抗BP230单克隆抗体的制备、鉴定和应用

目的探讨大疱性类天疱疮抗原1(BP230)作为皮肤与脑组织的共同抗原在大疱性类天疱疮(BP)合并神经系统疾病(ND)中的作用,制备抗BP230单克隆抗体(mAb)并进行鉴定和应用。方法采用抗原免疫Wistar大鼠,采用细胞融合技术制备并纯化mAb,检测效价和亲和常数,以免疫组化、免疫荧光法和免疫印迹进行鉴定。结果抗BP230 mAb效价在45 000～139 000之间;亲和常数为2.8×10~8L/mol;抗BP230 mAb可与鼠脑反应,主要反应部位为大脑皮质;抗BP230 mAb可与小鼠皮肤基底膜带反应;免疫印迹检测到抗BP230 mAb可与鼠皮肤及鼠脑230 kDa蛋白结合。结论成功制备了可识别小鼠大脑皮质、皮肤的抗鼠BP230 mAb,为进一步研究BP230在BP和ND中的作用提供了研究工具。     

大疱性类天疱疮抗原1各亚型在小鼠皮肤和神经组织中的表达

目的：明确大疱性类天疱疮抗原1（BPAg1）各亚型在不同组织中表达情况,探讨大疱性类天疱疮（bullous pemphigoid,BP）与神经系统疾病（neurological disease,ND）二者之间可能存在的病理机制联系。方法：采用反转录（RT）-PCR方法检测昆明小鼠BPAg1各亚型在大脑、心肌、皮肤和脊髓组织中的表达。结果：BPAg1的神经亚型（BPAg1-a）在小鼠大脑、脊髓、皮肤组织中均有表达;肌肉亚型（BPAg1-b）在大脑、脊髓、心肌、皮肤组织中均有表达;而皮肤亚型（BPAg1—e）仅在皮肤组织中有表达。结论：证实皮肤与神经组织中均存在BPAg1。由于BPAg1亚型的存在,免疫交叉反应可能在BP与神经系统疾病并发的机制中起一定的作用。     

131例神经系统疾病患者血清中抗BP180和抗BP230抗体的检测

目的：检测神经系统疾病患者血清中抗BP180、抗BP230和抗基底膜带抗体的阳性率。方法：收集神经系统疾病患者和正常对照血清,采用酶联免疫吸附试验（ELISA）和间接免疫荧光（IIF）检测两组血清中抗BPl80NC16A抗体和BP230抗体水平。结果：共收集到131例神经系统疾病患者血清(脑卒中109例,脑肿瘤17例,其他神经系统疾病19例)和131例正常对照血清。病例组中抗BP180NC16A1阳性率为1.45%,低于对照组的3.05%,差异具有统计学差异（P = 0.009）,病例组中抗BP230抗体阳性率5.34%与对照组（2.29%）比较,差异无统计学意义。IIF检测抗基底膜带抗体结果均为阴性。结论：抗BP180NC16A抗体在神经系统疾病患者中有较高的阳性率,可能与神经系统疾病患者合并大疱性类天疱疮相关。     

四种抗原在正常人和麻风愈后者中血清学活性及其趋势的比较研究

目的：比较ND－O－BSA（ND）、M.smegmatis(Ms)、LAM-B和α2四种抗原在正常人对照及愈后原麻风病人血清学活性及其态势,探索它们在疾病预测中的潜力。方法：用间接酶联免疫吸附试验（ELISA）检测正常人（192例）和愈后原麻风病人（666例,含8例复发者）血中抗体水平及其态势,计算各抗原检测结果间的个体符合率并加以比较。结果：用ND－IgM-ELISA检测愈后复发的原麻风患者,结果显示：在连续3年中,复发患者绝大多数抗体水平逐渐升高,仅个别持续高水平,或在抗体阴性水平时复发;用4种抗原在单纯检测抗麻风菌抗体有无时,方法间均显示很高的个体符合率,但在预测疾病时,仅ND－IgM-ELISA显示真好的结果。Ms-IgG-ELISA与之相近,ND－IgG-ELISA、α2－IgG-ELISA显示有抗体水平下降趋势。用LAM－B－IgG-ELISA未看到规律出现。结论：4种抗原单纯作为检测抗麻风菌抗体有无时方法间个体符合率高,但作为预测疾病方法时ND－IgM-ELISA显示最有潜力。Ms-IgG-ELISA显示类似的结果。     

