a Department of Microbiology , Rajendra Memorial Research Institute of Medical Sciences , Patna 800007 , India.
b Department of Bioinformatics , Rajendra Memorial Research Institute of Medical Sciences , Patna 800007 , India.
J Biomol Struct Dyn. 2017 Dec;35(16):3569-3580. doi: 10.1080/07391102.2016.1263240. Epub 2016 Dec 26.
Diagnosis of visceral leishmaniasis (VL) is often hindered by cross-reactions with antigens from other related parasite infections. This study aimed to develop an immunochromatographic test (ICT) which can detect the antigen present in circulating immune complexes (CICs) of VL patients using B-cell epitope-specific antibodies. MS analysis of six immunoreactive 2DE spots revealed two epitopes i.e. RFFVQGDGIGQHSLQEALERR (P) and RRVAVLVLLDRL (P) (From a hypothetical protein [Acc No: XP_003861458.1]). The epitope conservancy analysis suggested that the linear epitope (PP) is 97-100% conserved among Leishmania species and diverged from Homo sapiens (61% query coverage and 80% identity). Further, immunoinformatics analysis of hydrophilicity and flexibility confirmed the antigenicity of the peptide fragment. The linear epitope (PP) was synthesized (98% purity) and the purity was confirmed by high-performance liquid chromatography and MS. The indirect Enzyme linked immunosorbent assay results confirmed the presence of the corresponding antibody in VL patient's sera but not in those of healthy and other diseases. The result demonstrated a sensitivity 90%; Se Cl95% (82.16-96.27)% and specificity 100%; Sp Cl95% (84.56-100)% which indicated the possibility to be used as a diagnostic tool. Sensitivity, specificity, and diagnostic efficiency of colloidal gold conjugated anti-PP antibody ICT strip was 100, 95.2, and 96.7%, respectively, which is slightly better as compared to other ICT for VL. Though, our result indicated the utility of anti-PP antibody to detect CICs epitopes, a large-scale inspection in endemic and non-endemic area and in different ethnic population is needed for its validation and authentication.
内脏利什曼病（VL）的诊断常常受到与其他相关寄生虫感染的抗原交叉反应的阻碍。本研究旨在开发一种免疫层析试验（ICT），该试验使用 B 细胞表位特异性抗体检测 VL 患者循环免疫复合物（CIC）中的抗原。通过对六个免疫反应性 2DE 斑点的 MS 分析，揭示了两个表位，即 RFFVQGDGIGQHSLQEALERR（P）和 RRVAVLVLLDRL（P）（来自一个假设蛋白[Acc No: XP_003861458.1]）。表位保守性分析表明，线性表位（PP）在利什曼原虫种间高度保守，与智人（61%查询覆盖率和 80%同一性）不同。此外，亲水和柔性的免疫信息学分析证实了肽片段的抗原性。线性表位（PP）被合成（98%纯度），并通过高效液相色谱和 MS 确认了纯度。间接酶联免疫吸附试验结果证实了 VL 患者血清中存在相应的抗体，但在健康人和其他疾病患者的血清中不存在。结果表明，该试验具有 90%的敏感性；95%的置信区间（82.16-96.27）的特异性和 100%的特异性；95%的置信区间（84.56-100）的阳性预测值，表明其有可能成为一种诊断工具。胶体金偶联抗-PP 抗体 ICT 条的敏感性、特异性和诊断效率分别为 100%、95.2%和 96.7%，略优于其他用于 VL 的 ICT。尽管我们的结果表明抗-PP 抗体可用于检测 CIC 表位，但需要在流行地区和非流行地区以及不同种族人群中进行大规模检查，以验证和确认其效用。
