构建M23-AQP4稳定表达的HEK293细胞应用于血清抗AQP4抗体检测的研究  

Serum anti-AQP4 antibody detection based on HEK293 cells stably expressing M23-AQP4

作　　者：吴晓牧 符青青 刘诗英 项正兵 熊英琼 张昆南 

机构地区：[1]江西省神经病学研究所,330006 [2]南昌大学研究生院,330006

出　　处：《中国神经免疫学和神经病学杂志》2015年第6期418-423,共6页Chinese Journal of Neuroimmunology and Neurology

基　　金：江西省科技厅项目(20133BCB24017);江西省科技厅基金资助项目(20123BBA13050)

摘　　要：目的构建M23-AQP4稳定表达HEK293细胞(HEK293-M23-AQP4)并用于抗AQP4抗体检测,以探索临床可行的抗AQP4抗体检测方法。方法用磷酸钙转染试剂将pEGFP-N1-M23-AQP4质粒转入HEK293细胞,通过G418筛选HEK293-M23-AQP4,以细胞间接免疫荧光法(CBA)检测M23-AQP4表达及分布。以HEK293-M23-AQP4为底物的CBA法检测视神经脊髓炎(NMO)6例、多发性硬化(MS)16例、其他脱髓鞘疾病(视神经炎、脊髓炎、吉兰-巴雷综合征、急性播散性脑脊髓炎)30例、非脱髓鞘性疾病患者10例血清抗AQP4抗体及其滴度,并比较4组抗体阳性率,计算抗AQP4抗体诊断NMO的敏感性,分别以非NMO的脱髓鞘疾病和非脱髓鞘疾病作对照计算抗AQP4抗体诊断NMO的特异性。将HEK293-M23-AQP4细胞于室温、4℃、-20℃保存4周,分别作为底物检测经首次检测所得抗AQP4抗体阳性标本并随机选取5例抗体阴性标本的抗AQP4抗体及滴度,比较其阳性率及滴度变化;将上述首次检测所得抗AQP4抗体阳性标本及5例抗体阴性标本反复冻融3次后分别于室温、4℃、-20℃保存1周后,检测其抗AQP4抗体及滴度,比较阳性率和滴度变化。结果 HEK293-M23-AQP4构建成功,M23-AQP4主要表达在细胞膜上。NMO患者抗AQP4抗体阳性率达83.3%(5/6),显著高于MS患者〔6.3%(1/16)〕、其他脱髓鞘疾病〔3.3%(1/30)〕和非脱髓鞘性疾病〔0.0%(0/10)〕(均P<0.01);抗AQP4抗体诊断NMO的敏感性为83.3%(5/6),以非NMO的脱髓鞘疾病作对照时,其诊断NMO特异性为95.6%(44/46),以非脱髓鞘疾病作对照时,其诊断NMO特异性为100%(10/10)。HEK293-M23-AQP4于不同温度保存后所检测抗AQP4抗体阳性率和滴度与首次检测比较均无统计学意义(均P=1.0)。血清标本4℃及-20℃保存1周后所检测抗AQP4抗体阳性率和滴度与首次检测比较差异均无统计学意义(均P=1.0);室温保存1周后抗体滴度(1∶400、1∶400、1∶3200、1∶6400、1∶6400、1∶12800、1∶51200)与首次检测(1∶800、1�Objective To construct the M23-AQP4 stably expressing HEK293cells（HEK293-M23-AQP4）and use them for the detection of anti-AQP4 antibody in order to explore a clinically feasible assay for anti-AQP4 antibody detection.Methods pEGFP-N1-M23-AQP4 plasmid was transfected into HEK293 cells by using calcium phosphate transfection reagent.HEK293-M23-AQP4 was screened by G418.Expression and distribution of M23-AQP4 was examined by cell based assay（CBA）.52 serum samples from patients with demyelinating diseases,including 6 neuromyelitis optica（NMO）,16 multiple sclerosis（MS）and 30 other demyelinating diseases（optic neuritis,myelitis,Guillian-Barre,acute disseminated encephalomyelitis）,and 10 patients without demyelinating diseases as control were collected in our study.Serum anti-AQP4 antibody and its titer were detected by CBA method using HEK293-M23-AQP4 as substrate.The seropositive rates of the 4groups were compared and the sensitivity of anti-AQP4 antibody in the diagnosis of NMO was calculated.The specificity of anti-AQP4 antibody in the diagnosis of NMO was calculated by considering demyelinating disease（except NMO）and other diseases as control.The HEK293-M23-AQP4 that fixed with 4% paraformaldehyde and stored at room temperature,4℃ and-20℃ for 4 weeks was used to detect anti-AQP4 antibody of all seropositive specimens and 5randomly selected seronegative specimens.Anti-AQP4 antibody of all seropositive specimens and 5randomly selected seronegative specimens stored at room temperature,4℃ and-20℃ for 1week after 3freeze/thaw cycles were detected based on fresh HEK293-M23-AQP4.The positive rates and titer change were compared with previous detection.Results HEK293-M23-AQP4 was successfully constructed and M23-AQP4 mainly expressed in the cell membrane.The positive rate of the NMO patients（5/6,83.3%）was significantly higher than that of the MS patients（1/16,6.3%）,other demyelinating diseases（1/30,3.3%）and all non-demyelinating diseases patients（0/10,0.0%,P〈0.01,respectively�

关 键 词：视神经脊髓炎 HEK293 抗AQP4抗体 脱髓鞘性疾病 临床运用 

分 类 号：R744.5[医药卫生—神经病学与精神病学]