Generating fast-on rate reagents for lateral flow assays to detect HCV

生成用于侧向层析检测 HCV 的快速试剂

基本信息

批准号：
10697630
负责人：
BRIAN KENNETH KAY
金额：
$ 27.56万
依托单位：
TANGO BIOSCIENCES, INC.
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-04-13 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10697630
关键词：
Affinity Age American Antibodies Antigens Avidity Bacteriophage M13 Bacteriophages Binding Biological Biological Assay Biological Sciences Biometry Biotin Biotinylation Buffers C-terminal COVID-19 Capsid Proteins Chemistry Chicago Chinese Hamster Ovary Cell Chronic Hepatitis C Cirrhosis Code Collaborations Communicable Diseases Detection Development Diagnosis Diagnostic Reagent Disease Dissociation Early Diagnosis Engineering Epitopes FDA Emergency Use Authorization Genomics Goals Health Hepatic Lymphoma Hepatitis C Hepatitis C virus Home Horseradish Peroxidase Human Immunoglobulin Fragments In Vitro Individual Infection Injecting drug user Kinetics Label Laboratories Length Libraries Life Ligase Liver diseases Malignant neoplasm of liver Medical Metabolic Methods Mutagens Patients Peptides Performance Phage Display Phase Plasma Populations at Risk Pregnancy Tests Primary carcinoma of the liver cells RADx RNA Viruses Reagent Recombinant Antibody Recombinants Reporter Research Research Support Resource-limited setting Rural Community SARS-CoV-2 antigen Small Business Innovation Research Grant Source Specificity Techniques Technology Testing Translating United States National Institutes of Health Universities Virion With laterality Work antigen test combinatorial cost detection assay detection limit detector experience infection risk innovation instrumentation lateral flow assay manufacture medically underserved population nanoparticle novel overexpression particle pathogen point of care point of care testing programs prototype screening sex virus core young adult

Affinity Age American Antibodies Antigens Avidity Bacteriophage M13 Bacteriophages Binding Biological Biological Assay Biological Sciences Biometry Biotin Biotinylation Buffers C-terminal COVID-19 Capsid Proteins Chemistry Chicago Chinese Hamster Ovary Cell Chronic Hepatitis C Cirrhosis Code Collaborations Communicable Diseases Detection Development Diagnosis Diagnostic Reagent Disease Dissociation Early Diagnosis Engineering Epitopes FDA Emergency Use Authorization Genomics Goals Health Hepatic Lymphoma Hepatitis C Hepatitis C virus Home Horseradish Peroxidase Human Immunoglobulin Fragments In Vitro Individual Infection Injecting drug user Kinetics Label Laboratories Length Libraries Life Ligase Liver diseases Malignant neoplasm of liver Medical Metabolic Methods Mutagens Patients Peptides Performance Phage Display Phase Plasma Populations at Risk Pregnancy Tests Primary carcinoma of the liver cells RADx RNA Viruses Reagent Recombinant Antibody Recombinants Reporter Research Research Support Resource-limited setting Rural Community SARS-CoV-2 antigen Small Business Innovation Research Grant Source Specificity Techniques Technology Testing Translating United States National Institutes of Health Universities Virion With laterality Work antigen test combinatorial cost detection assay detection limit detector experience infection risk innovation instrumentation lateral flow assay manufacture medically underserved population nanoparticle novel overexpression particle pathogen point of care point of care testing programs prototype screening sex virus core young adult

