Table 2 Successive steps involved for the calibration and application of the prototype “IgHκ calibrator beads”.
Calibrator beads | QIFIkit (commercial) | Midi-QIFIkit (prototype) | Mini-QIFIkit (prototype) | µ-QIFIkit (prototype) | “IgHκ beads” (protoype) | “IgHκ beads” (prototype) |
|---|---|---|---|---|---|---|
Diameter | 10 µm | 3 µm | 3 µm | 1 µm | 1 µm | 1 µm |
Coated analyte | Mouse IgG1 | Biotin-IgG1 (mouse) | Biotin-IgG1 (mouse) | Biotin-IgG1 (mouse) | REAfinity* CD151-biotin (human IgG-k) | Trastuzumab on liposomes (human IgG-k) |
Coating method | Proprietary | Magnetic SA-beads | Magnetic SA-beads | Magnetic SA-beads | Magnetic SA-beads | Covalent coupling |
Calibration FCM instrument | FC500 and/or Gallios | Gallios | CytoFLEX-S | CytoFLEX-S | CytoFLEX-S | CytoFLEX-S |
Staining method | QIFI (indirect IF with unlabeled mouse IgGs) | QIFI (indirect IF with unlabeled mouse IgGs) | QIFI (indirect IF with unlabeled mouse IgGs) | QIFI (indirect IF with unlabeled mouse IgGs) | QIFI (indirect IF with unlabeled mouse IgGs) | Direct IF with L1C1-PE (anti-human kappa) |
Laser height at interrogation point | = or >10 µm, (depending on instrument) | 10 µm | 5 µm | 5 µm | 5 µm | 5 µm |
Excitation λ | 488 nm | 488 nm | 561 nm | 561 nm | 561 nm | 561 nm |
Calibration channel | FITC | FITC | PE | PE | PE | PE |
Calibration curve(s) | N.A. | Fig. S1a | Fig. S1b | Fig. S1c | Fig. 1 | Fig. 2 |
