Jung Jae-Wan, Jung Se-Hui, Yoo Je-Ok, Suh In-Bum, Kim Young-Myeong, Ha Kwon-Soo
Department of Molecular and Cellular Biochemistry and Vascular System Research Center, Chuncheon, Kangwon-Do 200-701, South Korea.
Biosens Bioelectron. 2009 Jan 1;24(5):1469-73. doi: 10.1016/j.bios.2008.08.048. Epub 2008 Sep 6.
We have developed a new, high-throughput, competition-based tagged-internal standard (TIS) assay to measure the levels of blood proteins in human serum. In this assay, target proteins in the sample serum compete with tagged-internal standard proteins for binding to an antibody array. Antibody arrays are fabricated by immobilizing a target protein-specific antibody on the carboxylate-modified latex bead surface of well-type arrays. A solution of Alexa 546-conjugated target protein is added to a sample of human serum and applied to the well-type antibody array. The array is then analyzed with a fluorescence scanner and the level of unlabeled target protein in the human sera is inferred from the amount of tagged protein bound to the array. We successfully applied this assay to measure the level of C-reactive protein (CRP) in 92 unlabeled human sera. The TIS assay was found to be specific and reproducible for the quantitative analysis of CRP. The antibody array data from the TIS assay correlate well with clinical laboratory data obtained using the commercialized latex-enhanced turbidimetry immunoassay (n=3, r=0.967, CV=0.32%). Thus, the antibody array-based TIS assay system is high-throughput, quantitative, and label-free and may be useful in the rapid serodiagnosis of human disease.
我们开发了一种新的、基于竞争的高通量标记内标（TIS）检测方法，用于测定人血清中的血液蛋白水平。在该检测方法中，样品血清中的目标蛋白与标记内标蛋白竞争结合抗体阵列。抗体阵列是通过将目标蛋白特异性抗体固定在孔型阵列的羧酸盐修饰乳胶珠表面制备而成。将Alexa 546偶联的目标蛋白溶液加入人血清样品中，并应用于孔型抗体阵列。然后用荧光扫描仪分析该阵列，并根据与阵列结合的标记蛋白量推断人血清中未标记目标蛋白的水平。我们成功地应用该检测方法测定了92份未标记人血清中的C反应蛋白（CRP）水平。发现TIS检测方法对CRP的定量分析具有特异性和可重复性。TIS检测方法的抗体阵列数据与使用商业化乳胶增强比浊免疫测定法获得的临床实验室数据相关性良好（n = 3，r = 0.967，CV = 0.32%）。因此，基于抗体阵列的TIS检测系统具有高通量、定量和无标记的特点，可能有助于人类疾病的快速血清学诊断。
