Molecule by molecule direct and quantitative counting of antibody-protein complexes in solution
Molecule by molecule direct and quantitative counting of antibody-protein complexes in solution
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DOI：
10.1021/ac049512c
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发表时间：
2004-08-01
影响因子：
7.4
通讯作者：
Klenerman, D
中科院分区：
文献类型：
--
作者：
Li, HT;Zhou, DJ;Klenerman, D
我们使用双色荧光符合检测来直接计数用一种或多种红色和蓝色激发抗体标记的蛋白 G 或单纯疱疹病毒的单个蛋白-抗体复合物。这使得可以对蛋白质-抗体复合物的浓度进行定量测量，其浓度降低了三个数量级，低至飞摩尔水平。稀释血清中的单分子测量也是可能的。样品制备简单，在溶液中进行，无需分离。抗体亲和力和复合物解离速率对于确定方法的灵敏度都很重要。目前，50 M 的灵敏度极限是由标记分析物与探针体积的相遇率决定的。该方法可用于检测和定量蛋白质，并测量低浓度下蛋白质-蛋白质复合物的化学计量、平衡常数和解离率。
We have used two-color fluorescence coincidence detection to directly count individual protein-antibody complexes of protein G or herpes simplex virus labeled with one or more red- and blue-excited antibodies. This allowed quantitative measurement of the concentration of the protein-antibody complexes over 3 orders of magnitude down to the femtomolar level. Single molecule measurements in diluted serum are also possible. The sample preparation is simple, takes place in solution, and requires no separation. Both the antibody affinity and complex dissociation rate are important in determining the sensitivity of the method. At present, the sensitivity limit of 50 M is determined by the encounter rate of the labeled analyte with the probe volume. This method can be used to detect and quantitate proteins and to measure the stoichiometry, equilibrium constant, and dissociation rate of protein-protein complexes at low concentrations.