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.     

Bullous pemphigoid antigen concentration in normal human skin in relation to body area and age

Summary The amount of bullous pemphigoid antigen (BPA) in normal human skin on different areas of the body and in bodies of different ages was determined by making endpoint titer estimations using the indirect immunofluorescence technique. Two sera with bullous pemphigoid antibodies were tested with 36 specimens of normal human skin from six cadavers. The greatest expression of BPA was found in plantar sites, whereas the lowest endpoint titers were seen in flexural arm biopsy specimens. Age-dependent differences in BPA expression were detected as well. The oldest individuals tested produced the highest endpoint titers in contrast to the lowest endpoint titers in the youngest. This study suggests that the amount of BPA depends not only on the body region where the biopsy specimen is taken but also on the age of the donor. This is important to know in connection with indirect immunofluorescence assays for BP antibodies on human skin.     

Sensitivity of indirect immunofluorescence test in the diagnosis of pemphigus

Indirect immunofluorescence testing of sera from patients with pemphigus produces a positive intercellular staining on a variety of epithelial substrates with different sensitivity. We aimed to determine the sensitivity of indirect immunofluorescence (IIF) test in detecting pemphigus antibodies, using two different substrates: guinea pig lip and human skin. IIF detected antibodies in 66 out of 109 patients with different types of pemphigus. Sensitivity of IIF performed with guinea pig lip was 40%, while with human skin it increased to 69%. However, we found that neither human skin nor guinea pig lip was sensitive enough to make an IIF test reliable for the diagnosis of pemphigus.     

Comparison of desmoglein ELISA and indirect immunofluorescence using two substrates (monkey oesophagus and normal human skin) in the diagnosis of pemphigus

A prospective study was performed to assess the usefulness of desmoglein enzyme-linked immunosorbent assay testing compared with indirect immunofluorescence in the diagnosis of new cases of pemphigus, as well as to compare the relative sensitivities of monkey oesophagus and normal human skin as substrates for indirect immunofluorescence. These tests were performed on the sera of 29 consecutive new cases of pemphigus diagnosed over a 2-year period based on clinical, histological and direct immunofluorescence findings. Desmoglein enzyme-linked immunosorbent assay was positive in all patients whereas indirect immunofluorescence was positive in only 25 of 29 patients. All four patients with negative indirect immunofluorescence had positive antinuclear antibodies or cytoplasmic fluorescence that could have masked the anti-intercellular antibodies. Desmoglein enzyme-linked immunosorbent assay appeared to reflect the disease activity better than indirect immunofluorescence in a few patients who had active disease of recent onset. Monkey oesophagus was found to be superior or equal to human skin as a substrate for indirect immunofluorescence in both pemphigus vulgaris and foliaceus.     

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.     

Circulating IgA anti-basement membrane antibodies in linear dermatitis herpetiformis (Duhring): immunofluorescence and immunoelectronmicroscopic studies.

IgA deposits were observed by direct immunofluorescence in linear distribution along the basement membrane zone in a case of dermatitis herpetiformis (Duhring). In addition, in the serum of the same patient circulating IgA antibasement membrane zone antibodies were detected by indirect immunofluorescence, utilizing normal human skin and monkey esophagus as substrates. The ultrastructural localization of in vivo-bound IgA and circulating IgA antibasement membrane zone antibodies fixed to substrate tissue in vitro was found to be in the uppermost strata of the dermis below the basal lamina.     

Calcium enhances the sensitivity of immunofluorescence for pemphigus antibodies

Indirect immunofluorescence (IF) to detect pemphigus and pemphigoid autoantibodies is commonly performed with monkey esophagus (ME) as substrate and phosphate-buffered saline (PBS) as a diluent. The purpose of this study was to evaluate comparative IF titers using human skin (HS) as substrate with variations in the buffers employed. Substrates (ME or HS) were incubated in PBS, Tris-acetate-buffered saline (TAS), TAS with 5 mM CaCl+2 (TAS-Ca+2), and PBS or TAS with 1 mM EDTA, prior to incubation with pemphigus or pemphigoid sera for indirect IF. We examined sera from 11 patients with pemphigus vulgaris (PV), 10 patients with Brazilian pemphigus foliaceus (BPF), and 4 patients with bullous pemphigoid. In 20 of 21 pemphigus sera, endpoint indirect IF titers were highest on normal skin with TAS-Ca+2. Six sera (2 PV and 4 BPF) had endpoints that were 5 double dilutions higher than the endpoints obtained with ME and PBS. Six sera (3 PV and 3 BPF) were 4 double dilutions higher, 7 sera (3 PV and 4 BPF) were 2-3 double dilutions higher, and 2 PV sera were equivalent with both substrate/buffers. Preincubation of either tissue with EDTA prior to indirect IF abolished PV and BPF antibody binding completely. Exposure to EDTA after the tissue was incubated with PV or BPF sera did not affect indirect IF titers. In the presence of Ca+2, the antigen was resistant to trypsin in concentrations of 0.001%; however, in the absence of added Ca+2 it was destroyed by 0.0001% trypsin. These differences were not observed with bullous pemphigoid sera; all 4 sera had similar endpoint indirect IF titers. This study shows a significant increase in the sensitivity of indirect IF assays for pemphigus autoantibodies by the use of Ca+2-supplemented buffers on human skin. This finding may also have implications for procedures designed to purify and/or detect pemphigus antigens.     

