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描述的最致命的物质。纳克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、灵敏地检测生物威胁毒素。此外，理想的诊断应该是稳定和简单的，足以供医疗培训有限的个人使用，例如现场的急救人员（警察和消防员）。目前的“金标准”检测方法耗时且依赖于样品，远远达不到理想的水平。我们的初步研究表明，模仿天然毒素受体的糖缀合配体可以用于快速和精确检测密切相关的毒素。作为原理证明，我们已经开发出定制的碳水化合物，可以区分志贺毒素的两种抗原形式，Stx1和Stx2。我们建议将这些研究扩展到开发诊断和治疗产品。在目标1中，我们将开发志贺、蓖麻毒素、肉毒杆菌、发酵梭菌毒素和肠毒素B葡萄球菌（SEB）特异性的第二代和第三代毒素配体。这些配体将最大限度地发挥协同结合作用，并优化每个毒素类变异之间的分化。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。偶联到预制链亲和素包被微滴板产生糖缀合微阵列药盒，理想的毒素在护理点设置快速测试。在Aim 3中，我们将验证和执行Aim 1中的先导化合物的临床前评估。我们将量化微阵列诊断的有效性及其在组织培养、小鼠模型和离体人类标本中的治疗潜力。灵敏度、特异性、快速性、坚固性、易用性和成本效益将进行评估，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.