Hello Mark, since two years we use the DAKO QIFIKIT (Code No. K0078) for quantitative analysis of indirect immunofluorescence staining in flow cytometry. The kit contains three vials, one with set-up (blank) beads (named A). The second vial contains calibration beads with 5 different mAb values (ABC, antibody binding capacity), i.e. 3000 (B), 9200 (C), 38000 (D), 150000 (E), 390000 (F), dependent on the lot you have purchased. The third vial contains a FITC-conjugated goat-anti mouse IgG (equivalent with DAKO Code No. F0479). Each kit is storeable at 2-8 dC and is desired for 10 calibrations. According to the manufacturer's protocol, in the first step you have to stain the cells with the mAb against the cell surface antigen. In the second step you have to stain in parallel the beads and the cells with the FITC-conjugated antibody. For analysis you must check the alignement of your flow cytometer to range the fluorescence intensity of the calibration beads and the compensation you need for your cells. Than you can start analysis saving all the data files, first running the set-up beads, than the compensation beads and than all other samples. The last samples should be the DAKO beads again to compare the calibration values. It is recommendable to stain the cells with various concentrations of both the first mAb and the second FITC-conjugated Ab to calculate the specific antibody binding capacity (SABC) with really saturated antibody amounts. For calculation DAKO offers a Windows-based software programm (TallyCAL for Windows, Code No. S2033, approx. USD750) which encompasses several protocols for differnt flow cytometers and their specific analysis software, which differ in uncalibrated range, arbitrary units and calculation methods. When we compared the results calculated with TallyCAL or MS-Excel we found some small differences (approx. 2 per cent) which even the DAKO rep could not explain. We used the kit to quantifiy antigens expressed on human cells by use of monoclonal antibodies (mAb), most of the mAb developed by ourself (S. Zahn et al., Eur. J. Imm., in press). Compared with experiments when iodinated Fab-antibodies or ligands were used we found similar results with much lower variations. Only when values are calculated over the calibration range (i.e. 2x10e6 SABC on transfected cells) the radioactive resultes were found to be lower (1x10e6 receptors) (unpublished results). Sigma also offers a kit for antigen quantitation in flow cytometry, the Quantum Simply Cellular Microbead Kit (QSC-20 for 20 test, or QSC-100 for 100 test including the software QuickCal for DOS, for Windows, QSC-20W or -100W). The kit "is a mixture of four highly uniform microbead populations which differ by their incremental capacities to bind mouse Ig" i.e. in the ABC range of 8000-150000, and a non-binding population is included. But we did not use the QSC kit. I hope, my brief description will give enough informations. Do not hesitate to directly contact me if you need some more informations. By e-mail, I also can send you some list mode files, obtained from calibrations done with a BD FACStar, FACScan or Coulters Elite which you can re-run on a PC with Joseph Trotters (many thanks to you!!) Windows-based software WinMDI. HP Literature describing the development of QIIF (equiv. QIFIKIT): P. Poncelet and P. Carayon, 1985 Cytofluorometric quantification of cell-surface antigens by indirect immunofluorescence using monoclonal antibodies. J. Imm. Meth. 85: 65-74 -------------------------------------------------------------------------- Dr. Hans-Peter Spengler Dept. of Immunology FAX: +49-551-395843 University of Goettingen Phone: +49-551-395819 Kreuzbergring 57 E-mail: hspengl@gwdg.de D-37075 Goettingen/Germany
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