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.     

Rat bladder epithelium: a sensitive substrate for indirect immunofluorescence of bullous pemphigoid

Serological diagnosis of bullous pemphigoid is based on immunoblotting or indirect immunofluorescence on normal human salt-split skin. These methods are expensive or time-consuming and not available as a routine test in all laboratories. We used rat bladder epithelium as substrate for indirect immunofluorescence and compared it with other substrates and with immunoblotting. Twenty-nine bullous pemphigoid sera were studied on rat bladder epithelium, monkey oesophagus, salt-split skin and with immunoblotting on human keratinocyte cultures. Indirect immunofluorescence on rat bladder epithelium proved to be more sensitive (72%) than on monkey oesophagus alone (45%) and less sensitive than on salt-split skin (97%). Rat bladder epithelium, when tested on 41 sera of a control group, showed a very high specificity: 2/41 (95%). In combination with immunoblotting on keratinocyte extracts, indirect immunofluorescence on rat bladder epithelium allowed 93% of sera to be recognized, a value close to the salt-split skin alone. Rat bladder epithelium appears to be a more sensitive substrate than monkey oesophagus for the diagnosis of bullous pemphigoid and, although less specific, it is easier and faster than using salt-split skin, which remains indispensable to distinguish bullous pemphigoid from epidermolysis bullosa acquisita.     

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.     

Substrate specificity of antinuclear antibodies in scleroderma.

Studies of antinuclear antibodies (ANA) were carried out in 39 cases of systemic scleroderma and for comparison in 19 cases of systemic lupus erythematosus (SLE) and 4 of mixed connective tissue disease (MCTD) using indirect immunofluorescence (IF) methods under standard conditions. The results on three different substrates--monkey esophagus, guineapig lip and rat liver--are reported. In 48.7% of scleroderma cases ANA showed a substrate specificity. The highest percentage of positive results in scleroderma was obtained on monkey esophagus (97.4%) and the lowest on rat liver (61.5%). In SLE and MCTD, in contrast, only about 13% of the sera displayed such specificity. If only sera with substrate specificity are considered, the positive results on monkey esophagus and rat liver are 94.7% and 21.1%, respectively. Titers of sera reacting positively on 2 or 3 substrates were mostly in agreement, although some sera both in systemic scleroderma and SLE showed higher titers on monkey esophagus. The IF pattern was usually the same regardless of the substrate, Tests for ANA in scleroderma should be performed on at least 2 substrates simultaneously.     

Comparison of different epithelial substrates useful for indirect immunofluorescence testing of sera from patients with active pemphigus.

A positive statistical correlation between intercellular antibody titer and disease severity was found for a group of 6 patients with active pemphigus vulgaris, irrespective of whether indirect IF testing was performed on 5 different epithelial substrates which included guinea pig esophagus, human foreskin, rabbit esophagus, monkey esophagus and monkey lip. In a group of 4 patients with pemphigus foliaceus a positive correlation between disease severity and titer was found only when rabbit esophagus was used for indirect fluorescence testing. In both groups of patients there were individual patients in whom the antibody titer was positive at a time when no disease was present and conversely, there were some patients in whom the antibody titer was negative at a time when extensive disease was present. Because of this inconsistency, the use of antibody titers to monitor disease activity and therapy in individual patients may not be justifiable.     

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.     

Presence of anti-basal cell antibodies in oral lichen planus.

