盐裂间接免疫荧光技术在大疱性类天疱疮鉴别诊断中的应用研究

目的:探讨盐裂皮肤间接免疫荧光(IIF)技术在大疱性类天疱疮(BP)鉴别诊断中的作用.方法:应用盐裂IIF技术检测78例常规方法诊断为BP的患者血清.结果:43例血清IgG沉积于表皮侧,7例IgG沉积于双侧,11例IgG沉积于真皮侧,另有17例双侧均未见抗体沉积.结论:盐裂IIF仅能用于BP的初步鉴别诊断.     

Comparison of an indirect immunofluorescence assay and a modified sensitive immunoblot assay for the study of the autoantibody in pemphigus vulgaris

Some patients with pemphigus vulgaris (PV) have positive direct immunofluorescence (DIF) but are negative by indirect immunofluorescence (IIF). The purpose of this study was (1) to compare the sensitivity of an IIF assay with an immunoblot (IB) assay, (2) to compare the IIF and the IB assay in PV patients in whom the clinical picture and DIF were consistent, but the IIF was negative and (3) to compare the IIF and the IB assay in patients in clinical remission for 3 years or more. A comparison was made of the titers of PV autoantibody in the IIF assay using monkey esophagus as substrate and the modified sensitive IB assay using preabsorbed normal human skin lysate and COLO-16 lysate as a substrate in the three groups of patients. The sensitivity of the Western blot was enhanced by modifications in the extraction procedure of the lysate, by absorption of lysate with normal human serum and by the use of an enzygraphic web. In group 1, comprising 23 PV patients with active generalized disease, the titers of the autoantibody in the IB assay were 2–4-fold higher than in the IIF assay. This difference was highly significant (P=0.0001). In group 2, comprising 10 patients with limited or minimal PV who were positive on DIF and negative on IIF, all the patients were positive in the IB assay. In group 3, comprising 9 patients clinically free of disease and off all therapy for at least 3 years and negative in IIF assay, all the patients were positive in the IB assay. An additional two such patients who had low titers in the IIF assay had significantly higher titers in the IB assay. In the IB assay normal human skin and COLO-16 cell lines produced similar results even though PV sera bound to a 130 kDa protein on normal human skin lysate and a 105 kDa protein on COLO-16 lysate. The availability of this modified sensitive IB assay will have significant clinical benefit in the diagnosis of PV patients when IIF is negative, and in the study of autoantibody production.     

Use of recombinant pemphigus vulgaris antigen in development of ELISA and IB assays to detect pemphigus vulgaris autoantibodies

Aim/Objective The objectives of this study are: (1) to measure the titers of pemphigus vulgaris (PV) autoantibody in the sera of patients with active disease, using three different assays: (a) Indirect immunofluorescence (IIF) using monkey esophagus as a substrate, (b) immunoblot (IB) and, (c) enzyme-linked immunosorbent assay (ELISA) using recombinant PV antigen (rPVA). (2) To compare the sensitivity of these three assays. Background The titer of PV autoantibodies and disease severity and extent do not always correlate. This could be due to the lack of consistency and specificity of the substrate. Different results are obtained using different substrates. A standard substrate with uniformly controlled source of antigen would be more useful and clinically beneficial. Methods In this study we studied 25 PV patients, six each with bullous pemphigoid (BP), ocular cicatricial pemphigoid (OCP), mucous membrane pemphigoid (MMP), and herpes gestationis (HG), and sera from 16 normal subjects. IIF was used to determine the PV autoantibody using monkey esophagus. IB assay was used according to standard protocol using normal human epidermis and rPVA as substrates. ELISA was performed using rPVA as antigens expressed in E. coli. Results Sera of all 25 PV patients showed binding to the rPVA, normal human sera and the sera from the six BP, six OCP, six MMP, and six HG patients did not show any binding. In addition, we used antisera from rabbits immunized with PVA peptides (Bos-1, Bos-6) which also showed binding to rPVA, whereas normal rabbit sera did not show any reactivity. ELISA and IB titers in all the patients were 2.5 to 160 times higher than with the conventionally used IIF assay. The titers of the PV specific autoantibody measured using the rPVA did not show statistically significant differences between the ELISA and IB assays. Conclusions IB and ELISA are superior to IIF in evaluating the antibody levels in PV patients. ELISA is more practical and is preferable to IB and is recommended for clinical use.     

