A peptide-based point-of-care vertical flow assay for the rapid diagnosis of Lyme disease

项目摘要 有一个明显的需要新的方法和更好的检测莱姆病的实验室诊断。 所有开发用于直接检测感染患者中伯氏疏螺旋体的实用测定的尝试都是在以下方面进行的： 失败.到目前为止，所有提出的血清学替代方法都是不成功的，不符合成本效益，或仍在进行中。 早期发展。改善早期疾病的血清学检测是最快和最有效的方法， 改善莱姆病患者预后。目前的血清诊断测定中使用的抗原靶标具有 相当大的缺陷。它们通常含有保守的表位，这些表位与针对其他抗原的抗体交叉反应。 抗原，降低特异性并需要使用保留特异性的两级血清测定范例 其代价是早期疾病检测的灵敏度差。肽作为血清诊断靶标的用途 显示出改善的功效，但使用含有单一表位的一种或两种肽仍然提供了再治疗。 在早期疾病中降低敏感性。把相同的旧抗原靶点放到新的平台上，无论如何- 创新的平台，将不会成功地改善莱姆病的血清诊断。目标抗- 基因和化验需要一种创新的方法。通过掺入含有线性表位的独特肽 对B高度特异。burgdorferi，转化为一种尖端的，多功能的，便携式的基于纸张的床旁诊断 通过使用具有成本效益的基于智能手机的阅读器的分析，我们的目标是改变莱姆病的诊断。 莱姆病的理想测试可以在一个单一的步骤中进行，并在现场产生一个答案，以支持 诊断和指导临床治疗过程。我们开发了一种创新的垂直流分析方法 其允许多重检测IgM和IgG结合的多达25个独立的抗原靶标， 定点护理环境。VFA设计允许样品均匀流过目标膜， 均匀的结合条件和最大化的显影信号。我们将这种设计与具有成本效益的 基于便携式智能手机的阅读器，允许定量测量抗体结合，消除主观性， ity。通过多重肽抗原，每个抗原含有1-2个B特有的表位。从多个不同的burgdorferi- 在哺乳动物感染期间的不同阶段表达的ent抗原，我们可以产生单一层，POC作为- 可以特异性和灵敏地检测疾病各个阶段的患者抗体。该测定可以是 在不到25分钟内完成，允许快速在办公室结果，以支持临床诊断，从而提高 患者结局。

Project Summary There is an obvious need for new approaches and better assays for the laboratory diagnosis of Lyme disease. All attempts to develop a practical assay for the direct detection of Borrelia burgdorferi in infected patients have failed. Thus far, all proposed alternatives to serology have been unsuccessful, not cost-effective, or are still in early development. Improving serological detection in early disease is the fastest and most effective way to improve patient outcomes in Lyme disease. The antigen targets utilized in current serodiagnostic assays have considerable defects. They often contain conserved epitopes that cross-react with antibodies raised to other antigens, reducing specificity and requiring the use of a two-tier seroassay paradigm that preserves specificity at the cost of poor sensitivity in the detection of early disease. The use of peptides as serodiagnostic targets demonstrate improved efficacy, but the use of one or two peptides containing single epitopes still provides re- duced sensitivity in early disease. Putting the same old antigen targets into new platforms, no matter how in- novative the platform, will not succeed in improving serodiagnostics for Lyme disease. Both the target anti- gens and assays need an innovative approach. By incorporating unique peptides containing linear epitopes highly specific to B. burgdorferi, into a cutting-edge, multiplex, portable paper-based point-of-care diagnostic assay that uses a cost-effective smartphone-based reader, we aim to transform the diagnosis of Lyme disease. An ideal test for Lyme disease could be performed in a single step and yield an answer on the spot to support diagnosis and direct the course of clinical treatment. We have developed an innovative vertical-flow assay which allows for the multiplexed detection of IgM and IgG binding of up to 25 independent antigen targets in a point-of-care setting. The VFA design allows for uniform flow of sample across the target membrane, creating uniform binding conditions and maximizing developed signal. We coupled this design with a cost-effective portable smart-phone based reader allowing for quantitative measure of antibody binding, eliminating subjectiv- ity. By multiplexing peptide antigens each containing 1-2 epitopes unique to B. burgdorferi from multiple differ- ent antigens expressed at different stages during mammalian infection we can generate a single tier, POC as- say that can specifically and sensitively detect patient antibody at all stages of the disease. The assay can be completed in less than 25 min allowing for rapid in office results to support clinical diagnosis, thereby improving patient outcomes.