【摘要】目的探讨用一种新建立的外加电场固定液相分子快速斑点免疫分析 (rapid electric field immobilizing liquid phase molecule dot blot，REILMD) 法检测自身免疫性疾病患者抗内耳抗体的临床意义。方法收集11种确诊的自身免疫性疾病患者共71例，突发性聋21例，非免疫性疾病患者48例（对照组）。检测内容包括一般免疫指标及自身抗体如抗smooth muscle (Sm)、抗ribonucleoprotein (RNP)、抗Sjgren's syndrome A (SSA)、抗Sjgren's syndrome B (SSB)、抗DNA topoisomerase I (Scl)70、抗histidyl-tRNA synthetase (Jo-1)、抗核糖体、抗双链DNA、抗组织和抗内耳抗体。抗内耳抗体检测先用REILMD筛选,再用免疫转印方法确定相对分子质量。结果类风湿性关节炎 (rheumatoid arthritis，RA)和系统性红斑狼疮(systemic lupus erythematosus，SLE)主要表现为血沉加快, 类风湿因子阳性, C-反应蛋白、IgG、循环免疫复合物升高,少数IgA、IgM及C₄升高。在RA中2/16抗双链DNA和抗线粒体及4/16抗核抗体阳性。在SLE中11/16抗核抗体、7/13抗RNP抗体、抗SSA抗体和抗双链DNA抗体,3/13抗Sm抗体、1/13抗ScL70抗体、抗横纹肌和抗胃壁细胞抗体阳性。强直性脊柱炎(ankylosing spondylitis，AS) 均为阴性。经REILMD法筛选共在9例自身免疫性疾病患者(占13%)、2例突发性耳聋患者(占10%)及1例对照(冠状动脉硬化心脏病占2%)中检出强阳性抗内耳抗体, 其中SLE抗体阳性率为31%(5/16),RA、AS、白塞病和链球菌感染后综合症各1例阳性。在所有抗内耳抗体阳性患者中,抗核抗体阳性率高达67%,抗RNP和抗dsDNA抗体阳性率为50%。用免疫转印法分析发现该抗体识别抗原分子的相对分子质量主要为52 000、36 000、31 000和15 000。结论用REILMD法筛选抗内耳抗体为一种临床实用的简便方法,数种自身免疫性疾病有内耳损害的可能,以SLE为主。

A novel method for screening anti-inner ear autoantibody in patients with autoimmune diseases

ZOU Jing^*，BI Aifang，WU Xingchen, et al.

(Bethune International Peace Hospital，Shijiazhuang 050082, China.)

Corresponding author：ZOU Jiang(zouj@126.com)

【Abstract】ObjectiveTo appraise the clinical value of a newly established method, rapid electric field immobilizing liquid phase molecule dot blot analysis (REILMD)， for screening anti-inner ear autoantibody in patients with autoimmune diseases.MethodsSeventy-one patients with 11 kinds of autoimmune diseases were chosen for the study. Both the general immunity and autoantibodies were tested. In the processes of detection of anti-inner ear autoantibody, REILMD was used for screening, and then the Western blot was used to define the molecular weight of inner ear antigen recognized by the positive autoantibody.Results Acceleration of erythrocyte sedimentation rate (ES), positive rheumatoid factor (RF), increases in C reactive protein (CRP), IgG and circulating immune complex (CIC) were found in most cases with rheumatoid arthritis (RA) and systemic lupus erythematodes (SLE). Some of these patients had increased IgA, IgM and C4. Two of 16 RA had anti- double-stranded DNA(dsDNA) and anti-mitochondria and 4/16 had anti-nucleus antibodies. Eleven of 16 SLE had anti-nucleus, 7/13 had anti-ribonucleoprotein (RNP), anti- Sjgren syndrome A(SSA) and anti-dsDNA, 3/13 had anti-smooth muscle (Sm) and 1/13 had anti- DNA topoisomerase I(Scl)-70, striated muscle and stomach acid cell antibodies. No autoantibody was detected in AS. Anti-inner ear autoantibody existed in 9 out of 71 patients (13%) with autoimmune diseases, in 2 of 21 patients (10%) with sudden deafness and only in 1 of 48 control subjects (2%, coronary heart disease). The anti-inner ear autoantibody was positive in 5 of 16 (31%) patients with SLE and 1 each in RA, AS,Behset's disease and streptococcus infection syndrome.In patients with positive anti- inner ear antibody,67% had anti-nucleus antibody,50% had anti-RNP and dsDNA antibody.The molecules reeognized by the positive anti-inner ear antibody were defined as 52 000, 36 000, 31 000 and 15 000 molecules of inner ear antigen.ConclusionREILMD is a feasible and easy method for screening anti-inner ear autoantibody.Several autoimmune diseases,particularly SLE,may be implicated in damage to the inner ear.

