High throughput screening of bacterial collections for surface proteins of commer

描述（由申请人提供）：人们认识到需要开发蛋白捕获工具，该工具将可靠地检测在广泛浓度范围内的各种靶标，用于人血浆和其他复杂样品混合物，用于诊断和治疗应用。我们的PEARL [病原体编码的适应性受体文库]技术的产品将产生抗体样分子，这些分子可以被工程化以证明其配体的亲和力比常规抗体高100- 10，000倍。这将允许将这些新型捕获试剂无缝地结合到现有的高通量测定程序和目前使用抗体作为捕获试剂的平台中;然而，由于PEARL产物的增强的亲和力和较慢的解离速率，将提高测定的灵敏度。这是特别重要的，当处理低浓度的目标配体在复杂的混合物与大摩尔过量的不相关的蛋白质，目前的瓶颈蛋白质组学研究在大多数医学亚专业。在本提案中，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，将发现携带独特抗体样表面蛋白的微生物，这些蛋白对正常或疾病状态受试者中发现的数百种血浆/血清组分之一显示出高亲和力。我们建议使用PEARL集合的一个子集来评估这一假设，如条形码 细菌和免疫球蛋白[IgG 1，IgG 3，IgM，伊加]，因为它们作为靶配体的医学和商业意义而被选择。 双盲实验将测试这一特定目标：开发和验证流式细胞术，用于评价已知携带能够特异性[抗体样]结合许多独特的不同生理学显著血浆组分的表面蛋白的细菌病原体的独特集合的成员。验证的关键步骤构成了本提案工作范围的基础。任务#1 开发并验证多参数流式细胞术筛选方法，以检测与任何靶向配体具有结合潜力的细菌。这将允许在几个小时内完成初始筛选，而不是几个月。任务#2 区分不同基因产物的结合活性和与单一基因产物的多功能结合相关的结合活性的方法。这将区分潜在的起始候选产品，并确定与客户提供的产品规格最相似的产品。 任务#3 -证明通过流式细胞术鉴定的阳性命中也可以通过配体印迹使用更传统的分析模式观察到。 公共卫生相关性：实际上，所有临床分析都受到这些特征中的一个或多个的限制：（a）所有所需测试的样本不足;（B）有限样本使用的灵敏度不足;和/或（c）供应商和实验室之间标准化的困难。这些限制是最流行的分析设计方法的不可避免的缺点;即，使用受抗原亲和力限制的抗体。所提出的PEARL方法保留了当前方法的优点，同时解决了几乎所有的问题。当成功实施时，将获得一种用于评估新的临床范例[如生物治疗药物的使用]以及改进旧分析[次优诊断免疫测定]的增强方法。

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.