Antibody Based Diagnosis for TB

描述（由申请人提供）：几十年来，人们一直在尝试设计一种结核病的血清诊断测试，但结果令人失望，主要是因为大多数被评估的抗原与人类免疫反应无关。在过去的7年里，我们描述了结核病患者在活动性疾病的不同阶段的体液免疫反应。这些研究为现在开发结核病血清诊断测试所需的研究奠定了基础。我们的研究清楚地表明，在活动性结核病的不同阶段，以及在hiv感染和非hiv结核病患者中，根据其免疫优势选择抗原，将为开发成功的诊断测试提供最大的机会。在这种情况下，我们已经在结核分枝杆菌培养滤过液中发现了大约12种蛋白质，这些蛋白质可在早期临床前结核病、非空腔结核病或空腔结核病患者中引发抗体。这一抗原亚群也在合并感染艾滋病毒和结核病的患者中引发抗体。该亚群中的两种蛋白已被克隆，并提供了一种灵敏度和特异性最高的血清诊断方法，这是迄今为止针对任何抗原所实现的。在目前的应用中，我们建议：(a)鉴定额外的免疫优势抗原。这将通过蛋白质组学和分子方法来完成；(b)将这些抗原基因克隆到大肠杆菌中，并评价重组蛋白与结核病不同阶段患者抗体的反应性；(c)通过多种策略在这些候选蛋白上绘制免疫优势表位；(d)确定这些抗原的免疫优势肽，这些抗原可被结核病患者的抗体识别；最后(e)选择和评估多肽组合，这些多肽组合将成为一种低成本、快速、高灵敏度和特异性的结核病即时诊断检测方法的基础。

DESCRIPTION (provided by applicant): Attempts to devise a serodiagnostic test for TB have been made for decades with disappointing results, primarily because most of the antigens evaluated are not relevant to the human immune response. During the last 7 years, we have characterized the humoral immune responses in TB patients at different stages of active disease. These studies form the basis for the research that is now needed to develop a serodiagnostic test for TB. Our studies clearly show that antigens chosen on the basis of their immunodominance during different stages of active TB, and in both HIV-infected and non-HIV TB patients, will provide the greatest opportunity to develop a successful diagnostic test. In this context, we have already identified approximately 12 proteins in culture filtrates of M. tuberculosis that elicit antibodies in patients with incipient pre-clinical TB, non-cavitary TB or cavitary TB. This subset of antigens also elicits antibodies in patients co-infected with HIV and TB. Two proteins of this subset have been cloned, and provide a serodiagnostic assay with the highest sensitivity and specificity that has yet been achieved with any antigens. In the current application, we propose to: (a) Identify the additional immunodominant antigens. This will be done both by proteomic and molecular approaches; (b) Clone the genes for these antigens into E.coli and evaluate the reactivity of the recombinant proteins with antibodies from TB patients at different stages of TB; (c) Map the immunodominant epitopes on these candidate proteins by a variety of strategies; (d) Identify the immunodominant peptides of these antigens that are recognized by antibodies from patients across the spectrum of TB; and finally (e) Select and evaluate the combinations of peptides that will be the basis of a low-cost, rapid, point-of-care diagnostic test for TB with high sensitivity and specificity.