用免疫金银法检测抗核抗体

抗核抗体(ANA)是诊断风湿性疾病的常用实验室指标。经典的检测方法为间接荧光法(IFA)~。但此法受荧光显微镜、荧光抗体、底物(抗原)片的种类与质量影响甚大,荧光又极易淬灭,标本片不能长期保存。我们建立了一种免疫金银法(IGSS)测定ANA,操作简便,结果清晰,无需特殊设备,一般实验室均可采用。     

用纸模的免疫金银法鉴定自制5—HT抗体的性质

本文用纸模的免疫金银法鉴定了自制5-HT抗体的性质。抗体的工作稀释度与其能显示的最小抗原量呈线性关系,1:10000稀释度的抗体能显示22.50ng的5-HT。该抗体能被过量的HSA-5-HT完全吸收,但不被过量的组织胺和色氨酸所吸收。以上结果表明表明,该抗体在免疫组化反应中有较高的敏感性和特异性。     

类风湿关节炎患者RA33／36抗体,角质蛋白抗体及抗核 …

目的 对类风湿关节炎（ＲＡ）患者血清中的ＲＡ３３／３６抗体，角质蛋白抗体（ＡＫＡ）及抗核周因子（ＡＰＦ）进行检测，并对这些抗体间的相关性进行探讨。方法 采用免疫印迹法检测血清ＲＡ３３／３６抗体；采用ＩＩＦ法检测ＡＫＡ及ＡＰＦ抗体；采用卡方检验及Ｆｉｓｈｅｒ＇ｓ检验对结果进行统计学比较。结果 ＲＡ患者血清中ＲＡ３３／３６抗体阳性率为３５．６％（１６／４５），ＡＫＡ（１：８０）阳性率为３１．１％（１４     

免疫金银染色法检测抗弓形虫抗体

免疫金银染色法检测抗弓形虫抗体张阳根解放军第１７５医院漳州３６３０００弓形虫病是一种广泛流行的人畜共患疾病，多数为隐性感染及临床症状的非典型性，使得其病原学诊断不易成功。目前常用的方法有ＥＬＩＳＡ．ＩＦＡＴ、ＩＨＡ等［１～３］，但都存在一定的局限性，...     

免疫金银法常见问题和对策探讨

免疫金银组织化学方法建立已有10年,实践证明此法具有敏感性高,试剂价廉,无毒等优点,适用于光镜和电镜的免疫组化检测。彩色免疫金银法更具有鲜明色彩,     

斑点金免疫渗滤法检测血清抗精子抗体

目的建立检测血清抗精子抗体的斑点金免疫渗滤法 (dot im munogold filtration assay,DIGFA)。方法　采用经 Sephadex G- 75 -抗人全血清柱亲和层析纯化的人精子抗原和胶体金标记的 SPA,根据免疫渗滤原理 ,建立抗精子抗体(ASAb)的斑点金免疫渗滤法检测法。结果　纯化的人精子抗原具有较好的特异性 ;斑点金免疫渗滤法与 EL ISA法比较 ,符合率为 97.7%。特异性为 96 .2 % (TN/ TN+ FP× 10 0 % ) ,敏感性为 88.9% (TP/ TP+ FN× 10 0 % ) ,阳性预测值 84.2 % (TP/TP+ FP) ,阴性预测值 97.2 % (TN/ TN+ FN) .结论　斑点金免疫渗滤法方法检测血清抗精子抗体有较好的特异性和敏感性 ,重复性好 ,试剂稳定 ,操作简便快速 ,具有临床应用价值     

类风湿关节炎患者RA33/36抗体，角质蛋白抗体及抗核周因子的相关性初步研究

目的对类风湿关节炎（ＲＡ）患者血清中的ＲＡ３３／３６抗体，角质蛋白抗体（ＡＫＡ）及抗核周因子（ＡＰＦ）进行检测，并对这些抗体间的相关性进行探讨。方法采用免疫印迹法检测血清ＲＡ３３／３６抗体；采用ＩＦ法检测ＡＫＡ及ＡＰＦ抗体；采用卡方检验及Ｆｉｓｈｅｒｓ检验对结果进行统计学比较。结果ＲＡ患者血清中ＲＡ３３／３６抗体阳性率为３５．６％（１６／４５），ＡＫＡ（１８０）阳性率为３１．１％（１４／４５），ＡＰＦ的阳性率为５３．３％（２４／４５）。结论虽然ＲＡ患者血清中可出现上述三种抗体，但三者间无相关性。     

