Goldman E R, Pazirandeh M P, Mauro J M, King K D, Frey J C, Anderson G P
Georgetown University Medical Center, Department of Biochemistry and Molecular Biology, Washington, DC 20007, USA.
J Mol Recognit. 2000 Nov-Dec;13(6):382-7. doi: 10.1002/1099-1352(200011/12)13:6<382::AID-JMR511>3.0.CO;2-W.
This study investigated the potential to utilize phage-displayed peptides as reagents in sensor applications. A library of random 12-mers displayed on phage was panned against staphylococcal enterotoxin B (SEB), a causative agent of food poisoning. Nine SEB binding phage clones were isolated, all of which share the consensus sequence Trp His Lys at their amino terminus. Binding of several of these phage was shown to be inhibited when they were assayed in a competitive enzyme-linked immunosorbent assay (ELISA) format with synthesized peptide corresponding to the peptide-encoding region of one of the clones. Whole phage were labeled with the dye Cy5, and incorporated into fluoroimmunoassays. Labeled phage were able to detect SEB down to a concentration of 1.4 ng/well in a fluorescence-based immunoassay. When incorporated into an automated fluorescence-based sensing assay, Cy5-labeled phage bound to probes coated with SEB generated a robust signal of about 10,000 pA, vs a signal of 1,000 pA using a control fiber coated with streptavidin. These results demonstrate the potential for development of phage-based sensor reagents.
本研究调查了利用噬菌体展示肽作为传感器应用试剂的潜力。针对作为食物中毒病原体的葡萄球菌肠毒素B（SEB），对展示在噬菌体上的随机12聚体文库进行淘选。分离出9个SEB结合噬菌体克隆，它们在氨基末端均具有共有序列Trp His Lys。当在竞争性酶联免疫吸附测定（ELISA）形式中用与其中一个克隆的肽编码区相对应的合成肽进行检测时，这些噬菌体中的几种的结合被证明受到抑制。用染料Cy5标记完整噬菌体，并将其纳入荧光免疫测定中。在基于荧光的免疫测定中，标记的噬菌体能够检测低至1.4 ng/孔浓度的SEB。当将其纳入基于荧光的自动传感测定中时，与使用涂有链霉亲和素的对照纤维时1000 pA的信号相比，与涂有SEB的探针结合的Cy5标记噬菌体产生了约10000 pA的强信号。这些结果证明了开发基于噬菌体的传感器试剂的潜力。
