QIFIKIT® is intended for the quantitative determination of cell surface antigens by flow cytometry using indirect immunofluorescence assay (1, 2).
QIFIKIT® consists of a series of 6 bead populations, approximately 10 µm in diameter and coated with different, but well-defined quantities of a mouse monoclonal antibody (Mab). The number of Mab molecules on the 6 bead populations ranges from 0 to 400 000-800 000. The precise values are provided with the kit. The beads mimic cells labeled with a specific primary mouse monoclonal antibody.
Briefly, the procedure for quantitation is as follows: Specimen cells are labeled with primary mouse Mab at saturating concentration. Under this condition the primary Mab binds to the cell surface antigen monovalently. Therefore, the number of bound antibody molecules corresponds to the number of antigenic sites. Then, the cells are incubated, in parallel with the QIFIKIT® beads, with Polyclonal Goat Anti-Mouse Immunoglobulins/FITC, Goat F(ab')2
, Code F0479, at saturating concentration.
A calibration curve is constructed by plotting the fluorescence intensity of the individual bead populations against the number of Mab molecules on the beads. The number of antigenic sites on the specimen cells are then determined by interpolation.
The kit is presented as two complementary bead cocktails: A 'Set-Up Cocktail' and a 'Calibration Cocktail', each containing 1 mL, enough for 10 calibrations. Also included in the kit is 200 µL Polyclonal Goat Anti-Mouse Immunoglobulins/FITC, Goat F(ab')2
, Code F0479.
The kit is economical in use, as different cell specimens may be labeled with different primary antibodies and then quantitated using the same set of calibration beads. The only requirement is that specimens and beads are incubated with the conjugate simultaneously.
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