SLE皮肤基底膜带自身抗体靶抗原的研究

目的：了解SLE皮肤基底膜带自身抗体（BMZ－Ab）靶抗原，并与自身免疫性大疱病相关抗原进行比较。方法：以人皮肤提取物为抗原，免疫印迹（IB）检测47例SLE及对照的14例大疱性类天疱疮（BP）、2例获得性大疱性表皮松解症（EBA）患者血清中IgG型自身抗体。结果：29／47例SLE血清IgG型自身抗体与表皮和／或真皮抗原反应，其中14／47例单纯结合180kD、145kD、130kD、97kD、85kD、75kD、66kD表皮抗原，8／47例单纯结合115kD、290kD真皮抗原，7／47例同时结合上述不同分子量表、表皮抗原。11／14例BP血清结合230kD、180kD、165kD、130kD、115kD、97kD表皮抗原。2例EBA血清结合290kD真皮抗原。结论：SLE BMZ－Ab靶抗原存在明显异质性。SLE与多种自身免疫性大疱BMZ抗原交叉，提示它们之间的内在相关性。     

基于热分离法制备的人表皮侧抗原的Western印迹实验对大疱性类天疱疮的诊断价值评估

【摘要】 目的 通过热分离方法制备人表皮侧蛋白,评价表皮侧蛋白Western印迹在大疱性类天疱疮（BP）诊断中的价值。方法 热分离健康人皮肤制备人表皮侧抗原,以此为底物,对2015年1月至2017年8月在中国医学科学院皮肤病医院住院的22例BP患者及25例非BP对照血清行Western印迹检测,同时对所有受试者血清行BP180 NC16A ELISA检测。应用统计软件SPSS22.0进行统计学分析。计数资料采用χ2检验及Fisher精确检验法分析。结果 表皮侧蛋白Western印迹、BP180 NC16A ELISA诊断BP的敏感性分别为86.36%（95％ CI为64.03% ~ 96.41％）、95.45%（95％ CI为75.11% ~ 99.76％）（P = 0.294）,特异性分别为100%（95％ CI为83.42% ~ 100％）、92%（95％ CI为75.11% ~ 99.76％）（P = 0.149）。BP患者表皮侧Western印迹条带分析显示,4例见相对分子质量（RMM）为230 000的蛋白条带,18例见180 000的蛋白条带,1例见120 000和1例见97 000蛋白条带,且为180 000蛋白条带的BP患者BP180 NC16A ELISA结果均大于50 U/ml。结论 对BP患者行表皮侧蛋白Western印迹检测,主要阳性条带RMM为180 000;表皮侧蛋白Western印迹诊断BP的敏感性低于BP180NC16A ELISA,当患者自身抗体滴度较低时表皮侧蛋白Western印迹结果往往为阴性。     

副肿瘤性天疱疮合并局灶性Castleman''s病和呼吸系统损害

副肿瘤性天疱疮（PNP）是一种与自身免疫相关的获得性大疱性疾病。本文报告4例同时合并局灶性Castleman‘s病和呼吸系统损害的PNP患者，其中3例合并闭塞性细支气管炎（BO）。他们的免疫学表现较为一致，血清中抗体可识别人角质形成细胞及边缘肺组织提取物中的210kD和190kD抗原。肿瘤切除后患者的皮肤逐渐消失，3例合并BO的患者仍有喘憋及咳嗽。     

