Obstetrics & Gynecology:
Immunoblotting and Enzyme-Linked Immunosorbent Assay for the Diagnosis of Pemphigoid Gestationis
Sitaru, Cassian MD*; Powell, Jenny MD†; Messer, Gerald MD‡; Bröcker, Eva-Bettina MD*; Wojnarowska, Fenella MD†; Zillikens, Detlef MD*
From the *Department of Dermatology, University of Würzburg, Würzburg, Germany; †Department of Dermatology, Oxford Radcliffe Hospital, United Kingdom; and ‡Department of Dermatology and Allergology, Ludwig–Maximilians University of Munich, Germany.
This study was supported by a grant from Else Kröner Fresenius-Stiftung (25-08-03), Bad Homburg, Germany (to D.Z.).
The authors thank Silvana Noll and Christa Knaus, University of Würzburg, for assistance with the ELISA studies.
Address reprint requests to: Detlef Zillikens, MD, Department of Dermatology, University of Würzburg, Josef-Schneider-Str. 2, 97080 Würzburg, Germany; e-mail: Zillikens_D@klinik.uni-wuerzburg.de.
Received July 24, 2003. Received in revised form December 4, 2003. Accepted December 11, 2003.
OBJECTIVES: To investigate the sensitivity of immunoblotting and enzyme-linked immunosorbent assay (ELISA) to detect autoantibodies to bullous pemphigoid antigen 180 in patients with pemphigoid gestationis and to correlate autoantibody serum levels with disease activity.
METHODS: In serum samples obtained from 44 pregnant patients before initiation of therapy and from the same number of healthy blood donors, the autoantibody reactivity was assayed by immunofluorescence microscopy on human skin sections as well as Western blot analysis and 2 different ELISAs by using recombinant forms of the immunodominant domain of BP180. In addition, ELISA reactivity with this autoantigen was assayed in 6 patients during the course of the disease, and its correlation with the clinical disease activity was estimated by applying the Spearman rank correlation test.
RESULTS: By indirect immunofluorescence microscopy, complement-fixing autoantibodies to the dermal-epidermal junction were found in 93% of patients’ sera. By immunoblotting and ELISA, autoantibodies to bullous pemphigoid antigen 180 were detected in 93% and 86.3% of pemphigoid gestationis patients, respectively, but in none of the healthy controls. Serum levels of autoantibodies as detected by ELISA paralleled the patients’ disease activity.
CONCLUSIONS: Our study shows that immunoblotting and ELISA are sensitive tools for the detection of autoantibodies to bullous pemphigoid antigen 180 in patients with pemphigoid gestationis. In addition, the ELISA is useful to monitor autoantibody serum levels.
LEVEL OF EVIDENCE: II-2
Pemphigoid gestationis, previously referred to as herpes gestationis, is a nonviral autoimmune subepidermal blistering disease associated with pregnancy. It is not related to herpes virus infections; the old term herpes gestationis rather reflects the occurrence of herpetiform lesions as part of the clinical picture.1 Most commonly, the onset of pemphigoid gestationis is in the second or third trimester and tends to exacerbate immediately after delivery. Intense pruritus is a characteristic feature of pemphigoid gestationis and may precede the polymorphic skin lesions by days or weeks. Initially, one may see erythematous papules, plaques, or targetoid lesions, and subsequently, vesicles and bullae may develop. However, blister formation can be absent during the entire course of the disease.1
