IMMUNODOTS/ELSA BASED ON RECOMBINANT BORRELIA ANTIGENS

莱姆病是美国最常见的节肢动物传播疾病， States.它是一种慢性，多系统，蜱传感染引起的， 伯氏疏螺旋体它的特点是复发性系统性 和局部表现，涉及皮肤，心脏，神经系统和 接头.血清学检测似乎是一种可行和实用的方法 来诊断B。伯氏菌感染然而，这种测定的发展 被B的复杂性所阻碍。伯氏抗原 不同基因种和地理分离株的抗原变异性 这种生物体，病人对它的免疫反应的异质性， 缺乏可靠和标准化的莱姆病实验室诊断测试 疾病，以及不确定到底是哪种B。伯氏抗原 应该出现在诊断测试中。作为B。burgdorferi似乎是 克隆的、染色体编码的B的基因产物。burgdorferi很可能 比质粒基因产物更稳定， 可能存在于这些细菌的所有基因型和分离株中。我们 已经用分子遗传技术鉴定出了一个B。burgdorferi 染色体编码的推定外表面脂蛋白，BmpC。我们将 测试这种蛋白质可用于开发诊断的假设 莱姆病的检测。为了证明这一假设，我们将 表达重组B。大肠杆菌中的伯氏菌BmpC作为天然的， 非融合蛋白，对其进行生物化学和免疫化学表征，以及 证实其在多种B中的存在和表达。伯氏菌属 并利用分子遗传学、生物化学和免疫化学 技术.免疫化学分析将使用多克隆和单克隆 针对BmpC和其它充分表征的B产生的抗体。burgdorferi proteins.最后，我们将描述人类对BmpC的免疫应答， 使用ELISA、免疫斑点和T细胞刺激试验检测其片段， 比较早期和晚期莱姆病患者的反应， 没有关节炎，有或没有神经系统并发症， 其他发热性传染病患者，包括 螺旋体疾病，以及健康对照受试者的那些。 拟议的商业应用：莱姆病是最常见的节肢动物 在美国传播的疾病。非常需要可靠的 血清学试验诊断该病，如B。布格多费里群岛 很难从病人身上培养出来，而目前的血清学检测 缺乏特异性和敏感性。血清学检测的总市场 用于诊断莱姆病的剂量估计为200万单位/ 这一年在这个国家和类似的数字在西欧和东欧。

Lyme disease is the most common arthropod-borne disease in the United States. It is a chronic, multisystem, tick-borne infection caused by the spirochete Borrelia burgdorferi. It is characterized by recurrent systemic and local manifestations involving the skin, heart, nervous system and joints. Serological assays appear to be a feasible and practical approach to diagnose B. burgdorferi infections. However, development of such assays has been hampered by the complexity of B. burgdorferi antigens, the antigenic variability of different genospecies and geographic isolates of this organism, the heterogeneity of patient immune response to it, the lack of reliable and standardized laboratory diagnostic tests for Lyme disease, and uncertainty about exactly which B. burgdorferi antigens should be present in a diagnostic test. As B. burgdorferi appear to be clonal, chromosomally-encoded gene products of B. burgdorferi are likely to be more genetically stable than plasmid gene products, and are more likely to be present in all genotypes and isolates of these bacteria. We have used molecular genetic technology to identify a B. burgdorferi chromosomally-encoded putative outer surface lipoprotein, BmpC. We will test the hypothesis that this protein can be used to develop a diagnostic assay for Lyme disease. To provide evidence for this hypothesis, we will express recombinant B. burgdorferi BmpC in Escherichia coli as a native, non-fused protein, characterize it biochemically and immunochemically, and confirm its presence and expression in a variety of B. burgdorferi genera and isolates using molecular genetic, biochemical and immunochemical techniques. Immunochemical analyses will use polyclonal and monoclonal antibodies raised to BmpC and other well characterized B. burgdorferi proteins. Finally, we will characterize human immune response to BmpC and its fragments using ELISA, immunodot and T-cell stimulation assays and compare responses of patients with early and late Lyme disease, with and without arthritis and with and without neurological complications, to those of patients with other febrile infectious diseases including spirochetal diseases, and to those of healthy control subjects. PROPOSED COMMERCIAL APPLICATION: Lyme disease is the most common arthropod borne disease in the United States. There is a great need for reliable serological assays to diagnose this disease, as B. burgdorferi is difficult to culture from patients, and the current serological assays lack specificity and sensitivity. The total market for serological assays for the diagnosis of Lyme disease is estimated to be 2,000,000 units per year in this country and a similar number in Western and Eastern Europe.