大疱性类天疱疮患者血清抗BP180自身抗体识别抗原表位的研究

目的分析大疱性类天疱疮(BP)患者血清抗BP180IgG和IgE自身抗体所识别的抗原表位。方法利用柱亲和层析的方法从10例BP患者血清中提取抗BP180-NC16A自身抗体,用免疫印迹技术对所获IgG和IgE自身抗体分别进行抗原表位鉴定。结果所有10例患者的自身抗体均识别aa507-aa532区域内(NC16A-2和NC16A-2.5)至少一个抗原表位,而且来自同一患者的IgG和IgE自身抗体在该区域所识别的表位完全一致。BP自身抗体与NC16A-1、NC16A-3和NC16A-4区域的结合率较低,与NC16A-5片段不结合。结论BP致病性自身抗体所识别的主要抗原表位可能位于BP180分子NC16A片段的aa507-aa532区域内。     

花斑癣患者血清中抗糠秕马拉色菌IgG、IgM、IgA抗体的检测及其意义

目的：探讨机体体液免疫在花斑癣发病中的作用和意义。方法：以糠秕马拉色菌（Ｍ．ｆｕｒｆｕｒ）整菌（ＷＭＦ）为抗原,用间接酶联免疫吸附试验（ＥＬＩＳＡ）方法,检测６８例花斑癣患者和４１例正常人血清中的抗ＷＭＦ抗体。结果：正常人血清中存在高滴度的抗ＷＭＦ抗体,花斑癣患者血清中抗ＷＭＦＩｇＧ抗体明显低于正常对照组（Ｐ〈０．０１）,男性患者血清中ＩｇＧ抗体低于女性患者（Ｐ〈０．０１）,病程１年以上者血清中特异的ＩｇＧ抗体低于病程不到１年者（Ｐ〈０．０１）。结论：机体血清抗Ｍ．ｆｕｒｆｕｒ抗体可能是人体内天然抗体,且特异的ＩｇＧ抗体具有保护作用。支持花斑癣的发病与免疫缺陷有关。     

银屑病患者血清抗角蛋白抗体的免疫荧光研究

为了探讨血清抗角蛋白抗体与银屑病的关系以及该抗体的生理、病理意义,本文以正常人皮肤冰冻切片为抗原,用IIF检测了35例银屑病患者的血清,并与其它皮肤病和内科疾患作了对照观察.银屑病患者血清抗角蛋白抗体阳性率IgG11.76%,IgM54.29%,IgA54.29%,与正常人(IgG66,67%,IgM96.43%,IgA100%)相比,均明显降低.但23例其它皮肤病IgM60.85%,IgA43%和25例内科疾患IgM63.38%也明显低于正常人.结果提示,抗角蛋白抗体血清阳性率的降低,并非银屑病的特异表现,两者之间并无特殊联系.     

大疱性类天疱疮230000和160000抗原的区域性分布

利用免疫印迹技术筛选出2份大疱性类天疱疮(BP)血清,一份只和230 000分子结合,一份只和160 000分子结合,分别和人体22处正常皮肤作间接免疫荧光(ⅡF),发现230 000抗原和160 000抗原的表达存在明显的区域性差异.230 000抗原在胸腹和大腿屈侧含量最高,头皮和足跖最低.160 000抗原在胸腹含量最高,头皮和颈部含量最低.230 000抗原和160 000抗原在同一部位的表达基本一致,但在腋窝、大腿伸侧和大腿屈侧三个部位明显高于后者.上述差异与临床上BP的好发部位有一定关联,靶抗原表达的区域性差异是造成自身免疫性皮肤病皮疹特异分布形式的一个重要原因.     

Identification of a 160-kD molecule as a component of the basement membrane zone and as a minor bullous pemphigoid antigen.

