High throughput screening of bacterial collections for surface proteins of commer

商业细菌表面蛋白的高通量筛选

• 批准号: 7997349
• 负责人: ROY H HAMMERSTEDT
• 金额: $ 9.75万
• 依托单位: APD LIFE SCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7997349
• 关键词: Address; Affinity; Albumins; Antibodies; Antigens; Avidin; Bacteria; Bar Codes; Binding; Binding Proteins; Biological Assay; Biological Markers; Biological Response Modifier Therapy; Biological Sciences; Biotin; Characteristics; Clinical; Code; Collection; Communities; Complex; Complex Mixtures; DNA; Development; Diagnostic; Disease; Double-Blind Method; Engineering; Enzyme-Linked Immunosorbent Assay; Excision; Family member; Flow Cytometry; Hour; Human; IgG1; IgG3; Immunoassay; Immunoglobulin A; Immunoglobulin Constant Region; Immunoglobulin M; Industry; Laboratories; Libraries; Ligands; Marketing; Medical; Membrane Proteins; Methods; Movement; Pharmaceutical Preparations; Plasma; Procedures; Process; Proteins; Proteomics; Reagent; Recombinant Proteins; Recombinants; Sales; Sampling; Screening procedure; Source; Specificity; Standardization; Surface; System; Technology; Testing; Therapeutic; Transferrin; Ursidae Family; Validation; Vendor; Work; base; commercialization; cytokine; design; feeding; high throughput screening; improved; innovation; member; microorganism; novel; pathogen; protein expression; public health relevance; receptor; repository; research study; tool; vaccine efficacy

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.

描述(申请人提供)：人们认识到有必要开发一种蛋白质捕获工具，该工具将可靠地检测各种浓度范围内的目标，用于诊断和治疗应用中的人体血浆和其他复杂样本混合物。我们珍珠[病原体编码的适应性受体文库]技术的产品将产生类似抗体的分子，这些分子可以被设计成与其配体的亲和力比传统抗体高100-10,000倍。这将允许这些新型捕获试剂无缝地整合到现有的高通量分析程序和平台中，这些程序和平台目前使用抗体作为捕获试剂；然而，由于珍珠产品的亲和力增强和失败率降低，分析的灵敏度将得到提高。当处理含有大量摩尔过剩的无关蛋白质的复杂混合物中的低浓度靶配体时，这一点尤其重要，这是目前大多数医学专业蛋白质组学研究的瓶颈。在这份提案中，我们解决了充分利用我们的平台技术的最后一个限制。选择流式细胞术来解决这一瓶颈，从而评估珀尔的关键原则[假设]：当筛选到足够大的病原体文库时，将发现具有独特的抗体样表面蛋白的微生物，这些蛋白与在正常或疾病状态的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。我们建议使用珍珠集合的子集作为条形码细菌和免疫球蛋白[IgG1、IgG3、IgM、IgA]来评估这一假设，因为它们具有医疗和商业意义作为目标配体。一项双盲实验将测试这一特定目标：开发并验证流式细胞术，以评估已知携带表面蛋白的独特细菌病原体集合的成员，这些表面蛋白能够与许多独特的不同生理意义的血浆成分结合。验证中的关键步骤构成了本提案工作范围的基础。任务1开发并验证一种多参数流式细胞仪筛选方法，以检测与任何目标配体具有结合潜力的细菌。这将使初始筛选在几个小时内完成，而不是几个月。任务2验证方法(S)区分不同基因产物的结合活性与与单一基因产物的多功能结合相关的结合活性。这将区分潜在的入门候选人，并确定与客户提供的产品规格最相似的一个(S)。任务3-证明使用更传统的分析模式通过配基杂交也可以观察到流式细胞仪确认的阳性命中。 与公共卫生相关：几乎所有临床分析都受到以下一个或多个特征的限制：(A)所有所需测试的样本不足；(B)用于限制样本的灵敏度不足；和/或(C)供应商和实验室之间难以实现标准化。这些限制是最流行的检测设计方法不可避免的缺点，即使用受抗原亲和力限制的抗体。拟议的珍珠办法保留了现行办法的优点，同时几乎解决了它的所有问题。一旦成功实施，将获得一种评估新的临床范例[如生物疗法的使用]以及改进旧的分析[次优诊断免疫分析]的改进方法。