大疱及疱疹性皮肤病

982191 免疫印迹技术检测天疱疮患者血清结合的天疱疮抗原/顾富祥（南京鼓楼医院皮肤科）…//中华皮肤科杂志.-1998,31（1）.-47～48 应用免疫印迹,以正常表皮提取物为抗原,检查18例天疱疮患者血清。结果显示4例寻常性天疱疮（PV）血清都与表皮提取物中130000分子反应,4例疱疹样天疱疮（PH）血清2例与表皮提取物中130000蛋白反应,另2例与表皮提取物中160000反应,1例落叶性天疱疮（PF）和9例红斑性天疱疮（PE）血清中有4例与表皮提取物160000蛋白反应。表明免疫印迹技术能准确地测定天疱疮患者血清中自身抗体所对应的自身抗原的分子量,从而准确地诊断天疱疮,并可进一步加以分型。4例PH患者血清反应不同,因为它既可以转变为PV,也可以转化为PF,说明免疫印迹对不典型的天疱疮亦能诊断。图2参5 （张小莲）     

用不同抗原来源进行免疫印迹法分析巴西落叶型天疱疮抗原

本文作者用人表皮提取物以及以牛鼻表皮提纯的桥粒体制剂为抗原,用免疫印迹法检查了27份巴西落叶型天疱疮(BPf)、13份日本散发性落叶型天疱疮(Pf)和7份寻常型天疱疮(Pv)血清.取其结果进行比较.作者用各型天疱疮血清进行间接免疫荧光法检     

正常包皮为底物的免疫印迹技术检测寻常型天疱疮抗体

目的建立以正常包皮为底物用免疫印迹技术检测寻常型天疱疮抗体的方法。方法环切的正常包皮分离表皮后提取天疱疮抗原,进行免疫印迹反应,对15份寻常型天疱疮(PV)患者血清进行检测,并与间接免疫荧光(IIF)比较。结果15份血清都检测到与表皮分离抗原结合的IgG抗体,且在约130KD抗原处出现条带,7份血清同时与分子量为160KD抗原结合,健康对照者中未检测出特异性天疱疮抗体。免疫印迹和间接免疫荧光结果差异无显著性意义(P=1)。结论免疫印迹技术在天疱疮的实验室诊断中具有重要的应用价值。     

免疫印迹技术检测天疱疮患者血清结合的天疱疮抗原

随着分子生物学技术的发展,应用免疫沉淀技术已测出表皮角朊细胞膜上寻常性天疱疮(PV)抗原和落叶性天疱疮(PF)抗原的结构[1,2],应用免疫印迹(Westernblotting)则已进一步测出PV抗原的分子量为130000,PF抗原为160000[3～5]。近年来国外学者已成功地应用免疫印迹对天疱疮进行诊断和鉴别诊断[5]。我们应用免疫印迹,以正常表皮提取物为抗原,检查18例天疱疮患者血清。一、材料和方法(一)血清:18例患者的血清,均来自1994～1996年在我科就诊的天疱疮患者,其中4例PV、4例疱疹样天疱疮(PH)、1例PF和9例红斑性天疱疮(PE)。所有患者皮损周围皮肤…     

在落叶型天疱疮中致病性自身抗体的抗原特异性免疫吸附

寻常型天疱疮(PV)和落叶型天疱疮(PF)组织学表现为表皮内水疱形成。患者血清中可检测到循环性自身抗体,在皮肤器官培养和新生鼠模型中它可导致角朊细胞粘连降低,免疫化学分析显示其相应抗原为130kD(PV)和160kD(PF)的糖蛋白,两者均属于粘合素钙粘     

