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

摘要 项目摘要莱姆病的实验室诊断显然需要新的方法和更好的检测方法。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失败了。到目前为止，所有提出的血清学替代方法都不成功，不符合成本效益，或仍处于早期开发阶段。改善早期疾病的血清学检测是改善莱姆病患者预后的最快和最有效的方法。目前的血清学诊断试验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产生的抗体交叉反应，降低了特异性，并需要使用两级血清测定范式，该范式以检测早期疾病的低灵敏度为代价来保留特异性。使用肽作为血清学诊断靶显示出改善的功效，但使用一种或两种含有单一表位的肽仍然在早期疾病中提供降低的敏感性。将相同的旧抗原靶标放入新平台中，无论该平台多么创新，都不会成功地改善莱姆病的血清学诊断。靶抗原和检测都需要创新的方法。通过掺入含有对B高度特异性的线性表位的独特肽。将莱姆病的诊断方法转化为使用具有成本效益的基于智能手机的阅读器的尖端、多重、便携式基于纸张的即时诊断测定，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步骤中进行，并在现场产生答案，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检测方法，可以在床旁环境中对多达25个独立抗原靶标的IgM和IgG结合进行多重检测。VFA设计允许样品均匀流过靶膜，从而产生均匀的结合条件并最大化所产生的信号。我们将该设计与基于成本有效的便携式智能手机的读取器相结合，允许定量测量抗体结合，消除主观性。通过多重肽抗原，每个肽抗原含有B特有的1 - 2个表位。从哺乳动物感染过程中不同阶段表达的多种不同抗原中分离伯氏螺旋体，我们可以产生一个单一的层，即POC，可以特异性和灵敏地检测疾病所有阶段的患者抗体。该分析可以在不到25分钟内完成，允许快速的办公室结果，以支持临床诊断，从而改善患者的结果。

Abstract 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.