抗核抗体的实验室检测进展

随着生物医学技术的迅速发展和抗核抗体(ANA)检测的临床推广应用,ANA的实验室检测新技术不断涌现,如间接免疫荧光法(IIF)检测ANA的自动化技术、其他免疫学技术检测ANA及其特异性自身抗体等。上述进展对ANA的实验室检测及其规范应用带来新的机遇和挑战。     

应用蛋白指纹图谱筛选肿瘤坏死因子抑制剂治疗类风湿关节炎的生物标志物

目的通过蛋白组学研究发现与肿瘤坏死因子(tumor necrosis factor,TNF)单克隆抗体治疗类风湿关节炎(rheumatoid arthritis,RA)疗效相关的新的血清生物标志物。方法入组患者为17例经传统改善病情抗风湿药治疗后病情仍为中至重度活动性RA患者,分别在第0、2、6、14和22周联合英夫利西单抗(200mg/次)静脉滴注。分别留取治疗前(第0周)、治疗中(第14周)及治疗后(第26周)血清,并进行详细临床病情评估。使用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)方法检测血清中蛋白组学。结果英夫利西单抗治疗后临床改善者13例(76.5%),无改善者4例(23.5%)。在治疗有效的患者中,蛋白组学分析共有10种小分子蛋白水平随治疗而下降;而治疗无效的患者上述10种蛋白水平无显著变化。此外,无论是治疗有效还是治疗无改善的患者血清中均有2种蛋白分子峰值随治疗升高。结论利用蛋白组学方法可发现与TNF抑制剂治疗相关的血清蛋白标志物,这些标志物可能有助于预测TNF抑制剂的有效性。     

类风湿关节炎相关自身抗体谱检测比对

目的了解类风湿关节炎相关自身抗体谱在我国的检测水平及各种自身抗体检测的准确率。方法由北京协和医院风湿免疫科向参与比对的实验室寄发比对品,各实验室在规定时间内将实验方法及检测结果回报。比对项目包括抗环瓜氨酸肽抗体、类风湿因子、抗角蛋白抗体、抗核周因子抗体。比对品发放和结果分析采用随机双盲法,由北京协和医院对各家医院检验结果进行分析并将结果反馈给各实验室。结果抗环瓜氨酸肽抗体、类风湿因子、抗角蛋白抗体、抗核周因子抗体正确率的平均值分别为98.6%、95.3%、73.4%及79.2%。结论目前全国大部分实验室开展了类风湿关节炎相关自身抗体谱的检测,其中抗环瓜氨酸肽抗体和类风湿因子的检测正确率高,而抗角蛋白抗体、抗核周因子抗体的检测水平有待提高。     

原发性胆汁性肝硬化患者调节性T细胞及其与肝功能损伤的研究

目的检测原发性胆汁性肝硬化（primary biliary cirrhosis，PBC）患者外周血CD4^＋ CD8^＋ T、CD4^＋ CD25^＋调节性T细胞亚群，探讨其与肝功能损伤、抗AMA—M2抗体产生的关系。方法采用流式细胞术检测北京协和医院住院和门诊PBC患者（n=27）外周血CD4^＋ CD8^＋ T细胞、CD4^＋ CD25^＋ T细胞群比例，以26例其他肝脏疾病患者作为疾病对照组，30例健康体检者作为正常对照，观察调节性T细胞亚群与患者的肝功能指标及自身抗体如AMA—M2、ANA的关系。结果PBC患者外周血CD4^＋ CD25^＋ T细胞比例与正常对照组比较结果差异无统计学意义（P〉0．05），但显著高于其他肝脏疾病组（P〈0．05）。CD4^＋ CD8^＋细胞群与正常人相比结果差异无统计学意义，其他肝病组明显高于正常对照组（P〈0．05）。PBC患者外周血NK细胞比例显著低于正常对照组（P〈0．05）。肝功严重损伤的PBC患者外周血CD4^＋ CD25^＋ T细胞比例明显高于肝功能轻度损伤者（P〈0．05）。PBC患者外周血CD4^＋ CD25^＋ T细胞数量与肝功能损伤指标ALB呈负相关（r=－0．338，P〈0．05），与TBIL、DBIL呈正相关（r分别为0．362，0．386，P〈0．05）。CD4^＋ CD25^＋ ／CD4^＋比例与GGT呈负相关（r=－0．335，P〈0．05），与TBIL、DBIL呈正相关（r分别为0．333，0．339，P〈0．05）。抗AMA—M2抗体阳性患者外周血CD4^＋ CD25^＋细胞比例明显高于AMA—M2阴性患者（P〈0．01）。未发现CD4^＋ CD25^＋ T、CD4^＋ CD8^＋T细胞比例在ANA^＋和ANA—PBC患者之间差异有统计学意义。结论PBC患者外周血CD4^＋ CD25^＋T细胞群比例与肝功能损害和抗AMA—M2抗体的产生密切相关，因此推测CD4^＋ CD25^＋T细胞的变化可能是导致病情发展以及肝脏损伤的关键环节之一。     

