Phage Display Tools for Automated Blood Typing

用于自动血型定型的噬菌体展示工具

• 批准号: 7110896
• 负责人: DONALD Lawrence SIEGEL
• 金额: $ 50.95万
• 依托单位: PHENOTECH, INC.
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7110896
• 关键词: agglutination reaction; antigen antibody reaction; biotechnology; blood groups; blood transfusion; complement; erythrocytes; high throughput technology; human tissue; immunoglobulin G; immunoglobulin M; immunohematology; immunologic substance development /preparation; laboratory rabbit; molecular cloning; monoclonal antibody; phage display; technology /technique development

DESCRIPTION (provided by applicant): Current technologies used in blood banking are extraordinarily labor intensive, prone to human error, and an order of magnitude more expensive per test that those in other clinical laboratories. Coupled with a growing shortage of skilled medical technologists, dwindling supplies of human plasma-derived phenotyping reagents, and an inherent difficulty in fully automating agglutination-based methodologies, the ability to perform rapid and accurate pre-transfusion testing in a cost-effective manner has become a significant challenge. PhenoTech's long-term objective is to use a set of novel molecular technologies to develop a new class of renewable, inexpensive, high-quality blood bank testing reagents that will function in a rapid, high- throughput, automatable assay system. At the core of the proposed technology are red blood cell antigen- specific monoclonal antibodies displayed on the surface of bacteriophage particles. Phase I studies proposed to exploit the naturally-occurring presence of unique DNA sequences within the particles to develop an assay system in which the phenotype of a cell is determined by assaying the genotype of the detecting reagent. Such a strategy would offer extraordinary sensitivity and specificity, would require minute amounts of testing materials and reagents, would be easily adapted to automation, and would be amenable to multiplexing strategies offering the possibility of simultaneous antigen profiling of a red cell sample in a single reaction vessel. This would make extended phenotyping and matching of blood beyond the traditional three-antigen A, B, and Rh(D) affordable and routine. Such a paradigm shift in the practice of transfusion medicine would then serve to reduce the incidence of red cell alloimmunization and occurrence of delayed hemolytic transfusion reactions, decrease the development of positive antibody screens and the costs in working them up, and improve the turn-around-time for the provision of immunologically-compatible blood. As reported in this Phase II proposal, PhenpTech was successful in demonstrating the feasibility of multiplexing red cell typing reactions using its novel reagent genotyping methodology. Reactions required less than 1/100,000 the number of RBCs of a conventional agglutination assay and used attogram equivalents of conventional antibody reagents. In addition, the ability to use phage particles which express anti-globulin-like molecules for performing indirect antiglobulin tests, the most labor-intensive and costly group of pre-transfusion tests, was also shown. Having demonstrated the feasibility of phage reagent genotyping methods, PhenoTech's Phase II program will now focus on the development of a suite of phage reagents to clinically important blood group antigens required for further development of its blood testing technologies. Combined with its concurrent development of a microfluidics-based "lab-on-a-chip" platform, PhenoTech will be able to provide the tools necessary to ensure the safe practice of transfusion medicine in the future by addressing the technical and fiscal limitations of conventional blood bank testing methods.

描述（由申请人提供）：目前在血库中使用的技术是非常劳动密集型的，容易出现人为错误，并且每次测试的成本比其他临床实验室高一个数量级。再加上熟练的医疗技术人员日益短缺，人类血浆来源的表型试剂供应不断减少，以及基于凝集的方法完全自动化的固有困难，以具有成本效益的方式进行快速准确的输血前检测的能力已成为一项重大挑战。PhenoTech的长期目标是使用一套新颖的分子技术来开发一类新的可再生、廉价、高质量的血库检测试剂，这些试剂将在快速、高通量、自动化的分析系统中发挥作用。该技术的核心是显示在噬菌体颗粒表面的红细胞抗原特异性单克隆抗体。第一阶段研究建议利用颗粒内自然存在的独特DNA序列来开发一种检测系统，其中通过分析检测试剂的基因型来确定细胞的表型。这样的策略将提供非凡的灵敏度和特异性，将需要少量的测试材料和试剂，将很容易适应自动化，并将适用于多路复用策略，提供在单个反应容器中同时抗原分析红细胞样本的可能性。这将使超出传统的三抗原A、B和Rh(D)的扩展表型和血液匹配变得负担得起和常规。输血医学实践中的这种范式转变将有助于减少红细胞异体免疫的发生率和延迟溶血性输血反应的发生，减少阳性抗体筛查的发展和开展这些筛查的成本，并改善提供免疫相容血液的周转时间。正如这项II期提案所报道的那样，PhenpTech成功地证明了使用其新型试剂基因分型方法进行多路红细胞分型反应的可行性。反应所需的红细胞数量少于传统凝集试验的1/100,000，并使用了相当于传统抗体试剂的图解。此外，还显示了使用表达抗球蛋白样分子的噬菌体颗粒进行间接抗球蛋白测试的能力，这是输血前测试中最费力和最昂贵的一组。在证明了噬菌体试剂基因分型方法的可行性之后，PhenoTech的II期项目现在将重点开发一套噬菌体试剂，用于进一步开发其血液检测技术所需的临床重要血型抗原。结合其同时开发的基于微流体的“芯片实验室”平台，PhenoTech将能够通过解决传统血库测试方法的技术和财政限制，为确保未来输血医学的安全实践提供必要的工具。