. Anal Chem, 2017. 89(7): p. 4007-4012.
6. Inglesby, T.V., et al., Plague as a biological weapon: medical and public health management. Working Group on Civilian Biodefense. JAMA, 2000. 283(17): p. 2281-90.
7. Tsuzuki, S., et al., Dynamics of the pneumonic plague epidemic in Madagascar, August to October 2017. Euro Surveill, 2017. 22(46).
8. Hill, J., et al., Regions of Yersinia pestis V antigen that contribute to protection against plague identified by passive and active immunization. Infect Immun, 1997. 65(11): p. 4476-82.
9. Zauberman, A., et al., Neutralization of Yersinia pestis-mediated macrophage cytotoxicity by anti-LcrV antibodies and its correlation with protective immunity in a mouse model of bubonic plague. Vaccine, 2008. 26(13): p. 1616-25.
10. Levy Y, F.Y., Zauberman A, Tidhar A, Aftalion M, Lazar S, Protection against plague afforded by treatment with polyclonal LcrV and F1 antibodies, in The challenge of highly pathogenic microorganisms, O.A. Shafferman A, Velan B, Editor. 2010, Springer: Eilat.
11. Mueller CA, B.P., Muller SA, Ringler P, Erne-Brand F, Sorg I, The V-antigen of Yersinia forms a distinct structure at the tip of injectisome needles. Science 2005. 310(5748): p. 674-676.
12. Cabanel, N., et al., Plasmid-mediated doxycycline resistance in a Yersinia pestis strain isolated from a rat. Int J Antimicrob Agents, 2018. 51(2): p. 249-254.
13. Flashner, Y., et al., The search for early markers of plague: evidence for accumulation of soluble Yersinia pestis LcrV in bubonic and pneumonic mouse models of disease. FEMS Immunol Med Microbiol, 2010. 59(2): p. 197-206.
14. Leary, S.E., et al., Active immunization with recombinant V antigen from Yersinia pestis protects mice against plague. Infect Immun, 1995. 63(8): p. 2854-8.
15. Aida, Y. and M.J. Pabst, Removal of endotoxin from protein solutions by phase separation using Triton X-114. J Immunol Methods, 1990. 132(2): p. 191-5.
16. Lane, R.D., A short-duration polyethylene glycol fusion technique for increasing production of monoclonal antibody-secreting hybridomas. J Immunol Methods, 1985. 81(2): p. 223-8.
17. Freshney, R.I. Culture of Animal Cells: A Manual of Basic Technique. 1994. 389-391.
18. Galfre, G. and C. Milstein, Preparation of monoclonal antibodies: strategies and procedures. Methods Enzymol, 1981. 73(Pt B): p. 3-46.
19. Staros, J.V., R.W. Wright, and D.M. Swingle, Enhancement by N-hydroxysulfosuccinimide of water-soluble carbodiimide-mediated coupling reactions. Anal Biochem, 1986. 156(1): p. 220-2.
20. Hermanson, G.T., Bioconjugate Techniques. 2013: Elsevier.
21. Jones E, O.E., Peterson P et al., SciPy: Open Source Scientific Tools for Python. 2001.
22. Crosnier, C., N. Staudt, and G.J. Wright, A rapid and scalable method for selecting recombinant mouse monoclonal antibodies. BMC Biol, 2010. 8: p. 76.