Immunopathologic hallmarks of pemphigoid gestationis are linear deposition of C3 and, less frequently, of immunoglobulin G along the cutaneous basement membrane zone as detected by direct immunofluorescence microscopy.1,2 By using an indirect complement fixation immunofluorescence technique, circulating immunoglobulin G autoantibodies, termed herpes gestationis factor, is identified in the majority of pemphigoid gestationis sera.1 Immunoelectron microscopy shows deposition of immunoreactants to the upper portion of the lamina lucida, just beneath the plasma membrane of basal keratinocytes.1 The autoimmune response in pemphigoid gestationis is mainly directed to the bullous pemphigoid antigen of 180 kd.3,4 This autoantigen is a transmembrane hemidesmosomal glycoprotein consisting of an intracellular N-terminal portion, a transmembrane region, and a C-terminal ectodomain.5 The extracellular portion of bullous pemphigoid antigen 180 contains 15 collagenous and 16 noncollagenous domains harboring different antigenic sites recognized by autoantibodies from patients with various subepidermal blistering diseases, including bullous pemphigoid, mucous membrane pemphigoid, and linear immunoglobulin A disease.6 The 16th noncollagenous A domain of bullous pemphigoid antigen 180 is the major target of autoantibodies in pemphigoid gestationis,7–9 the antigenic sites are clustered within the membrane-proximal portion of this domain.8–10
The pathogenic relevance of autoantibodies to bullous pemphigoid antigen 180 is supported by several lines of evidence: 1) in pemphigoid gestationis, autoantibodies passively transferred from the mother to the fetus may induce transient skin blistering of the newborn;11,12 2) serum levels of autoantibodies to bullous pemphigoid antigen 180 correlate with disease activity in patients with bullous pemphigoid;13 3) autoantibodies to bullous pemphigoid antigen 180 from patients with bullous pemphigoid recruit leukocytes to the dermal–epidermal junction and induce dermal–epidermal separation in cryosections of human skin;14 and 4) rabbit antibodies that were generated against murine and hamster bullous pemphigoid antigen 180 induce subepidermal blisters when passively transferred into neonatal mice or newborn hamsters, respectively.15,16
The diagnosis of pemphigoid gestationis is based on the demonstration of tissue-bound immunoreactants or circulating complement-fixing antibodies to the basement membrane zone by immunofluorescence microscopy.17 However, patients with pemphigoid gestationis may often be hesitant to undergo a skin biopsy. In addition, indirect immunofluorescence analysis may depend on the quality of the substrate(s), is associated with a subjective assessment of reactivity, necessitates specialized personnel, is relatively time-consuming, and titers of autoantibodies as revealed by the indirect immunofluorescence complement-fixation technique do not correlate well with disease activity.18 Moreover, indirect immunofluorescence microscopy does not distinguish between reactivity to bullous pemphigoid antigens 180 or 230, which represent the 2 major autoantigens in pemphigoid gestationis. In contrast to autoantibodies to bullous pemphigoid antigen 180, clinical and experimental data do not support the pathogenic relevance of autoantibodies to bullous pemphigoid antigen 230.13–16,19
In recent years, immunoblotting and enzyme-linked immunosorbent assay (ELISA) tests for the detection of antibodies to recombinant bullous pemphigoid antigen 180 have been described.20–23 In contrast to immunoblotting, ELISA is less time consuming and allows for obtaining quantitative results. Recently, an ELISA using a recombinant form of the immunodominant region of bullous pemphigoid antigen 180 has become commercially available.24 However, in pemphigoid gestationis patients, an ELISA using recombinant bullous pemphigoid antigen 180 has not yet been evaluated in a larger number of patients. In the present study, we examined sera from 44 consecutive patients with pemphigoid gestationis by both immunoblot analysis and ELISA and demonstrated that these assays are sensitive tools for the detection of autoantibodies to bullous pemphigoid antigen 180 in pemphigoid gestationis. In addition, the ELISA was shown to be useful to monitor disease activity.
