Zhao Ning, Spencer John, Schmitt Margaret A, Fisk John D
Department of Chemical and Biological Engineering, Colorado State University, Fort Collins, CO 80523, United States.
Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, United States.
Anal Biochem. 2017 Mar 15;521:59-71. doi: 10.1016/j.ab.2016.12.021. Epub 2017 Jan 3.
Tuberculosis is the leading cause of death from infectious disease worldwide. The low sensitivity, extended processing time, and high expense of current diagnostics are major challenges to the detection and treatment of tuberculosis. Mycobacterium tuberculosis ornithine transcarbamylase (Mtb OTC, Rv1656) has been identified in the urine of patients with active TB infection and is a promising target for point-of-care diagnostics. Specific binding proteins with low nanomolar affinities for Mtb OTC were selected from a phage display library built upon a hyperthermostable Sso7d scaffold. Phage particles displaying Sso7d variants were utilized to generate a sandwich ELISA-based assay for Mtb OTC. The assay response is linear between 2 ng/mL and 125 ng/mL recombinant Mtb OTC and has a limit of detection of 400 pg/mL recombinant Mtb OTC. The assay employing a phage-based detection reagent is comparable to commercially-available antibody-based biosensors. Importantly, the assay maintains functionality at both neutral and basic pH in presence of salt and urea over the range of concentrations typical for human urine. Phage-based diagnostic systems may feature improved physical stability and cost of production relative to traditional antibody-based reagents, without sacrificing specificity and sensitivity.
结核病是全球传染病致死的主要原因。当前诊断方法灵敏度低、处理时间长且费用高昂,是结核病检测和治疗面临的主要挑战。在活动性结核感染患者的尿液中已鉴定出结核分枝杆菌鸟氨酸转氨甲酰酶(Mtb OTC,Rv1656),它是即时诊断的一个有前景的靶点。从基于超嗜热Sso7d支架构建的噬菌体展示文库中筛选出了对Mtb OTC具有低纳摩尔亲和力的特异性结合蛋白。利用展示Sso7d变体的噬菌体颗粒开发了一种基于夹心ELISA的Mtb OTC检测方法。该检测方法对重组Mtb OTC的响应在2 ng/mL至125 ng/mL之间呈线性,重组Mtb OTC的检测限为400 pg/mL。采用基于噬菌体的检测试剂的检测方法与市售的基于抗体的生物传感器相当。重要的是,在人尿液典型浓度范围内存在盐和尿素的情况下,该检测方法在中性和碱性pH条件下均保持功能。相对于传统的基于抗体的试剂,基于噬菌体的诊断系统可能具有更好的物理稳定性和更低的生产成本,同时不牺牲特异性和灵敏度。