| ObjectiveWith availability of monoclonal antibodies against human leukocyte cluster of differentiation antigens (CD antigens), CD antigens detection is technically feasible for determining the cell lineage and developmental stage of leukemic cells, which boosts leukemia immunophenotyping research and improves the diagnosis accuracy. Nowadays, immunohistochemical technique and immunofluorescent assay are commonly used for leukemia immunophenotyping, but these two methods have too many shortcomings. The former can not be used for multiple antigen detection; and furthermore, it shows other disadvantages including high costs due to excessive monoclonal antibody consumption in single antigen detection, complicated processing steps and excessive time consumption. The latter is only available for detecting a limited list of antigen and the detection procedure necessitates expensive equipments such as fluorescence microscopy and flow cytometry. Meeting the demand of high throughput cell-based screening and combining with microarray technology, cell chip has been developed. The emergence of this new type of detection technology platform, free of the above-mentioned shortcomings, makes leukemia immunopheno- typing more efficient, convenient and high-throughput.Cell chip for leukemia immunophenotyping, a newly achievement of our lab, is based on specificity of antigen-antibody reactions and prepared by fixing monoclonal antibodies against CD antigens(CD antibodies) on the micrarray supports , forming antibody array. Different monoclonal antibodies can automatically identify and capture the leukemic cell with corresponding HLDAs, thus leukemia immunophenotyping is achieved.Choosing optimal microarray supports is the first key step in the cell chip preparation process. How to choose the optimal support material and surface modification that can make monoclonal antibody fixation stronger and more specific while maintaining protein activity on the solid surface to the utmost extent is critical for the successful application of this detection technology. This experiment aims to explore the optimal cell chip support and surface modification.Methods1. Microarray support preparationFive kinds of microarray supports were prepared. Glass slides were surface-modified with 3-APTMS-glutaraldehyde, chitosan-amino, chitosan- glutaraldehyde, mercapto-group respectively, and polystyrene slide was cleaned thoroughly in an ultrasonic cleaner with distilled water.2. Determination of optimal buffer pH for spottingPhosphate buffer solution of pH 6.0,7.2,8.4 and carbonate buffer solution of pH 9.6 were chosen for spotting buffers (also containing 3% glycerol). Monoclonal CD antibodies were diluted 1 : 4 and immobilized on the five kinds of supports, which was followed by routine steps (hydration, blocking and rinsing). Isolated from the 2ml blood of healthy donor by using the self-prepared leukocyte separation medium, peripheral blood leukocytes (>1×10~6/ml) were applied to cell chips. After acridine orange staining and proper rinsing, cell chips were incubated for 45 min. After thorough PBS rinse till clean background was obtained, cell chips were scanned to determine optimal buffer pH for spotting.3. Determination of optimal saturation points of the immobilized CD antibodiesMonoclonal CD antibodies were diluted at different rates (1∶2, 1∶4, 1∶8, 1∶16, 1∶32, 1∶64) and immobilized on five kinds of supports in the descending order of antibody concentration. After 30 min incubation with FITC-conjugated goat anti-mouse IgG solution (100μg/ml), optimal saturation points of the immobilized CD antibodies on different microarray supports were determined.4. Determination of optimal antibody immobilization time on different microarray supportsMonoclonal CD antibodies were immobilized on the five microarray supports. After hydration in a moist chamber at 4℃for different duration (0.5,1,2,4,12,24,48h), cell chips were incubated with FITC-conjugated IgG solution before optimal antibody immobilization time was determined.5. Detection of protein-binding capacity of different microarray supportsFITC- conjugated antibodies were immobilized on different chip supports and the average fluorescence intensity was measured as well as fluorescence signal intensity in different parts.6. Protein activity detection on different microarray supports and protein activity duration determinationProtein activity was determined by fluorescent antibody technique or fluorescence-based cell assay on day 1 and at the end of the first month, third month and sixth month after preparation.Results1. Under either of the surface modification techniques, protein immobilization is best achieved when buffer pH value was set at 7.2 for glass slide and 9.6 for polystyrene slide.2. With antibody dilution ratio of 1:4 or antibody concentration of 50μg/ml, the immobilized antibodies on five kinds of supports reached saturation point.3. For optimal efficacy, protein immobilization in moist chamber at 4℃should be more than 12 hours.4. 3-APTMS-glutaraldehyde modification showed the highest immobilization efficacy and the better uniformity, and meanwhile, fluorescence signal intensity variation at different parts is less than 5%. 5. 3-APTMS-glutaraldehyde modification showed the highest efficacy in preserving the immobilized antibody activity and slowest activity decay rate as time passed. For example, only slight decay was present after an interval of six months.ConclusionWith buffer pH of 7.2, antibody concentration of 50μg/ml and immobilization time over 12 h, 3-APTMS-glutaraldehyde modified glass slide shows the highest efficacy of antibody immobilization and antibody activity.preservation.In conclusion, 3-APTMS-glutaraldehyde modified glass slide is an optimal choice for leukemia immunophenotyping cell chip.
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