Department of Laboratory Medicine, Shenzhen Institute of Translational Medicine, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, 518035, Shenzhen, China.
School of Public Health, Dongguan Key Laboratory of Environmental Medicine, Guangdong Medical University, 523808, Guangdong, China.
Virol J. 2023 Jul 27;20(1):166. doi: 10.1186/s12985-023-02132-w.
Chikungunya virus (CHIKV) and Dengue virus (DENV) have similar clinical symptoms, which often induce misdiagnoses. Therefore, an antigen detection diagnostic system that can clearly identify these two viruses is desirable.
In this study, we developed a novel peptide with high affinity and specificity to CHIKV, and further constructed peptide aptamer-based TRFIA assay to efficiently detect CHIKV. Peptide aptamer B2 (ITPQSSTTEAEL) and B3 (DTQGSNWI) were obtained through computer-aided design and selected as CHIKV-specific peptide aptamers based on their high binding affinity, strong hydrogen bonding, and RMSD of molecular docking. Then, a sandwich-Time-Resolved Fluoroimmunoassay (TRFIA) was successfully constructed for the detection of the interaction between peptide aptamers and viruses.
When using B2 as the detection element, highly specific detection of CHIKV E2 was achieved with detection limits of 8.5 ng/ml in PBS solution. Variation coefficient between inter-assay showed the disturbances received from the detection of clinical fluid specimens (including serum and urine), were also within acceptable limits. The detection limits for 10-fold dilution serum and urine were 57.8 ng/mL and 147.3 ng/mL, respectively. The fluorescent signal intensity exhibited a good linear correlation with E2 protein concentration in the range of 0-1000 ng/mL, indicating the potential for quantitative detection of E2 protein.
These results demonstrate that the construction of peptide aptamers with high affinity and specificity provides an excellent method for rapid diagnostic element screening, and the developed peptide aptamer B2 contributed to better detection of CHIKV viral particles compared to traditional antibodies.
基孔肯雅热病毒（CHIKV）和登革热病毒（DENV）具有相似的临床症状，这往往会导致误诊。因此，需要一种能够明确区分这两种病毒的抗原检测诊断系统。
本研究中，我们开发了一种对 CHIKV 具有高亲和力和特异性的新型肽，并进一步构建了基于肽适体的 TRFIA 检测法，以有效检测 CHIKV。通过计算机辅助设计获得肽适体 B2（ITPQSSTTEAEL）和 B3（DTQGSNWI），并根据其高结合亲和力、强氢键和分子对接 RMSD 选择作为 CHIKV 特异性肽适体。然后，成功构建了夹心时间分辨荧光免疫分析（TRFIA），用于检测肽适体与病毒之间的相互作用。
当使用 B2 作为检测元件时，可实现对 CHIKV E2 的高度特异性检测，在 PBS 溶液中的检测限为 8.5ng/ml。在测定不同批次之间，从检测临床体液标本（包括血清和尿液）中获得的变异系数也在可接受范围内。对 10 倍稀释血清和尿液的检测限分别为 57.8ng/mL 和 147.3ng/mL。荧光信号强度与 E2 蛋白浓度在 0-1000ng/ml 范围内呈良好线性关系，表明具有定量检测 E2 蛋白的潜力。
这些结果表明，构建具有高亲和力和特异性的肽适体为快速诊断元件筛选提供了一种极好的方法，与传统抗体相比，开发的肽适体 B2 有助于更好地检测 CHIKV 病毒颗粒。
