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（由适用提供）：细菌内毒素，即脂多糖（LPS），是许多药物，医疗装置和食品的常见污染物，它们可以引起人和动物中非常强烈的免疫剂，有时会导致脓毒症。因此，对医疗和食品中内毒素的监测至关重要，每年60亿美元。当前检测内毒素的方法复杂，昂贵且难以标准化。因此，对快速，简单的评估的需求未满足，可以用来快速评估现场或护理点中的内毒素水平。基于侧流的评估（LFA）非常适合经常，快速和直接内毒素测试的最终用户。 LFAS所需的样品很少，没有设备或其他试剂，并且简单，健壮且每次生产$ 1-2。我们将开发一种新型的乳沟耦合竞争性横向流量测定法（C3-LFA），该测定法将实现不需要外部仪器或熟练人员使用的廉价侧向流动设备。测定技术的基础是一种新型的肽构建体，该肽结合了内毒素激活的蛋白酶的裂解位点，这是在薄弱的宠物中，这些蛋白酶被特定的单克隆抗体特别识别。该构建体的完整二聚体形式对抗体具有高亲和力，但裂解形式的单价亲和力被认为较弱。该活性可以揭开抗体的抗原结合位点，以前通过二聚体肽构建体通过裂解对低亲和力单体阻塞。该阶段I应用程序的具体目的是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横向流动免疫测定，这些侧向流免疫测定可以快速确定高于或以下阈值的内毒素水平。我们以前已经开发了这些Mimetope Petides作为其同源抗体的基于侧向流动的传感器，因此被充分毒化，可以通过横向流动来利用该技术来解决酶活性的独特问题。