The antigens in normal human skin defined by antibodies in patients with bullous pemphigoid (BP) were studied by Western immunoblots. Eighteen (90%) of 20 BP sera reacted to a 230-kD antigen. Seven (35%) of the sera reacted to a 160-kD antigen. Two of these reacted only to the 160-kD antigen and five also reacted to the 230-kD antigen. Antibodies to the 160-kD antigen were not present in 25 control sera obtained from normal individuals or patients with other bullous diseases. The 160-kD antigen was present in epidermal extracts of four different specimens of normal human skin but not in dermal extracts or extracts of control cells including melanoma, fibroblasts, lung carcinoma, and colon carcinoma. Monospecific sera with antibodies to either the 230-kD or to the 160-kD antigen reacted solely to their respective target antigens, but not to both, in extracts of epidermis that contained both antigens. The 160-kD antigen broke down to a 140-kD fragment, while the 230-kD antigen was unchanged in the absence of protease inhibitors. Western blot affinity purified antibody to the 160-kD antigen bound only to the basement membrane zone on the epidermal side of 1M NaCl split skin. These results indicate that a 160-kD antigen is a normal component of the basement membrane zone of human skin. The antigen is located on the epidermal side of skin split with 1M NaCl. It is a minor BP antigen, antibodies to which are present in some patients with BP.     

Comparative sensitivity of indirect immunofluorescence to immunoblot assay for the detection of circulating antibodies to bullous pemphigoid antigens 1 and 2

Summary The sera of 263 patients with bullous pemphigoid (BP) were tested by indirect immunofluorescence (IIF) on salt-split skin (SSS) and immunoblot (IB) assay, in order to assess the diagnostic sensitivity of these techniques. Among the 263 sera tested. 198 sera (75%) contained antibasement membrane zone antibodies demonstrable by IIF reacting to the epidermal (98%) or both the dermal and epidermal sides (2%) of SSS. One hundred and eighty-two of the 263 sera (69%) reacted by IB with BP antigens (Ag), most commonly the BPAgl (93 cases, 51%), and a complex of BPAgl and the 180kDa minor BP antigen (BPAg2) (47 cases, 26%). BPAg2 alone was found in 42 cases (23%). A good correlation was found between the detection of autoantibodies by IIF and labelling of BPAg1 and/or BPAg2 by IB assay, in which 152 of 198 sera with an epidermal pattern in IIF identified a BP antigen. IB analysis of the 65 sera negative by IIF yielded positive results in 30 cases (46%). Thirty-one per cent (13 of 42) of sera recognizing by IB BPAg2, were negative by IIF, as compared with 12% (11 of 93) of those recognizing BPAg1 (P < 0.01). Comparing the sensitivity of the two tests, IIF (75%) was found to be more sensitive than IB (69%). Thirty-five of the 263 sera (13%) remained negative by both techniques. It can be concluded from this study that IIF on SSS appears to be a sensitive and reliable assay for screening BP;IB should be performed for the sera that are negative by IIF as it may reveal circulating antibodies, particularly to BPAg2.     

大疱性系统性红斑狼疮的皮肤基底膜带相关抗原

免疫印迹和盐裂皮肤间接免疫荧光检测 ５例大疱性系统红斑狼疮（ＢＳＬＥ）血清，对照为 ５例获得性大疤性表皮松解症（ＥＢＡ）、２０例类天疱疮（ＢＰ）、２０例ＳＬＥ和１０例正常人血清。结果表明，３例（３／５）ＢＳＬＥ血清结合盐裂皮肤真皮侧和真皮提取物中２９０ ０００抗原，其中２例ＢＳＬＥ血清也结合表皮提取物中 １６５ ０００抗原，结果与 ＥＢＡ和部分ＢＰ血清相同。ＳＬＥ血清未结合 ２９０ ０００和 １６５ ０００抗原。提示ＢＳＬＥ血清中存在ＥＢＡ和ＢＰ抗体，推测ＥＢＡ和ＢＰ抗原可能是ＢＳＬＥ的皮肤基底膜带相关抗原。     

