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:
- 金额:$ 27.56万
- 依托单位:
- 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:
- 关键词:AffinityAgeAmericanAntibodiesAntigensAvidityBacteriophage M13BacteriophagesBindingBiologicalBiological AssayBiological SciencesBiometryBiotinBiotinylationBuffersC-terminalCOVID-19Capsid ProteinsChemistryChicagoChinese Hamster Ovary CellChronic Hepatitis CCirrhosisCodeCollaborationsCommunicable DiseasesDetectionDevelopmentDiagnosisDiagnostic ReagentDiseaseDissociationEarly DiagnosisEngineeringEpitopesFDA Emergency Use AuthorizationGenomicsGoalsHealthHepatic LymphomaHepatitis CHepatitis C virusHomeHorseradish PeroxidaseHumanImmunoglobulin FragmentsIn VitroIndividualInfectionInjecting drug userKineticsLabelLaboratoriesLengthLibrariesLifeLigaseLiver diseasesMalignant neoplasm of liverMedicalMetabolicMethodsMutagensPatientsPeptidesPerformancePhage DisplayPhasePlasmaPopulations at RiskPregnancy TestsPrimary carcinoma of the liver cellsRADxRNA VirusesReagentRecombinant AntibodyRecombinantsReporterResearchResearch SupportResource-limited settingRural CommunitySARS-CoV-2 antigenSmall Business Innovation Research GrantSourceSpecificityTechniquesTechnologyTestingTranslatingUnited States National Institutes of HealthUniversitiesVirionWith lateralityWorkantigen testcombinatorialcostdetection assaydetection limitdetectorexperienceinfection riskinnovationinstrumentationlateral flow assaymanufacturemedically underserved populationnanoparticlenoveloverexpressionparticlepathogenpoint of carepoint of care testingprogramsprototypescreeningsexvirus coreyoung adult
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RNA病毒,可引起丙型肝炎和某些形式
肝癌和淋巴瘤许多感染者没有症状,这使得早期
诊断挑战在这项工作中,我们将应用噬菌体展示技术来产生亲合力-
用于捕获和检测HCV的加强和快速结合的重组亲和试剂
核心Ag在侧流测定(LFA)中的表达。这个项目是Tango Biosciences,
以及休斯顿大学理查德·威尔森教授的实验室。Tango是一家初创公司,
在组合PEP的噬菌体展示和亲合力工程中采用创新技术-
潮汐、抗体片段和抗体替代物。威尔逊博士拥有广泛的技术和
对于特定目标#1,Tango将产生重组亲和力,
通过噬菌体展示以高亲和力和快速结合HCV核心抗原(cAg)的试剂,
rates. Tango将发现单抗体和人类单链可变片段(scFvs),
通过Megaprimer Shuffled Tandem Affinity Reagents(MegaSTAR)结合cAg的不同表位。
Tango将诱变单个单抗体/scFv的编码区,并选择用于快速启动。
速率(LFA中的关键)和缓慢的解离速率。最佳单体的串联高亲合力形式/
scFv将用不同长度的接头构建,用于噬菌体展示和转化成LFA
报告纳米颗粒。我们将针对以下性能指标来优化亲和性
试剂:结合速率为108 M-1 sec-1,解离常数≤ 1 pM。在具体目标#2中,Viri-
优化亲和试剂的离子和可溶形式将由Willson博士的团队进行格式化
作为噬菌体LFA中的捕获和检测试剂。系统检测缓冲液后,
和阻断化学物质,目标将是达到cAg的检测限(LOD)(加标到
人血浆)的0.5 pM(10 pg/mL),其灵敏度足以检测85%的HCV感染,
选择。我们将通过在一系列浓度范围内加标cAg来验证优化的LFA格式
将来自不同性别、不同年龄和不同性别的商业化、去识别化人血浆,
缓解和代谢背景,以检测任何共同的生物变量。最后,最好的
将在HCV专家的实验室中评估亲和试剂和原型LFA。Suc-
这项工作的顺利完成将导致HCV cAg的原型LFA,并建立一个新的
适用于开发用于检测感染的上级LFA的搜索范例。
-1-
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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BRIAN KENNETH KAY其他文献
BRIAN KENNETH KAY的其他文献
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{{ truncateString('BRIAN KENNETH KAY', 18)}}的其他基金
High-throughput profiling of proteases with phage and arrays
使用噬菌体和阵列对蛋白酶进行高通量分析
- 批准号:
10602245 - 财政年份:2023
- 资助金额:
$ 27.56万 - 项目类别:
Building a pipeline to generate affinity reagents to phosphothreonine epitopes
建立生产磷酸苏氨酸表位亲和试剂的管道
- 批准号:
10481540 - 财政年份:2022
- 资助金额:
$ 27.56万 - 项目类别:
Enzyme-delivery scaffold technology for targeted cancer killing.
用于靶向杀死癌症的酶递送支架技术。
- 批准号:
8311639 - 财政年份:2011
- 资助金额:
$ 27.56万 - 项目类别:
Technology Development for Recombinant Affinity Reagents
重组亲和试剂技术开发
- 批准号:
8335435 - 财政年份:2011
- 资助金额:
$ 27.56万 - 项目类别:
Technology Development for Recombinant Affinity Reagents
重组亲和试剂技术开发
- 批准号:
8218358 - 财政年份:2011
- 资助金额:
$ 27.56万 - 项目类别:
Enzyme-delivery scaffold technology for targeted cancer killing.
用于靶向杀死癌症的酶递送支架技术。
- 批准号:
8518269 - 财政年份:2011
- 资助金额:
$ 27.56万 - 项目类别:
Enzyme-delivery scaffold technology for targeted cancer killing.
用于靶向杀死癌症的酶递送支架技术。
- 批准号:
8034009 - 财政年份:2011
- 资助金额:
$ 27.56万 - 项目类别:
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