【Key words】Immunoassay；Autoimmune diseases； Autoantibodies； Labyrinth

检索Medline得知1967年以来全球共在有影响的杂志上发表有关内耳免疫的论文 554篇。因此,内耳免疫研究历史悠久,已经受到全世界广泛关注。虽然已经有许多免疫检测方法用于内耳免疫研究,但是,由于特异性、灵敏度以及操作难等诸多因素的影响,大多数方法或者已经被淘汰,或者不能作为一种常规检查而广泛应用。临床上急需一种特异性较好、操作简便、快捷、经济的检测方法。我们经过不断探索,逐步完善,寻找出一种内耳抗原抗体检测方法,将该方法命名为外加电场固定液相分子快速斑点免疫分析（rapid electric field immobilizing liquid phase molecule dot blot，REILMD）法（详细资料已另文总结，投中华微生物免疫学杂志）。现将用该方法检测临床确诊的自身免疫性疾病和突发性聋患者抗内耳抗体的结果报告如下。

材料与方法

一、材料

1.临床资料：11种确诊的自身免疫性疾病患者来自我院中医科、肾病科和皮肤科，共71例，男33例，女38例；年龄10～70岁，平均36岁。其中类风湿性关节炎（rheumatoid arthritis，RA）19例，男3例，女16例；幼年类风湿性关节炎2例，男女各1例；强直性脊柱炎（ankylosing spondylitis，AS）19例，男18例，女1例；系统性红斑狼疮（systemic lupus erythematosus，SLE）16例，男4例，女12例；白塞病2例，男女各1例；干燥综合症2例，均为女性；成人斯替尔病2例，男女各1例；多发性肌炎1例，女性；链球菌感染后综合症1例，女性；多发性大动脉炎1例，女性；肾病综合症6例，男5例，女1例。各病诊断依据国际标准及全国标准。突发性聋患者来自我院耳鼻咽喉科，共21例，男15例，女6例，年龄20～56岁，平均32岁。对照组48例，为我院各科明确非自身免疫性疾病的其它疾病患者。

2．主要试剂：HRP-羊抗人IgG为卫生部北京生物制品研究所产品。用于体外检测抗核抗原抗体的自身免疫检查试剂盒-6（AUTO6）为美国GenBio公司产品。羊抗人IgG荧光抗体为卫生部上海生物制品研究所产品。

3．主要仪器：77000X Easy-TiterTM ELIFA System为美国Pierce公司生产,改造后用于REILMD分析。Eagle Eye TM Ⅱ 型电泳图像分析系统为美国Cold Spring Harbor公司产品。7150型全自动生化分析仪为日立公司生产。其它各种电泳仪器为北京六一仪器厂产品。

二、方法

一般项目检测包括血沉、类风湿因子(rheumatoid factor，RF)、IgG、IgA、IgM、C₃、C₄、C-反应蛋白（C-reactive protein，CRP）、循环免疫复合物(circulating immune complex，CIC)、抗链球菌溶血素“O”等。检测免疫球蛋白和补体用免疫比浊法,检测CRP用琼脂单扩散法,检测CIC用 PEG沉淀法,检测RF和抗链球菌溶血素“O”用胶乳凝集法。

自身抗体检测采用以下几种方式：

1.抗多肽抗体检测：按试剂盒说明书分别检测抗smooth muscle (Sm)抗体、抗ribonucleoprotein (RNP)抗体、抗Sjgren syndrome A (SSA)抗体、抗Sjgren syndrome B （SSB）抗体、抗DNA topoisomerase I （Scl）-70抗体、抗histidyl-tRNA synthetase (Jo-1)抗体和抗核糖体抗体。

2.抗双链DNA和抗组织抗体：用鼠各种组织切片作底抗原片,采用间接免疫荧光法检测抗双链DNA和抗各种组织抗体。

3.抗内耳抗体检测：用 REILMD方法筛选，首先使用外加电场快速吸附膜迷路蛋白：先将一铂丝经ELIFA System排液孔插入缓冲液池,并弯曲成蛇形,其表面盖上滤纸,池内倒满转移电泳缓冲液(0.025 mol/L Tris,0.192 mol/L甘氨酸,20%甲醇,pH 8.35),再按原说明书安装样品池底板,继续向缓冲液池注入缓冲液,直至其经96孔溢出,彻底驱赶96孔内气泡(该步骤非常重要),小心将预先浸泡该缓冲液的硝酸纤维素膜（nitrocellulose，NC膜）放于底板硅胶片表面,勿使其下面留任何气泡。盖好样品池顶板并拧紧螺栓固定(另一硅胶片盖于NC膜表面)。将样品池96孔内注满缓冲液,勿留气泡。用样品缓冲液(0.063 mol/L Tris-HCl，pH6.8,2%十二烷基磺酸钠,0.1%溴酚蓝,5%2-巯基已醇,10%蔗糖)将膜迷路蛋白稀释为0.9 mg/ml,每孔加入50 μL样品,小心将多层滤纸盖在上盖顶板96孔表面，滤纸间夹入蛇形弯曲的铂丝。上方铂丝接负极，缓冲液池铂丝接正极,200 mA电泳10 min。取下滤纸，丢弃缓冲液，然后筛选抗体：用蒸馏水冲洗微孔,每孔加200 μL PBS-T并在37℃摇