‡Department of Chemistry, Penn State University, University Park, Pennsylvania 16802, United States.
§Department of Biochemistry and Molecular Biology, Penn State University, University Park, Pennsylvania 16802, United States.
Anal Chem. 2015 Jul 21;87(14):7163-70. doi: 10.1021/acs.analchem.5b00999. Epub 2015 Jun 30.
Herein, we developed a new separation-based detection method that is capable of simultaneously identifying multiple competitively binding proteins for the same ligand on supported lipid bilayers (SLBs). This strategy used unlabeled target analyte proteins that bind to fluorescently tagged, lipid-conjugated ligands within the SLB. The protein-ligand binding complexes were then focused under an applied potential to different locations within the SLB based on each protein's size and charge. Both protein identity and relative surface concentration information could be obtained, simultaneously. Specifically, the competitive binding of streptavidin and goat anti-biotin for biotin-conjugated lipids was explored. It was found that streptavidin could inhibit the binding of goat anti-biotin antibodies for biotin-cap-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)(biotin-cap-NBD-PE) lipids and that streptavidin more effectively outcompeted the anti-biotin antibody at lower protein concentrations. Also, modulating the chemical composition of the membrane helped control the ultimate focusing position and separation of the streptavidin-bound biotin, anti-biotin-bound biotin, and free biotin-conjugated lipid bands. The assay developed herein provides a simple and convenient strategy for simultaneously monitoring target analytes that bind to the identical ligand and may ultimately be useful in developing assays that help overcome problems associated with cross-reactivity.
在此，我们开发了一种新的基于分离的检测方法，能够同时识别同一配体在支持脂质双层（SLB）上结合的多种竞争结合蛋白。该策略使用未标记的靶标分析物蛋白，这些蛋白与 SLB 内荧光标记的脂质缀合配体结合。然后，根据每种蛋白的大小和电荷，将蛋白-配体结合复合物在施加的电势下聚焦到 SLB 内的不同位置。可以同时获得蛋白的身份和相对表面浓度信息。具体来说，研究了链霉亲和素和山羊抗生物素与生物素缀合脂质的竞争性结合。结果发现，链霉亲和素可以抑制生物素帽-1,2-二棕榈酰-sn-甘油-3-磷酸乙醇胺-N-（7-硝基-2-1,3-苯并恶二唑-4-基）（生物素帽-NBD-PE）脂质上的山羊抗生物素抗体与生物素的结合，并且在较低的蛋白浓度下，链霉亲和素更有效地与抗生物素抗体竞争。此外，调节膜的化学组成有助于控制链霉亲和素结合的生物素、抗生物素结合的生物素和游离生物素缀合脂质带的最终聚焦位置和分离。本文开发的测定方法为同时监测与相同配体结合的靶标分析物提供了一种简单方便的策略，最终可能有助于开发有助于克服交叉反应性相关问题的测定方法。
