On Tue 09 Feb, Rafael Nunez wrote: > > Dear Jeffrey, just try U937, is very handy and has a lot of such > receptors, cheers Rafael > > Greetings to all. I am new to this listing and flow cytometry. I > > am a researcher at a small start-up biotech company, Sunol Molecular > > Corp. in Miramar, Florida. I want to do some experimentas to > > investigate the interaction of a humanized antibody(IgG1 and 3 /Kappa)w > > with human Fc Gamma receptors on the cell surface. I was wondering if > > anyone out there could recommend a stable cell line expressing high > > levels of human CD16 and/or CD32 on the surface. I tried search in the > > ATCC on line but without success. I'd really appreciate any help or > > advise, thanks. Jeff jeffreystinson@sunolmolecular.com > > I've just spotted this in the list and I think that U937 may not be suitable for your purposes. One problem with U937 is that like many human cell lines it expresses multiple FcR at the same time and so it is difficult to get clean results. Certainly U937 does express CD32 and it can be induced to express CD16 with gamma interferon treatment. However it also expresses the high affinity CD64 receptor. I have faced this problem myself several times when trying to characterise recombinant and also monoclonal antibodies such as our CAMPATH-1H (CD52) antibody. In the end I have resorted to using functional assays such as ADCC or phagocytosis with several different human donors as well as comparing these results with those obtained for binding to transfectants expressing single human FcR at a time. Using these approaches it has also been possible to identify differences with regard to allelic differences in FcR and Ig allotype. You might like to look at Redpath, S., Michaelsen, T.E., Sandlie, I. & Clark, M.R. (1998), Human Immunology 59, 720-727 The influence of the hinge region length in binding of human IgG to human Fc gamma receptors. Haagen,I-A., Geerars,A.J.G., Clark,M.R., & van de Winkel,J.G.J. (1995), Journal of Immunology 154, 1852-1860 Interaction of human monocyte Fc gamma receptors with rat IgG2b: A new indicator for the Fc gamma RIIA (R-H131) polymorphism Greenwood,J., Clark,M. & Waldmann,H. (1993), Eur. J. Immunol. 23, 1098-1104 Structural motifs involved in human IgG antibody effector functions Clark, M (1997), Chemical Immunology 65, 88-110 Antibody Engineering IgG Effector Mechanisms There is also some more information on my antibody structure and function website see <URL:http://www.path.cam.ac.uk/~mrc7/> Mike Clark, -- o/ \\ // || ,_ o M.R. Clark, PhD. Division of Immunology <\__,\\ // __o || / /\, Cambridge University, Dept. Pathology "> || _`\<,_ // \\ \> | Tennis Court Rd., Cambridge CB2 1QP ` || (_)/ (_) // \\ \_ Tel.+44 1223 333705 Fax.+44 1223 333875
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