免疫印迹法检测抗核糖体抗体及其临床意义

抗核糖体抗体主要见于 SLE,对 SLE的诊断具有一定特异性。本文用免疫印迹法(IBT)证实兔胸腺 ENA 中38,16.5,15KD 多肽具有核糖体抗原表位,应用兔胸腺ENA代替胞浆纯化核糖体用 IBT检测抗核糖体抗体,结果表明该抗体在40例 SLB中的阳性率为 15％,在抗Sm和/或抗 RNP抗体阳性的 SLE患者中,阳性率可达 30.8％,而在 143例非 SLE风湿病患者中阳性率仅1.4％,差别显著(p<0.01)。本文SLE中抗核糖体阳性与阴性二组在主要临床特征上无显著差别(P>0.05)。     

免疫胶体金检测不孕症患者血清抗精子抗体

目的:建立快速测定抗人精子抗体(AsAb)的新方法。方法:采用人精子膜抗原,以胶体金颗粒结合的羊抗人IgG为标记抗体,根据免疫胶体金技术原理,建立斑点金免疫渗滤法(DIGFA)检测人血清AsAb。共测检137份不孕者血清并与酶联吸附试验(ELISA)作对比。结果:人精子膜抗原与患者血清中的特异性AsAb通过渗滤法在硝酸纤维素膜上反应, 6min内即可直接观察结果。在137份血清标本中,不孕女方阳性21 9%,不孕男方阳性13 19%。与ELISA的结果基本一致。符合率为96 1%,特异性为95 4%,敏感性为90 2%,交叉试验与重复试验结果显示,DIGFA具有良好的特异性及稳定性。结论:DIGFA可快速测定AsAb,具有推广使用的价值。     

类风湿关节炎患者血清和滑液中抗组蛋白抗体的检测

类风湿关节炎患者血清和滑液中抗组蛋白抗体的检测陈常胜，韩宽英，顾幸振，梁俊林济南军区总医院肾内科济南２５００３１施桂英（解放军总医院风湿科）抗组蛋白抗体（ＡＨＡ）是针对暴露于核小体组蛋白表位的自身抗体。已知类风湿关节炎（ＲＡ）患者血清中的ＡＨＡ与其活...     

Demonstration of anti-keratin antibodies by ELISA using keratin or thiol-containing compounds in urea as antigens

During the development of an enzyme-linked immunosorbent assay (ELISA) for the demonstration of anti-keratin antibodies (AKA) it was observed that rabbit anti-keratin antisera also reacted with polystyrene surfaces treated with beta-mercaptoethanol in 8 M urea (ME-urea). Sera from non-immunized rabbits or rabbits immunized with antigens unrelated to keratin failed to react. The specificity of the reaction was further assessed by absorption experiments and by testing affinity-purified AKA. IgM activity against ME-urea could be demonstrated in 62.5% of sera from patients with infectious mononucleosis and in 37.5% of sera from patients with rheumatoid arthritis, and there was a good correlation to the presence of AKA. Coating of the solid phase with compounds containing free SH groups in 4-8 M urea generated the antigen of this ELISA. The exact molecular configuration of this presumptive synthetic antigen is obscure, but the ME-urea ELISA seems to provide a simple way to detect anti-keratin antibodies of a certain specificity.     

Subclass distribution of IgG antibodies to the rat oesophagus stratum corneum (so-called anti-keratin antibodies) in rheumatoid arthritis.

Serum IgG, labelling the stratum corneum of the rat oesophagus epithelium, so-called anti-keratin antibodies (AKA) constitute the most specific marker for the diagnosis of rheumatoid arthritis. In this study, we investigated 31 IgG AKA-positive rheumatoid sera and 21 control sera from patients with non-rheumatoid inflammatory rheumatic diseases. The serum level of IgG1,2,3 and 4 was determined by radial immunodiffusion and the subclass distribution of IgG AKA by a three-step semi-quantitative immunofluorescence assay using standard monoclonal antibodies specific for each of the four human IgG subclasses. In the rheumatoid sera, the serum level of IgG1 was found to be significantly increased and the level of IgG2 significantly decreased with regard to the control sera, while the levels of IgG3 and 4 as well as total IgG were in the normal range. IgG1,2,3, and 4 AKA were detected in 27 (87%), 6 (19%), 4 (13%) and 11 (35%) of the 31 rheumatoid sera, respectively, and were found to be independent of the clinical and biological indices of the disease. In spite of inter-individual heterogeneity, two predominant profiles were distinguished: IgG1 (alone) and IgG(1 + 4), which together represented 18 sera (58%). The large predominance of IgG1 AKA and the quasi-absence of IgG2 AKA suggest that the recognized antigen may be partly comprised of protein. Moreover, the high frequency of occurrence of IgG4 AKA might result from chronic exposure to the eliciting antigen, which could be a genuine autoantigen since we demonstrated that it is also present in the stratum corneum of human epidermis.     

