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目的 观察 β2 糖蛋白I( β2 GPI)基因转染的HEp 2细胞 (HEp β2 GPI)中 β2 GPI融合蛋白的过度表达。探讨以HEp β2 GPI为底物间接免疫荧光 (IIF)法检测抗β2 GPI抗体的可行性。方法 应用逆转录聚合酶链反应 (RT PCR)扩增人源 β2 GPIcDNA ,克隆入pEGFP C1 ,并转染HEp 2细胞。通过RT PCR、共焦荧光显微镜、免疫印迹法 (IBT)及IIF鉴定β2 GPI 绿色荧光蛋白 (GFP)融合蛋白的表达和抗原性。以转染细胞为底物IIF检测 1 9份疑诊继发性抗磷脂综合征 (APS)病人、1份原发性APS病人及 1 0份正常人血清的IgG β2 GPI,与同时进行的ELISA法检测IgG ACL、IgG β2 GPI的结果作比较。结果 ( 1 )获得的HEp β2 GPI细胞传十几代后仍具有较强的 β2 GPI GFP表达 ,融合蛋白保持 β2 GPI的抗原性 ,其IIF表型具有特征性。 ( 2 )IIF法测血清IgG β2 GPI的结果显示 :7份病人血清出现特征性免疫荧光表型 ,而正常人血清均无荧光染色。 3种方法的比较性研究显示IIF法测IgG β2 GPI与ELISA法测IgG β2 GPI的一致性程度最佳 (Kappa值 0 .886 )。 ( 3)HEp β2 GPI保持了原型HEp 2细胞检测抗核抗体 (ANA)的荧光特性 ,且检出 2例原型HEp 2细胞IIFANA是阴性的血清。结论 HEp β2 GPI转染细胞株可用于IIF检测抗 β2 GPI,而且仍可作?
Objective To observe the overexpression of β2 GPI fusion protein in HEp 2 cells transfected with β2 glycoprotein I (β2 GPI) gene (HEp β2 GPI). To investigate the feasibility of detecting anti-β2 GPI antibody by using indirect immunofluorescence (IIF) with HEp β2 GPI as a substrate. Methods Human β2 GPI cDNA was amplified by reverse transcription polymerase chain reaction (RT PCR), cloned into pEGFP C1 and transfected into HEp 2 cells. The expression and antigenicity of the β2 GPI green fluorescent protein (GFP) fusion protein were identified by RT PCR, confocal fluorescence microscopy, immunoblotting (IBT) and IIF. The transfected cells were used as substrate IIF to detect IgG β2 GPI in serum of 19 suspected secondary antiphospholipid syndrome (APS) patients, 1 primary APS patient and 10 normal persons, France IgG IgG IgG, IgG2 GPI results for comparison. Results (1) The HEp β2 GPI cells obtained after a few generations still had strong β2 GPI GFP expression. The fusion protein maintained the antigenicity of β2 GPI, and the IIF phenotype was characteristic. (2) Serum IgG β2 GPI measured by IIF method showed that the characteristic immunofluorescence phenotypes were found in serum of 7 patients, but no serum was detected in normal human. A comparative study of the three methods showed that IgG2 GPI measured by IIF method had the best agreement with IgG beta2 GPI by ELISA (Kappa value 0.886). (3) HEp β2 GPI retained the fluorescence characteristics of the prototype HEp 2 cells for the detection of anti-nuclear antibody (ANA), and two cases of IIFANA-negative serum of the prototype HEp 2 cells were detected. Conclusion HEp β2 GPI transfected cell line can be used to detect anti-β2 GPI in IIF,