A cytometric dual-bead array for analyzing PML-raralpha fusion protein

Abstract

This study investigated a cytometric dual-bead array used to analyze PML-RARalpha fusion protein. The carboxylated and aminated polystyrene beads were prepared and barcoded with either high or low brightness using a fluorescein isothiocyanate (FITC) penetration method. Using anti-RARalpha antibody, homotype control antibody, barcoded beads and phycoerythrin (PE)-labeled antibody, the analytical capability of the cytometric dual-bead array was tested with two cell models containing a NB4 cell line. Fusion protein levels were normalized using a PE mean fluorescence intensity (MFI) ratio (PE MFI of high brightness beads/PE MFI of low brightness beads). In flow cytometry, the cytometric dual-bead array could analyze PML(L)-RARalpha protein with high specificity, and this assay possessed analytical sensitivity of at least 0.6%. A dilution experiment also demonstrated a concordant result between the logarithm of the PE MFI ratio and the logarithm of the NB4 cell concentration (R2 = 0.9936). When compared with a cytometric single-bead assay, this array exhibited a lower relative standard deviation (R.S.D.) and a lower relative error (R.E.). In conclusion, using the MFI ratio can attenuate data error in fusion protein cytometric analysis, and the cytometric dual-bead array presented here may be of use in the quantification of PML-RARalpha fusion protein.