Cleavage Coupled Lateral Flow Immunoassay for Rapid Endotoxin Testing

 描述（由申请人提供）：细菌内毒素，即脂多糖（LPS），是许多药品、医疗器械和食品的常见污染物，可在人和动物中引起非常强烈的免疫反应，有时导致败血性休克。因此，监测医疗和食品中的内毒素是必不可少的，每年有60亿美元的市场。目前用于检测内毒素的方法复杂、昂贵且难以标准化。因此，对于可用于在现场或护理点环境中快速评估内毒素水平的快速、简单的测定存在未满足的需求。基于侧向流的检测试剂盒（LFA）非常适合需要频繁、快速和简单的内毒素检测的最终用户。LFA需要很少的样品，没有设备或额外的试剂，并且简单，耐用，大规模生产每个1 -2美元。我们将开发一种新的裂解偶联竞争性侧流测定（C3-LFA），这将使廉价的侧流装置，不需要外部仪器或熟练的人员使用。该测定技术的基础是一种新的肽构建体，其在由特定单克隆抗体特异性识别的模拟肽之间掺入内毒素活化蛋白酶的切割位点。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，活性可以通过切割成低亲和力单体来暴露先前被二聚体肽构建体阻断的抗体的抗原结合位点。该I期申请的具体目标是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能够快速测定内毒素水平高于或低于设定阈值的侧向流动免疫测定法。我们先前已经开发了这些模拟肽作为其同源抗体的基于侧向流动的传感器，因此很好地准备利用该技术来解决通过侧向流动的酶活性的独特问题。

 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.