构建白血病免疫学分型细胞芯片的片基选择及最佳修饰方法的研究

发布时间：2018-05-21 01:34

本文选题：细胞芯片 + 载体　；参考：《中国医科大学》2007年硕士论文

【摘要】： 目的随着大量抗白细胞表面分化抗原单克隆抗体的问世，应用其来检测白血病细胞表面分化抗原，以确定其细胞系列来源及发育阶段，推动了白血病免疫学分型的研究，提高了白血病诊断的准确性。目前常用的进行白血病免疫学分型的方法有免疫组化法、免疫荧光法，但这两种方法存在很多缺陷。前者不能同时进行多种抗原检测；检测一种抗原需要的单克隆抗体量较多，不经济；处理步骤多，费时。后者同时检测抗原种类有限；检测所需的荧光显微镜或流式细胞仪设备昂贵。对细胞高通量研究的要求与芯片技术的结合使细胞芯片应运而生，这种新型的检测技术平台的出现，克服了上述方法的缺陷，使白血病的免疫学分型高通量诊断更快捷、方便。本实验室构建的用于白血病免疫学分型的细胞芯片就是利用抗原、抗体特异性结合的原理，将白细胞表面分化抗原单克隆抗体固定于载体上，形成抗体点阵。不同的单克隆抗体能够自动识别和捕获表达相应表面分化抗原的细胞，从而对白血病进行免疫学分型。载体选择是芯片制备过程中的第一个关键步骤。选择何种载体及如何修饰才能使单克隆抗体更牢固、更特异性地固定在载体表面，同时使固相支持物表面的蛋白保持最大的活性，是影响该技术检测成功率的关键。本实验欲探索制备细胞芯片的最佳载体及载体的最佳修饰方法。方法 1、制备5种片基分别对玻片进行氨基硅烷-醛基修饰、壳聚糖-氨基修饰、壳聚糖-醛基修饰、巯基修饰；聚苯乙烯板蒸馏水超声清洗备用。 2、确定5种片基的最佳点样缓冲液pH值分别用含3％甘油的pH值为6.0、7.2、8.5的磷酸盐缓冲液和pH值为9.6的碳酸盐缓冲液，1：4稀释CD单抗，在5种片基上点制抗体点阵，水化、封闭、清洗。取新鲜正常人的外周血2ml，用本实验室自配的全白细胞分离液提取白细胞(1×10~6/ml)，丫叮橙染色、清洗，孵育芯片45分钟，PBS清洗至背景干净，，扫描，分析不同片基的最佳点样缓冲液pH值。 3、确定5种片基固定抗体的饱和浓度将原浓度是200μg/ml的CD单抗分别按1：2、1：4、1：8、1：16、1：32、1：64稀释后，在5种片基上按浓度下降的次序点样，FITC标记的羊抗鼠IgG溶液(浓度100μg/ml)孵育30分钟，分析不同片基固定抗体的饱和浓度。 4、确定5种片基固定抗体的最佳时间在5种片基上固定CD单抗，分别在湿盒中4℃水化0.5、1、2、4、12、24、48小时，FITC标记IgG溶液孵育，分析不同片基固定抗体的最佳时间。 5、检测5种片基的蛋白结合力在5种片基上固定FITC标记的荧光抗体，比较不同片基的平均荧光信号强度及不同部位荧光信号强度的变异率。 6、检测5种片基固定的蛋白活性及保存时间对蛋白活性的影响在芯片制备后1天、1个月、3个月和6个月，分别用荧光标记抗体和荧光标记细胞来检测蛋白的活性。结果 1、不同方法修饰的玻片和聚苯乙烯片基分别在点样缓冲液pH值为7.2、9.6时，蛋白固定效果最好。 2、抗体稀释比例达到1：4时，即抗体浓度达到50μg/ml时，5种片基固定的抗体均达到饱和浓度。 3、在湿盒中4℃条件下固定时间超过12小时，蛋白的固定效果最好。 4、氨基硅烷-醛基玻片蛋白固定效率最高，均匀度好，不同部位的荧光信号强度变异率5％。 5、氨基硅烷-醛基玻片固定的抗体活性最高，随着保存时间的延长抗体活性衰减最慢，保存6个月时仅有轻微的衰减。结论氨基硅烷-醛基玻片在以pH值为7.2的磷酸盐缓冲液作为点样缓冲液，固定蛋白的浓度为50μg/ml，固定时间大于12小时的条件下，对蛋白的固定效率及固定蛋白的反应性最好，且明显优于其他片基，可做为制备白血病免疫学分型细胞芯片的最佳载体。
[Abstract]:objective
With the advent of a large number of monoclonal antibodies against leukocyte surface differentiation antigen, it is used to detect the surface differentiation antigen of leukemic cells, in order to determine the source and development stage of its cells, promote the study of leukemia immunology classification and improve the accuracy of leukemia diagnosis. There are immunofluorescence and immunofluorescence, but there are many defects in these two methods. The former can not detect multiple antigens at the same time; the amount of monoclonal antibodies needed to detect a kind of antigen is more and less economical; the treatment steps are more time-consuming. The latter is used to detect the limited type of antigen at the same time; the detection of the required fluorescence microscope or flow cytometry equipment The combination of the requirement of high flux research with the cell technology and the combination of chip technology make the cell chip come into being. The emergence of this new detection technology platform overcomes the defects of the above methods and makes the leukemia immunological classification high throughput diagnosis faster and convenient.
The cell chip used in this laboratory is to use the antigen and the principle of antibody specific binding to immobilize the monoclonal antibody against the leukocyte surface differentiation antigen on the carrier and form an antibody lattice. Leukaemia is immunologically typed.
The selection of carrier is the first key step in the process of chip preparation. What carrier and how to modify to make the monoclonal antibody stronger, more specifically fixed to the surface of the carrier, and keep the protein on the surface of the solid phase support to maintain the maximum activity, is the key to the success rate of the test. The best carrier and the best modification method of the chip.