大疱及疱疹性皮肤病

20 0 0 2 56 2　大疱性皮肤病自身抗体靶抗原定位研究 /杨春俊 (安徽医大一附院皮肤科 )…∥安徽医科大学学报 .- 1999,34(6 ) .- 4 51为进一步确定存在分岐的靶抗原在细胞间或皮肤基底膜带中的位置。验证有关大疱性皮肤病自身抗体靶抗原定位的不同研究结果。采用包埋后金标记直接和间接免疫电镜技术 ,结合免疫印迹技术分别对 5例天疱疮皮损组织中 Ig G的沉积部位、8例天疱疮患者血清中自身抗体 Ig G结合正常人皮肤的部位进行超微水平的研究。同样对 5例大疱性类天疱疮皮损组织中 Ig G的沉积部位及 8例大疱性类天疱疮患者血清中自身抗体 …     

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。     

间接免疫荧光和免疫印迹诊断天疱疮的比较研究

为了在常规诊断为寻常型天疱疮(PV)、落叶型天疱疮(PF)的患者中,比较间接免疫荧光(IIF)和免疫印迹(IB)两种方法,作者回顾性地研究了54例天疱疮,其中PV 40例、PF 14例。年龄40～70岁。     

天疱疮抗体结合靶抗原的定位研究

目的 研究天疱疮抗体铺皮细胞间抗原在超微结构水平的部位。方法 采用ＬＲＷｈｉｔｅ树脂为包埋剂,用金标记包一直接和间接免疫电镜技术,观察天疱疮患者皮损中ＩｇＧ的沉积部位和患者血清中ＩｇＧ型自身抗体结构正常人皮肤的部位。结果 寻常型天疱疮和落叶型天疱疮的直接和间接免疫电镜均在表皮细胞间的桥粒部位觅金颗粒沉积,在非桥数部位的角质形成细胞间未金颗粒沉积。结论寻常型天疱疮和落叶型天疱疮的靶抗原均是桥粒成分,     

异质性大疱性类天疱疮抗体:当间接免疫荧光阴性时,用免疫印迹技术检测及其特性

作者以热分离的正常人表皮的提取物作为抗原来源,用Western免疫印迹技术分析蛋白区带,对59例大疱性类天疱疮(BP)患者的血清进行了研究。59例BP患者中,30例血清用间接免疫荧光(IIF)检查循环抗基底膜带(BMZ)抗体阳性,     

Immunoblot analyses of Brazilian pemphigus foliaceus antigen using different antigen sources

Summary We investigated the Brazilian pemphigus foliaceus (BPf) antigen applying the immunoblotting method to two different antigen sources using 27 patients' sera. Twelve BPf sera reacted specifically with a 150 kD protein in extract of dispase separated human epidermis, while 18 sera yielded a similar protein band in bovine muzzle desmosomal preparation. The diversity of staining intensities between the two samples suggested the heterogeneity of BPf antigens in terms of epitopes. Japanese sporadic pemphigus foliaceus (Pf) sera showed similar results but Japanese pemphigus vulgaris (Pv) sera recognized different antigens of 130 kD or 135 kD, suggesting that BPf is similar to Japanese Pf but is distinct from Pv in respect to the antigenic substance. Furthermore, the present study showed that immunoblot analysis using different antigen sources should be a valuable tool to determine clinical types of pemphigus.This work was supported in part by grant from the Ministry of Health and Welfare of Japan. Dr. Marilia M. Ogawa was an awardee of the Uehara Memorial Foundation Research Fellowship Program (1988)     

Immunoblot and immunoelectronmicroscopic analysis of endemic Tunisian pemphigus

Tunisian pemphigus is a newly described form of endemic pemphigus whose clinical, histological and epidemiological characteristics have recently been detailed. The objective of this study was to analyse the binding properties of autoantibodies present in sera from patients with endemic Tunisian pemphigus using immunoblotting and indirect immunoelectron microscopy (IEM). Thirty patients with pemphigus foliaceus (PF) and six with pemphigus vulgaris (PV) seen in the dermatology department of Tunis Hospital between 1992 and 1994 were selected for this study. Seven of 30 (23%) and six of 12 (50%) PF sera tested bound to the 160 kDa band of desmoglein 1 when tested on bovine tongue and human epidermal extracts, respectively. Two of six and two of three PV sera tested bound to the 130 kDa desmoglein 3 in these two extracts. Immunoblot and indirect IEM showed that 24 of 30 (80%) PF sera contained IgG1, IgG3 or IgG4 antibodies that bound to a 185-kDa polypeptide localized on the desmosomal plaque. This immunological analysis showed that most endemic Tunisian pemphigus sera correspond to PF sera and are characterized by a high frequency of autoantibodies directed against a recently identified 185-kDa antigen of the desmosomal plaque.     

