Division of Molecular Neuroimmunology, Department of Neurology, University of Heidelberg, Heidelberg, Germany.
J Neurol Sci. 2010 Apr 15;291(1-2):52-6. doi: 10.1016/j.jns.2010.01.002. Epub 2010 Feb 1.
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.
To develop an easy-to-use, recombinant immunofluorescence assay (rIFA) suitable for standardized and high-throughput detection of NMO-IgG/AQP4-Ab.
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.
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.
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.
最近，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，称为 NMO-IgG，水通道蛋白 4（CNS 中最丰富的水通道）被确定为靶抗原。已经描述了几种用于检测 NMO-IgG/AQP4-Ab 的方法。基于重组人 AQP4 的检测已反复证明比以前的金标准检测方法，即小鼠脑组织免疫组化（IHC）更敏感。然而，所应用的复杂技术将其可用性限制在全球少数实验室中。
开发一种易于使用的、基于重组免疫荧光的检测方法（rIFA），用于标准化和高通量检测 NMO-IgG/AQP4-Ab。
在大规模培养皿中用全长重组人 AQP4 转染 HEK293 细胞。将固定细胞的盖玻片切成毫米大小的碎片，并转移到显微镜载玻片上。对 151 份来自视神经脊髓炎谱系疾病（NMOSD）患者和对照者的血清样本，分别用标准 IHC 检测和新开发的 rIFA 进行分析。
与 IHC 检测相比，rIFA 检测中 25/32（78.1%）例临床确诊的 NMO 患者和 36/51（70.6%）例 NMOSD 患者均为 NMO-IgG/AQP4-Ab 阳性，而阳性率分别为 65.6%和 58.8%。
本文介绍的重组 IFA 为熟悉间接免疫荧光显微镜的实验室提供了一种高度敏感且可重复的诊断工具，用于标准化检测 AQP4 抗体。这种新方法可以使具有重要临床意义的 AQP4-Ab 检测更广泛地应用。
