Molecular Recognition Research Center, Korea Institute of Science and Technology (KIST), Seoul 02792, Korea.
Department of Materials Science and Engineering, Hallym University, Chuncheon-si, Korea and Integrative Materials Research Institute, Hallym University, Chuncheon-si, Korea.
Analyst. 2018 Jul 7;143(13):3141-3147. doi: 10.1039/c8an00714d. Epub 2018 Jun 7.
We developed an assay for diagnosing rheumatoid arthritis (RA) by capillary electrophoresis with the laser-induced fluorescence detection (CE-LIF) of antibodies against cyclic citrullinated peptides (CCPs) using fluorescein isothiocyanate-labeled F-CCP11A and F-CCP. The CCPs were incubated overnight with a purified antibody against CCP and then injected into the CE-LIF system to estimate complex concentration. The immunocomplexes and free peptides were separated within 5 min; the LOD and LOQ values of the immunoassay are 0.1 μg mL and 0.3 μg mL, respectively. The calibration curves for the immunocomplex had a coefficient of determination of 0.98. The reproducibility of the CE-LIF based immunoassay when using F-CCP11A and F-CCP was less than 5% and the accuracy ranged between 89 and 103%. The applicability of the assay was evaluated using serum samples from RA patients and healthy control subjects. We found that the sensitivity and specificity for RA diagnosis were 100% and 83.3%, respectively, for F-CCP11A and 100% and 90%, respectively, for F-CCP. These results demonstrate that the CE-LIF method can be useful for quantitative immunoassay development.
我们开发了一种通过毛细管电泳与激光诱导荧光检测（CE-LIF）来诊断类风湿关节炎（RA）的方法，该方法使用荧光素异硫氰酸酯标记的 F-CCP11A 和 F-CCP 检测针对环瓜氨酸肽（CCP）的抗体。将 CCP 与针对 CCP 的纯化抗体孵育过夜，然后注入 CE-LIF 系统以估计复合物浓度。免疫复合物和游离肽在 5 分钟内分离；免疫测定的 LOD 和 LOQ 值分别为 0.1 μg mL 和 0.3 μg mL。免疫复合物的校准曲线的决定系数为 0.98。使用 F-CCP11A 和 F-CCP 时，CE-LIF 基于免疫测定的重现性小于 5%，准确性在 89%至 103%之间。使用来自 RA 患者和健康对照者的血清样本评估了该测定法的适用性。我们发现，F-CCP11A 对 RA 诊断的灵敏度和特异性分别为 100%和 83.3%，F-CCP 分别为 100%和 90%。这些结果表明，CE-LIF 方法可用于定量免疫测定的开发。