Antigenic specificity of fogo selvagem autoantibodies is similar to North American pemphigus foliaceus and distinct from pemphigus vulgaris autoantibodies

Fogo selvagem (FS) is clinically, histologically, and immunopathologically similar to sporadic pemphigus foliaceus (PF, as seen in North America and Europe), although the epidemiology of these 2 diseases differs markedly. It has been proposed that FS is identical to PF but, for some reason, occurs in an endemic focus in central Brazil. If this hypothesis is correct, the autoantibodies in FS and PF should have similar antigenic specificities. We studied sera from 13 patients with FS from central Brazil, and compared them with 20 sera from patients with PF from the United States. All these sera had circulating antibodies that bound the cell surface of epithelial cells in identical patterns by indirect immunofluorescence on monkey esophagus or normal human skin. In immunoprecipitation studies none of the 13 FS sera precipitated the pemphigus vulgaris (PV) antigen from radiolabeled extracts of cultured human keratinocytes. This is similar to the findings with PF sera of which 19 of 20 sera did not react with PV antigen, but in sharp contrast to the results with PV sera which, as previously reported, all immunoprecipitate the PV antigen. Immunoblotting performed on extracts of normal human epidermis demonstrated that 7 of 20 PF sera specifically and intensely bound an approximately 160 kD polypeptide, previously identified as desmoglein I, a desmosomal glycoprotein. Similarly, 3 of 13 FS sera specifically bound a 160 kD polypeptide. Thirteen normal sera from North America, 8 normal and disease control sera from central Brazil, 11 PV sera, and 12 bullous pemphigoid sera did not specifically bind this polypeptide. Two-dimensional gel electrophoresis confirmed that the 160 kD polypeptides identified by the subgroup of PF and FS sera were identical. These studies demonstrate that, although the exact molecular specificities of the majority of PF and FS sera remain to be determined, FS autoantibodies do not bind the PV antigen and a subgroup of FS autoantibodies have molecular specificity identical to that of a subgroup of PF autoantibodies.     

Pemphigus foliaceus with anti-intercellular and anti-basement membrane zone antibodies. Blocking immunofluorescence and Western immunoblotting studies

In a patient with clinical, histologic, and routine direct immunofluorescence (IF) findings compatible with pemphigus foliaceus (PF), anti-intercellular and anti-basement membrane zone (BMZ) antibodies were found on indirect IF. Blocking IF studies using the biotin-avidin method revealed that intercellular staining of this patient's serum was significantly decreased by sera of PF patients, however, reaction of this patient's serum was not blocked by sera of PV and BP patients. Western immunoblot analysis using the SDS extracts of normal human epidermis demonstrated that this patient's serum reacted with 370-, 220-, 170-, 130- and 21-kD proteins. Another PF serum reacted with 170- and 21-kD proteins. BP sera reacted with 220-, 68-, 46 and 21-kD proteins. Considering the results of the previous reports and this study, 220- and 170-kD protein bands are specific for BP and PF, respectively, and this is the first case of PF with both anti-intercellular and anti-BMZ circulating antibodies including immunoblotting analysis.     

