Department of Chemical and Biomolecular Engineering, University of Connecticut, Storrs, CT, 06269, USA.
Department of Biomedical Engineering, University of Connecticut, Storrs, CT, 06269, USA.
Anal Bioanal Chem. 2021 Feb;413(5):1493-1502. doi: 10.1007/s00216-020-03120-7. Epub 2021 Jan 19.
The study of protein-solid surface binding as well as blocking efficiency of blocking agents plays an important role in the development of high-performance immunoassays. Although conventional colorimetric based assays are widely employed to monitor protein non-specific binding on the surface of microplate wells and evaluate the performance of blocking agents, there is still a great need to develop new methods to achieve the same goal from a new perspective. In this study, an innovative whole area scanning (WAS)-enabled direct-counting strategy was developed and validated through studying the blocking efficiency of different blocking agents on the non-specific binding of streptavidin-alkaline phosphatase conjugate (Strep-ALP, a model protein) to the surface of 96-well microplates. After non-specific binding of Strep-ALP in wells with or without blocking agents' treatment and loading of ELF™ 97 phosphate (ELFP), ALP in Strep-ALP conjugates converts ELFP to water-insoluble ELF™ 97 alcohol (ELFA), which precipitates locally, self-assembles into large needle structures, and glows green fluorescence upon excitation. After quenching the reaction, WAS of the whole wells allows us to directly count the number of individual fluorescent precipitates, which can be used to calculate and compare the blocking efficiency of three commonly used blocking agents (BSA, casein, and dry milk) based on mitigating the non-specific binding of Strep-ALP. WAS-enabled counting of individual needle-type precipitates opens a new avenue to investigate protein-solid surface binding as well as the efficiency of blocking agents with high sensitivity.
研究蛋白质-固体表面结合以及封闭剂的封闭效率在高性能免疫分析的发展中起着重要作用。尽管基于传统比色法的测定法广泛用于监测微板孔表面上蛋白质的非特异性结合并评估封闭剂的性能，但仍需要开发新的方法从新的角度来实现相同的目标。在这项研究中，通过研究不同封闭剂对链霉亲和素-碱性磷酸酶缀合物（Strep-ALP，一种模型蛋白）与 96 孔微板表面非特异性结合的封闭效率，开发并验证了一种创新的全区域扫描（WAS）启用的直接计数策略。在有或没有封闭剂处理和加载 ELF™ 97 磷酸盐（ELFP）的情况下，Strep-ALP 在孔中非特异性结合后，Strep-ALP 缀合物中的 ALP 将 ELFP 转化为不溶于水的 ELF™ 97 醇（ELFA），ELFA 局部沉淀、自组装成大针状结构，并在激发时发出绿色荧光。反应淬灭后，整个孔的 WAS 允许我们直接计数单个荧光沉淀物的数量，这可以用于基于减轻 Strep-ALP 的非特异性结合来计算和比较三种常用封闭剂（BSA、酪蛋白和干奶）的封闭效率。WAS 启用的单个针状沉淀物的计数为研究蛋白质-固体表面结合以及封闭剂的效率提供了一种新的途径，具有高灵敏度。
