FCM Microbeads Immunoassay For APL Cellular Line

APL is discovered to be associated with a reciprocal translocation between PML genes in chromosomes 15 and retinoic acid in chromosomes 17. This reciprocal translocation leads to the production of PML/RARαfusion protein that is APL marker molecule. Detection of PML/RARαaberrations is important to APL diagnosis, classification and treatment. PML/RARαaberrations are currently detected by karyotyping, ?uorescence in situ hybridization (FISH) or PCR techniques, which are time-consuming and have many shortcomings. This paper developed a simple ?ow microsphere immunoassay for detection of PML/RARαfusion proteins within NB4 cell.Highly monodisperse polystyrene (PS) microspheres with a 3.5–7.0μm diameter were synthesized via dispersion polymerization. It was observed that 5.4μm carboxylated polystyrene microspheres can be successfully prepared, when polymerization mixture containing 6 g stabilizer, 80 g absolute enthanol, 1 g initiator, 0.3 g crosslinker and 21 g styrene was reacted in three-mouth flask at N2 gas atmosphere for 12 h.In microspheres conjugation experiment, this paper studied the effect of the mount of EDC, pH, reaction time, and antibody concentration on the conjugation of capture antibody to carboxylated microspheres. This study elicited that capture antibody can efficiently bind to carboxylated microspheres, when 20000 microspheres were incubated with 1 mg EDC and 1μg capture antibody, pH 8.0, for 2 h.In this study, microspheres coated with anti-PML antibody (capture antibody) can capture RARαpeptide epitope in fusion protein specifically, while a anti-RARαantibody as report antibody can recognise RARαpeptide epitope specifically. Using flow microspheres immunoassay, the value of PML/RARαfusion protein was represented as PE mean ?uorescenc intensity (MFI), which was examined by flow cytometer. Compared to one step method, multi-steps approach to microspheres immunoassay exhibited the higher sensitive and the more timesaving characterics. In one step approach, the topgallant value of protein extraction, report antibody and reaction time was 50μL, 1.25μL and 1 h respectively.Results from this study demonstrated that flow microsphere immunoassay can efficiently detect PML/RARαfusion protein within NB4 cells. And this method might be useful for APL clinical diagnosis.