Diagnostic features of pemphigus vulgaris in patients with bullous pemphigoid. Molecular analysis of autoantibody profile

BACKGROUND: The simultaneous presence of features of pemphigus vulgaris (PV) in patients with bullous pemphigoid (BP) has previously been reported in the literature. OBJECTIVE: The purpose of this retrospective study is to present 13 patients with an initial diagnosis of BP, who subsequently demonstrated coexistent serological features of both BP and PV. METHODS: The following information on each patient was documented, at the time of initial diagnosis: clinical profile on presentation, histology, direct immunofluorescence, indirect immunofluorescence (IIF) using monkey esophagus as substrate, salt-split skin (SSS) and an immunoblot assay. Since all 13 patients failed to respond to conventional systemic therapy, intravenous immunoglobulin (IVIg) was used as an alternative treatment modality. Prior to initiating IVIg therapy, in all 13 patients, serological studies were performed. In addition to IIF using monkey esophagus, an immunoblot assay and SSS, an enzyme-linked immunosorbent assay (ELISA) was performed to detect antibodies to desmogleins. These different assays were done to identify pathological autoantibodies typical of BP and PV. A control group of 25 healthy normal individuals, 37 patients with BP, 17 patients with PV and 12 patients with pemphigus foliaceus were used for comparison of serological studies. RESULTS: At the time of initial presentation, histological and immunopathological studies confirmed the diagnosis of BP in all 13 patients. Prior to the initiation of IVIg therapy, results of IIF using monkey esophagus as substrate demonstrated high levels of anti-intercellular cement substance (anti-ICS) or antikeratinocyte cell surface antibody. Sera of all 13 patients on SSS bound to the epidermal side of the split. In an immunoblot, using bovine gingival lysate as substrate, sera of 6 patients bound to both a 230-kD (BP Ag1) and 180-kD protein (BP Ag2), while 7 sera bound to only a 230-kD protein. All 13 patients had high levels of antibodies to desmoglein 3 on ELISA. In a pilot experiment, the anti-ICS antibody in sera from 6 random patients was found to be predominantly of the IgG4 subclass. Use of IVIg resulted in an effective clinical response and the maintenance of a prolonged clinical remission. CONCLUSION: In patients with BP, who are nonresponsive to conventional therapy, the presence of two autoimmune diseases or a dual diagnosis should be considered.     

Place of human amniotic membrane immunoblotting in the diagnosis of autoimmune bullous dermatoses

Background Fine analysis of antiskin autoantibodies can contribute to the differential diagnosis of autoimmune bullous dermatoses. Objectives To develop a high‐performance immunoblotting method using human amniotic membrane as the antigen source, and to compare it with current laboratory methods. Methods Sera from 113 patients were tested by immunoblotting (IB), rat and monkey oesophagus and salt‐split skin indirect immunofluorescence (IIF), and enzyme‐linked immunosorbent assay (ELISA) quantification of anti‐BP180‐NC16a and anti‐BP230, or antidesmoglein (Dsg) 1 and 3 antibodies. There were 56 cases of bullous pemphigoid (BP), 22 cases of mucous membrane pemphigoid (MMP), eight cases of epidermolysis bullosa acquisita (EBA), two cases of bullous systemic lupus erythematosus (BSLE), 17 cases of pemphigus vulgaris (PV), and four cases each of pemphigus foliaceus (PF) and paraneoplastic pemphigus (PNP). Results In BP, the three methods had similar sensitivity (84–89%) for both anti‐BP180‐NC16a and anti‐BP230 antibody detection. In MMP, autoantibodies (mainly directed against BP180 or laminin 332 subunits) were detected in 77% of patients by IB, compared with only 9% by IIF on rat and monkey oesophagus and 36% on salt‐split skin, and 14% by anti‐BP180‐NC16a and anti‐BP230 ELISA. In patients with pemphigus, ELISA had 92% sensitivity for anti‐Dsg1 and 3, but IB and rat bladder IIF were necessary to confirm PNP by revealing specific and rare patterns (antidesmoplakin I/II, antienvoplakin and antiperiplakin antibodies). IB also revealed anticollagen VII antibodies in 60% of patients with EBA and BSLE, and antibodies to BP180, BP230 and Dsg3 in a few patients who were negative using the other two techniques. Conclusion Amniotic membrane immunoblotting is an interesting diagnostic tool for bullous diseases, as the entire panel of autoantibodies can be detected with a single extract. This method improves the identification of complex and heterogeneous autoimmune processes in conjunction with IIF and ELISA, and is particularly useful for MMP characterization.     