Erythrodermic bullous pemphigoid is a clinical variant of bullous pemphigoid

Bullous pemphigoid (BP) is an autoimmune blistering disease of the skin. Several variants olBP have been described but until recently the relationship of these variants to generalized BP was unclear. Several studies have shown that pemphigoid nodularis, pemphigoid vegetans, localized BP and vesicular pemphigoid are true variants of BP as the circulating antibodies in these patients recognize the same 230kDa BP antigen as found in patients with generalized BP. Erythrodermic BP is a very unusual variant characterized by an erythroderma along with blister formation. We describe the third known patient to develop erythrodermic BP and characterize the antigenic specificity of the circulating antibodies in both our newly reported patient with erythrodermic BP and in one of the two other previously reported cases of erythrodermic BP. Both patients with erythrodermic BP had circulating IgG antibodies which bound to the epidermal side of salt-split human skin in a pattern identical to two patients with immunopathologically proven generalized BP. Sera from four erythrodermic patients without blisters and from a healthy normal volunteer, as controls, failed to demonstrate detectable circulating IgG autoantibodies. Immunoprecipitation studies revealed that both patients with erythrodermic BP had circulating IgG autoantibodies which recognized, to varying degrees, the same 230 and 180kDa BP antigens as recognized by sera from two patients with immunopathologically proven generalized BP. Sera from four erythrodermic patients without blisters and from a healthy normal volunteer, as controls, failed to recognize any specific polypeptides. These observations demonstrate that erythrodermic BP is a distinct clinical variant of BP.     

Major antigenic epitopes of bullous pemphigoid 230 kDa antigen map within the C-terminal end of the protein. Evidence using a 55 kDa recombinant protein

In order to obtain greater insight into the nature of B-cell epitopes in bullous pemphigoid (BP), we generated a BP recombinant protein of 55 kDa M_r (rBP 55) from a cDNA sequence encoding for the carboxyterminal region of the 230 kDa BP antigen. Serum IgG from guinea-pigs immunized with rBP 55 stained the basement membrane zone of normal human skin and immunoprecipitated the rBP 55 protein, and also the 230 kDa BP antigen recovered from extracts of cultured keratinocytes, thus confirming that the rBP 55 amino acid sequence is present in native BP antigen. The reactivity of sera from 60 patients with BP was analysed using an immunoblot assay on epidermal protein extracts and on the rBP 55 protein. Forty of the 60 BP sera (66%) contained autoantibodies to the 230 kDa polypeptide in an epidermal extract, and 37 of these 40 sera (92%) recognized the rBP 55 protein. In contrast, no reactivity against rBP 55 was detected with 20 BP sera devoid of autoantibodies against the 230 kDa antigen. Likewise, sera from patients with autoimmune blistering skin disorders other than BP (epidermolysis bullosa acquisita or pemphigus vulgaris), and control sera, were unreactive to rBP 55. These results clearly demonstrate the immunogenicity and antigenicity of the C-terminal end of the 230 kDa BP antigen. They confirm that this 555 amino acid segment, corresponding to rBP 55, contains major epitopes which can bind BP patients' autoantibodies, and suggest that the rBP 55 protein could be useful for further characterization of these B-cell epitopes.     

The major cicatricial pemphigoid antigen is a 180-kD protein that shows immunologic cross-reactivities with the bullous pemphigoid antigen.

Recent studies have shown that sera from patients with cicatricial pemphigoid (CP) contained autoantibodies against epidermal antigens of molecular weight 230 kD and/or 180 kD by immunoblotting, similar to those recognized by bullous pemphigoid (BP) sera. Previous immunoprecipitation studies have shown that BP sera only precipitated the 230-kD antigen. To characterize the CP antigen(s) we tested 10 CP sera, 10 BP sera, and four controls by both immunoprecipitation of radiolabeled cells and immunoblotting of epidermal extracts. For immunoprecipitation, we used 0.5% NP-40 extracts of both normal human keratinocytes and Pam cells. All CP sera precipitated a 180-kD protein that co-migrated with the BP180 antigen precipitated by some individual BP sera. Two of these CP sera also faintly bound a 230-kD protein of similar molecular weight as the major BP230 antigen. CP and BP sera with an immunoblotting pattern of 180 kD immunoprecipitated a co-migrating 180-kD protein. CP sera reacting by immunoblotting with the 230-kD antigen precipitated the 180-kD and/or the 230-kD antigen. In contrast, BP sera reacting with the 230-kD antigen only precipitated this antigen. In further experiments, labeled 0.5% NP-40 extracts from Pam cells were first preabsorbed with a reference BP serum and then immunoprecipitated with CP sera. Under these conditions, CP sera that immunoprecipitated both 180-kD and 230-kD proteins with the standard procedure no longer precipitated these proteins. Our results suggest that a 180-kD protein is the major CP target-antigen that demonstrated immunologic cross-reactivities with the BP180 and the BP230 antigens.     

