Development of a Prototype Lateral Flow Immunoassay (LFI) for the Rapid Diagnosis of Melioidosis
Development of a Prototype Lateral Flow Immunoassay (LFI) for the Rapid Diagnosis of Melioidosis
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DOI：
10.1371/journal.pntd.0002727
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发表时间：
2014-03-01
影响因子：
3.8
通讯作者：
AuCoin, David P.
中科院分区：
文献类型：
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作者：
Houghton, Raymond L.;Reed, Dana E.;AuCoin, David P.
类鼻疽伯克霍尔德氏菌是一种土壤细菌，是类鼻疽病的病原体。分离B。临床样本中的假鼻疽是诊断类鼻疽病的“金标准”;结果可能需要3-7天才能产生。另外，基于抗体的检测由于流行地区血清阳性个体的高百分比而具有低特异性。显然需要开发一种用于诊断类鼻疽的快速床旁抗原检测测定。以前，我们采用体内微生物抗原发现（InMAD）来识别潜在的B。类鼻疽诊断生物标志物。B。类鼻疽荚膜多糖（CPS）和许多蛋白质抗原被鉴定为潜在的候选物。在这里，我们描述了基于CPS检测的诊断免疫测定的发展。在生产CPS特异性单克隆抗体（mAb）后，开发了抗原捕获免疫测定法，以测定一组类鼻疽患者血清和尿液样本中的CPS浓度。使用相同的mAb制备原型活动性类鼻疽检测侧流免疫测定法（AMD LFI）; CPS的LFI检测限与抗原捕获免疫测定法相当（类似于0.2 ng/ml）。当针对大样本组B进行检测时，AMD LFI的分析反应性（包容性）为98.7%（76/77）。类鼻疽分离株。分析特异性（交叉反应性）检测确定97.2%的B。邻近种的类鼻疽（35/36）无反应。非反应性B。假鼻疽菌株和反应性近邻菌株之间的差异可以通过基因序列分析来解释。重要的是，我们表明AMD LFI能够检测各种患者样本中的CPS。LFI目前正在泰国和澳大利亚进行评价;重点是在启动大型多中心临床前评价之前优化和验证类鼻疽患者样本的检测程序。
Burkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. Isolation of B. pseudomallei from clinical samples is the "gold standard'' for the diagnosis of melioidosis; results can take 3-7 days to produce. Alternatively, antibody-based tests have low specificity due to a high percentage of seropositive individuals in endemic areas. There is a clear need to develop a rapid point-of-care antigen detection assay for the diagnosis of melioidosis. Previously, we employed In vivo Microbial Antigen Discovery (InMAD) to identify potential B. pseudomallei diagnostic biomarkers. The B. pseudomallei capsular polysaccharide (CPS) and numerous protein antigens were identified as potential candidates. Here, we describe the development of a diagnostic immunoassay based on the detection of CPS. Following production of a CPS-specific monoclonal antibody (mAb), an antigen-capture immunoassay was developed to determine the concentration of CPS within a panel of melioidosis patient serum and urine samples. The same mAb was used to produce a prototype Active Melioidosis Detect Lateral Flow Immunoassay (AMD LFI); the limit of detection of the LFI for CPS is comparable to the antigen-capture immunoassay (similar to 0.2 ng/ml). The analytical reactivity (inclusivity) of the AMD LFI was 98.7% (76/77) when tested against a large panel of B. pseudomallei isolates. Analytical specificity (cross-reactivity) testing determined that 97.2% of B. pseudomallei near neighbor species (35/36) were not reactive. The non-reactive B. pseudomallei strain and the reactive near neighbor strain can be explained through genetic sequence analysis. Importantly, we show the AMD LFI is capable of detecting CPS in a variety of patient samples. The LFI is currently being evaluated in Thailand and Australia; the focus is to optimize and validate testing procedures on melioidosis patient samples prior to initiation of a large, multisite pre-clinical evaluation.