A case of linear IgA disease in a child with IgA and IgG circulating antibodies directed to BPAg2

A 7-year-old Caucasian girl had multiple bullae on the trunk, upper and lower limbs (Fig. 1a,b) for 10 days. The lesions were large, tense, and asymptomatic, and mucosae were not involved. Laboratory findings were all normal. Histopathology revealed a subepidermal blister containing numerous neutrophils, eosinophils, and fibrin. Direct immunofluorescence of perilesional skin disclosed linear deposition of IgA and slight linear deposits of IgM at the basement membrane zone (Fig. 2). Indirect immunofluorescence on monkey esophagus and on human salt-split skin was negative. Immunoblot assay (IB), performed with an epidermal extract, revealed IgA and IgG antibodies directed to BPAg2 antigen (Fig. 3), while it was negative when performed on dermal extract. Enzyme-linked immunosorbent serologic assay using a commercial kit (MBL, Naka-Ku Nagoya, Japan) with the noncollagenous domain (NC16A) of the BPAg2 antigen was negative for both IgA and IgG. A diagnosis of linear IgA disease (LAD) was made and a treatment with dapsone (50 mg/day) and prednisolone (30 mg/day) was initiated. One month later, lesions had cleared.     

Enzyme‐linked immunosorbent assay (ELISA) and indirect immunofluorescence testing in a bullous pemphigoid and pemphigoid gestationis

Enzyme‐linked immunosorbent assay (ELISA) is an excellent tool for detection of circulating antibodies against the NC16A portion of BP180 antigen. We compared the sensitivity and specificity of a commercially available BP180‐NC16a domain ELISA with that of an indirect immunofluorescence (IIF) testing in the evaluation of bullous pemphigoid (BP) and pemphigoid gestationis (PG), and analyzed the relationship between ELISA results and the presence of IgG deposition, in an epidermal or combined pattern, on direct immunofluorescence (DIF) testing of salt‐split skin. ELISA was performed on serum from 28 patients (24 BP, 4 PG) and 50 controls. IIF testing was performed on serum from 27 patients and 98 controls. For the group of 28 patients with BP or PG, ELISA had a sensitivity of 93% and specificity of 96% (P < 0.001), while sensitivity was 74% and specificity 96% (P < 0.001) for IIF testing. In these patients, ELISA has a higher sensitivity than IIF testing, but similar specificity. Evaluation of controls who had IgG deposition on the dermal side of salt‐split skin on DIF testing showed specificity for the ELISA of 100% (all four cases negative) and 80% for IIF testing (one of five positive). Positive ELISA correlated with a diagnosis of BP or PG only in patients who had IgG at the basement membrane zone (BMZ) by DIF testing. Overall, ELISA appears to have greater sensitivity and specificity for BP or PG than does IIF testing.     