MATERIALS AND METHODS
The study included serum samples from 44 consecutive white female patients with pemphigoid gestationis seen at the departments of dermatology in Würzburg, Oxford, and Munich over a period of 10 years, and from 44 controls randomly selected from healthy blood donors. All patients were pregnant at the time of their inclusion in this study and demonstrated tissue-bound immunoreactants or circulating complement-fixing antibodies to the basement membrane zone as revealed by direct or indirect immunofluorescence microscopy, respectively. With approval from the ethics committee of the University of Würzburg, serum samples were collected from these patients before the initiation of treatment. In addition, in 6 randomly selected patients, serum samples were collected during the course of their disease. All of these 6 patients received systemic treatment with prednisolone. The initial dose ranged from 0.3 to 0.5 mg/kg body weight prednisolone, depending on the extent of the disease. Subsequently, the dose of the corticosteroid was tapered according to the individual disease activity. In these patients, 2 investigators recorded the number of blisters/erosions or urticarial plaques on each visit. More than 10 blisters, erosions, or urticarial plaques corresponded to an activity score of 3 and 1–10 lesions to a score of 2; when no skin lesions were detected but the patient was still receiving therapy, the score was 1; a disease activity of 0 was assigned to patients free of lesions and not receiving medication.
The complement-fixation test was performed on cryosections of 1 M NaCl-split skin by using 10-fold diluted serum. Cryosections of human salt-split skin were incubated with patients’ serum at 37°C for 30 minutes followed by washing with phosphate-buffered saline (pH 7.2) twice for 10 minutes. Subsequently, sections were treated with fresh human serum as a source of complement, diluted 1:5 with veronal buffered saline (pH 7.4; containing 150 mM NaCl, 3 mM 5,5′-diethylbarbituric acid, 1 mM sodium 5,5′-diethylbarbiturate, 0.15 mM CaCl2, and 0.5 mM MgCl2), at 37°C for 30 minutes. After washing, complement deposition was visualized with a goat anti–human C3 antibody (Kallestad Diagnostics, Austin, TX).
For preparation of a recombinant form of the immunodominant domain of bullous pemphigoid antigen 180 and immunoblot analysis, glutathione-S-transferase fusion protein containing the 16th noncollagenous A domain of bullous pemphigoid antigen BP180 was expressed in Escherichia coli DH5α and purified by glutathione agarose affinity chromatography.7,25 The recombinant protein was subjected to 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose.25 Blots were blocked for 45 minutes in 3% solution of skimmed milk powder in Tris-buffered saline plus tween-20 buffer (0.02 M Tris-[hydromethyl]-aminomethane, 0.14 M NaCl, tween-20 0.01%, pH 7.5; Sigma Chemical, St. Louis, MO). Human sera were diluted 100-fold in tween-20 0.01% in Tris-buffered saline plus 1% bovine serum albumin. To abolish non-specific reactivity with the glutathione-S-transferase moiety, diluted sera were preadsorbed with recombinant glutathione-S-transferase following a liquid phase protocol described previously.25 The sensitivity and specificity of the immunoblot assay were estimated to be 90% and 100%, respectively.21
ELISA reactivity with the recombinant immunodominant domain of bullous pemphigoid antigen 180 was determined as described.13 The calculated sensitivity and specificity of this assay were 97% and 99%, respectively.21 The nonadjusted intraassay and interassay coefficient of variation were below 10.7% and 15%, respectively. After adjustment of the data based on the internal standard curves, the coefficients of variation were reduced to below 1% and 3.9%, respectively.21 The commercially available ELISA was purchased from Medical and Biological Laboratories (Nagoya, Japan) and performed by following the manufacturer's instructions. The sensitivity and specificity of this ELISA were 84.4% and 98.9%, respectively. The non adjusted intraassay and interassay coefficient of variation were below 20% and 15%, respectively.24
Sensitivities of the immunoassays were estimated by using the Wilson score method.26,27 Statistical analysis comparing reactivities of patients’ sera obtained by complement fixation with those obtained by immunoblotting and ELISA was performed with the Fisher exact test. To estimate the correlation between levels of autoantibodies to bullous pemphigoid antigen 180 measured by the 2 ELISAs, the Pearson product moment correlation coefficient was used. To estimate the correlation between autoantibody serum levels, as detected by ELISA, and disease activity, the Spearman rank correlation test was applied. All statistical tests were performed using SPSS 7.5 software (SPSS, Chicago, IL).
