Department of Chemistry, University of Utah, Salt Lake City, UT, 84112, USA; Nano Institute of Utah, University of Utah, Salt Lake City, UT, 84112, USA.
Department of Chemical Engineering, University of Utah, Salt Lake City, UT, 84112, USA; Nano Institute of Utah, University of Utah, Salt Lake City, UT, 84112, USA.
Anal Chim Acta. 2019 Jan 10;1046:140-147. doi: 10.1016/j.aca.2018.09.037. Epub 2018 Sep 18.
The development of an accurate and rapid diagnostic test for tuberculosis (TB) to use at point of need is vital to efforts aimed at reducing the global burden from this disease. This paper builds on our previous studies of mannose-capped lipoarabinomannan (ManLAM) as a serum biomarker for active TB infection by means of a heterogeneous immunoassay. That work found that complexation with components in serum (e.g., proteins) sterically hindered the capture and/or labeling of ManLAM in an immunoassay at levels <10 ng mL, compromising the clinical utility of this biomarker for detection of active TB infection. We also showed that the acidification of ManLAM-containing serum samples with perchloric acid improved the detectability of ManLAM by 250× by complex disruption when compared to measurements of untreated serum. The present study examined what effects the PCA treatment of serum samples may have on the recovery and structural integrity of ManLAM, owing to its potential susceptibility to acid hydrolysis. Recovery was assessed with an enzyme-linked immunosorbent assay (ELISA). The possible impact of acid hydrolysis on the ManLAM structure was investigated by gas chromatography-mass spectrometry and carbohydrate chemical degradation methods. The ELISA study indicated that while the signal strength for ManLAM in the serum spike-in experiments was significantly stronger after PCA pretreatment when compared to untreated human serum, it was only ∼20% of the ManLAM measured in physiological buffer. This loss in detectability was shown by structural analysis to arise mainly from the acid-induced degradation of the arabinan domains of ManLAM that are targeted by antibodies used for antigen capture and/or tagging. The implications of these findings in terms of the detection of this important biomarker for TB are also discussed.
开发一种准确、快速的诊断测试方法，用于在需要时进行检测，对于降低全球结核病负担的努力至关重要。本文基于我们之前关于甘露糖封端的脂阿拉伯甘露聚糖（ManLAM）作为一种用于检测活动性结核病感染的血清生物标志物的研究，采用异质免疫测定法进行研究。该研究发现，在血清中与成分（如蛋白质）络合，在免疫测定中阻碍了 ManLAM 的捕获和/或标记，在<10ng/ml 时，该生物标志物用于检测活动性结核病感染的临床实用性受到限制。我们还表明，与未处理的血清相比，用高氯酸酸化含 ManLAM 的血清样品通过络合破坏，可将 ManLAM 的可检测性提高 250 倍。本研究考察了 PCA 处理血清样品可能对 ManLAM 的回收率和结构完整性产生的影响，因为它可能容易受到酸水解的影响。采用酶联免疫吸附测定（ELISA）评估回收率。通过气相色谱-质谱和碳水化合物化学降解方法研究了酸水解对 ManLAM 结构的可能影响。ELISA 研究表明，与未经 PCA 预处理的人血清相比，血清加标实验中 ManLAM 的信号强度在 PCA 预处理后显著增强，但仅为生理缓冲液中测量的 ManLAM 的 20%左右。结构分析表明，这种检测能力的丧失主要是由于阿拉伯聚糖结构域的酸诱导降解所致，该结构域是用于抗原捕获和/或标记的抗体的靶标。还讨论了这些发现对 TB 重要生物标志物检测的影响。
