Multiplexed protein profiling on microarrays by rolling-circle amplification
Multiplexed protein profiling on microarrays by rolling-circle amplification
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DOI：
10.1038/nbt0402-359
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发表时间：
2002-04-01
影响因子：
46.9
通讯作者：
Kingsmore, SF
中科院分区：
文献类型：
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作者：
Schweitzer, B;Roberts, S;Kingsmore, SF
微阵列上的荧光夹心免疫分析对蛋白质组学研究具有吸引力，因为设备和抗体随时可用，而且分析简单、可扩展和可重复性。然而，要获得足够的灵敏度和特异度，需要一种通用的免疫分析扩增方法。为此，我们描述了等温滚环扩增(RCA)与通用抗体的偶联。采用RCA信号放大技术，在玻璃阵列上同时检测75种细胞因子，具有高特异性、高灵敏度、3对数定量范围、样品消耗经济等特点。用51个特征的RCA细胞因子玻璃阵列检测内毒素(LPS)或肿瘤坏死因子-α(TNF-α)诱导的人树突状细胞(DC)的分泌。正如预期的那样，内毒素诱导炎症细胞因子的快速分泌，如巨噬细胞炎症蛋白(MIP)-1β、白细胞介素8(IL)-8和干扰素诱导蛋白(IP)-10。我们发现Eoaxin-2和I-309由内毒素诱导，另外，巨噬细胞衍生的趋化因子(MDC)、胸腺和活化调节的趋化因子(TARC)、可溶性白介素6受体(sIL-6R)和可溶性肿瘤坏死因子受体I(sTNF-RI)由肿瘤坏死因子-α(TNF-α)诱导。由于微阵列可以容纳1000个这种类型的夹心免疫分析，相对少量的RCA微阵列似乎为蛋白质组研究提供了一种容易处理的方法。
Fluorescent-sandwich immunoassays on microarrays hold appeal for proteomics studies, because equipment and antibodies are readily available, and assays are simple, scalable, and reproducible. The achievement of adequate sensitivity and specificity, however, requires a general method of immunoassay amplification. We describe coupling of isothermal rolling-circle amplification (RCA) to universal antibodies for this purpose. A total of 75 cytokines were measured simultaneously on glass arrays with signal amplification by RCA with high specificity, femtomolar sensitivity, 3 log quantitative range, and economy of sample consumption. A 51-feature RCA cytokine glass array was used to measure secretion from human dendritic cells (DCs) induced by lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-alpha). As expected, LPS induced rapid secretion of inflammatory cytokines such as macrophage inflammatory protein (MIP)-1beta, interleukin (IL)-8, and interferon-inducible protein (IP)-10. We found that eotaxin-2 and I-309 were induced by LPS; in addition, macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC), soluble interleukin 6 receptor (sIL-6R), and soluble tumor necrosis factor receptor I (sTNF-RI) were induced by TNF-alpha treatment. Because microarrays can accommodate 1,000 sandwich immunoassays of this type, a relatively small number of RCA microarrays seem to offer a tractable approach for proteomic surveys.