类风湿因子及相关自身抗体对类风湿关节炎的诊断价值

目的探讨类风湿因子（RF）、抗环瓜氨酸肽抗体（抗CCP）及抗角蛋白抗体（AKA）联合检测对类风湿关节炎（RA）的临床诊断价值。方法对80例RA和65例非RA的其他自身免疫病患者检测RF、RF-IgM、抗CCP及AKA 4种指标。结果自身免疫性疾病女性发病较高,以RA组为甚;RA组的RF、RF-IgM、抗CCP及AKA高于其他自身免疫性疾病组;单指标检测RA诊断的敏感性为RF〉RF-IgM〉AKA〉抗CCP,特异性为RF-IgM〉抗CCP〉RF〉AKA。阳性预测值为抗CCP〉RF〉RF-IgM、AKA,阴性预测值为RF〉RF-IgM〉AKA〉抗CCP;以并联或串联方式联合检测均以RF、RF-IgM及抗CCP三联及RF、RF-IgM、抗CCP及AKA四联检测为佳,并联检测的敏感性分别为93%及97%,串联检测的特异性分别为98%及99%。结论 RF、RF-IgM、抗CCP及AKA 4种指标联合检测可提高对RA诊断的敏感性和特异性,对RA的早期诊断有较高价值。     

Human monoclonal rheumatoid factors augment arthritis in mice by the activation of T cells

In order to investigate the in vivo role of rheumatoid factor (RF), the effects of the administration of human monoclonal (m) IgM-RF and IgG-RF on the development of arthritis in mice were examined. The administration of human mRFs into mice immunized with type II collagen (CII) markedly enhanced the clinical score and paw swelling. The severity of arthritic joint disease with a marked infiltration of lymphoid cells, proliferation of synovial membrane, pannus formation and destruction of articular cartilage was significantly enhanced in both groups receiving RF (RF-enhanced arthritis). Skin ulcers were also observed in some of these RF-enhanced arthritis mice, whereas no such signs were observed in CII-immunized mice without mRFs. Both IgM-RF and IgG-RF increased CII-specific IgG antibodies in circulation, and the severity of arthritis correlated with the production of high titres of anti-CII antibodies. In vivo treatment of RF-enhanced arthritis mice with an anti-CD4 MoAb or an anti-CD8 MoAb inhibited the induction and progression of arthritis in these mice. Administration of RF to severe combined immunodeficient (SCID) mice with arthritis developed by the transfer of spleen cells from CII-immunized mice, prolonged the arthritis and enhanced the severity. This murine model of RF-enhanced arthritis may provide a useful tool for analysing the pathogenesis of rheumatoid arthritis in RF-positive patients.     

Array 360和乳胶法检测类风湿因子的诊断意义

目的 对速率散射比浊法和乳胶凝集法进行比较。方法 对４７例类风湿关节炎（ＲＡ）患者进行ＲＦ检测，并用比浊法检测了３２例ＳＬＥ患者，４３例病毒性肝炎患者臁２５名正常人的ＲＦ。结果 ４７例ＲＡ中比浊法阳性３７例，阳性率为７８．８％；交法阳性２５便，阳性率为５３．２％，两法检测结果呈平行关系。ＳＬＥ及肝炎患者可有ＲＦ的升高，但是低滴度与ＲＡ患者的ＲＦ含量有性差别。结论 比浊法明显优于乳胶法，速率散射比浊     

RF、AKA和抗CCP抗体联检对类风湿关节炎诊断的临床价值

目的:探讨类风湿因子(Rheum atoid factor,RF)、抗角质蛋白抗体(antikeratin antibody,AKA)及抗环瓜氨酸肽(anti-cyc lic c itru llinated peptide,CCP)抗体对类风湿关节炎(rheum atoid arthritis,RA)的临床意义和早期应用价值。方法:对40例类风湿关节炎患者、30例系统性红斑狼疮和30名正常健康体检者进行RF、AKA、抗CCP抗体检测,应用速率散射比浊法测定RF,间接免疫荧光法检测AKA,ELISA法测定抗CCP抗体。结果:40例RA患者血清中,RF灵敏度和特异性分别为70.0%、90.0%,AKA灵敏度和特异性分别为35.0%、96.7%,抗CCP抗体灵敏度和特异性分别为85.0%、93.3%,联检RF、AKA及抗CCP抗体灵敏度和特异性分别为97.07%、99.8%。结论:RF、AKA和抗CCP抗体可作为诊断RA比较特异的血清学指标,三项指标联检可在一定程度上弥补RF对RA的诊断不足,提高RA的阳性诊断率,且有助于疾病的预后判断。     

