免疫荧光检测人血清视神经脊髓炎免疫球蛋白G的实验方法

目的 建立检测神经脊髓炎免疫球蛋白G(NMO-IgG)的间接免疫荧光方法,描述其注意事项.方法 选择C57小鼠小脑、中脑、肾脏和胃冰冻切片作为基质.人血清经稀释、豚鼠肝粉预处理后,滴加于封闭液处理的上述切片,4℃过夜后滴加荧光素结合的羊抗人IgG的抗体,封片、荧光显微镜观察.实验通过荧光双标技术确定NMO-Igc结合部位与AQP4表达位置是否一致.结果 测定了182份人血清,通过间接免疫荧光法证实部分NMO、MS、脊髓炎、视神经炎患者血清有NMO-IgG结合到小鼠的软脑膜、脑膜下和脑、脊髓的毛细血管部位.荧光双标技术显示NMO-IgG的结合位置与AQP4表达的位置完全一致.豚鼠肝粉-PBS液对照及空白对照无特异荧光.结论 以C57小鼠的小脑、肾脏和胃冰冻切片作为基质,通过间接免疫荧光法能成功检测出人血清中的NMO-IgG抗体,有助于我们对NMO进行诊断与鉴别诊断.

Neuromyelitis optica-IgG detection in serum by indirect immunofluorescence assay

Objective To establish a method to detect neuromyelitis optiea (NMO)-IgG in patients serum using indirect immunofluorescence assay (IFA). Methods The normal tissues (cerebellum/ midbrain, kidney and stomach) from C57 mice were cryosectioned onto microscope slides as detective substrate. For NMO-IgG detection, isolated serum from patient with NMO, multiple sclerosis (MS), optic neuritis or myelitis was incubated with the tissue sections on the slide at 4℃ overnight and subsequently incubated with a fluorochrome-cojugated lgG specific for human. For double immunostaining with aquaporius-4 (AQP4), the slides were incubated with primary antibody of AQP4 and secondary antibody of IgG-TRITC. Detection of NMO-IgG and its co-localization with AQP4 was analyzed using fluorescence microscope. Results All 182 serum samples from patients were tested using IFA. Some samples revealed a characteristic immunohistochemical staining of NMO-IgG in mouse CNS tissues, predominately in pia and subpia, and capillaries in white and grey matter in the cerebellum, midbrain, and spinal cord. Double immunostaining with AQP4 demonstrated the co-localization of NMO-IgG with AQP4. Conclusions We established an IFA using a substrate from C57 mouse cerebellum/midbrain, kidney and stomach tissue to detect NMO-IgG in patient serum. This method is specific and efficient in detection and may be useful in diagnosis and differential diagnosis of neuromyelitis optica.