Evaluation of compound interference in immobilized metal ion affinity-based fluorescence polarization detection with a four million member compound collection
Evaluation of compound interference in immobilized metal ion affinity-based fluorescence polarization detection with a four million member compound collection
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DOI：
10.1089/154065803322163768
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发表时间：
2003-06-01
影响因子：
1.8
通讯作者：
Auld, DS
中科院分区：
文献类型：
--
作者：
Beasley, JR;Dunn, DA;Auld, DS
IMAP（TM）是一种非分离、抗体独立的FP检测方法，可用于多种类型的蛋白激酶和磷酸酶。该技术目前正用于整个行业的许多高通量筛选活动。在该技术中，荧光标记的肽底物被磷酸化，然后被捕获在固定的金属（M-III）纳米颗粒上，这种相互作用在低pH（pH 5.5）下增强。使用FP检测磷酸化肽与纳米颗粒的结合。IMAP与其他FP格式的不同之处在于，极化信号是抗体无关的，并且涉及在低pH下检测到的金属配位络合物。在这里，使用1，536孔测定设计对4，000，000个成员的化合物集合评估该技术，该设计没有酶，因此仅测量化合物对检测系统的干扰。微型化的测定1,536孔板进行了讨论。未观察到由于磷酸肽与M-III纳米颗粒结合的抑制而引起的化合物干扰。此外，可以得出结论，对于大多数化合物集合，荧光化合物干扰水平与典型FP格式相似。
IMAP(TM) is a non-separation-based, antibody-independent, FP assay that can be applied to many types of protein kinases and phosphatases. This technology is currently being used in many high-throughput screening campaigns throughout the industry. In this technology, a fluorescently labeled peptide substrate is phosphorylated and then captured on immobilized metal (M-III) nanoparticles, an interaction that is enhanced at low pH (pH 5.5). The binding of the phosphorylated peptide to the nanoparticles is detected using FP. IMAP differs from other FP formats in that the polarization signal is antibody-independent and involves metal coordination complexes detected at low pH. Here, this technology is evaluated against a 4,000,000-member compound collection using a 1,536-well assay design that is devoid of enzymes so that only interference of the compounds with the detection system is measured. Miniaturization of the assay to 1,536-well plates is discussed. Compound interference due to inhibition of phosphopeptide binding to the M-III nanoparticles is not observed. Additionally, it is concluded that the level of fluorescence compound interference is similar to typical FP formats for the majority of the compound collection.