On Fri 26 Apr, Karim Vermaelen wrote: > > Hello, > Does anyone know why some mouse Ab isotypes give more background than > others when staining human cells? We stained single cell suspensions form > human lung enzymatic digests and have a lot of problems with mouse IgG2a > antibodies: the isotype control stains very bright. Mouse IgG1 and IgG2b > are less of a problem... Could someone explain this selective phenomenon > and/or recommend an optimal blocking strategy to get rid of the problem? > Thanks a lot, Karim > In humans, mice and rats there are multiple IgG subclasses. There are also multiple classes of FcgR. However in some species such as rabbit there are no subclasses of IgG. In those species which have multiple subclasses, different FcgR have evolved to discriminate different subclasses of IgG. Clearly selection for this evolutionary adaption takes place within a species not between species. If you compare the sequences of human IgGs and of human FcgRs with mouse you will see that both the ligand and the receptor are changing in sequence. When you mix IgGs from one species with FcgR bearing cells from another it is likely that you will see different patterns of cross reactivity. Rat IgG2b and the related mouse IgG2a seem to react with human FcgR particularly strongly compared with other rat and mouse subclasses. see for example Hale,G., Clark,M., & Waldmann,H. (1985), J. Immunol. 134, 3056-3061 Therapeutic potential of rat monoclonal antibodies: Isotype specificity of antibody-dependant cell-mediated cytotoxicity with human lymphocytes. Bruggemann,M., Teale,C., Bindon,C., Clark,M., & Waldmann,H. (1989), J. Immunol. 142, 3145-3150 A matched set of rat/mouse chimeric antibodies. 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 Cheers, Mike <URL:http://www.path.cam.ac.uk/~mrc7/> -- M.R. Clark, PhD. Division of Immunology Cambridge University, Dept. Pathology Tennis Court Rd., Cambridge CB2 1QP Tel.+44 1223 333705 Fax.+44 1223 333875
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