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: 7254809
• 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 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7254809
• 关键词: Address; Agglutination; Alloimmunization; Amino Acid Sequence; Antibodies; Antigen Receptors; Antigens; Automation; Bacteriophages; Biological Assay; Blood; Blood Banks; Blood Group Antigens; Blood Tests; Blood typing procedure; Cells; Class; Clinical; Collection; Compatible; Complement; Complement 3d; Coombs' Test; Coupled; DNA; DNA Sequence; Detection; Development; Ensure; Erythrocytes; Funding; Future; Genotype; Globulins; Human; Immunoglobulin G; Immunoglobulin M; Incidence; Inovirus; Laboratories; Length; Link; MNSs Blood-Group System; Manuals; Materials Testing; Medical; Medicine; Methodology; Methods; Microfluidics; Molecular; Monoclonal Antibodies; Mus; Nucleic Acids; Numbers; Oryctolagus cuniculus; Performance; Phage Display; Phase; Phase I Clinical Trials; Phenotype; Plasma; Reaction; Reagent; Reporting; Sampling; Scheme; Sensitivity and Specificity; Services; Specificity; Surface; System; Technology; Testing; Time; Transfusion; Tube; United States Food and Drug Administration; Work; antigen antibody binding; base; cell type; cost; improved; instrumentation; micro-total analysis system; novel; nucleic acid detection; particle; programs; reagent testing; receptor; tool

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，并且每次检测的成本比其他临床实验室高出一个数量级。再加上熟练的医疗技术人员日益短缺，人血浆衍生的表型分析试剂的供应减少，以及完全自动化基于凝集的方法的固有困难，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经成为一个重大挑战。PhenoTech的长期目标是使用一套新的分子技术来开发一类新的可再生的、廉价的、高质量的血库检测试剂，这些试剂将在快速、高通量、自动化的检测系统中发挥作用。这项技术的核心是展示在噬菌体颗粒表面的红细胞抗原特异性单克隆抗体。I期研究提出利用颗粒内天然存在的独特DNA序列来开发测定系统，其中通过测定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，将需要微量的测试材料和试剂，将易于适应自动化，并且将适合于多路复用策略，提供在单个反应容器中同时进行红细胞样品的抗原谱分析的可能性。这将使血液的扩展表型和匹配超出传统的三抗原A、B和Rh（D），并且负担得起和常规。输血医学实践中的这种范式转变将有助于减少红细胞同种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工作成本，并改善提供免疫相容性血液的周转时间。正如第二阶段提案中所报告的那样，PhenpTech成功地证明了使用其新型试剂基因分型方法进行多重红细胞分型反应的可行性。反应所需的RBC数量少于传统凝集试验的1/100，000，并且使用了传统抗体试剂的Attogram等效物。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试验的能力，间接抗球蛋白试验是输血前试验中最劳动密集型和最昂贵的一组。在证明了噬菌体试剂基因分型方法的可行性之后，PhenoTech的第二阶段计划现在将专注于开发一套噬菌体试剂，以进一步开发其血液检测技术所需的临床重要血型抗原。结合其基于微流体的“芯片实验室”平台的同步开发，PhenoTech将能够提供必要的工具，通过解决传统血库检测方法的技术和财政限制，确保未来输血医学的安全实践。