Lee Minki, Rafiq Sayyed Danishmalik, Kim Hyejeong, Sanchez Jean-Charles, Sik Hong Sung, Choi Sehee, Kim Hyunghoon, Han Eunhee, Won Kang Hye, Min Kim Jeong, Joan Montaner, Kim Hanshin, Chae Hyojin, Park Jong-Myeon
Precision Biosensor, 306, Techno 2-ro, Yuseong-gu, Daejeon 34036, South Korea.
Department of Laboratory Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea.
Clin Chim Acta. 2024 Apr 15;557:117872. doi: 10.1016/j.cca.2024.117872. Epub 2024 Mar 11.
The goal of this study is to create a highly sensitive time-resolved fluorescence lateral flow immunoassay (TRF-LFIA) capable of concurrently measuring glial fibrillary acidic protein (GFAP) and the N-terminal fragment of B-type natriuretic peptide precursor (NT-proBNP). This assay is designed as a diagnostic tool and aims to provide an algorithm for stroke management, specifically for distinguishing between Ischemic stroke (IS) and Hemorrhagic stroke (HS). However, LFIA to quantify simultaneous serum NT-proBNP and GFAP are not yet available. We have developed and validated a novel TRF-LFIA for the simultaneous quantitative detection of NT-proBNP and GFAP. The sensitivity and reproducibility of the immunoassay were significantly improved by employing specific monoclonal antibodies linked to europium nanoparticles (EuNPs) that specifically target NT-proBNP and GFAP. The detection area on the nitrocellulose membrane featured sandwich-style complexes containing two test lines for NT-proBNP and GFAP, and one Control line. The fluorescence intensity of these test lines and control line was measured using an in-house developed Exdia TRF-Plus analyzer. As proof-of-concept, we enrolled patients suspected of having a stroke who were admitted within a specific time frame (6 h). A small amount of clinical specimen (serum) was used. To optimize the LFIA, an EuNPs conjugated antibodies were investigated to improve the detection sensitivity and decrease the background signal as well shorten the detection time. The Exdia TRF-LFIA cartridge offers a wide linear dynamic detection range, rapid detection, high sensitivity, and specificity. The limit of detection was determined to be 98 pg/mL for NT-proBNP and 68 pg/mL for GFAP, with minimal cross-reactivity. There were 200 clinical human serum samples that were used to evaluate this platform with high correlation. By combining the results of NT-proBNP and GFAP, we formulated an algorithm for the clinical assessment of Ischemic Stroke (IS) and Hemorrhagic Stroke (HS). According to our proposed algorithm, the combination of GFAP and NT-proBNP emerged as the most effective biomarker combination for distinguishing between IS and HS. Exdia TRF-LFIA shows great potential as a supplemental method for in vitro diagnostics in the laboratory or in other point-of-care testing (POCT) applications. Its development substantially decreases the diagnosis time for IS and HS. The proposed algorithm not only minimizes treatment delays but also lowers medical costs for patients.
本研究的目标是创建一种高灵敏度的时间分辨荧光侧向流动免疫分析方法（TRF-LFIA），能够同时检测胶质纤维酸性蛋白（GFAP）和B型利钠肽前体的N端片段（NT-proBNP）。该分析方法被设计为一种诊断工具，旨在提供一种用于中风管理的算法，特别是用于区分缺血性中风（IS）和出血性中风（HS）。然而，用于同时定量检测血清NT-proBNP和GFAP的LFIA尚未出现。我们已经开发并验证了一种用于同时定量检测NT-proBNP和GFAP的新型TRF-LFIA。通过使用与铕纳米颗粒（EuNPs）连接的特异性单克隆抗体，该免疫分析方法的灵敏度和重现性得到了显著提高，这些抗体特异性靶向NT-proBNP和GFAP。硝酸纤维素膜上的检测区域具有夹心式复合物，包含两条用于NT-proBNP和GFAP的检测线以及一条对照线。使用自行开发的Exdia TRF-Plus分析仪测量这些检测线和对照线的荧光强度。作为概念验证，我们纳入了在特定时间范围内（6小时）入院的疑似中风患者。使用了少量临床标本（血清）。为了优化LFIA，研究了EuNPs偶联抗体以提高检测灵敏度、降低背景信号并缩短检测时间。Exdia TRF-LFIA试剂盒具有宽线性动态检测范围、快速检测、高灵敏度和特异性。NT-proBNP的检测限确定为98 pg/mL，GFAP的检测限为68 pg/mL，交叉反应最小。使用了200份临床人血清样本对该平台进行了高度相关性评估。通过结合NT-proBNP和GFAP的结果，我们制定了一种用于缺血性中风（IS）和出血性中风（HS）临床评估的算法。根据我们提出的算法，GFAP和NT-proBNP的组合成为区分IS和HS最有效的生物标志物组合。Exdia TRF-LFIA作为实验室体外诊断或其他即时检测（POCT）应用的补充方法显示出巨大潜力。它的开发大大缩短了IS和HS的诊断时间。所提出的算法不仅最大限度地减少了治疗延迟，还降低了患者的医疗成本。
