本文選題：細(xì)胞芯片 + 載體��； 參考：《中國醫(yī)科大學(xué)》2007年碩士論文

【摘要】： 目的 隨著大量抗白細(xì)胞表面分化抗原單克隆抗體的問世，應(yīng)用其來檢測白血病細(xì)胞表面分化抗原，以確定其細(xì)胞系列來源及發(fā)育階段，推動了白血病免疫學(xué)分型的研究，提高了白血病診斷的準(zhǔn)確性。目前常用的進(jìn)行白血病免疫學(xué)分型的方法有免疫組化法、免疫熒光法，但這兩種方法存在很多缺陷。前者不能同時進(jìn)行多種抗原檢測；檢測一種抗原需要的單克隆抗體量較多，不經(jīng)濟(jì)；處理步驟多，費(fèi)時。后者同時檢測抗原種類有限；檢測所需的熒光顯微鏡或流式細(xì)胞儀設(shè)備昂貴。對細(xì)胞高通量研究的要求與芯片技術(shù)的結(jié)合使細(xì)胞芯片應(yīng)運(yùn)而生，這種新型的檢測技術(shù)平臺的出現(xiàn)，克服了上述方法的缺陷，使白血病的免疫學(xué)分型高通量診斷更快捷、方便。 本實(shí)驗(yàn)室構(gòu)建的用于白血病免疫學(xué)分型的細(xì)胞芯片就是利用抗原、抗體特異性結(jié)合的原理，將白細(xì)胞表面分化抗原單克隆抗體固定于載體上，形成抗體點(diǎn)陣。不同的單克隆抗體能夠自動識別和捕獲表達(dá)相應(yīng)表面分化抗原的細(xì)胞，從而對白血病進(jìn)行免疫學(xué)分型。 載體選擇是芯片制備過程中的第一個關(guān)鍵步驟。選擇何種載體及如何修飾才能使單克隆抗體更牢固、更特異性地固定在載體表面，同時使固相支持物表面的蛋白保持最大的活性，是影響該技術(shù)檢測成功率的關(guān)鍵。本實(shí)驗(yàn)欲探索制備細(xì)胞芯片的最佳載體及載體的最佳修飾方法。 方法 1、制備5種片基 分別對玻片進(jìn)行氨基硅烷-醛基修飾、殼聚糖-氨基修飾、殼聚糖-醛基修飾、巰基修飾；聚苯乙烯板蒸餾水超聲清洗備用。 2、確定5種片基的最佳點(diǎn)樣緩沖液pH值 分別用含3％甘油的pH值為6.0、7.2、8.5的磷酸鹽緩沖液和pH值為9.6的碳酸鹽緩沖液，1：4稀釋CD單抗，在5種片基上點(diǎn)制抗體點(diǎn)陣，水化、封閉、清洗。取新鮮正常人的外周血2ml，用本實(shí)驗(yàn)室自配的全白細(xì)胞分離液提取白細(xì)胞(1×10~6/ml)，丫叮橙染色、清洗，孵育芯片45分鐘，PBS清洗至背景干凈，，掃描，分析不同片基的最佳點(diǎn)樣緩沖液pH值。 3、確定5種片基固定抗體的飽和濃度 將原濃度是200μg/ml的CD單抗分別按1：2、1：4、1：8、1：16、1：32、1：64稀釋后，在5種片基上按濃度下降的次序點(diǎn)樣，F(xiàn)ITC標(biāo)記的羊抗鼠IgG溶液(濃度100μg/ml)孵育30分鐘，分析不同片基固定抗體的飽和濃度。 4、確定5種片基固定抗體的最佳時間 在5種片基上固定CD單抗，分別在濕盒中4℃水化0.5、1、2、4、12、24、48小時，F(xiàn)ITC標(biāo)記IgG溶液孵育，分析不同片基固定抗體的最佳時間。 5、檢測5種片基的蛋白結(jié)合力 在5種片基上固定FITC標(biāo)記的熒光抗體，比較不同片基的平均熒光信號強(qiáng)度及不同部位熒光信號強(qiáng)度的變異率。 6、檢測5種片基固定的蛋白活性及保存時間對蛋白活性的影響 在芯片制備后1天、1個月、3個月和6個月，分別用熒光標(biāo)記抗體和熒光標(biāo)記細(xì)胞來檢測蛋白的活性。 結(jié)果 1、不同方法修飾的玻片和聚苯乙烯片基分別在點(diǎn)樣緩沖液pH值為7.2、9.6時，蛋白固定效果最好。 2、抗體稀釋比例達(dá)到1：4時，即抗體濃度達(dá)到50μg/ml時，5種片基固定的抗體均達(dá)到飽和濃度。 3、在濕盒中4℃條件下固定時間超過12小時，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，均勻度好，不同部位的熒光信號強(qiáng)度變異率5％。 5、氨基硅烷-醛基玻片固定的抗體活性最高，隨著保存時間的延長抗體活性衰減最慢，保存6個月時僅有輕微的衰減。 結(jié)論 氨基硅烷-醛基玻片在以pH值為7.2的磷酸鹽緩沖液作為點(diǎn)樣緩沖液，固定蛋白的濃度為50μg/ml，固定時間大于12小時的條件下，對蛋白的固定效率及固定蛋白的反應(yīng)性最好，且明顯優(yōu)于其他片基，可做為制備白血病免疫學(xué)分型細(xì)胞芯片的最佳載體。
[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.
【學(xué)位授予單位】：中國醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R329;R733.7

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