Analysis And Testing Of On-Microbead Binding Assay For APL By Flow Cytometry

APL with the translocation of chromosome of t(15,17) is one kind of acute leukemia. It produces a fusion gene called PML/RARαthat encodes PML/RARαfusion protein. A novel diagnosis method might be developed through detecting the level of PML/RARαfusion protein in patient with APL.Firstly, we adjusted the quantities of monomer, the temperature of reaction as well as response time to make sure the optimal reaction condition.The optimal reaction condition is:temperature 70℃,AIBN 1g,St 21g. Polystyrene microbeads possessing active carboxyl surface and 5.4μm in mean diameter were successfully prepared in this study. Scanning electric microscope confirmed that microbeads had smooth surface, good dispersion and 5.4μm mean diameter. Microbeads were barcoded by fluoresceine isothiocyanate fluorescence as high brightness, medium brightness and low brightness populations. Triplexed microbeads array was tested by flow cytometer. High, medium and low brightness microbeads were coated with N-19 antibody, c-abl 24-11 antibody and G6 antibody respectively, in order to capture PML/RARαfusion protein, C-ABL control protein and BCR-ABL fusion protein. Low brightness microbead population was utilized as a negative control. Three microbead populations were mixed and incubated with NB4 whole cell extract. Washed and resuspended in phosphate buffer, microbead was incubated with RARαC-20 antibody for reporting PML/RARαprotein and c-abl H-300 antibody for reporting C-ABL and BCR/ABL protein. Phycoerythrin(PE)-labelled antibody was next employed to display the level of report antibody binding to protein. Values were numbered via PE fluorescence intensity accessed by flow cytometry. Data were acquired on the FACSCalibur cytometer using Cell Quest Pro software.Results demonstrated that triplexed microbeads immunoassay array can specially detect PML/RARαfusion protein in a concentration-dependent manner, and might be useful for APL clinical diagnosis.