抗环瓜氨酸肽抗体检测对类风湿关节炎的临床诊断价值

目的探讨抗环瓜氨酸肽抗体（抗CCP抗体）检测对类风湿关节炎（RA）诊断的意义。方法采用酶联免疫吸附试验（ELISA）检测115份人血清的抗CCP抗体,同时采用免疫透射比浊法定量检测类风湿因子（RF）,包括40例RA患者,45例其它风湿病患者,30名正常人;并分析抗CCP抗体与RF实验结果之间的相关性。结果在40例RA病人中,抗CCP抗体的阳性率为80.0%,在其它风湿病人中的阳性率为7.0%,抗CCP抗体对RA的敏感性和特异性分别为80.0%、96.0%,其敏感性高于RF,但差异无统计学意义（P〉0.05）,特异性明显高于RF（P〈0.05）。联合应用抗CCP抗体与RF进行诊断,二者均阳性时敏感性为65.0%,特异性为97.3%。抗CCP抗体与RF实验结果之间无相关性。结论抗CCP抗体对RA具有较好的敏感性和很高的特异性,可与RF相互补充,联合检测可提高对RA早期诊断的准确性。     

链替换技术提高抗角蛋白人Fab抗体的亲和力

目的通过链替换技术对一株人源性抗角蛋白Fab单抗进行体外亲和力成熟。方法分离、扩增多样性人免疫球蛋白轻链基因和IgG/IgM重链Fd段基因,分别构建轻链库和重链库。先以原始克隆的重链与轻链库随机搭配进行轻链替换,对形成的换链库采用解离速率筛选法（off-rate selection）选取亲和力提高的克隆;然后再将获得的轻链基因与重链库基因重组进行重链替换,筛选亲和力进一步提高的克隆并进行亲和力测定、可变区基因序列分析等一系列鉴定。结果替换轻链后所获抗体的亲和力提高到原来的10倍;替换重链后亲和力进一步提高达到7.9×10     

检测抗聚丝蛋白抗体在类风湿关节炎诊断中的意义

目的探讨抗聚丝蛋白抗体(AFA)对类风湿关节炎(RA)诊断的价值,并比较与RA的其他早期诊断指标的相关性。方法从人表皮细胞中提取并部分纯化聚丝蛋白(filaggrin)抗原,用于免疫印迹检测(Western blot)103例RA血清标本和140例对照血清,包括系统性红斑狼疮(SLE)、干燥综合征(SS)、骨性关节炎(OA)95份及正常人45份。APF和AKA用间接免疫荧光法检测。结果103例RA病人的AFA阳性率、特异性分别为35.9%,93.7%,显著高于疾病对照组和正常人(P<0.001)。在AFA阳性的37例中,AKA也阳性的为26例,重叠率为70.3%;APF亦阳性的为31例,重叠率为83.8%,AFA与AKA、APF之间存在相关性。结论AFA对RA具有很高的特异性。AFA的检测可用于RA的临床诊断。AFA与APF及AKA有相关性,但不能完全取代它们的检测。     

抗角蛋白16单克隆抗体对角质形成细胞增殖活性的影响

目的研究抗角蛋白单克隆抗体(mAb)对体外培养的角质形成细胞增殖活性与细胞周期的影响.方法以无血清培养基体外原代培养角质形成细胞.应用3H-TdR掺入DNA合成测定法,观察抗角蛋白16mAb(anti-K16mAb)对角质形成细胞代谢增殖的影响.用流式细胞仪分析anti-K16mAb作用后角质形成细胞的细胞周期变化.结果mAb作用后,角质形成细胞样品的cpm降低,并呈抗体剂量依赖方式.流式细胞仪分析表明,处于S期的细胞比例由27%上升至42.2%,同时处于G1期与G2期的细胞比例分别相应下降.结论anti-K16mAb可抑制体外培养的角质形成细胞的增殖活性,且将细胞阻滞于S期,无法进入G2期与M期分裂增殖.