Gonzalez Rachel M, Seurynck-Servoss Shannon L, Crowley Sheila A, Brown Marty, Omenn Gilbert S, Hayes Daniel F, Zangar Richard C
Pacific Northwest National Laboratory, 902 Battelle Boulevard, P7-56, Richland, Washington 99354, USA.
J Proteome Res. 2008 Jun;7(6):2406-14. doi: 10.1021/pr700822t. Epub 2008 Apr 19.
Sandwich enzyme-linked immunosorbent assay (ELISA) microarrays are emerging as a strong candidate platform for multiplex biomarker analysis because of the ELISA's ability to quantitatively measure rare proteins in complex biological fluids. Advantages of this platform are high-throughput potential, assay sensitivity and stringency, and the similarity to the standard ELISA test, which facilitates assay transfer from a research setting to a clinical laboratory. However, a major concern with the multiplexing of ELISAs is maintaining high assay specificity. In this study, we systematically determine the amount of assay interference and noise contributed by individual components of a multiplexed 24-assay system. We find that nonspecific reagent cross-reactivity problems are relatively rare. We did identify the presence of contaminant antigens in a "purified antigen". We tested the validated ELISA microarray chip using paired serum samples that had been collected from four women at a 6-month interval. This analysis demonstrated that protein levels typically vary much more between individuals than within an individual over time, a result which suggests that longitudinal studies may be useful in controlling for biomarker variability across a population. Overall, this research demonstrates the importance of a stringent screening protocol and the value of optimizing the antibody and antigen concentrations when designing chips for ELISA microarrays.
夹心酶联免疫吸附测定（ELISA）微阵列正成为多重生物标志物分析的有力候选平台，因为ELISA能够定量检测复杂生物流体中的稀有蛋白质。该平台的优势在于高通量潜力、检测灵敏度和严格性，以及与标准ELISA检测的相似性，这便于检测方法从研究环境转移到临床实验室。然而，ELISA多重检测的一个主要问题是保持高检测特异性。在本研究中，我们系统地确定了一个24重检测系统中各个组件造成的检测干扰和噪声量。我们发现非特异性试剂交叉反应问题相对较少。我们确实在一种“纯化抗原”中鉴定出了污染性抗原的存在。我们使用从四名女性身上每隔6个月采集的配对血清样本对经过验证的ELISA微阵列芯片进行了测试。该分析表明，蛋白质水平在个体之间的差异通常远大于个体内部随时间的变化，这一结果表明纵向研究可能有助于控制人群中生物标志物的变异性。总体而言，这项研究证明了严格筛选方案的重要性以及在设计ELISA微阵列芯片时优化抗体和抗原浓度的价值。
