Cleavage Coupled Lateral Flow Immunoassay for Rapid Endotoxin Testing

用于快速内毒素检测的裂解偶联侧向层析免疫分析

• 批准号: 8905383
• 负责人: BRADLEY T MESSMER
• 金额: $ 21.44万
• 依托单位: ABREOS BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-01 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8905383
• 关键词: Affinity; Animals; Antibodies; Antibody Affinity; Antigens; Avidity; Binding; Binding Sites; Biological Assay; Blood Cells; Cleaved cell; Coagulation Process; Complex; Coupled; Development; Devices; Endotoxemia; Endotoxins; Enzymes; Equipment; Food; Goals; Gold; Gold Colloid; Health; Human Resources; Immune response; Immunoassay; Kinetics; Lateral; Libraries; Limulus; Link; Lipopolysaccharides; Marketing; Medical Device; Methods; Monitor; Monoclonal Antibodies; Peptide Hydrolases; Peptides; Phage Display; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Population; Reagent; Recombinants; Sampling; Septic Shock; Site; Structure; Techniques; Technology; Testing; Therapeutic Monoclonal Antibodies; Validation; Work; Xiphosura; antigen binding; arm; base; cost; detector; dimer; endotoxin binding protein; enzyme activity; factor C; glutamylalanine; instrumentation; medical food; monomer; novel; point of care; pressure; prototype; reagent testing; rituximab; sensor

 DESCRIPTION (provided by applicant): Bacterial endotoxins, namely lipopolysaccharide (LPS), are common contaminants of many pharmaceutical, medical device, and food products that can elicit very strong immune responses in people and animals, sometimes resulting in septic shock. As such, monitoring for endotoxin in medical and food products is essential and a $6 billion annual market. Current methods for detecting endotoxin are complex, expensive, and difficult to standardize. Hence, there is an unmet need for rapid, simple assays that can be used to quickly assess endotoxin levels in field or point of care settings. Lateral flow based assays (LFAs) are ideally suited for end-users that require frequent, rapid, and straightforward endotoxin testing. LFAs require very little sample, no equipment or additional reagents, and are simple, robust and produced for $1-2 each at scale. We will develop a novel cleavage coupled competitive lateral flow assay (C3-LFA) that will enable inexpensive lateral flow devices that require no external instrumentation or skilled personnel to use. The basis of the assay technology is a novel peptide construct that incorporates the cleavage site for an endotoxin-activated protease between mimetope peptides that are specifically recognized by a particular monoclonal antibody. The intact dimer form of this construct has high affinity for the antibody but the monovalent affinity of the cleaved form is considerably weaker. Thus activity can unmask the antibody's antigen binding sites previously blocked by the dimer peptide construct by cleavage to low affinity monomers. The specific aims of this Phase I application are to 1) Optimize the dimer peptide and 2) Develop the LPS C3- LFA prototype. The long term goal of this project is to develop lateral flow immunoassays that can rapidly determine endotoxin levels above or below set thresholds. We have previously developed these mimetope peptides as lateral flow based sensors for their cognate antibody and are thus well poised to leverage that technique for the unique problem of enzyme activity via lateral flow.

 描述（由申请人提供）：细菌内毒素，即脂多糖（LPS），是许多药品、医疗器械和食品中的常见污染物，可以在人和动物中引起非常强烈的免疫反应，有时会导致败血性休克。因此，监测医疗和食品中的内毒素至关重要，并且每年的市场价值达 60 亿美元。目前检测内毒素的方法复杂、昂贵且难以标准化。因此，对可用于快速评估现场或护理点环境中内毒素水平的快速、简单测定的需求尚未得到满足。侧向层析检测 (LFA) 非常适合需要频繁、快速和直接内毒素检测的最终用户。 LFA 需要的样品非常少，不需要设备或额外的试剂，而且简单、稳定，大规模生产时每个 LFA 的成本为 1-2 美元。我们将开发一种新型裂解耦合竞争性侧流测定（C3-LFA），该方法将实现不需要外部仪器或技术人员即可使用的廉价侧流装置。该检测技术的基础是一种新型肽构建体，该构建体在被特定单克隆抗体特异性识别的模拟表位肽之间整合了内毒素激活蛋白酶的切割位点。该构建体的完整二聚体形式对抗体具有高亲和力，但切割形式的单价亲和力相当弱。因此，活性可以通过裂解成低亲和力单体来揭示先前被二聚体肽构建体阻断的抗体的抗原结合位点。该第一阶段申请的具体目标是 1) 优化二聚体肽和 2) 开发 LPS C3-LFA 原型。该项目的长期目标是开发侧流免疫测定法，可以快速确定高于或低于设定阈值的内毒素水平。我们之前已经开发了这些模拟表位肽作为其同源抗体的基于侧流的传感器，因此准备好利用该技术通过侧流解决酶活性的独特问题。