Antibody Based Diagnosis for TB

描述(申请人提供)：几十年来一直试图设计一种结核病的血清诊断测试，但结果令人失望，主要是因为大多数评估的抗原与人类免疫反应无关。在过去的7年中，我们对活动性疾病不同阶段的结核病患者的体液免疫反应进行了表征。这些研究构成了现在开发结核病血清诊断测试所需的研究的基础。我们的研究清楚地表明，在活动性结核病的不同阶段，以及在感染艾滋病毒和非艾滋病毒结核病患者中，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机会。在此背景下，我们已经在结核分枝杆菌培养滤液中鉴定了大约12种在临床前早期结核病、非空洞性结核病或空洞性结核病患者中引起抗体的蛋白质。这一亚组抗原还能在同时感染艾滋病毒和结核病的患者中产生抗体。这个亚组的两个蛋白已经被克隆，并提供了一种具有任何抗原所达到的最高灵敏度和特异度的血清诊断试验。在目前的应用中，我们建议：(A)鉴定额外的免疫优势抗原。这项工作将通过蛋白质组和分子手段完成；(B)将这些抗原的基因克隆到大肠杆菌中，并评估重组蛋白与结核病患者不同阶段抗体的反应性；(C)通过各种策略绘制这些候选蛋白上的免疫优势表位图；(D)确定这些抗原的免疫优势表位；(D)确定这些抗原的免疫优势表位；以及(D)确定这些抗原的免疫优势表位；以及(E)选择和评估将作为低成本、快速和高敏感性和特异度的结核病定点诊断试验基础的肽的组合。

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.