去唾液酸糖蛋白受体H1亚单位基因片段的克隆表达及其检测自身免疫性肝炎的价值

Objective To clone and express the human asialoglycoprotein receptor(ASGPR) H1 subunit, purify and identify the immunoreactivity of the recombinant protein, and establish the enzyme linked immunosorbent assay (ELISA) to detect anti-ASGPR antibodies in diagnosis of autoimmune hepatitis. Methods The CRDHI cDNA (435 bp) was subcloned into eukaryotic vector PEGH, and the recombinant protein expression was induced by D (+)-Galactose. The recombinant CRDH1 was purified with Glutathione Sepharose 4B, and its immunoreactivity was identified by SDS-PAGE and western blot as well as MALDI-TOF. ELISA was established to detect the anti-ASGPR antibodies in serum samples of 45 patients with AIH, 30 patients with SLE, 30 patients with RA, 10 patients with SS and 30 normal controls. Results The sequencing of recombinant plasmid showed the CRDH1 gene was successfully inserted to the eukaryotic expression vector with correct sequence and open reading frame. The fusion protein showed a molecular weight of 42 500 Da on SDS-PAGE gel and confirmed to be the human ASGPR by MALDI-MS through peptide mass fingerprint analysis with Mascot in human protein database. It shared 98. 34% homology with ASGPR H1 subunit. Western blot analysis showed that the fusion protein had the same immunoreactivity as human ASGPR. The results of ELISA indicated that the positive rate of anti-ASGPR was 35.6% ( 16/45 ), but the ELISA was negative in other control. There was significant difference of positivity of the autoantibodies between AIH and non-AIH controls (χ2 = 31.85,P < 0. 01 ). Conclusions The human plasmid containing ASGPR is successfully clone into Saccharomyces cerevisiae Y258. The recombinant autoantigen owns good antigenicity and specificity. ELISA established with the purified protein shows good specificity for diagnosis of AIH.     

组氨酰转移核糖核酸合成酶自身抗原的原核表达及初步临床应用

目的 通过克隆人组氨酰转移核糖核酸合成酶自身抗原Jo-1基因,构建重组表达质粒,获得具有免疫活性的纯化重组蛋白,建立间接ELISA法,并探讨其在检测多发性肌炎/皮肌炎(PM/DM)中的抗Jo-1抗体的价值.方法 构建重组表达载体,在大肠杆菌DH5 α和BL21(DE3)中表达;融合蛋白经Ni-NTA树脂柱进行亲和层析纯化,并通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和免疫印迹(WB)进行免疫活性鉴定;应用表达蛋白建立间接ELISA法.同时用该方法检测30名正常献血者,30例SLE,30例类风湿关节炎(RA),10例原发性干燥综合征(SS),75例PM/DM患者血清中抗Jo-1抗体.结果 经重组质粒测序和酶切结果证实,Jo-1目的基因已正确插入原核表达载体中,基因序列正确,符合表达框架;经SDS-PAGE检测显示,表达产物在相对分子质量55 000处有一明显的蛋白表达条带;WB分析表明,重组蛋白具有人Jo-1抗原反应性;间接ELISA法检测标本血清结果显示,PM/DM组中抗Jo-1抗体的阳性率为28%,非PM/DM组(疾病对照组及正常对照组)均为阴性,其差异均有统计学意义(x2=31.84,均P＜0.01).结论 成功克隆了人组氨酰转移核糖核酸合成酶自身抗原Jo-1基因,其可在大肠杆菌中表达,且重组自身抗原具有较好的抗原性和特异性.应用纯化融合蛋白建立的间接ELISA法检测PM/DM抗Jo-1抗体具有较好的特异性.     

