LAMPoles for Dengue Diagnosis

 描述登革热是热带和亚热带地区的主要公共卫生威胁，发病率和死亡率相当高。它是继疟疾之后最广泛的病媒传播疾病，目前不能通过接种疫苗预防。登革热可以从良性发热疾病迅速发展为登革出血热（DHF）和/或登革休克综合征（DSS）和死亡;支持性护理对于限制并发登革热的发病率和死亡率很重要。因此，早期和准确诊断登革热的能力对于该疾病的临床管理非常重要。该提案的创新之处在于将3种强大的技术- LAMP、“蝌蚪”和纳米抗体（Nbs）-联系起来，以创建一种动态的基于蛋白质-DNA嵌合体的DF/DHF诊断方法，与现有技术相比具有高特异性和灵敏度。我们成功地创建了蛋白质-DNA嵌合体，称为“LAMPole”，通过融合基于植物酶基因序列的DNA寡核苷酸尾与先前描述的蛋白质L/G-DNA嵌合体，其蛋白质部分结合大多数种类的哺乳动物抗体，以创建用于超灵敏检测基本上任何哺乳动物IgG的蛋白质L/G LAMPole。为了验证概念，我们表明蛋白LG-LAMPoles增强了ELISA检测血液中宿主抗非洲锥虫抗体的灵敏度几百倍，从而导致我们的假设，即改善急性登革热的检测也是可能的。被称为纳米抗体（Nb）的单链骆驼抗体片段的发现是抗体工程的重大突破，其将使得即使在宿主Ab存在下也能够测量DV Ag。通过从靶向人IgM和DV抗原的Nbs产生LAMPoles，我们将产生用于诊断急性登革热的超灵敏和特异性测定。我们的团队已将马来西亚、斯里兰卡和尼加拉瓜确诊和排除登革热患者的血清和血液存档。我们将从对DV抗原（Ag）具有特异性的骆驼纳米抗体（Nbs）产生LAMPoles，并使用存档的临床样品评估LAMPoles相对于最先进的急性登革热诊断（相对于过去和无登革热）的灵敏度和特异性。

 DESCRIPTION (provided by applicant): Dengue is a major public health threat throughout the tropics and subtropics will considerable morbidity and mortality It is the most widespread vector-borne disease after malaria and cannot be prevented by vaccination now Dengue can progress rapidly from benign febrile illness to dengue hemorrhage fever (DHF) and/or dengue shock syndrome (DSS) and death; supportive care is important to limit morbidity and mortality from complicated dengue. Hence, the ability to diagnose dengue early and accurately is important for clinical management of the disease. This proposal is innovative in that links 3 powerful technologies - LAMP, `tadpoles' and Nanobodies (Nbs) - to create a dynamic protein-DNA chimera-based diagnostic for DF/DHF with high specificity and sensitivity compared with existing technologies. We successfully created protein-DNA chimeras, termed `LAMPoles', by fusing DNA oligonucleotide tails based on a sequence of a plant enzyme gene with a previously described protein L/G-DNA chimera whose protein moiety binds most classes of mammalian antibodies to create a protein L/G LAMPole for ultrasensitive detection of essentially any mammalian IgG. For proof-of-concept we show that protein LG-LAMPoles enhanced the sensitivity of ELISA to detect host anti-African trypanosome antibodies in blood several hundred-fold leading to our hypothesis that improved detection of acute dengue is also possible. The discovery of single chain camelid antibody fragments called Nanobodies (Nb) is a major breakthrough in antibody engineering that will enable measurement of DV Ags even in the presence of host Abs. By creating LAMPoles from Nbs targeting both human IgM and DV antigens, we will create an ultrasensitive and specific assay for the diagnosis of acute dengue. Our team has archived serum and blood from patients in Malaysia, Sri Lanka, and Nicaragua with confirmed and excluded dengue. We will generate LAMPoles from camelid nanobodies (Nbs) specific for DV antigens (Ags) and evaluate the sensitivity and specificity of LAMPoles versus state-of-the-art diagnosis of acute dengue (vs. past and no dengue) using archived clinical samples.