Standardized method for the detection of antibodies to aquaporin-4 based on a highly sensitive immunofluorescence assay employing recombinant target antigen
Standardized method for the detection of antibodies to aquaporin-4 based on a highly sensitive immunofluorescence assay employing recombinant target antigen
复制标题
DOI：
10.1016/j.jns.2010.01.002
复制
发表时间：
2010-04-15
影响因子：
4.4
通讯作者：
Wandinger, K. P.
中科院分区：
文献类型：
--
作者：
Jarius, S.;Probst, C.;Wandinger, K. P.
背景：近年来，在视神经脊髓炎患者中发现了一种高度特异性的血清自身抗体NMO-IgG，并确定了中枢神经系统中最丰富的水通道水通道蛋白-4为其靶抗原。已有几种检测NMO-IgG/AQP4-Ab的方法。基于重组人AQP4的试验已被反复证明比以前的金标准试验（即小鼠脑组织免疫组织化学（IHC））更敏感。然而，所应用的复杂技术限制了它们在世界范围内的少数实验室的可用性。目的：建立一种简便易行的重组免疫荧光法（rIFA），用于标准化、高通量检测NMO-IgG/AQP4-Ab。方法：用重组人AQP4全长基因大规模转染HEK293细胞。将固定的细胞切成毫米大小的碎片并转移到显微镜载玻片上。采用标准免疫组化法和新开发的rIFA对NMO谱系障碍（NMOSD）患者和对照组的151份血清样本进行了分析。结果：25/32（78.1%）的临床明确NMO患者和36/51（70.6%）的NMOSD患者在rIFA中NMO- igg /AQP4-Ab阳性，而在IHC中分别为65.6%和58.8%。结论：本文提出的重组IFA为熟悉间接免疫荧光显微镜的实验室提供了一种高灵敏度和可重复性的诊断工具，用于AQP4抗体的标准化检测。这种新方法可以使AQP4-Ab检测这一具有较高临床相关性的检测方法得到更广泛的应用。(C) 2010 Elsevier B.V.版权所有
Background: Recently, a highly specific serum autoantibody was discovered in patients with neuromyelitis optica, called NMO-IgG, and aquaporin-4, the most abundant water channel in the CNS, was identified as the target antigen. Several assays for the detection of NMO-IgG/AQP4-Ab have been described. Tests based on recombinant human AQP4 have been repeatedly demonstrated to be more sensitive than the previous gold standard assay, i.e. immunohistochemistry (IHC) on mouse brain tissue. However, the sophisticated techniques applied restrict their availability to few laboratories worldwide.Objective: To develop an easy-to-use, recombinant immunofluorescence assay (rIFA) suitable for standardized and high-throughput detection of NMO-IgG/AQP4-Ab.Methods: HEK293 cells seeded on cover glasses were transfected with full-length recombinant human AQP4 at large scale. Cover glasses with the immobilized cells were cut into millimetre-sized fragments and transferred to microscopy slides. 151 serum samples from patients with NMO spectrum disorders (NMOSD) and controls were analysed both in the standard IHC assay and in the newly developed rIFA.Results: 25/32 (78.1%) patients with clinically definite NMO and 36/51 (70.6%) of total patients with NMOSD were positive for NMO-IgG/AQP4-Ab in the rIFA compared to 65.6% and 58.8%, respectively, in the IHC assay.Conclusion: The recombinant IFA presented here provides laboratories familiar with indirect immunofluorescence microscopy with a highly sensitive and reproducible diagnostic tool for standardized detection of antibodies to AQP4. This new approach could make AQP4-Ab testing, which is of high clinical relevance, more widely available. (C) 2010 Elsevier B.V. All rights reserved.