Abstract
In vitro immunization involves the exposure of spleen cells to antigen in tissue culture rather than the antigenic stimulation of spleen cells via immunization of mice. The production of monoclonal antibodies (MAbs) to highly conserved molecules, such as enzymes (1,2), is possible using in vitro immunization. MAbs to such “self”-antigens often are not possible to make using traditional in vivo methods owing to immune suppression or tolerance. Utilizing in vitro immunization, it is possible to elicit the formation of MAbs in response to picogram quantities of antigen (3–6). Although certain protocols (1,7) indicate a minimum requirement of from 30–100 µg antigen for in vitro immunization, we have found that the nanogram or picogram quantities of antigen available from blotted polyacrylamide gels provide sufficient antigen for the preparation of MAbs by in vitro immunization (3,5). Additional advantages of in vitro immunization include shortening the immunization procedure from the 5 or 6 wk required for in vivo immunization to 4 d, allowing defined antigen concentrations, and controlling antigen degradation (3).
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Pardue, R. L., Brady, R. C., Perry, G. W., and Dedman, J. R. (1983) Production of MAbs against calmodulin by in vitro immunization of spleen cells. J. Cell Biol. 96, 1149–1154.
Glad, C., Wennerstrom, G., and Fredlund, B. M. (1988) Optimization of murine in vitro immunization against different antigens, in in vitro immunization, in Hybridoma Technology (Borrebaeck, C. A. K., ed.), Elsevier, Amsterdam, pp. 295–301.
Schelling, M. E. (1986) Increase of hybridoma formation by addition of dextran sulphate to in vitro immunization system. Hybridoma 5(2), 159–161.
Schelling, M. E., Hawker, J. R., and Granger, H. (1987) Immunochemical comparison of peptide angiogenic factors. Tissue & Cell 19(4), 463–467.
Venkateswaran, S., Blanckaert, V., and Schelling, M. E. (1992) Production of anti-fibroblast growth factor receptor monoclonal antibodies by in vitro immunization. Hybridoma 11(6), 729–739.
Luben, R., Brazeau, P., Bohlen, P., and Guillemin, R. (1982) Monoclonal antibodies to hypothalamic growth hormone-releasing factor with picomoles of antigen. Science 218, 887–898.
Jonak, Z. L. and Kennett, R. H. (1984) In vitro immunization of mouse spleen cells, in Monoclonal Antibodies and Functional Cell Lines (Kennett, R. H., Bechtol, K. B., and McKearn, T. J., eds.), Plenum, New York, pp. 368–370.
Pollock, B. J. and d’Apice, A. J. F. (1988) Production of human monoclonal antibodies against specific antigens by in vitro immunization, in In Vitro Immunization in Hybridoma Technology (Borrebaeck, C. A. K., ed.), Elsevier, Amsterdam, pp. 277–284.
Dinarello, C. A. and Krueger, J. M. (1986) Induction of IL-1 by synthetic and naturally occuring muramyl peptides. Fed. Proc. 45, 2545–2548.
Bahr, G. M. and Chedid, L. (1986) Immunological activities of muramyl peptides. Fed. Proc. 45, 2541–2544.
Jacot-Guillarmod, H. (1988) Human monoclonal antibodies obtained by fusion with a heteromyeloma, in In Vitro Immunization in Hybridoma Technology (Borrebaeck, C. A. K., ed.), Elsevier, Amsterdam, pp. 295–301.
Farrar, J. J., Fuller-Farrar, J., Simon, P. L., Hilfiker, M. L., Stadler, B. M., and Farrar, W. L. (1980) Thymoma production of T-Cell growth factor (Interleukin 2). J. Immunol. 125, 2555–2558.
Borrebaeck, C. A. K. and Moller, S. A. (1986) In vitro immunization. Effect of growth and differentiation factors on antigen-specific B cell activation and production of monoclonal antibodies to autologous antigens and weak immunogens. J. Immunol. 136, 3710–3715.
Martin, D., Brodeur, B. R., Larose, Y., Faucher, S., and Hamel, J. (1988) Production of human monoclonal antibodies against Haemophilus influenzae type B using a heteromyeloma, in In Vitro Immunization in Hybridoma Technology (Borrebaeck, C. A. K., ed.), Elsevier, Amsterdam, pp. 295–301.
Kennett, R. (1980) Fusion protocols by centrifugation of cells suspended in polyethylene glycol, in Monoclonal Antibodies (Kennett, R., McKearn, T., and Bechtol, K., eds.), Plenum, New York, pp. 365–367.
Oi, T. and Herzenberg, L. (1980) Immunoglobulin-producing hybrid cell lines, in Selected Methods in Cellular Immununology (Mishell, B. and Shiigi, S., eds.), Freeman, San Francisco, CA, pp. 351–372.
McKearn, T. (1980) Cloning of hybridoma cells by limiting dilution in fluid phase, in Monoclonal Antibodies (Kennett, R., McKearn, T., and K. Bechtol, K., eds.), Plenum, New York, pp. 374,375.
Davis, W. C. (1988) Enhancement of myeloma-B-cell hybridoma outgrowth in primary cultures with B cell mitogens. Periodicum Biologorum 90(3), 367–374.
Harris, J. F., Hawley. R. G., Hawley, T. S., and Crawford-Sharpe, G. C. (1992) Increased frequency of both total and specific monoclonal antibody producing hybridomas using a fusion partner that constitutively expresses recombinant IL-6. J. Immunol. Methods 148, 199–207.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1995 Humana Press Inc.
About this protocol
Cite this protocol
Schelling, M.E. (1995). Methods of Immunization to Enhance the Immune Response to Specific Antigens In Vitro. In: Davis, W.C. (eds) Monoclonal Antibody Protocols. Methods in Molecular Biology™, vol 45. Humana Press. https://doi.org/10.1385/0-89603-308-2:9
Download citation
DOI: https://doi.org/10.1385/0-89603-308-2:9
Publisher Name: Humana Press
Print ISBN: 978-0-89603-308-5
Online ISBN: 978-1-59259-532-7
eBook Packages: Springer Protocols