系统性硬皮病抗核抗体谱的检测分析

目的 通过对系统性硬皮病（SSc）患者抗核抗体谱（ANAs）检测结果的分析,探讨这些抗核抗体（ANA）对系统性硬皮病的诊断价值。方法 分别采用间接免疫荧光法（IIF）和免疫印迹法（IB）对62例SSc患者和20例健康对照者血清中15种抗核抗体进行测定。结果 IIF检测ANA结果显示,SSc患者中ANA的阳性率为98.4%（61/62）,其荧光染色模型主要为均质核仁型;IB检测15种ANA结果显示,SSc患者中的ANA以抗Scl-70抗体、抗SS-A抗体和抗Ro-52抗体为主,在46例弥漫性硬皮病（dSSc）患者中抗Scl-70抗体的阳性率高达67%,特异性为100%。结论 多种自身抗体同时检出,可能提示SSc患者同时伴随有其他的自身免疫性疾病,或者有发生其他自身免疫性疾病的危险;此外,欧蒙免疫印迹试剂盒,对ANAs检测的阳性率和特异性均较高,对SSc的诊断帮助很大。     

去唾液酸糖蛋白受体H1亚单位基因片段的克隆表达及其检测自身免疫性肝炎的价值

Objective To clone and express the human asialoglycoprotein receptor(ASGPR) H1 subunit, purify and identify the immunoreactivity of the recombinant protein, and establish the enzyme linked immunosorbent assay (ELISA) to detect anti-ASGPR antibodies in diagnosis of autoimmune hepatitis. Methods The CRDHI cDNA (435 bp) was subcloned into eukaryotic vector PEGH, and the recombinant protein expression was induced by D (+)-Galactose. The recombinant CRDH1 was purified with Glutathione Sepharose 4B, and its immunoreactivity was identified by SDS-PAGE and western blot as well as MALDI-TOF. ELISA was established to detect the anti-ASGPR antibodies in serum samples of 45 patients with AIH, 30 patients with SLE, 30 patients with RA, 10 patients with SS and 30 normal controls. Results The sequencing of recombinant plasmid showed the CRDH1 gene was successfully inserted to the eukaryotic expression vector with correct sequence and open reading frame. The fusion protein showed a molecular weight of 42 500 Da on SDS-PAGE gel and confirmed to be the human ASGPR by MALDI-MS through peptide mass fingerprint analysis with Mascot in human protein database. It shared 98. 34% homology with ASGPR H1 subunit. Western blot analysis showed that the fusion protein had the same immunoreactivity as human ASGPR. The results of ELISA indicated that the positive rate of anti-ASGPR was 35.6% ( 16/45 ), but the ELISA was negative in other control. There was significant difference of positivity of the autoantibodies between AIH and non-AIH controls (χ2 = 31.85,P < 0. 01 ). Conclusions The human plasmid containing ASGPR is successfully clone into Saccharomyces cerevisiae Y258. The recombinant autoantigen owns good antigenicity and specificity. ELISA established with the purified protein shows good specificity for diagnosis of AIH.     

树突状细胞与系统性红斑狼疮

树突状细胞(dendriticcell,DC)是一种具有刺激初级免疫反应、调节免疫应答、诱导免疫耐受的专职抗原提呈细胞(antigenpmsenllngcell,APC)。它也是唯一能够刺激静息型T细胞活化的APC,对调节T细胞免疫反应有重要作用。近年来的研究表明DC与自身免疫阡疾病特别是系捷性红斑狼疮(sy