Glycoconjugate based diagnostics for bacterial toxins

描述(由申请人提供)：Biothreat毒素包括所描述的最致命的毒剂。纳克量的雾化或注射毒素会导致死亡。为了防止生命损失，生物毒素的检测必须快速、准确和高度灵敏。此外，理想的诊断应该足够稳定和简单，供医疗培训有限的个人使用，例如现场使用的急救人员(警察和消防员)。目前的“金标准”检测方法费时费力且依赖于样品，远远达不到理想的检测结果。我们的初步研究表明，模拟天然毒素受体的糖共轭配体可以用于快速准确地检测密切相关的毒素。作为原理的证明，我们开发了量身定制的碳水化合物，可以区分志贺毒素的两种抗原型，STX1和STX2。我们建议扩大这些研究，以开发诊断和治疗产品。在目标1中，我们将开发针对志贺氏菌、蓖麻毒素、肉毒杆菌、产气荚膜梭菌表型毒素和葡萄球菌肠毒素B(SEB)的第二代和第三代毒素配体。这些配体将最大限度地协同结合并优化每一类毒素变异体之间的差异。在目标2中，我们将开发和评估不同的诊断平台。生物素将以共价方式连接到配体上。与预制的链霉亲和素涂层微滴定板结合可产生糖共轭微阵列色谱盒，非常适合于在护理点设置中快速检测毒素。在目标3中，我们将验证和执行来自目标1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、小鼠模型和体外人类样本中的治疗潜力。将对敏感性、特异性、快速性、坚固性、易用性和成本效益进行评估，以确定原则证据。

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.