Point-of-care immunoassay for early diagnosis of pertussis

用于早期诊断百日咳的即时免疫分析

• 批准号: 9302246
• 负责人: Amanda Burnham-Marusich
• 金额: $ 35.13万
• 依托单位: DXDISCOVERY, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-01 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9302246
• 关键词: Affinity; Animal Model; Annual Reports; Antigen Targeting; Antigens; Aspirate substance; Bedside Testings; Bioinformatics; Biological Assay; Biological Markers; Bordetella pertussis; Case Study; Cells; Clinic; Clinical; Clinical Sensitivity; Coughing; Country; Data; Detection; Diagnosis; Diagnostic; Disease; Disease Outbreaks; Early Diagnosis; Early treatment; Economics; Enzyme-Linked Immunosorbent Assay; Epitopes; Equipment; Evaluation; Goals; Health Personnel; Hospitals; Immunoassay; Infant; Infection; Lateral; Libraries; Measures; Modeling; Monoclonal Antibodies; Morbidity - disease rate; Papio; Patient Care; Patients; Performance; Pertussis; Phase; Protein Isoforms; Public Health; Reproducibility; Respiratory Tract Infections; Sampling; Sensitivity and Specificity; Severity of illness; Ships; Streptococcal Infections; Swab; Symptoms; Target Populations; Technology; Test Result; Testing; Time; United States; Vaccines; base; clinically relevant; commercialization; cost; design; factor A; illness length; improved; innovation; large scale production; pathogen; pediatrician; point of care; point-of-care diagnostics; polyclonal antibody; prevent; protein aminoacid sequence; prototype; rapid diagnosis; sample collection; transmission process; urgent care

Project Summary Pertussis is rapidly re-emerging as a serious public health threat in the United States. Despite high vaccine coverage nationally, annual reported cases in the U.S. have been increasing recently, with a 57-year high of 48,000 in 2012. Moreover, reported cases represent a large underestimate of pertussis infections. Diagnosis of early pertussis (catarrhal stage; prior to paroxysmal cough) is particularly challenging because its symptoms are non-specific and because there are no assays that can diagnose pertussis at the point of patient care. There is a critical need to develop improved pertussis diagnostics to fill this gap because pertussis treatment reduces disease severity and duration, but only if treatment begins prior to paroxysmal cough. The goal of this project is to develop a rapid (<15min), point-of-care (POC) immunoassay to detect Bordetella pertussis during early disease. The target population will be infants with the non-specific, respiratory tract infection symptoms of early-stage pertussis. The approach will be detection of B. pertussis antigens from nasopharyngeal (NP) samples by lateral flow immunoassay (LFI). The POC diagnostic that we propose is innovative because it will change the current clinical status quo: a pertussis LFI will give healthcare providers immediate access to actionable and relevant information, which will enable rapid and appropriate patient care. In Phase I, we demonstrated the feasibility of our LFI approach and achieved every milestone of our Phase I Specific Aims. Specifically, we used an innovative bioinformatics-based strategy to develop epitope-specific polyclonal antibodies (pAbs) against a B. pertussis antigen that has both cell-associated and secreted isoforms. We then validated our pAbs for reactivity with both isoforms in sensitive and specific LFI prototypes. The limit of detection of our current pAb-based LFI is 1.6 x 105 CFU, which is well below the typical bacterial burden of infant NP washes (107 to 1010 CFU/ml) or swabs (106 CFU). In Phase II, we will build on our Phase I results and develop an advanced monoclonal antibody (mAb) based LFI commercial diagnostic. We will focus on developing mAbs against the validated biomarker epitopes (Aim 1) and incorporating these mAbs into an LFI with sensitivity and commercialization potential superior to that of the Phase I pAb-based prototypes. We will i) optimize the mAb-based LFI for analytical sensitivity in NP samples (Aim 2), ii) determine clinical sensitivity and specificity at different disease stages in a clinically relevant, baboon infection model (Aim 3), and iii) determine the LFI's limit of detection with patient samples (Aim 4). Together, this data will guide our FDA 510(k) Pre-Submission at the end of Phase II. Successful completion of our milestones will ultimately yield a rapid, affordable, POC immunoassay that will dramatically increase early pertussis diagnosis, which will i) initiate prompt treatment, ii) reduce disease severity and duration, iii) limit outbreaks by preventing unnecessary transmission, and iv) save infant lives.

项目摘要 百日咳正迅速在美国重新成为严重的公共卫生威胁。尽管疫苗价格很高 在全国范围内，美国的年度报告病例最近一直在增加，达到57年来的最高水平 2012年为4.8万人。此外，报告的病例大大低估了百日咳感染。诊断 早期百日咳(卡他期；阵发性咳嗽之前)尤其具有挑战性，因为它的症状 是非特异性的，因为在病人护理时没有可以诊断百日咳的检测方法。 迫切需要开发改进的百日咳诊断方法来填补这一空白，因为百日咳的治疗 减少疾病的严重程度和持续时间，但只有在阵发性咳嗽之前开始治疗。 该项目的目标是开发一种快速(15分钟)、护理点(POC)免疫分析方法来检测波尔德氏菌 疾病早期的百日咳。目标人群将是患有非特异性呼吸道疾病的婴儿 早期百日咳感染症状。该方法将检测百日咳杆菌抗原。 鼻咽(NP)标本采用侧向流动免疫分析(LFI)。我们建议的PoC诊断是 创新是因为它将改变目前的临床现状：百日咳LFI将为医疗保健提供者提供 立即获得可采取行动的相关信息，这将使快速和适当的患者护理成为可能。 在第一阶段，我们证明了我们LFI方法的可行性，并实现了第一阶段的每一个里程碑 明确的目标。具体地说，我们使用了一种创新的基于生物信息学的策略来开发表位特异性 抗百日咳杆菌细胞相关抗原和分泌型抗原的多克隆抗体 异构体。然后，我们验证了我们的Pabs在敏感和特定的LFI原型中与两种异构体的反应性。 我们目前基于PAb的LFI的检测极限是1.6×105CFU，远远低于典型的细菌 婴儿NP负担洗涤(107至1010 cfu/ml)或拭子(106 Cfu)。 在第二阶段，我们将在第一阶段结果的基础上，开发一种先进的基于单抗的 LFI商业诊断。我们将重点开发针对经过验证的生物标记表位的单抗(目标1) 并将这些单抗整合到LFI中，其灵敏度和商业化潜力优于 第一阶段以PAB为基础的原型。我们将优化基于单抗的LFI在NP样品中的分析灵敏度 (目标2)，二)确定临床相关疾病不同阶段的临床敏感性和特异性， 狒狒感染模式(目标3)，以及三)确定LFI对患者样本的检测下限(目标4)。 总而言之，这些数据将指导我们在第二阶段结束时提交FDA 510(K)。成功完成 我们的里程碑最终将产生一种快速、负担得起的POC免疫分析，它将在早期大幅增加 百日咳的诊断，这将：一)迅速开始治疗，二)减少疾病的严重程度和病程，三)限制 通过防止不必要的传播爆发疫情，以及iv)拯救婴儿的生命。