Bullous pemphigoid in Liguria: A 2-year survey

BACKGROUND: The epidemiology of bullous pemphigoid (BP) is not clear because of the heterogeneity of the disease, and its possible association with internal malignancies has been under debate for many years. We report the findings of a 2-year study on incident BP cases in the Liguria region of Italy. SUBJECTS AND METHODS: Thirty-two patients with BP were collected over the 2-year period. Diagnosis was made based on clinical findings and confirmed by histology, direct immunofluorescence (DIF) and indirect immunofluorescence (IIF) with salt-split skin and monkey oesophagus, and immunoblotting (IB). All patients were thoroughly investigated for possible malignancies and all were followed up for 6 months to monitor the response to treatment. RESULTS: DIF showed linear deposits at the dermoepidermal junction in all but one patient. IIF gave positive findings for 15 sera tested with monkey oesophagus and 20 tested with salt-split skin. IB gave positive findings in 19 cases. There was a malignancy in six cases, but no clinical or immunological features that could be considered to predict this occurrence. CONCLUSIONS: The findings of this study are in accordance with most of the data found in the literature, including the fact that IgG serum levels did not predict the course of the disease. Contrary to previous indications, IgE levels were not indicative of disease course either. Mucosal lesions, erythema multiform-like lesions, negative IIF findings and antibodies to AgPB2 were not a prediction for the development of malignancy.     

Heterogeneous bullous pemphigoid antibodies: detection and characterization by immunoblotting when absent by indirect immunofluorescence

We studied sera from 59 patients with bullous pemphigoid (BP) and 25 control subjects (normal volunteers, patients with pemphigus, psoriasis, eczema, or other dermatoses) by western blotting analysis on protein bands from normal human heat-separated epidermis. BP sera reacted with four protein bands that were not detected by control sera: two major bands at 220-240 and 165 kD and two faint bands at 190 and 95 kD. Three of these bands were significantly associated with BP: 220-240 kd (51% of the BP patients; p less than 0.001), 165 kD (49%; p less than 0.001) and 190 kD (20%; p less than 0.05). These results are consistent with a molecular heterogeneity of BP antibodies, because each individual BP serum showed a distinctive pattern of reactivity. Thirty out of the 59 BP sera contained anti-basement membrane zone antibodies demonstrable by indirect immunofluorescence (IIF). All these IIF positive BP sera reacted by immunoblotting with at least one protein band: 23 (77%) with the 220-240-kD band and 21 (70%) with the 165-kD band. Furthermore, 45% of the 29 IIF negative BP sera showed a reactivity with the 220-240-kD band and/or the 165-kD band. These results indicate that western immunoblotting might be a more sensitive method for the detection of circulating BP antibodies than IIF techniques, including IIF on salt split skin.     

IgE and its related phenomena in bullous pemphigoid

This study was designed to analyse IgE and its related phenomena in bullous pemphigoid (BP). We analysed 17 BP sera by indirect immunofluorescenee (IIF) and immunoblotting (IB) using a monoclonal antibody to IgE. In addition, inflammatory cells in lesional skin from 11 patients with BP were analysed by the alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique using monoclonal antibodies to IgE and Fc?RII/CD23. IgE class anti-basement membrane zone (BMZ) autoantibody was detected in nine of 17 sera (52.9%) by IIF. IgG class anti-BMZ antibody could block the BMZ-binding reactivity of IgE class antibody. Titres of IgE class autoantibody in the sera ranged from 1:40 to 1:320, and statistically correlated with serum IgF levels. Two of 11 sera contained an IgE class autoantibody which recognized a 230-kDa BP antigen by IB. By radio-allergosorbent test (RAST), IgE-specific antibodies to an extended series of common inhalant and food allergens were detectable in six sera with higb concentrations of total IgF(over 3300 IU/ml). IgE-bearing and Fc?RII-expressing cells were demonstrated in the upper dermis and along the BMZ in seven of 11 biopsy specimens by tbe APAAP technique. The distribution and number of IgE-bearing cells in the lesions were similar to those of the FcEERII-expressing cells. Tbese results suggest that botb IgE-mediated immune responses and autoimmunity characterize BP as distinctive features.     

免疫即迹法与分离人皮肤IIF洁检测类天疱疮抗体的比较研究

采用１ｍｏｌ／ＬＮａＣｌ分离正常人皮肤为底物的ＩＩＦ法和免疫印迹技术检测１２２例类天疱疮患者血清中抗基底膜抗体，比较研究上述两种方法对检测类天疱疮抗体的灵敏度及价值。结果分离皮肤ＩＩＦ法阳性８９例（７２．９５％）。免疫印迹法检测，与２３０ＫＤ、１８０ＫＤ和１６０ＫＤ分子量蛋白抗原结合阳性９２例（７５．４１％）。两法合用总阳性率为８３．６１％。分离皮肤ＩＩＦ法适合于常规采用，而免疫印迹法是前者的进一步补充。     

