重症肌无力相关抗agrin抗体细胞检测方法的建立与应用。

IF 2.9 3区 医学 Q2 CLINICAL NEUROLOGY
Seon Hui Kim, Hye Yoon Chung, MinGi Kim, Seung Woo Kim, Ha Young Shin
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引用次数: 0

摘要

背景和目的:最近在重症肌无力(MG)患者中发现了抗agrin抗体(agrin Abs),有时与针对乙酰胆碱受体(AChR)、肌肉特异性酪氨酸激酶(MuSK)或低密度脂蛋白受体相关蛋白4的抗体(Abs)结合。本研究旨在开发一种基于内部细胞的检测方法(CBA),用于检测agrin Abs,并测试其在MG患者血清样本中的应用。方法:将Agrin互补DNA克隆到pCMV6-AC-GFP载体中,转染人胚胎肾293T (HEK293T)细胞。转染后的HEK293T细胞与患者血清和与红色荧光染料结合的抗人免疫球蛋白G Ab孵育。使用CBA测定389份血清样本中的Agrin Ab水平:340份来自MG患者,36份来自其他神经肌肉疾病患者,13份来自健康对照。利用荧光显微镜根据荧光强度和共定位来确定agrin Ab的存在。结果:用逆转录聚合酶链反应和Western blotting分别检测转染HEK293T细胞中agrin mRNA和蛋白的表达水平。免疫细胞化学进一步证实了细胞中Agrin的表达。340例MG患者中有2例(0.6%)检测出agin Ab阳性:191例achr阳性患者中有1例,54例musk阳性患者中有1例。结论:我们开发并验证了一种用于检测agin Abs的新型CBA,该CBA成功地用于检测MG患者血清样品中的agin Abs。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Development and Application of a Cell-Based Assay for Detecting Anti-Agrin Antibodies Associated With Myasthenia Gravis.

Background and purpose: Anti-agrin antibodies (agrin Abs) have recently been identified in patients with myasthenia gravis (MG), sometimes in conjunction with antibodies (Abs) to the acetylcholine receptor (AChR), muscle-specific tyrosine kinase (MuSK), or low-density lipoprotein receptor-related protein 4. This study aimed to develop an in-house cell-based assay (CBA) for detecting agrin Abs, and to test its application to serum samples collected from individuals diagnosed with MG.

Methods: Agrin complementary DNA as cloned into a pCMV6-AC-GFP vector, which was subsequently transfected into human embryonic kidney 293T (HEK293T) cells. Transfected HEK293T cells were incubated with patient serum and antihuman immunoglobulin G Ab conjugated with a red fluorescent dye. Agrin Ab levels were measured using the CBA in 389 serum samples: 340 from patients with MG, 36 from patients with other neuromuscular diseases, and 13 from healthy controls. The presence of agrin Ab was determined based on the fluorescence intensity and colocalization using fluorescence microscopy.

Results: The expression levels of agrin mRNA and protein in transfected HEK293T cells were confirmed using the reverse-transcription polymerase chain reaction and Western blotting, respectively. Agrin expression in cells was further confirmed by immunocytochemistry. Two (0.6%) of the 340 patients with MG tested positive for agrin Ab: 1 of 191 AChR-positive patients and 1 of 54 MuSK-positive patients.

Conclusions: We have developed and validated a novel CBA for detecting agrin Abs. This CBA was successfully applied to detect agrin Abs in serum samples obtained from individuals with MG.

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来源期刊
Journal of Clinical Neurology
Journal of Clinical Neurology 医学-临床神经学
CiteScore
4.50
自引率
6.50%
发文量
0
审稿时长
>12 weeks
期刊介绍: The JCN aims to publish the cutting-edge research from around the world. The JCN covers clinical and translational research for physicians and researchers in the field of neurology. Encompassing the entire neurological diseases, our main focus is on the common disorders including stroke, epilepsy, Parkinson''s disease, dementia, multiple sclerosis, headache, and peripheral neuropathy. Any authors affiliated with an accredited biomedical institution may submit manuscripts of original articles, review articles, and letters to the editor. The JCN will allow clinical neurologists to enrich their knowledge of patient management, education, and clinical or experimental research, and hence their professionalism.
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