血清载脂蛋白B椭偏光蛋白质芯片技术定量检测的条件优化  

Optimization of ellipsometry protein-chip technique in quantitative detection of serum apolipoprotein B

作　　者：宁翔[1] 王娇娇[2] 王娟[1] 毕少杰[1] 盛林[1] NING Xiang WANG Jiaojiao WANG Juan BI Shaojie SHENG Lin(The Second Hospital of Shandong University, Jinan 250033, China)

机构地区：[1]山东大学第二医院,济南250033 [2]山东大学齐鲁医院

出　　处：《山东医药》2016年第47期17-20,共4页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(81500232);山东省医药卫生科技发展计划项目(2014WS0155);山东省自然科学基金-联合专项资助项目(2014ZRE27456)

摘　　要：目的优化椭偏光蛋白芯片技术定量检测血清载脂蛋白B(ApoB)的条件,并观察最佳条件下椭偏光蛋白芯片技术定量检测血清ApoB的准确性。方法用椭偏光蛋白芯片技术检测含有0.2、0.3、0.4、0.5、0.6 mg/m L的载脂蛋白B抗体(anti ApoB)和200μg/m L ApoB标准品的溶液,测得灰度值最高者选为最佳anti-ApoB浓度。用椭偏光蛋白芯片技术检测5、10、20、50、100、200μg/m L ApoB标准品溶液,anti-ApoB浓度为0.3 mg/m L,结果以灰度值表示。然后在上述标准品溶液中加入蛋白G使其终浓度为0.1 mg/m L,再检测一次灰度值。比较两次检测的灰度值,确定是否应该加入0.1 mg/m L蛋白G。最佳条件下椭偏光蛋白芯片技术检测0、5、10、20、50、100、200μg/m L ApoB标准品溶液,计算上述ApoB标准品溶液灰度值与不含ApoB的空白探针溶液(含anti ApoB和蛋白G)灰度值的差值。对上述ApoB标准品溶液浓度和灰度值差值的相关性进行分析,以ApoB标准品溶液浓度为横坐标,灰度值差值为纵坐标描画标准曲线并建立方程。最佳条件下用椭偏光蛋白芯片技术检测16份40倍稀释的已知ApoB浓度(采用免疫比浊法测定)血清的灰度值两次。用上述方程计算16份血清中ApoB浓度,并与其实际ApoB浓度比较。结果最佳anti-ApoB浓度为0.3 mg/m L。与不应用蛋白G者相比,应用0.1 mg/m L蛋白G的椭偏光蛋白芯片技术测得样品的灰度值高(P均<0.05)。以ApoB浓度为横坐标x、灰度差值为纵坐标y描画标准曲线,Maple9.5软件得出的回归方程为:y=-23.116Ln(1-x/60),R2=0.936 4。16份血清ApoB浓度和第一次、第二次用椭偏光蛋白芯片技术检测测得血清ApoB浓度相比P均>0.05。结论椭偏光蛋白芯片技术检测血清ApoB的最佳条件为anti-ApoB浓度0.3 mg/m L,蛋白G浓度0.1 mg/m L。此条件下椭偏光蛋白芯片技术检测血清ApoB结果准确。Objective To optimize the conditions for quantitatively detecting human serum apolipoprotein B via ellip-sometry protein-chip technique and to evaluate the accuracy of this technique under optimum conditions.Methods In our study,we detected the gray values of solutions containing 0.2,0.3,0.4,0.5 and 0.6 mg/mL anti-ApoB and 200 μg/mL ApoB with ellipsometry protein-chip technique,and the optimum concentration was the one with the highest gray value.We detected the gray values of solutions containing 0.3 mg/mL anti-ApoB and 5,10,20,50,100,200 μg/mL ApoB before and after protein G was added in those solutions until its final concentration was 0.1 mg/mL.By comparing the grey val-ues,we determined whether protein G should be added or not.We detected the grey values of solutions containing 0,5, 10,20,50,100,200 μg/mL ApoB via ellipsometry protein-chip technique under optimum conditions and calculated the difference between them and the gray values of the solutions without ApoB （consisted of anti-ApoB and protein G）.Then we analyzed the correlations between the concentrations and the differences by drawing the curve and establishing the equa-tion of it with concentration as X-axis and gray value as Y-axis.At last,we detected the gray values of 16 serum samples diluted 40 times twice through ellipsometry protein-chip technique and immunoturbidimetry under the optimum conditions. Then we calculated the concentrations of ApoB in the samples with the equation and compared them with its practical con-centration.Results The optimum concentration of anti-ApoB was 0.3 mg/mL.The gray values in protein G group were higher than that of the contrast group （all P〈0.05）.The regression equation was y=-23.116Ln（1 -x/60）,R2 =0.936 4 by Maple 9.5 software according to the curve.No significantly statistical difference was found between the ApoB concentra-tions detected via ellipsometry protein-chip technique and 16 cases of serum apoB concernttations （all P〉0.05）.Conclu-sions The optimal conditions for detection of A

关 键 词：椭偏光蛋白芯片技术 光学蛋白质芯片 椭偏光生物显微成像技术 载脂蛋白B 载脂蛋白B抗体 蛋白G 

分 类 号：R446.11[医药卫生—诊断学]