The subclass distribution of IgG autoantibodies in cicatricial pemphigoid and epidermolysis bullosa acquisita

To study the subclass distribution of autoantibodies and their complement-fixing capacity in cicatricial pemphigoid (CP) and epidermolysis bullosa acquisita (EBA) we studied the sera from 23 patients by both indirect immunofluorescence (IIF) on 4-microns cryostat sections of normal human skin and immunoblotting of epidermal or dermal extracts. Monoclonal antibodies of strict specificity for human IgG subclasses were used. Sera from 20 patients with BP served as controls. In addition, total IgG subclass levels were determined by indirect competitive ELISA in all sera. Complement binding capacity was studied by IIF using antibodies to C3 after incubation of skin section with autoantibodies and source of fresh complement. CP autoantibodies reacting with the 230-240 kD and/or the 180-kD epidermal bands showed an IgG4/IgG1 subclass restriction, with a predominance of IgG4 in 10 cases, of IgG1 in four. In BP sera, IgG4 and IgG1 autoantibodies were detected with a similar frequency (100% and 83%, respectively). In EBA sera, autoantibodies reacting with the 290 kD and 145 kD dermal bands also showed an IgG1/IgG4 restriction. Concordant results were obtained by IIF. However, the IIF method had a lower sensitivity for the detection of IgG4 CP antibodies and IgG1 EBA antibodies than immunoblotting. Finally, when CP antibodies were analyzed for their complement-binding activity, it was found that sera containing IgG4 autoantibodies alone never fixed complement whereas all complement-fixing CP sera had IgG1 autoantibodies, suggesting that only this subclass of antibodies is capable of fixing complement.     

自身免疫性表皮下大疱病基底膜带自身抗体的检测

目的 比较免疫印迹（ＩＢ）和盐裂皮肤间接免疫荧光（ＩＩＦ）检测自身免疫性表皮下大疱病（ＳＡＢＤ）基底膜带自身抗体（ＢＭＺ－Ａｂ）的敏感性和特异怀。方法 分另应用ＩＩＦ和ＩＢ技术对９７例ＳＡＢＤ患者血清中ＩｇＧ型或ＩｇＡ型ＢＭＺ－Ｚｂ进行检测。结果 ＩＩＦ法阳性率７５．３％,免疫印迹法阳性率７９．４％,两者在检测灵敏度上无显著性差异。结论 二者均可用于ＳＡＢＤ中ＢＭＺ－Ａｂ的检测,二者联用对于提高Ｂ     

Role of BP180NC16a-enzyme-linked immunosorbent assay (ELISA) in the diagnosis of bullous pemphigoid in China

Background Autoantibodies of bullous pemphigoid (BP) patients react with two components of the hemidesmosome of stratified epithelia: the BP antigen 230 (BP230) and the BP antigen 180 (BP180). Recently, strong evidence has been provided that autoantibodies to BP180 play a key role in subepidermal blister formation in BP patients, and NC16A contains an important antigen determinant of BP. Objective To study the role of BP180NC16a enzyme‐linked immunosorbent assay (BP180NC16a‐ELISA) in the diagnosis of BP in China. Methods Sera from BP patients (n = 42) and control subjects (normal controls, n = 24; pemphigus patients, n = 18) were measured by BP180NC16a‐ELISA. All BP sera were obtained at presentation from patients who had not received previous systemic treatment. The values of immunoglobulin G (IgG) antibody levels measured by ELISA were compared with those measured by indirect immunofluorescence (IIF) (gold standard for the diagnosis of BP) on salt‐split skin. Results Using BP180NC16a‐ELISA, 41 of the 42 BP sera were positive, whereas only one of the serum samples from 24 normal controls was positive and all the pemphigus sera showed a negative result. Thus, the sensitivity and specificity of BP180NC16a‐ELISA were both 97.62%. There was no correlation between the mean ELISA values and IIF titers. The ELISA and IIF results were further compared and analyzed using a 2 × 2 contingency table, which showed that they were not significantly different. Conclusions It is suggested that BP180NC16a‐ELISA is a useful tool for the diagnosis of BP.     