BACKGROUND: An autoimmune hypothesis for oral lichen planus (OLP) has been proposed, but no anti-basal cell antibodies (anti-BCA) have been found in the sera of patients with OLP. OBJECTIVE: Our purpose was to test whether the negative results of anti-BCA assays in sera have been due to the insensitivity of the substrates. Rat esophagus, monkey esophagus, as well as human oral mucosa and skin were used to detect anti-BCA in sera of OLP patients. METHODS: By indirect immunofluorescence technique, the rat esophagus was found to be the most sensitive substrate. Therefore it was used as the only substrate to test the presence of anti-epithelial cell antibodies (anti-ECA) in a large group of patients with OLP and other oral mucosal diseases or normal control subjects. RESULTS: The results showed that anti-ECA were detected in 54% (34 of 63) of patients with OLP, 71% (15 of 21) of patients with aphthous ulcers, 29% (6 of 21) of patients with oral carcinoma, 20% (2 of 10) of patients with traumatic ulcer, and 7% (1 of 15) of patients with periodontitis but none of the healthy control subjects (n = 41). The presence of anti-BCA in OLP patients' sera was persistent and lasted for a few months or years. There was a decrease in the serum anti-BCA titers in six of eight anti-BCA-positive OLP patients after topical application of triamcinolone. CONCLUSION: These anti-BCA that persist longer in OLP patients' sera may be autoantibodies that are raised against altered basal cell-specific antigens.     

The use of chemically split tissue in the detection of circulating anti-basement membrane zone antibodies in bullous pemphigoid and cicatricial pemphigoid

Human skin and mucous membranes were used to detect circulating auto-antibodies by indirect immunofluorescence in 20 patients with bullous pemphigoid and eight with cicatricial pemphigoid. The tissue substrate was used intact and after chemical separation through the basement membrane zone (BMZ) by incubation with I M NaCl. Chemically split skin and oral mucosa provided a more sensitive assay for demonstrating circulating anti BMZ antibodies. Use of a battery of substrates increased the number of positives in bullous pemphigoid from 30% detected on monkey oesophagus to 100% (tissue battery). In cicatricial pemphigoid there was an increase in the proportion of positive sera from 13% (monkey oesophagus) to 88% (tissue battery). In addition, a different class of antibody was frequently detected on split tissue substrate suggesting that new antigens are exposed by this procedure.     

Serum levels of autoantibodies to BP180 correlate with disease activity in patients with bullous pemphigoid

Background The 180‐kDa transmembrane hemidesmosomal protein (BPAG2) has been identified as an important autoantigen in bullous pemphigoid (BP). Using the NC16A domain as the antigenic target, a highly sensitive and specific enzyme‐linked immunosorbent assay (ELISA) for the detection of autoantibodies to BP180 was developed. Aim To investigate the correlation of clinical severity and ELISA indices in BP. Methods Antibody titers in the sera from 20 patients were measured using BP180NC16a‐ELISA, and an analysis of the correlation of ELISA indices with disease activity was performed. Serum was obtained from each patient with BP at least three times: before the initiation of treatment, during complete disease control just before the decrease in corticosteroid, and when the dosage of corticosteroid was successfully decreased to half the initial dose. Of the 20 patients, three showed recurrence at a later stage, caused by their decision to stop treatment; serum was obtained at the early stage of recurrence. Results ELISA indices were significantly decreased after successful therapy, although indirect immunofluorescence titers did not always show apparent correlations. Indices measured using BP180NC16a‐ELISA were well correlated with disease activity. Three patients decided to stop taking their medication; subsequently (within 1–2 weeks), blisters recurred, and the levels of antibodies to BP180 increased to close to those before the initiation of treatment. Conclusion BP180 antibody titers showed a closer correlation than indirect immunofluorescence titers to disease activity. The titer of BP180 antibody may be a useful tool for the evaluation of disease activity and for the assessment of the effectiveness of treatments in BP.     

Desmoglein 3-ELISA: a pemphigus vulgaris-specific diagnostic tool

BACKGROUND: Pemphigus vulgaris (PV) is an autoimmune-blistering disease of the skin and mucous membranes caused by autoantibodies against desmoglein 3 (Dsg3), an epidermal desmosomal adhesion protein of the cadherin family. Cloning of the Dsg3 gene and expression of the protein in a native conformation enabled the recent development of a specific and sensitive enzyme-linked immunosorbent assay (ELISA) for the detection of PV autoantibodies. OBJECTIVES: To evaluate serum samples from patients with PV and other dermatologic diseases for anti-Dsg3 antibodies. To compare ELISA values with autoantibody titers obtained by classic indirect immunofluorescence (IIF). DESIGN: Serum samples from patients with PV and various other bullous and nonbullous skin diseases were tested for anti-Dsg3 reactivity by ELISA. SETTING: Ambulatory and hospitalized patients from a university hospital. PATIENTS: Fifty-two serum samples from 11 patients with PV, and serum samples from 11 patients with bullous pemphigoid, 12 patients with other bullous diseases, 22 patients with various nonbullous skin disorders, and 10 healthy individuals were tested. RESULTS: Forty-seven (98%) of 48 serum samples from patients with PV that were positive by IIF on monkey esophagus were also reactive by Dsg3-ELISA, whereas 4 of 4 IIF-negative PV serum samples showed no reactivity by ELISA. In addition, negative ELISA results were obtained from 11 of 11 serum samples from patients with bullous pemphigoid, 10 of 12 serum samples from patients with other bullous skin disorders, 7 of 9 serum samples from patients with autoimmune-connective tissue diseases, and 13 of 13 serum samples from patients with other nonbullous skin diseases. Interestingly, 1 patient with paraneoplastic pemphigus had positive ELISA results. There was a positive correlation (r = 0.654) between ELISA values and IIF titers within the whole population with PV. In addition, when multiple serum samples from 1 patient with PV sampled over a 2-year period were tested, ELISA reactivity paralleled both the IIF titers and the clinical course. CONCLUSION: The Dsg3-ELISA is a sensitive, objective, and PV-specific test that should be considered as an adjunct test for the management of patients with PV.     

