 |
 |
 |
 |
 |
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were found to be lower (1x10e6 receptors) (unpublished results).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
|
|
|
|
|
|
CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web