UVB与钙离子对体外培养人角质形成细胞表达天疱疮抗原的影响

目的 研究中波紫外线(UVB)照射以及不同钙离子浓度对角质形成细胞表达天疱疮抗原的影响.方法 通过改变培养基中的钙离子浓度以及采用不同剂量UVB照射体外培养的人角质形成细胞,在不同时间段以寻常型天疱疮(PV)或落叶型天疱疮(PF)血清作为一抗进行免疫荧光检测,观察寻常型天疱疮抗原(PVA)和落叶型天疱疮抗原(PFA)的表达情况;提取细胞或皮肤表皮组织蛋白质,用PV和PF血清进行免疫印迹检测.结果 无论是否增加钙离子浓度,体外培养人角质形成细胞间隙均可以检测到PV血清特异性着色.只有增加培养基中钙离子浓度后,形成的复层化角质形成细胞间隙才可以检测到PF血清特异性荧光着色.不同剂量UVB照射后的角质形成细胞均不产生PF血清的特异性着色.PF血清与160000条带反应,PV血清可与130000和160000两条带反应.结论 体外培养的单层或复层角质形成细胞均可以表达PVA,提高培养基中钙离子浓度可以诱导培养人角质形成细胞复层化并表达PFA,而UVB照射不能促使人角质形成细胞在体外培养条件下表达PFA.     

Detection of pemphigus vulgaris and pemphigus foliaceus antigens by immunoblot analysis using different antigen sources

In an immunoblot analysis with human epidermal extract as a source of antigens, all (28/28) pemphigus vulgaris (Pv) sera showed a specific reactivity with a 130-kD protein. Several, but not all, Pv sera reacted with similar antigens in both a bovine muzzle desmosome preparation and extract of cultured human squamous carcinoma cells. On the other hand, some pemphigus foliaceus (Pf) sera exhibited reactivity with a 150-kD protein, which is most likely desmoglein I, in both the human epidermal extract and the bovine desmosome preparation, but no Pf serum reacted with this antigen in the squamous carcinoma cell extract. Furthermore, 4/16 Pv sera also reacted with a 150-kD protein in the desmosome preparation, which seemed to be the same as Pf antigen. These results show a relationship between antigens of both Pf and Pv and desmosomes, as well as heterogeneities of both Pv and Pf antigens in terms of antigenic molecules or epitopes. Furthermore, this study presents the possibility that immunoblot analysis can be routinely used for differentiation of Pv and Pf antibodies.     

Distinct types of IgG and IgA anti-intercellular autoantibodies from patients with pemphigus and vesiculopustular dermatosis

The pattern of binding of two different types of IgA class pemphigus-like antibodies was compared with that of the true IgG class pemphigus antibody. The IgG antibodies from pemphigus vulgaris (PV) and pemphigus foliaceus (PF) sera bound to the intercellular spaces in normal human epidermis, whereas only the PV antibody reacted with monolayers of keratinocytes derived from human foreskin. Both IgG and IgA antibodies from a patient with PF were found in the intercellular spaces of the epidermis and only the IgA antibody reacted with the cultured keratinocytes. The IgA antibody from a patient with a vesiculopustular eruption and whose serum contained IgA intercellular space antibody alone, bound to the upper epidermis but not to the monolayers of keratinocytes. These findings indicate that PV but not PF antigens can be expressed by monolayers of cultured human keratinocytes and that there are at least two distinct types of IgA intercellular antibodies.     

Comparative study of autoantigens for various bullous skin diseases by immunoblotting using different dermo-epidermal separation techniques

We investigaged the reactivity of pemphigus vulgaris (PV), pemphigus vegetans, pemphigus foliaceus (Pf), Brazilian Pf, bullous pemphigoid (BP), and epidermolysis bullosa acquisita (EBA) sera with an immunoblot analysis using human epidermal and dermal extracts as a source of antigen. To obtain epidermal and dermal extracts three different dermo-epidermal separation methods were used: namely, ethylenediaminetetraacetic acid (EDTA) separation, heat separation, and dispase separation. All the 15PV and the seven pemphigus vegetans sera demonstrated a 130-kDa PV antigen in epidermal extracts obtained by all the three methods. Furthermore, three PV sera also showed a 160-kDa Pf antigen, desmoglein. Ten of 14 Pf sera and six of 15 Brazilian Pf sera reacted with desmoglein in the same pattern in all the three epidermal extracts. Fifteen of the 22 BP sera showed reactivity with 230-kDa BP antigen in the same pattern in all the three epidermal extracts, whereas 14 BP sera delected the 180-kDa BP antigen in extracts of EDTA-and heat-separated epidermis but not in dispase-separated epidermal extract. Dermal extracts were obtained by EDTA- and heat-separated dermis, and all six EBA sera labelled a 290-kDa EBA antigen in both samples. These results suggest that heat-separated skin is as useful as EDTA-separated skin for detecting various autoantigens, but heat separation is preferable because the preparation time is shorter.     

