Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions.
Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions.
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DOI：
10.1186/gb-2001-2-2-research0004
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发表时间：
2001
期刊：
影响因子：
12.3
通讯作者：
Brown PO
中科院分区：
文献类型：
--
作者：
Haab BB;Dunham MJ;Brown PO
我们已经开发并测试了一种打印蛋白质微阵列的方法，并在比较荧光测定中使用这些微阵列来测量复杂溶液中许多特定蛋白质的丰度。使用机器人装置在衍生化的显微镜载玻片表面上以阵列形式打印数百种特异性抗体或抗原溶液。两个复杂的蛋白质样品，一个作为比较定量的标准品，另一个代表实验样品，其中的蛋白质量进行测量，通过共价连接的光谱可分辨的荧光染料进行标记。特异性抗体-抗原相互作用将复杂混合物的特异性组分定位于阵列中限定的同源点，其中代表实验样品和参考标准的荧光信号的相对强度提供了实验样品中每种蛋白质丰度的量度。为了测试该测定的特异性、灵敏度和准确性，我们分析了115个抗体/抗原对的性能。50%的阵列抗原和20%的阵列抗体分别在或低于0.34 μg/ml和1.6 μg/ml的浓度下提供了其同源配体的特异性和准确测量。一些抗体/抗原对允许在低于1 ng/ml的绝对浓度和1/106的部分浓度下检测同源配体，灵敏度足以测量患者血液样品中的许多临床重要蛋白质。这些结果表明，蛋白质微阵列可以提供一种实用的手段来表征在临床或研究应用中的数十万种不同蛋白质的变异模式。
We have developed and tested a method for printing protein microarrays and using these microarrays in a comparative fluorescence assay to measure the abundance of many specific proteins in complex solutions. A robotic device was used to print hundreds of specific antibody or antigen solutions in an array on the surface of derivatized microscope slides. Two complex protein samples, one serving as a standard for comparative quantitation, the other representing an experimental sample in which the protein quantities were to be measured, were labeled by covalent attachment of spectrally resolvable fluorescent dyes. Specific antibody-antigen interactions localized specific components of the complex mixtures to defined cognate spots in the array, where the relative intensity of the fluorescent signal representing the experimental sample and the reference standard provided a measure of each protein's abundance in the experimental sample. To test the specificity, sensitivity and accuracy of this assay, we analyzed the performance of 115 antibody/antigen pairs. 50% of the arrayed antigens and 20% of the arrayed antibodies provided specific and accurate measurements of their cognate ligands at or below concentrations of 0.34 μg/ml and 1.6 μg/ml, respectively. Some of the antibody/antigen pairs allowed detection of the cognate ligands at absolute concentrations below 1 ng/ml, and partial concentrations of 1 part in 106, sensitivities sufficient for measurement of many clinically important proteins in patient blood samples. These results suggest that protein microarrays can provide a practical means to characterize patterns of variation in hundreds of thousands of different proteins in clinical or research applications.