A-347 用于同时定量 GFAP 和 NT-proBNP 以区分缺血性和出血性脑卒中的 Exdia TRF-LFIA 综合方法

IF 7.1 2区医学 Q1 MEDICAL LABORATORY TECHNOLOGY

Clinical chemistry Pub Date : 2024-10-02 DOI:10.1093/clinchem/hvae106.341

D R Sayyed, M Lee, S Hong, H Kim, H Kim, J Sanchez, S Choi, E Han, H Kang, J Kim, M Joan, H Kim, H Chae, J Park

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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 three test lines for NT-proBNP, GFAP, and Control. The fluorescence intensity of these test lines on the nitrocellulose membrane 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 hours). A small amount of clinical specimen (serum) was used with a well-assembled cartridge for the measurement of GFAP and NT-proBNP. Results 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. 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引用次数: 0

摘要

背景本研究的目标是建立一种高灵敏度的时间分辨荧光侧向流免疫分析法（TRF-LFIA），能够同时测量神经胶质纤维酸性蛋白（GFAP）和 B 型钠尿肽前体 N 端片段（NT-proBNP）。这种检测方法是作为一种诊断工具设计的，旨在为中风治疗提供一种算法，特别是用于区分缺血性中风（IS）和出血性中风（HS）。然而，目前还没有同时量化血清 NT-proBNP 和 GFAP 的 LFIA。方法我们开发并验证了一种新型 TRF-LFIA 法，用于同时定量检测 NT-proBNP 和 GFAP。通过采用与铕纳米粒子（EuNPs）相连的特异性单克隆抗体，这种免疫测定的灵敏度和重现性得到了显著提高。硝酸纤维素膜上的检测区域采用三明治式复合物，包含 NT-proBNP、GFAP 和对照组的三条检测线。使用内部开发的 Exdia TRF-Plus 分析仪测量硝酸纤维素膜上这些检测线的荧光强度。作为概念验证，我们招募了在特定时间（6 小时）内入院的疑似中风患者。少量临床标本（血清）与组装好的试剂盒一起用于测量 GFAP 和 NT-proBNP。结果为了优化 LFIA，研究人员研究了一种 EuNPs 连接抗体，以提高检测灵敏度、降低背景信号并缩短检测时间。Exdia TRF-LFIA 试剂盒具有线性动态检测范围宽、检测速度快、灵敏度高和特异性强等特点。经测定，NT-proBNP 的检测限为 98 pg/mL，GFAP 的检测限为 68 pg/mL，交叉反应极小。有 200 份临床人类血清样本被用于评估该平台，其相关性很高。结合 NT-proBNP 和 GFAP 的检测结果，我们制定了缺血性中风（IS）和出血性中风（HS）的临床评估算法。根据我们提出的算法，GFAP 和 NT-proBNP 的组合是区分 IS 和 HS 最有效的生物标志物组合。结论 Exdia TRF-LFIA 作为实验室体外诊断或其他护理点检测 (POCT) 应用的补充方法，显示出巨大的潜力。它的开发大大缩短了 IS 和 HS 的诊断时间。所提出的算法不仅能最大限度地减少治疗延误，还能降低患者的医疗费用。

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A-347 A Comprehensive Exdia TRF-LFIA for Simultaneous Quantification of GFAP and NT-proBNP in Distinguishing Ischemic and Hemorrhagic Stroke

Background 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. Methods 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 three test lines for NT-proBNP, GFAP, and Control. The fluorescence intensity of these test lines on the nitrocellulose membrane 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 hours). A small amount of clinical specimen (serum) was used with a well-assembled cartridge for the measurement of GFAP and NT-proBNP. Results 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. Conclusions 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.

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来源期刊

Clinical chemistry 医学-医学实验技术

CiteScore

11.30

自引率

4.30%

发文量

212

审稿时长

1.7 months

期刊介绍： Clinical Chemistry is a peer-reviewed scientific journal that is the premier publication for the science and practice of clinical laboratory medicine. It was established in 1955 and is associated with the Association for Diagnostics & Laboratory Medicine (ADLM). The journal focuses on laboratory diagnosis and management of patients, and has expanded to include other clinical laboratory disciplines such as genomics, hematology, microbiology, and toxicology. It also publishes articles relevant to clinical specialties including cardiology, endocrinology, gastroenterology, genetics, immunology, infectious diseases, maternal-fetal medicine, neurology, nutrition, oncology, and pediatrics. In addition to original research, editorials, and reviews, Clinical Chemistry features recurring sections such as clinical case studies, perspectives, podcasts, and Q&A articles. It has the highest impact factor among journals of clinical chemistry, laboratory medicine, pathology, analytical chemistry, transfusion medicine, and clinical microbiology. The journal is indexed in databases such as MEDLINE and Web of Science.