Abdo Michael, Irving Bob, Hudson Peter, Zola Heddy
CSIRO Division of Health Sciences and Nutrition, 343 Royal Parade, Parkville, Victoria 3052, Australia.
J Immunol Methods. 2005 Oct 20;305(1):3-9. doi: 10.1016/j.jim.2005.07.008. Epub 2005 Aug 18.
Protein microarrays combine aspects of DNA microarrays and ELISA for the parallel interrogation of a biological sample using a multiplex of protein biomarkers. Here we report the development of a protein microarray consisting of a subset of CD antibodies and CRP. Several preparations (culture supernatant, ascites fluid and purified Ig) of each antibody were used in a forward phase protein microarray. Microarrays were fabricated using a non-contact printer delivering 300 pL (+/-30 pL) to specific locations on polyacrylamide gel-based substrates. Following production, microarrays were blocked for non-specific binding and incubated with sera conjugated directly with Cy3. Using CRP as a control biomarker, 12 clinical samples (inflammatory conditions and controls) were interrogated using the protein microarray format and results compared to CRP measured by conventional immunoassay. The data obtained from the microarray correlated with CRP assessed by immunoassay. Subsequently CRP 'positive' samples were interrogated for CD antigen expression; which revealed CD25 and CD45RO expression in all samples. Whilst this study focussed on a subset of CD antibodies, it is anticipated that this array could be expanded to include a larger number of CD antibodies and allow screening of sera from multiple conditions in order to identify disease markers.
蛋白质微阵列结合了DNA微阵列和酶联免疫吸附测定（ELISA）的特点，可利用多种蛋白质生物标志物对生物样品进行平行检测。在此，我们报告了一种由CD抗体和C反应蛋白（CRP）的一个子集组成的蛋白质微阵列的开发情况。在正向蛋白质微阵列中使用了每种抗体的几种制剂（培养上清液、腹水和纯化的免疫球蛋白）。使用非接触式打印机将300皮升（±30皮升）的液体输送到基于聚丙烯酰胺凝胶的底物上的特定位置，从而制作微阵列。制作完成后，对微阵列进行封闭以防止非特异性结合，并与直接用Cy3标记的血清一起孵育。以CRP作为对照生物标志物，使用蛋白质微阵列形式检测了12份临床样品（炎症状态和对照），并将结果与通过传统免疫测定法测得的CRP进行比较。从微阵列获得的数据与通过免疫测定法评估的CRP相关。随后，对CRP “阳性” 的样品检测CD抗原表达；结果显示所有样品中均有CD25和CD45RO表达。虽然本研究聚焦于CD抗体的一个子集，但预计该阵列可扩展以纳入更多的CD抗体，并能够筛选来自多种病症的血清，以便识别疾病标志物。
