Glycoconjugate based diagnostics for bacterial toxins

描述（由申请人提供）：生物威胁毒素包括有史以来最致命的制剂。纳克数量的雾化或注射毒素会导致死亡。为了防止生命损失，必须快速、准确和灵敏地检测生物威胁毒素。此外，理想的诊断方法应该足够稳定且简单，适合医疗培训有限的个人，例如急救人员（警察和消防员）在现场使用。目前的“金标准”检测分析是耗时的并且依赖于样品，并且远远达不到理想。我们的初步研究表明，模拟天然毒素受体的糖缀合物配体可用于快速和精确检测密切相关的毒素。作为原理的证明，我们已经开发了定制的碳水化合物，可以区分滋贺毒素的两种抗原形式，Stx 1和Stx 2。我们建议将这些研究扩展到开发诊断和治疗产品。目标一：研制第二代和第三代毒素配体，分别针对滋贺、蓖麻毒素、肉毒杆菌、产气荚膜梭菌毒素和葡萄球菌肠毒素B（SE B）。这些配体将使协同结合最大化并优化每种毒素类别中变体之间的区分。在目标2中，我们将开发和评估不同的诊断平台。生物素将与配体共价连接。结合到预制的链霉亲和素包被的微量滴定板产生一个糖缀合物微阵列盒，理想的快速测试毒素在一个点的护理设置。在目标3中，我们将对目标1中的先导化合物进行验证和临床前评价。我们将量化微阵列诊断的有效性及其在组织培养、小鼠模型和人体标本中作为治疗剂的潜力。将对灵敏度、特异性、快速性、耐用性、易用性和成本效益进行评价，以建立原理证明。

DESCRIPTION (provided by applicant): The Biothreat toxins include the most lethal agents ever described. Nanogram amounts of aerosolized or injected toxins cause death. To prevent loss of life, detection of biothreat toxin must be made rapidly, accurately and with exquisite sensitivity. In addition, an ideal diagnostic should be stable and simple enough for individuals with limited medical training, such as first responders (police and fire-fighters) to use on site. Current "gold standard" detection assays are time consuming and sample dependent, and fall far short of the ideal. Our preliminary studies have shown that glycoconjugate ligands mimicking the natural toxin receptor can be used for rapid and precise detection of closely related toxins. As proof of principle, we have developed tailored carbohydrates that can discriminate between the two antigenic forms of Shiga toxin, Stx1 and Stx2. We propose to extend these studies to develop diagnostic and therapeutic products. In Aim 1, we will develop 2nd and 3rd generation toxin ligands specific for Shiga, Ricin, Botulinum, Clostridium perfingens epsilon toxins and Staphylococcus enterotoxin B (SEB). These ligands will maximize cooperative binding and optimize differentiation between variants in each toxin class. In Aim 2 we will develop and evaluate different diagnostic platforms. Biotin will be covalently linked to the ligands. Conjugation to prefabricated streptavidin coated microtiter plates yields a glycoconjugate microarray cartridge, ideal for rapid testing of toxins in a point-of-care setting. In Aim 3, we will validate and perform pre-clinical evaluation of lead compounds from Aim 1. We will quantify the effectiveness of the microarray diagnostics and their potential as therapeutics in tissue culture, in mouse models, and in human specimens ex vivo. Sensitivity, specificity, rapidity, ruggedness, ease-of-use and cost-effectiveness will be evaluated to establish proof of principle.