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。 各州。它是一种慢性、多系统、由壁虱传播的传染病，由 伯氏疏螺旋体。以全身性反复发作为特征。 以及累及皮肤、心脏、神经系统和 关节。血清学检测似乎是一种可行和实用的方法。 来诊断伯氏杆菌感染。然而，这种检测方法的发展 已经被复杂的伯氏杆菌抗原所阻碍， 猪瘟病毒不同基因种和地理分离株的抗原性变异 这种微生物，患者对它的免疫反应的异质性， 莱姆病缺乏可靠和标准化的实验室诊断检测 疾病，以及不确定到底是哪些伯氏杆菌抗原 应该出现在诊断测试中。就像伯格多尔费氏杆菌 伯氏杆菌的克隆、染色体编码的基因产物很可能是 在遗传上比质粒基因产品更稳定，而且更多 可能存在于这些细菌的所有基因类型和分离株中。我们 已经使用分子基因技术鉴定了一株伯氏杆菌 染色体编码的推测的外表面脂蛋白，BmpC。我们会 测试这种蛋白质可以用来开发诊断程序的假设 莱姆病化验。为了为这一假设提供证据，我们将 重组伯氏杆菌BmpC在大肠杆菌中的原生表达， 非融合蛋白，对其进行生物化学和免疫化学表征，以及 证实其在多种伯氏杆菌属中的存在和表达 和使用分子遗传学、生化和免疫化学的分离株 技巧。免疫化学分析将使用多克隆和单克隆 针对BmpC和其他具有良好特征的伯氏杆菌的抗体 蛋白质。最后，我们将描述人类对BmpC和 用酶联免疫吸附试验、免疫斑点试验和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， 患有其他发烧传染病的病人，包括 螺旋体疾病，以及健康对照受试者的。 建议商业应用：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 诊断这种疾病的血清学检测，就像伯氏杆菌一样 很难从患者身上培养，而且目前的血清学检测 缺乏特异性和敏感性。血清学检测的总市场 用于莱姆病诊断的单位估计为200万单位 这一数字在这个国家是每年，在西欧和东欧也有类似的数字。

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.