High throughput screening of bacterial collections for surface proteins of commer

描述（由申请人提供）：目前公认需要开发蛋白质捕获工具，以可靠地检测各种靶标，在广泛的浓度范围内，用于人血浆和其他复杂的样品混合物，用于诊断和治疗应用。我们的PEARL（病原体编码适应性受体文库）技术产品将产生抗体样分子，其对配体的亲和力比传统抗体高100-10,000倍。这将允许这些新的捕获试剂无缝地纳入现有的高通量分析程序和平台，目前使用抗体作为捕获试剂；然而，由于PEARL产品的亲和力增强，脱失率较慢，因此测定的灵敏度将得到提高。这在处理低浓度的靶配体与大量无关蛋白质的复杂混合物时尤其重要，这是目前大多数医学亚专业蛋白质组学研究的瓶颈。在这个提议中，我们解决了充分利用我们平台技术的最后一个限制。选择流式细胞术来解决瓶颈，从而评估PEARL的关键原则[假设]：当筛选足够大的病原体文库时，将发现微生物具有独特的抗体样表面蛋白，这些蛋白对正常或疾病状态受试者中发现的数百种血浆/血清成分中的一种具有高亲和力。我们建议使用PEARL收集的一个子集来评估这一假设，作为条形码细菌和免疫球蛋白[IgG1， IgG3, IgM, IgA]，因为它们作为靶配体的医学和商业意义而被选择。一项双盲实验将验证这一特定目标：开发并验证流式细胞术，以评估一种独特的细菌病原体集合的成员，这种细菌病原体已知具有能够与许多独特的不同生理上重要的血浆成分特异性[抗体样]结合的表面蛋白。验证过程中的关键步骤构成了本建议工作范围的基础。开发并验证一种多参数流式细胞术筛选方法，以检测具有任何靶向配体结合潜力的细菌。这将允许在几个小时内完成初始筛选，而不是几个月。验证区分不同基因产物与单一基因产物的多功能结合的结合活性的方法。这将区分潜在的候选产品，并确定最接近客户提供的产品规格的候选产品。任务#3 -证明流式细胞术鉴定的阳性命中也可以用更传统的配体印迹分析模式观察到。

DESCRIPTION (provided by applicant): There is a recognized need for development of protein capture tools that will reliably detect a variety of targets, over a broad range of concentrations, for use with human plasma and other complex sample mixtures for diagnostic and therapeutic applications. The products from our PEARL [Pathogen-Encoded Adaptable Receptor Library] technology will yield antibody-like molecules that can be engineered to demonstrate 100-10,000 fold higher affinity for their ligand than conventional antibodies. This will allow these novel capture reagents to be seamlessly incorporated into existing highthru- put assay procedures and platforms that currently use antibodies as the capture reagent; however because of the enhanced affinity and slower off-rate of the PEARL products and the sensitivity of assays will be improved. This is particularly important when dealing with low concentrations of target ligands in complex mixtures with a large molar excess of irrelevant proteins, a current bottleneck for proteomic studies in most medical subspecialties. In this proposal we address the last limit to full exploitation of our platform technology. Flow cytometry was selected to solve the bottleneck and thus evaluate the key tenet [hypothesis] of PEARL: When a sufficiently large pathogen library is screened, microorganisms will be found that bear unique antibody-like surface proteins that display high affinity toward one of hundreds of plasma/serum components found in either normal or disease-state subjects. We propose to evaluate this hypothesis using a subset of the PEARL collection, as bar coded bacteria, and immunoglobins [IgG1, IgG3, IgM, IgA] selected because of their medical and commercial significance as the target ligands. A double blind experiment will test this specific aim: Develop and validate flow-cytometry for evaluating members of a unique collection of bacterial pathogens known to bear surface proteins capable of specific [antibody-like] binding to many uniquely different physiologically significant plasma components. The key steps in that validation form the basis of the work scope of this proposal. Task #1 Develop and validate a multiparameter flow cytometric screening method to detect bacteria with binding potential for any targeted ligand. This will allow an initial screen to be completed in hours, not months. Task #2 Validate method(s) to distinguish between binding activities due to different gene products from those associated with multi functional binding of a single gene product. This will distinguish between potential starting candidates and identify the one(s) most closely resembling product specifications provided by the customers. Task #3 - Demonstrate that positive hits identified by flow cytometry are also observed using more traditional modes of analysis via ligand blot. PUBLIC HEALTH RELEVANCE: Virtually all clinical analyses are limited by one-or-more of these features: (a) insufficient sample for all needed tests; (b) insufficient sensitivity for use with limiting samples; and/or (c) difficulties in standardization across vendors and laboratories. These limits are an unavoidable downside of the most popular approach to assay design; i.e., use of antibodies which are limited by the affinity for antigen. The proposed PEARL approach retains the advantages of the current method while addressing virtually all of its problems. When successfully implemented, an enhanced method for assessing both new clinical paradigms [such as use of biotherapeutics] as well as improving old analyses [suboptimal diagnostic immunoassays] will be attained.