Qin Shuzhen, Qiu Weiliang, Ehrlich Joshua R, Ferdinand Angeline S, Richie Jerome P, O'leary Michael P, Lee Mei-Ling Ting, Liu Brian C-S
Molecular Urology Laboratory, Division of Urology, Brigham and Women's Hospital, Harvard Medical School, 221 Longwood Avenue, Boston, MA 02115, USA.
Proteomics. 2006 May;6(10):3199-209. doi: 10.1002/pmic.200500673.
Diagnosing cancers based on serum profiling is a particularly attractive concept. However, the technical challenges to analysis of the serum proteome arise from the dynamic range of protein amounts. Cancer sera contain antibodies that react with a unique group of autologous cellular antigens, which affords a dramatic amplification of signal in the form of antibodies relative to the amount of the corresponding antigens. The serum autoantibody repertoire from cancer patients might, therefore, be exploited for antigen-antibody profiling. To date, studies of antigen-antibody reactivity using microarrays have relied on recombinant proteins or synthetic peptides as arrayed features. However, recombinant proteins and/or synthetic peptides may fail to accurately detect autoantibody binding due to the lack of proper PTMs. Here we describe the development and use of a "reverse capture" autoantibody microarray. Our "reverse capture" autoantibody microarray is based on the dual-antibody sandwich immunoassay platform of ELISA, which allows the antigens to be immobilized in their native configuration. As "proof-of-principle", we demonstrate its use for antigen-autoantibody profiling with sera from patients with prostate cancer and benign prostate hyperplasia.
基于血清谱诊断癌症是一个特别有吸引力的概念。然而，血清蛋白质组分析的技术挑战源于蛋白质含量的动态范围。癌症血清中含有与一组独特的自体细胞抗原发生反应的抗体，相对于相应抗原的量，这使得抗体形式的信号显著放大。因此，癌症患者的血清自身抗体库可用于抗原-抗体谱分析。迄今为止，使用微阵列对抗原-抗体反应性的研究依赖于重组蛋白或合成肽作为阵列特征。然而，由于缺乏适当的翻译后修饰，重组蛋白和/或合成肽可能无法准确检测自身抗体结合。在此，我们描述了一种“反向捕获”自身抗体微阵列的开发和应用。我们的“反向捕获”自身抗体微阵列基于ELISA的双抗体夹心免疫测定平台，该平台允许抗原以其天然构象固定。作为“原理验证”，我们展示了其用于前列腺癌和良性前列腺增生患者血清的抗原-自身抗体谱分析。