Subacute prurigo variant of bullous pemphigoid: autoantibodies show the same specificity compared with classic bullous pemphigoid

We describe a 76-year-old white woman with a 6-month history of intensive pruritus and excoriated papules resembling subacute prurigo. Histopathology showed signs of chronic dermatitis, whereas findings by direct and indirect immunofluorescence microscopy were compatible with bullous pemphigoid (BP). The patient's serum contained IgG autoantibodies that recognized epitopes on both BP180 and BP230 by Western blot analysis of epidermal extracts. In addition, we found strong reactivity with recombinant NC16A, an immunodominant region of BP180 targeted in the majority of BP sera, whereas no antibodies against the keratinocyte-derived soluble BP180 ectodomain (LAD-1) or the recombinant intracellular domain of BP180 were detected. The patient's disease responded well to oral methylprednisolone and mycophenolate mofetil. Disease activity correlated with enzyme-linked immunosorbent assay reactivity of antibodies to BP180 but not with titers of antibodies to the dermoepidermal junction as determined by indirect immunofluorescence on salt-split skin. Our findings suggest that the subacute prurigo form of BP is a true variant of BP.     

Differentiating anti-lamina lucida and anti-sublamina densa anti-BMZ antibodies by indirect immunofluorescence on 1.0 M sodium chloride-separated skin

Sixty-one bullous disease sera containing IgG anti-BMZ antibodies were examined by indirect immunofluorescence on intact skin and skin separated through the lamina lucida by incubation in 1.0 M NaCl. All sera produced an indistinguishable pattern of linear immunofluorescence on intact skin at dilutions of 1:10 or higher. On separated skin, antibodies bound to either the epidermal (epidermal pattern), dermal (dermal pattern), or epidermal and dermal (combined pattern) sides of the separation. The binding patterns were consistent on separated skin from several donors and titers of anti-basement membrane zone antibodies on separated skin were comparable to those on intact skin. Sera from 3 patients with herpes gestationis (HG), 36 patients with bullous pemphigoid (BP), and 1 patient with clinical and histologic features of epidermolysis bullosa acquisita (EBA) showed an epidermal pattern. Sera from 9 patients with BP showed a combined pattern and sera from 6 patients with EBA and 6 patients with clinical and histologic features of BP showed a dermal pattern. Indirect immunoelectron microscopy of selected sera showed antibodies producing the epidermal and combined patterns were anti-lamina lucida antibodies and those producing the dermal pattern were anti-sublamina densa antibodies. These results show indirect immunofluorescence on separated skin is a dependable method for differentiating bullous disease anti-lamina lucida and anti-sublamina densa antibodies and that differentiating between the antibodies is essential for accurate diagnosis in some patients. The results also suggest BP anti-lamina lucida antibodies may have more than one antigenic specificity.     

Intercellular and circulating antibodies in patients with dyskeratosis follicularis, Darier's disease.

Skin biopsies from 6 patients, and biopsies of the palatal mucosa of 4 of these patients with dyskeratosis follicularis (DF) (Dariers disease) were examined for in vivo bound antibodies by means of a direct immunofluorescence (IF) technique. Antibodies located in the intercellular substance of the epidermis were found in the skin lesions of all patients. Immunoglobulins of the classes IgG, IgA and IgM as well as C3 were found in all lesions. No antibodies reacting with the palatal mucosa were found. Sera from 6 patients with DF and 10 control persons were tested by an indirect IF technique for circulating antibodies. Guinea pig lip and normal oral mucosa and skin were used as antigens. All patients sera and one control serum reacted with the basal cell of the guinea pig lip. Three DF sera--but no control sera--reacted with the basal cells of human oral mucosa. None of the sera reacted with human skin.