Abstract
Immunoaffinity protocols offer a rapid, efficient, and simple way of obtaining a protein of interest. Almost all of the complement components, receptors, and regulators have been successfully isolated by immunoaffinity protocols. The use of these methods is limited only by the availability of suitable antibodies in sufficient quantities (1–6). Immunoaffinity methods are compatible with most detergents used in the solubilization of membrane proteins and are thus well suited to the purification of membrane regulators of complement and complement receptors (3,7,8). Proteins isolated by immunoaffinity methods, particularly on monoclonal antibody solid phases, are usually of high purity and require either no downstream processing or simple “polishing” steps, such as gel filtration to remove aggregates. Elution of the bound protein from the antibody-solid phase is commonly achieved by subjecting the column to extremes of pH to disrupt antibody-antigen interaction. Some complement proteins are labile at these pH extremes and methods must be modified accordingly (Table 1). Here, I will describe the steps followed in establishing an immunoaffinity protocol for a complement protein. These differ little from those used for any other target protein. Example protocols will be provided for a serum complement protein and a membrane bound complement regulator.
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References
Morgan B. P., Daw R. A., Siddle K., et al. (1983) Immunoaffinity purification of human complement component C9 using monoclonal antibodies. J. Immunolog. Meth. 64,269–281.
Fowler S. R. and Kolb W. P. (1984) Utilization of immunoaffinity and high performance liquid chromatography for the isolation of human component complement C5: mediation of human monocyte chemotaxis by trypsin activated C5. Federat. Proc. 43, 1460–1461.
Nemerow G. R., Siaw M. F. E., and Cooper N. R. (1986) Purification of the Epstein-Barr virus/C3d complement receptor of human B lymphocytes: antigenic and functional properties of the purified protein. J. Virology 58,709–712.
Abraha A., Morgan B. P., and Luzio J. P. (1988) The preparation and characterization of monoclonal antibodies to human complement component C8 and their use in purification of C8 and C8 subunits. Biochem. J. 251,285–292.
Misasi R., Huemer H. P., Schwaeble W., Solder E., Larcher C., and Dierich M. P. (1989) Human complement factor H: An additional gene product of 43 kDa isolated from human plasma shows cofactor activity for the cleavage of the third component of complement. Europ. J. Immunol. 19,1765–1768.
Seya T., Hara T., Iwata K., Kuriyama S. I., Hasegawa T., Nagase Y., et al. (1995) Purification and functional properties of soluble forms of membrane cofactor protein (CD46) of complement: Identification of forms increased in cancer patients’ sera. Intl. Immunol. 7, 727–736.
Davies A., Simmons D. L., Hale G., Harrison R. A., Tighe H., Lachmann P. J., and Waldmann H. (1989) CD59, an LY-6-like protein expressed in human lymphoid cells, regulates the action of the complement membrane attack complex on homologous cells. J. Experiment. Med. 170,637–654.
Purcell D. F. J., Deacon N. J., Andrew S. M., and McKenzie I. F. C. (1990) Human non-lineage antigen, CD46 (HuLy-m5): Purification and partial sequencing demonstrates structural homology with complement-regulating glycoproteins. Immunogenetics 31, 21–28.
Axen R., Porath J., and Ernback S. (1967) Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides. Nature 214,1302–1304.
Karlsson G. B. and Platt F. M. (1991) Analysis and isolation of human transferrin receptor using the OKT-9 monoclonal antibody covalently crosslinked to magnetic beads. Analyt. Biochem. 199,219–222.
Chiong M., Lavandero S., Ramos R., Aguillon J. C., and Ferreira A. (1991) Octadecyl silica: A solid phase for protein purification by immunoadsorption. Analyt. Biochem. 197,47–51.
Morgan B. P., Daw R. A., Siddle K., Luzio J. P., and Campbell A. K. (1983) Immunoaffinity purification of human complement component C9 using monoclonal antibodies. J. Immunolog. Meth. 64, 269–281.
van den Berg C. W., Harrison R. A., and Morgan B. P. (1993) The sheep analogue of human CD59: purification and characterization of its complement inhibitory activity. Immunology 78, 349–357.
van den Berg C. W., Perez de la Lastra J. M., Llanes D., and Morgan B. P.(1997) Purification and characterization of the pig analogue of human membrane cofactor protein (CD46/MCP). J. Immunol. 158, 1703–1709.
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© 2000 Humana Press Inc., Totowa, NJ
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Morgan, B.P. (2000). Immunoaffinity Methods for Purification of Complement Components and Regulators. In: Morgan, B.P. (eds) Complement Methods and Protocols. Methods in Molecular Biology, vol 150. Humana Press. https://doi.org/10.1385/1-59259-056-X:53
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DOI: https://doi.org/10.1385/1-59259-056-X:53
Publisher Name: Humana Press
Print ISBN: 978-0-89603-654-3
Online ISBN: 978-1-59259-056-8
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