Point-of-care immunoassay for early diagnosis of pertussis

描述(申请人提供)：百日咳是由百日咳杆菌引起的一种严重且可能危及生命的呼吸道疾病。在世界范围内，每年有1840万人患上百日咳，其中25.4万人死亡。尽管美国的疫苗覆盖率很高，但2012年美国报告的百日咳病例仍超过4.8万例。百日咳的快速诊断至关重要，因为只有在疾病早期开始治疗和控制暴发才有效。目前，还没有能够快速准确地检测早期百日咳的检测方法。其目标是开发一种快速、护理点(POC)免疫分析方法，以检测疾病早期的百日咳杆菌感染。目标人群将是出现早期百日咳非特异性呼吸道感染症状的婴儿(在发作性咳嗽之前)。该方法将通过侧向流动免疫分析(LFI)检测鼻咽拭子中的百日咳杆菌抗原。该产品将是一种与i)美国儿科医生办公室和ii)低资源环境下的国际诊所相关的检测方法：一种敏感和特异的检测方法，无需专门设备或用户专业知识，即可在5-10分钟内以极低的成本得出结果。在初步研究中，我们使用生物信息学分析来鉴定五种高优先级百日咳杆菌蛋白抗原，以供进一步研究。具体目标1将开发检测百日咳杆菌抗原的表位特异性多克隆抗体(PAB)。将对PAB进行筛选，以选择I)与百日咳杆菌蛋白反应的那些， Ii)与百日咳杆菌反应，但不能与鼻咽中发现的其他细菌反应，iii)可以成对发挥作用，以夹心形式结合构建LFI所需的抗原，以及iv)检测患者鼻咽样本中的抗原。只有满足所有标准的PAB才会进入特定目标2的LFI原型开发。特定目标2将通过优化LFI原型的物理和生物成分来确定LFI检测百日咳杆菌的下限。根据先前确定的婴儿NP拭子中每个拭子106个细菌的百日咳杆菌载量，敏感度的基准是每个拭子检测105个cfu。总而言之，第一阶段的实验将确定哪些百日咳杆菌抗原表位可以产生具有所需灵敏度的LFI原型的PAb对。第二阶段研究将开发针对已验证表位的单抗，将它们合并到LFI中，并进行详细的临床前研究。第三阶段全面临床试验的成功导航最终将产生一种POC免疫分析，该方法可以显著提高百日咳的早期诊断，限制百日咳在社区暴发期间的传播，并挽救患者的生命。

DESCRIPTION (provided by applicant): Pertussis is a serious and potentially life-threatening respiratory disease caused by the bacterium Bordetella pertussis. Worldwide, 18.4 million people become ill with pertussis each year, and 254,000 of these patients die. Despite high vaccine coverage in the United States, there were still over 48,000 pertussis cases reported in the U.S. in 2012. Rapid diagnosis of pertussis is critical because treatment and outbreak containment are only effective if initiated early in the disease. Currently, there are no assays that can rapidly and accurately detect early-stage pertussis. The goal is to develop a rapid, point-of-care (POC) immunoassay to detect B. pertussis during early disease. The target population will be infants who present with the non-specific, respiratory tract infection symptoms of early pertussis (prior to onset of paroxysmal cough). The approach will be detection of B. pertussis antigens from nasopharyngeal swabs by lateral flow immunoassay (LFI). The product will be an assay relevant to i) U.S. pediatrician offices, and ii) international clinics in low-resource settings: a sensitive and specific test that delivers results in 5-10min at very low cost without specialized equipment or user expertise. In preliminary studies, we used a bioinformatics analysis to identify five high priority B. pertussis protein antigens for further stdy. Specific Aim 1 will develop epitope-specific polyclonal antibodies (pAbs) for detection of B. pertussis antigens. pAbs will be screened to select those that i) react with B. pertussis proteins, ii) react with B. pertussis but not other bacteria found in the nasopharynx, iii) can function in pairs to bind the antigen in the sandwich format necessary for LFI construction, and iv) detect antigen in patient nasopharyngeal samples. Only pAbs that meet all the criteria will be advanced to LFI prototype development in Specific Aim 2. Specific Aim 2 will determine the limit of detection of B. pertussis by LFI by optimizing the physical and biological components of the LFI prototypes. Based on previously determined B. pertussis loads in infant NP swabs of 106 bacteria per swab, the benchmark for sensitivity is detection of 105 CFU per swab. Together, the experiments of Phase I will determine which B. pertussis antigen epitopes can produce pAb pairs that yield LFI prototypes with the required sensitivity. A Phase II study would develop monoclonal antibodies (mAbs) against the validated epitopes, incorporate them into an LFI, and perform detailed pre-clinical studies. Successful navigation of a full clinical trial in Phase III would ultimately yield a POC immunoassay that could dramatically increase early pertussis diagnosis, limit pertussis transmission during community outbreaks, and save patient lives.