Mark, Few references describe calibrated measurement of binding affinity using flow cytometry. Though, this does not mean that flow will not yield an accurate value of the dissociation rate constant (kdiss), which is often the dominant variable. Though direct on-rate measurement is a problem, the equilibrium KD can be estimated for moderate to high affinity interactions using a series of antibody/antigen concentrations. I have found affinity measurements/rankings of cell surface displayed antibodies to be both quick and reliable (see refs). Most likely an appropriate assay would involve covalent or biotin-streptavidin based immobilization of your antigen onto non-fluorescent polymeric beads (2-20 um in diameter). Your antibody could then be covalently labeled with BODIPY or another high quantum yield fluorescent probe. A similar protocol has been described: Daugherty, P. S., Chen, G., Olsen, M. J., Iverson, B. L., and Georgiou, G. (1998). Antibody affinity maturation using bacterial surface display. Protein Engineering 11, 101-108. see also: Nolan, J. P., and Sklar, L. A. (1998). The emergence of flow cytometry for sensitive, real-time measurements of molecular interactions. Nat Biotechnol 16, 633-8. Kieke, M. C., Cho, B. K., Boder, E. T., Kranz, D. M., and Wittrup, K. D. (1998). Isolation of anti-T cell receptor scFv mutants by yeast surface display. Prot. Eng. 10, 1303. Hope this helps. Patrick At 09:00 AM 2/4/00 +1100, you wrote: > >Hello everybody, > >I was wondering if anybody could furnish me with a reference for measuring >antibody affinity by flow cytometry. Any help would be appreciated. > >Regards, > >Mark Gauci >Biotechnology Frontiers >Sydney, Australia. > > ____________________________________________ Patrick S. Daugherty, Ph.D. Fred Hutchinson Cancer Research Center Division of Molecular Medicine, C2-023 1100 Fairview Avenue North P.O. Box 19024 Seattle, Washington 98109-102 Phone: 206-667-3672 FAX: 206-667-6523 email: pdaugher@fhcrc.org ____________________________________________
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