Comparative study of indirect immunofluorescence and immunoblotting for the diagnosis of autoimmune pemphigus

The diagnosis of pemphigus relies on immunopathological criteria including the detection of circulating autoantibodies to desmosomal components. In the present work we compared the usefulness of immunoblotting (IB) and indirect immunofluorescence (IIF) in the diagnosis of pemphigus using monkey oesophagus (MO) and rabbit lip (RL) as epithelial substrates. Among 54 sera from patients with well-documented pemphigus (40 pemphigus vulgaris, PV, and 14 pemphigus foliaceus, PF), 46 (85%) proved positive by IFF (46 on MO and 41 on RL) as compared with 44 (81.5%) positive by IB. IIF and IB were equally sensitive (90%) for the diagnosis of PV whereas IIF (on RL) was more sensitive (71%) than IB (57%) for the detection of PF autoantibodies. However, when the two techniques were considered in combination, the sensitivity of the detection of pemphigus autoantibodies rose to 94.5%. An IB study would therefore be warranted in the presence of an (alleged) pemphigus serum that was IIF-negative since approximately 10% of these were found to be positive. Furthermore, the pattern of IB reactivity may assist in classification, since the 130- and the 160-kDa antigens seem specifically correlated with PV and PF, respectively.     

Two groups of bullous pemphigoid antigens are identified by affinity-purified antibodies

The bullous pemphigoid antigen was originally described as a 240-kD protein extracted from human epidermis, but a subsequent report has described patients' sera which react with epidermal proteins of molecular masses 240, 200, 180, 97, and 77 kD. We have evaluated the heterogeneity of the pemphigoid antigens identified by the sera of 10 patients with clinically typical bullous pemphigoid. We used indirect immunofluorescence and Western immunoblots of epidermal extracts prepared from epidermis separated by either 1 M salt or 20 mM EDTA to characterize the reactivity of both crude sera and affinity-purified antibodies. Affinity purification of antibodies was performed with either normal human epidermis or protein bands blotted onto nitrocellulose as immunoabsorbents. The anti-basement membrane antibody titers determined by indirect immunofluorescence on the saline- and EDTA-separated epidermis were identical. Despite this, Western blots of extracts prepared from EDTA-separated epidermis demonstrated greater amounts of the 240-kD antigen than saline split skin. Multiple antigens were recognized in epidermal extracts on Western blots by most crude BP sera, including bands at 240, 200, 160, and 100 kD. Different sera reacted with these antigens with a markedly different intensity, falling into two major groups, those bearing antibodies to the 240-200-kD antigens and those with antibodies to the 160-100-kD components. When epidermis was used as a substrate for affinity purification of bullous pemphigoid antibodies, the eluted antibodies reacted with multiple bands on Western blots, demonstrating the reactivity of anti-basement membrane zone antibodies with multiple proteins. Antibodies eluted from several individual bands of immunoblots were found to react with the basement membrane on indirect immunofluorescence. When these nitrocellulose-purified antibodies were reapplied to Western blots, they cross-reacted within two groups, the 240-200 kD antigens and the 160-100 kD antigens. We conclude that pemphigoid antigens are best demonstrated when EDTA-split skin is used for extraction and that different pemphigoid sera may contain antibodies to two separate groups of basement membrane zone antigens.