大疱性类天疱疮与某些神经系统疾病间发病机制的研究

目的 探讨大疱性类天疱疮（BP）与某些神经系统疾病（ND）之间可能存在的相关病理机制。 方法 分别以正常人皮肤组织和正常小鼠脑组织蛋白提取物作为抗原，以BP合并ND患者（12例）血清作为一抗，通过免疫印迹方法明确患者血清中抗体是否与脑组织发生反应。同时以单纯BP患者（42例）、单纯ND患者（15例）、正常人（30例）及抗BPAg1多抗作为对照。 结果 以正常人皮肤组织提取蛋白为底物，大部分BP合并ND组与单纯BP组患者可出现230 000、180 000或165 000抗原条带。BP合并ND组10例患者血清可识别脑组织中抗原条带（83%）；单纯BP组患者中5例可识别脑组织中抗原条带（12%），单纯ND组患者中2例可识别脑组织中抗原条带（13%），正常人对照组无条带出现。3例合并脑出血的BP患者血清抗体可同时识别皮肤和脑组织中的BPAg1。 结论 BP患者血清中识别脑组织抗原的抗体可能在BP与神经系统疾病合并发生的机制中起到一定作用。     

Sensitivity of indirect immunofluorescence and immunoblotting for the detection of intercellular antibodies in endemic pemphigus foliaceus (fogo selvagem)

BACKGROUND: Two assays are available to detect anti-skin antibodies in patients with fogo selvagem (FS): indirect immunofluorescence (IIF) and immunoblotting (IB). This study was conducted to compare the sensitivity of these assays in detecting FS antibodies. DESIGN: Eighty-nine serum samples of 48 patients with FS and control serum from 15 normal individuals were tested concurrently for the presence of FS antibodies by IIF and IB. IIF studies were conducted using four different substrates: human skin, monkey and guinea pig esophagus, and bovine tongue. RESULTS: FS antibodies were detected much more commonly by IIF than by IB, i.e. in 71% vs. 28% of serum samples respectively. By IIF, the antibodies reacted most strongly against human skin. CONCLUSIONS: IIF is a more sensitive assay than IB for detecting antibodies associated with FS. The sensitivity of the test is maximized by using human skin as a substrate.     

Anti-basement membrane zone antibodies in elderly patients with pruritic disorders and diabetes mellitus

Anti-basement membrane zone (anti-BMZ) antibodies are detectable in a low percentage of elderly subjects without clinical signs of bullous pemphigoid (BP). BP may initially mimic other pruritic dermatoses and may be more common in patients with diabetes mellitus (DM), since DM is frequently associated with pruritic disorders. The aim of the present study was to analyse a possible association of BP and DM and to detect subclinical BP among elderly patients with pruritic dermatoses. Ninety elderly patients (78.6 +/- 4.7 years) treated for dermatologic conditions were divided into four groups: I. DM+/pruritus+, II. DM-/pruritus+, III. DM+/pruritus-, and IV. DM-/pruritus-. Patients' sera were tested by indirect immunofluorescence (IIF) on monkey oesophagus; positive or dubious results were further evaluated by ELISA with human recombinant BP180 and BP230 proteins and purified laminin 5. Positive results were found in 1 of 21 (4.8%) patients in group I, 6/31 (19.3%) patients in group II, 1/18 (5.5%) patients in group III, 3/20 (15%) patients in group IV. In the whole cohort positive anti-BMZ antibodies of linear or basal cell cytoplasmic and membrane pattern were found in 11 cases (12.2%). ELISA was positive in 11/29 (37.9%) tested sera for at least one antigen (BP180, BP230 and laminin 5 ELISA was positive in 7, 5, and 2 sera, respectively). Positive IIF corresponded with positive ELISA in 6/11 (54.5%) cases (ELISA with BP180, BP230, laminin 5 was positive in 5, 3, and 1 serum, respectively). Thus, by IIF, a significant portion of elderly patients had anti-BMZ antibodies and these findings were confirmed by ELISA. There was no statistically significant difference in the presence of anti-BMZ antibodies among the groups I-IV. Thus, the association of anti-BMZ antibodies with age overrules the potential association with DM and/or pruritus.     

