Aguilar-Mahecha Adriana, Cantin Christiane, O'Connor-McCourt Maureen, Nantel Andre, Basik Mark
Lady Davis Institute for Biomedical Research, Department of Oncology, Montréal, Québec, H3T 1E2, Canada.
Biotechnology Research Institute, National Research Council of Canada, Montréal, Québec, H4P 2R2, Canada.
Proteome Sci. 2009 Apr 6;7:15. doi: 10.1186/1477-5956-7-15.
Many putative disease blood biomarkers discovered in genomic and proteomic studies await validation in large clinically annotated cohorts of patient samples. ELISA assays require large quantities of precious blood samples and are not high-throughput. The reverse phase protein microarray platform has been developed for the high-throughput quantification of protein levels in small amounts of clinical samples.
In the present study we present the development of reverse-phase protein microarrays (RPPMs) for the measurement of clusterin, a mid-abundant blood biomarker. An experimental protocol was optimized for the printing of serum and plasma on RPPMs using epoxy coated microscope slides and a non-denaturing printing buffer. Using fluorescent-tagged secondary antibodies, we achieved the reproducible detection of clusterin in spotted serum and plasma and reached a limit of detection of 780 ng/mL. Validation studies using both spiked clusterin and clinical samples showed excellent correlations with ELISA measurements of clusterin.
Serum and plasma spotted in the reverse phase array format allow for reliable and reproducible high-throughput validation of a mid-abundant blood biomarker such as clusterin.
在基因组和蛋白质组学研究中发现的许多假定的疾病血液生物标志物有待在大量带有临床注释的患者样本队列中进行验证。酶联免疫吸附测定（ELISA）需要大量珍贵的血液样本，且不是高通量的。反相蛋白质微阵列平台已被开发用于对少量临床样本中的蛋白质水平进行高通量定量。
在本研究中，我们展示了用于测量clusterin（一种中等丰度血液生物标志物）的反相蛋白质微阵列（RPPM）的开发。使用环氧涂层显微镜载玻片和非变性打印缓冲液，优化了在RPPM上打印血清和血浆的实验方案。使用荧光标记的二抗，我们在点样的血清和血浆中实现了对clusterin的可重复检测，检测限达到780 ng/mL。使用添加了clusterin的样本和临床样本进行的验证研究表明，与clusterin的ELISA测量结果具有良好的相关性。
以反相阵列形式点样的血清和血浆能够对clusterin这种中等丰度血液生物标志物进行可靠且可重复的高通量验证。