Method
1, the preparation of 5 kinds of sheet base
The glass slides were modified by amino silane aldehyde group, chitosan amino modification, chitosan aldehyde modified, sulfhydryl modification, and polystyrene board distilled water for ultrasonic cleaning.
2, determine the best point sample buffer pH value for 5 kinds of sheet base
The phosphate buffer solution containing 3% glycerol with pH value of 6.0,7.2,8.5 and 9.6 carbonate buffer solution with pH value, 1:4 diluted CD monoclonal antibody, on the 5 kinds of substrate, the antibody lattice, hydration, sealing and cleaning were taken to extract the peripheral blood 2ml from fresh normal people, and the leukocyte (1 x 10~6/ml) was extracted with the self matched total white cell separation solution and dyed orange staining. Clean and incubate the chip for 45 minutes. PBS clean to clean background and scan. Analyze the best pH buffer value of different substrate.
3, determine the saturation concentration of 5 kinds of fragment fixed antibodies
The CD McAbs with the original concentration of 200 mu g/ml were diluted by 1:2,1:4,1:8,1:16,1:32,1:64 respectively, and on the 5 substrates, the order samples were reduced by concentration, and the FITC labeled Sheep anti rat IgG solution (100 micron g/ml) was incubated for 30 minutes, and the saturation concentration of the different fragment fixed antibodies was analyzed.
4, determine the best time for 5 sheet based immobilized antibodies
CD McAbs were immobilized on 5 substrates, respectively, in wet boxes at 4 degrees centigrade for 0.5,1,2,4,12,24,48 hours, and incubated with FITC labeled IgG solution, and the optimum time for different fragment fixed antibodies was analyzed.
5, detection of protein binding ability of 5 kinds of sheet
FITC labeled fluorescent antibodies were fixed on the 5 substrates. The mean fluorescence intensity of different substrates and the variation rate of fluorescence intensity at different locations were compared.
6, to detect the protein activity and the effect of storage time on the protein activity of 5 tablets.
After 1 days, 1 months, 3 months and 6 months, the activity of protein was detected by fluorescent labelled antibody and fluorescent labeled cells respectively.
Result
1, different methods of modified glass and polystyrene tablets were the best protein immobilized when the sample buffer pH was 7.2,9.6.
2, when the antibody dilution ratio reaches 1:4, that is, when the antibody concentration reaches 50 mu g/ml, 5 kinds of antibody immobilized on the substrate are saturated.
3, in the wet box, the immobilization time is better than 4 hours at 4 OC.
4, amino silane aldehyde glass slide protein has the highest fixation efficiency and good uniformity, and the fluorescence intensity variation rate of different parts is 5%.
5, the activity of antibody immobilized on amino silane aldehyde glass was the highest. With the prolongation of storage time, the activity of antibody decreased the slowest and only slight attenuation at 6 months.
conclusion
Amino silane aldehyde based glass slides with a pH value of 7.2 phosphate buffer solution as point sample buffer, fixed protein concentration of 50 mu and fixed time for more than 12 hours, have the best reactivity to protein fixation efficiency and fixed protein, and obviously better than other tablets, which can be used as the preparation of leukemia immunological type cell chip. The best carrier.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329;R733.7

【参考文献】