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项目摘要

Hepatitis C virus (HCV) is a small, enveloped RNA virus that causes hepatitis C and some forms of liver cancer and lymphoma. Many infected individuals are asymptomatic, which makes early diagnosis challenging. In the proposed work, we will apply phage-display to produce avidity- boosted and fast on-rate recombinant affinity reagents for use in capture and detection of HCV core Ag in lateral flow assays (LFAs). This project is a collaboration between Tango Biosciences, and the University of Houston laboratory of Prof. Richard Willson. Tango is a start-up specializ- ing in innovative techniques in the phage-display and avidity engineering of combinatorial pep- tides, antibody fragments, and antibody surrogates. Dr. Willson has extensive technical and translational experience with LFAs.For Specific Aim #1, Tango will generate recombinant affinity reagents by phage-display that bind the HCV core antigen (cAg) with high affinity and fast on- rates. Tango will discover monobodies and human single-chain variable fragments (scFvs) that bind different epitopes of cAg via Megaprimer Shuffled Tandem Affinity Reagents (MegaSTAR). Tango will mutagenize the coding regions of individual monobodies/scFvs and select for fast on- rates (critical in LFA) and slow off-rates. Tandem high-avidity forms of the best monobodies/ scFvs will be constructed with different length linkers for phage-display and conversion into LFA reporter nanoparticles. We will aim for the following performance metrics for optimized affinity reagents: on-rates of 108 M-1 sec-1 and dissociation constants of ≤ 1 pM. In Specific Aim #2, viri- ons and soluble forms of the optimized affinity reagents will be formatted by Dr. Willson's team as capture and detector reagents in phage LFAs. After systematically testing buffers, conjugate and blocking chemistries, the goal will be to reach a limit of detection (LOD) of cAg (spiked into human plasma) of 0.5 pM (10 pg/mL), which is sensitive enough to detect 85% of HCV infec- tions. We will validate the optimized LFA format by spiking cAg over a range of concentrations into commercial, de-identified human plasma from both sexes, a range of ages, and varied dis- ease and metabolic backgrounds, to detect any cofounding biological variables. Finally, the best affinity reagents and prototype LFA will be evaluated in the laboratory of an HCV expert. Suc- cessful completion of this work will lead to a prototype LFA for HCV cAg and establish a re- search paradigm applicable for the development of superior LFAs for detecting infections. -1-

丙型肝炎病毒（HCV）是一种小的，包膜的RNA病毒，引起丙型肝炎和某些形式肝癌和淋巴瘤。许多受感染的人是不对称的，这使得诊断挑战。在拟议的工作中，我们将应用噬菌体播放来产生亲和力 - 提高速率快速的重组亲和力试剂用于捕获和检测HCV 侧流分析中的核心AG（LFA）。这个项目是探戈生物科学之间的合作，理查德·威尔森教授休斯顿大学实验室。探戈是一家专业的启动 - 组合PEP-的噬菌体播放和亲戚工程中的创新技术潮汐，抗体片段和抗体替代物。威尔森博士拥有广泛的技术和 lfas的翻译经验。对于特定的目标＃1，探戈将产生重组亲和力通过噬菌体显示的试剂结合具有高亲和力的HCV核心抗原（CAG）的试剂费率。探戈会发现单链和人类的单链变量片段（SCFV）通过巨拟二聚体洗牌串联亲和力试剂（Megastar）结合不同的CAG表位。探戈将诱变单个单体组合/SCFV的编码区域，并选择快速on- 费率（在LFA中至关重要）和偏移速度缓慢。最佳单体组合的串联高避免形式/ SCFV将使用不同的长度接头构建，用于噬菌体 - 播放，然后转换为LFA 记者纳米颗粒。我们将旨在制定以下性能指标以优化亲和力试剂：108 m-1 sec-1的率和分离常数≤1pm。在特定的目标＃2中，viri- 优化亲和力试剂的ONS和固体形式将由Willson博士的团队格式化作为噬菌体LFA中的捕获和检测器试剂。系统测试缓冲液后，共轭并阻止化学物质，目标是达到CAG的检测限（LOD）人血浆）为0.5 pm（10 pg/ml），足够敏感，可以检测85％的HCV Infec- tions。我们将通过在浓度范围内尖峰CAG来验证优化的LFA格式从性别，一系列年龄段以及各种各样的疾病中进入商业化的，取消识别的人血浆轻松和代谢背景，以检测任何共同的生物变量。最后，最好亲和力试剂和原型LFA将在HCV专家的实验室中进行评估。成功这项工作的结束完成将导致HCV CAG的原型LFA，并建立一个重新搜索范式适用于开发用于检测感染的优质LFA。 -1-