The study of the participation of basement membrane zone antibodies in the formation of the lupus band in systemic lupus erythematosus

BACKGROUND: The lupus band test (LBT) is an important auxiliary method in the diagnosis of systemic lupus erythematosus (SLE), but the mechanism of its formation is still unknown. There are many kinds of autoantibodies, such as basement membrane zone autoantibodies (BMZ-Abs), in patients with SLE. AIM: To detect whether skin BMZ-Abs participated in the formation of the lupus band in SLE. Methods Immunoglobulin G (IgG)-type BMZ-Abs in 15 SLE patients were detected by means of immunofluorescence (IF), immunoblotting (IB), and immunoelectron microscopy (IEM). RESULTS: Direct immunofluorescence (DIF) on salt-split skin showed epidermal fluorescence in 12 of the 15 SLE patients. Two of the 12 patients also showed dermal fluorescence. Indirect immunofluorescence (IIF) on salt-split skin revealed that, in 12 of the 15 (80%) sera, antibodies were bound to the epidermal roof of the salt-split skin. IB showed that, in 14 SLE sera, autoantibodies reacted to 230, 200, 180, 130, and 97 kDa epidermal extracts and 75 kDa dermal extracts. Direct (DIEM) and indirect (IIEM) immunoelectron microscopy showed that gold particles were directed to every region of the BMZ, including hemidesmosomes, lamina lucida, lamina densa, and sublamina densa. CONCLUSIONS: BMZ-Abs in SLE sera may participate in the formation of the lupus band.     

Use of sodium-chloride separated human skin in detection of circulating anti-basement membrane zone antibodies

The sensitivity of the indirect immunofluorescence (IIF) technique for detection of circulating basement membrane zone (BMZ) antibodies was evaluated, employing NaCl-separated human skin and intact skin as substrate. Consecutive serum samples from 12 patients with clinically, histologically and immunohistologically verified bullous pemphigoid (BP) were investigated in parallel on both substrates, in dilutions ranging from 1:10 to 1:1,280. All BP sera showed linear deposits of IgG at the BMZ on intact skin, with titres ranging from 10 to 160. On NaCl-separated skin, all BP sera produced a linear epidermal fluorescent band for IgG, with titres ranging from 80 to 1,280. None of the sera showed deposits of IgM anti-BMZ antibodies. Sera from 5 healthy donors (dilutions 1:10) produced no fluorescence, either on intact or on NaCl-separated skin. The serum-titres of circulating anti-BMZ IgG antibodies in 2 patients with corticosteroid-resistant BP were significantly reduced (from 160 to less than 10) during treatment with plasmapheresis, when using NaCl-separated skin as substrate for IIF, whereas the serum-titres showed insignificant reduction (from 20 to less than 10), when using intact skin as substrate. We conclude that the IIF method is more sensitive for detection of circulating anti-BMZ antibodies, when NaCl-separated skin as compared with intact human skin is employed as substrate.     

Anatomical distribution and immunological characteristics of epidermolysis bullosa acquisita antigen and bullous pemphigoid antigen

A Japanese patient with epidermolysis bullosa acquisita (EBA) was autopsied, and direct immunofluorescence (DIF) testing was performed. Using this patient's serum (EBA serum) and three bullous pemphigoid (BP) sera, the anatomical distribution and immunological characteristics of EBA antigen and BP antigen were investigated by indirect immunofluorescence (IIF). EBA antigen showed the same anatomical distribution as BP antigen in DIF and IIF studies; both antigens were limited to the skin, tongue, oesophagus, trachea, cornea and bladder. EBA antigen was located on the dermal side of both NaCl and PBS-separated skin, whereas BP antigen was limited to the epidermal side. Ethanol fixation abrogated the antigenic stability of BP antigen, but not that of EBA antigen. No difference was found when acetone or formalin fixation was used. The separation methods and prefixation in ethanol could be useful techniques applicable to the classification of the bullous disorders which manifest circulating anti-BMZ antibodies.