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(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，可在人和动物中引发非常强烈的免疫反应，有时会导致感染性休克。因此，监测医疗和食品产品中的内毒素是必不可少的，这是一个每年60亿美元的市场。目前检测内毒素的方法复杂、昂贵、难以标准化。因此，对快速、简单的检测方法的需求尚未得到满足，这些检测方法可用于在现场或护理场所快速评估内毒素水平。基于侧向流动的检测(LFA)非常适合需要频繁、快速和直接的内毒素检测的最终用户。LFA需要很少的样品，不需要设备或额外的试剂，而且简单、坚固，每台规模生产成本为1-2美元。我们将开发一种新的裂解耦合竞争侧向流动分析(C3-LFA)，它将使不需要外部仪器或熟练人员使用的廉价侧向流动装置成为可能。该检测技术的基础是一种新的多肽结构，该结构结合了内毒素激活的蛋白酶在特定单抗特异性识别的模拟表位多肽之间的切割位点。该结构的完整二聚体形式与抗体有很高的亲和力，但切割形式的单价亲和力要弱得多。因此，活性可以揭开抗体的抗原结合部位，先前通过裂解低亲和力单体而被二聚体多肽结构阻断。这一阶段应用的具体目标是1)优化二聚肽和2)开发LPSC3-LFA原型。该项目的长期目标是开发能够快速确定高于或低于设定阈值的内毒素水平的侧向流动免疫分析方法。我们以前已经开发了这些模拟表位多肽作为基于横向流动的传感器来检测它们的同源抗体，因此可以很好地利用这一技术通过横向流动来解决酶活性的独特问题。