Single molecule enzyme-linked immunosorbent assays: theoretical considerations.
Single molecule enzyme-linked immunosorbent assays: theoretical considerations.
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DOI：
10.1016/j.jim.2012.02.011
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发表时间：
2012-04-30
影响因子：
2.2
通讯作者：
Duffy DC
中科院分区：
文献类型：
--
作者：
Chang L;Rissin DM;Fournier DR;Piech T;Patel PP;Wilson DH;Duffy DC
我们开发了一种高度灵敏的免疫测定法（称为数字 ELISA），它基于对密封在飞升孔阵列中的珠子上的单一酶联免疫复合物的检测。数字 ELISA 旨在高效捕获目标蛋白、标记这些蛋白以及在单分子阵列 (SiMoA) 中检测它们；本质上，检测的目标是“捕获每一个分子，检测每一个分子”。在这里，我们为该测定的设计提供了理论基础，该测定源自基于双分子相互作用的简单方程。利用这些方程和试剂浓度、相互作用时间以及测定每个步骤的分子相互作用的开启和解离速率的知识，可以预测 SiMoA 形成和检测的免疫复合物的数量。 SiMoA 具有计算单个免疫复合物并确定每个珠子平均酶数 (AEB) 的独特能力，使得可以直接将实验检测到的分子数与理论预测的分子数进行比较。这些预测与前列腺特异性抗原 (PSA) 数字 ELISA 生成的实验数据相当。数字 ELISA 过程在一系列抗体亲和力 (KD ~ 10−11–10−9 M) 中都是有效的，并且预计具有高结合率 (kon > 105 M−1 s−1) 的抗体表现最佳。数字 ELISA 的高效率和 SiMoA 对酶标记的敏感性也使得降低标记试剂的浓度、降低背景并提高方法的特异性成为可能。还描述了处理抗体复合物随时间解离的策略，该解离可能影响测定中的信号。
We have developed a highly sensitive immunoassay—called digital ELISA—that is based on the detection of single enzyme-linked immunocomplexes on beads that are sealed in arrays of femtoliter wells. Digital ELISA was designed to be highly efficient in the capturing of target proteins, labeling of these proteins, and their detection in single molecule arrays (SiMoA); in essence, the goal of the assay is to “capture every molecule, detect every molecule”. Here we provide the theoretical basis for the design of this assay derived from simple equations based on bimolecular interactions. Using these equations and knowledge of the concentrations of reagents, the times of interactions, and the on- and off-rates of the molecular interactions for each step of the assay, it is possible to predict the number of immunocomplexes that are formed and detected by SiMoA. The unique ability of SiMoA to count single immunocomplexes and determine an average number of enzymes per bead (AEB), makes it possible to directly compare the number of molecules detected experimentally to those predicted by theory. These predictions compare favorably to experimental data generated for a digital ELISA for prostate specific antigen (PSA). The digital ELISA process is efficient across a range of antibody affinities (KD ~ 10−11–10−9 M), and antibodies with high on-rates (kon > 105 M−1 s−1) are predicted to perform best. The high efficiency of digital ELISA and sensitivity of SiMoA to enzyme label also makes it possible to reduce the concentration of labeling reagent, reduce backgrounds, and increasing the specificity of the approach. Strategies for dealing with the dissociation of antibody complexes over time that can affect the signals in an assay are also described.
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影响因子：
4.5
作者：
Lepor, Herbert;Cheli, Carol D.;Partin, Alan W.
通讯作者：
Partin, Alan W.
影响因子：
8.8
作者：
Miedouge, Marcel;Greze, Monique;Izopet, Jacques
通讯作者：
Izopet, Jacques
影响因子：
2.2
作者：
Song, Linan;Hanlon, David W.;Duffy, David C.
通讯作者：
Duffy, David C.
DOI：
10.1039/c0an00204f
发表时间：
2010-10
期刊：
The Analyst
影响因子：
--
作者：
Rusling JF;Kumar CV;Gutkind JS;Patel V
通讯作者：
Patel V
影响因子：
9.3
作者：
Wilson, David H.;Hanlon, David W.;Duffy, David C.
通讯作者：
Duffy, David C.