Characterization of the antibody response in oesophageal cicatricial pemphigoid

Cicatricial pemphigoid (CP) is a subepidermal, autoimmune bullous dermatosis. It is classified as a clinical subset of bullous pemphigoid (BP). However, it differs from BP in some significant ways: (i) in CP mucosal involvement with clinical scarring is prominent; (ii) there is a prominent IgA class antibody response alone or in addition to the IgG class antibody response; and (iii) there is a heterogeneous antibody response in CP, whereas in BP the majority of the antibodies are directed against a 180-kDa hemidesmosomal protein, bullous pemphigoid antigen 2 (BPAg2). Oesophageal involvement in CP is a rare, but often devastating manifestation. In this study we examined the humoral autoimmune response in oesophageal CP, in an attempt to characterize the autoantibody reactivity profile. We used direct and indirect immunofluorescence and Western immunoblotting using normal human skin and oesophagus substrates. We studied patient sera over time in order to search for evidence of epitope spreading in these patients. All patients had positive direct immunofluorescence of perilesional oesophageal epithelium. All patients had positive circulating antibasement membrane zone autoantibody titres. There was a significant IgA class in addition to an IgG class autoantibody response. IgA and IgG antibodies demonstrated significant reactivity with BPAg2 and the 97 kDa linear IgA disease antigen on Western immunoblot suggesting intraprotein epitope spreading. There was no evidence of interprotein epitope spreading over time. Our findings suggest that there is a heterogeneous antibody response in oesophageal CP with the predominant antigen being BPAg2.     

Specific detection of anti-cell surface antibodies in herpes gestationis sera

Abstract We examined 42 herpes gestationis sera with immunofluorescence of normal human skin sections, and found that anti-keratinocyte cell surface antibodies were detected specifically in 10 herpes gestationis sera. The diagnosis of these herpes gestationis cases was confirmed by detecting antibodies against the 180 kD bullous pemphigoid antigen with immunoblotting of its fusion protein. The results of immunoadsorption assay using baculoproteins of both pemphigus vulgaris and pemphigus foliaceus antigens indicated that the herpes gestalionis sera did not recognize common pemphigus antigens. Immunoblotting of human epidermal extracts and immunofluorescence of various tissues also suggested that the sera did not recognize any other desmosomal components or paraneoplastic pemphigus antigens. The significance of this reactivity is unclear. However, because no control bullous pemphigoid sera showed this reactivity, it may suggest a different pathophysiology between herpes gestationis and bullous pemphigoid.     

IgA basement membrane zone autoantibodies in bullous pemphigoid detect epidermal antigens of 270–280 kDa, 230 kDa, and 180 kDa molecular weight by immunoblotting

Bullous pemphigoid (BP) is an acquired subepidermal blistering disease characterized by circulating IgG autoantibodies binding to the 230 and 180 kDa hemidesmosomal proteins. Associated basement membrane zone (BMZ) autoantibodies of the IgA class have been reported in few BP patients. The incidence and clinical relevance of these IgA antibodies, as well as their target antigens are unknown. Sera of 26 patients with BP were analysed for circulating IgG- and IgA-anti-BMZ autoantibodies by indirect immunofluorescence on salt-split human skin. All of the patients had circulating IgG autoantibodies and, in addition, nine (35%) also had circulating anti-BMZ IgA antibodies, that bound to the epidermal side of salt-split skin. By immunoblotting, IgA antibodies in seven of nine sera recognized either the 180 kDa, the 230 kDa, or both BP antigens. Moreover, IgA anti-BMZ antibodies in seven sera also detected an epidermal protein of 270-280 kDa. IgA antibodies did not identify specific bands on immunoblots of dermal extracts. There was no clinical difference between BP patients with or without circulating anti-BMZ-IgA.