Immunoblot analysis detects autoantibodies to bullous pemphigoid antigen 180 in the vast majority of pemphigoid gestationis sera. In serum from 41 of 44 (93.3%) pemphigoid gestationis patients, antibodies to the 16th noncollagenous domain of bullous pemphigoid antigen 180 were detected by immunoblot analysis (Table 1). Complement-fixing antibodies to the basement membrane zone were also present in 41 of 44 of pemphigoid gestationis patients as revealed by indirect immunofluorescence microscopy on 1 M NaCl-split skin. None of the controls showed specific reactivity by immunoblotting or indirect immunofluorescence microscopy. Complement-fixing autoantibodies were detected in 2 patients that were negative by immunoblotting and ELISA with recombinant protein (pemphigoid gestationis 23 and 24). Autoantibodies in these 2 sera were shown to immunoblot a full-length form of cell-derived bullous pemphigoid antigen 180 in extracts of cultured keratinocytes and may therefore bind to epitopes outside the immunodominant region of this autoantigen. With regard to clinical presentation, these 2 patients were not different from the other 42 patients.
ELISA is a sensitive tool for detection of autoantibodies to bullous pemphigoid antigen 180 in serum of pemphigoid gestationis patients. By using our previously described ELISA protocol,13 autoantibodies to bullous pemphigoid antigen 180 were detected in 38 of the 44 PG sera, but in none of the healthy controls (Figure 1). For the diagnosis of pemphigoid gestationis, sensitivity and specificity of this test were estimated as 88% and 100% (95% confidence interval), respectively. Autoantibodies in 2 of the 6 pemphigoid gestationis sera that did not react with the immunodominant region of bullous pemphigoid antigen 180 by ELISA also failed to recognize this recombinant protein by immunoblotting (Table 1). One pemphigoid gestationis serum that was negative by immunoblotting exhibited reactivity with the same recombinant protein by ELISA. Subsequently, both our previously published assay and the now commercially available ELISA comparatively tested pemphigoid gestationis sera. The sensitivity of this commercial test was, as in our assay, 88%. However, as shown in Table 1, discrepancies between the 2 tests were found in 4 patients (3, 28, 31, and 33). There was a strong correlation of levels of autoantibodies to bullous pemphigoid antigen 180 as detected by the 2 different ELISAs (correlation coefficient 0.752; P < .05). In 2 pemphigoid gestationis patients (23 and 24), complement-fixing autoantibodies were not reactive with the recombinant protein by immunoblotting or the 2 ELISAs. The difference in reactivities of pemphigoid gestationis patients’ sera as measured by indirect immunofluorescence microscopy compared with immunoblotting and ELISA was shown not be significant using the Fisher exact test (P = .24).
To quantitatively monitor serum levels of autoantibodies to bullous pemphigoid antigen 180 during the course of the disease, reactivity with the immunodominant domain was assayed in 6 pemphigoid gestationis patients by using our own ELISA protocol. Serum samples were obtained at the onset of the disease before the initiation of treatment as well as 4 and 8 weeks later. The results of this analysis are summarized in Table 2. By intercorrelating the serum levels of autoantibodies to bullous pemphigoid antigen 180 as measured by ELISA at 3 time points with the Spearman rank correlation test, the autoantibody levels showed a tendency to correlate with disease activity in these patients (r = 0.62; P = .116).
Recently, immunoblot assays and ELISA-based systems have been developed to detect autoantibodies to bullous pemphigoid antigen 180 and are currently used for the diagnosis of bullous pemphigoid.20–24 Pemphigoid gestationis is also associated with an autoimmune response to bullous pemphigoid antigen 180; in fact, autoantibodies to this target antigen appear to be pathogenically relevant in this disease.7–9,11,12 Therefore, we assessed the usefulness of immunoblotting and ELISA in a large number of patients with this relatively rare disease.
Circulating autoantibodies, detected in serum from the majority of our patients, were shown to recognize the immunodominant 16th noncollagenous domain of bullous pemphigoid antigen 180 by Western blot analysis. This finding is in line with previous observations that most pemphigoid gestationis sera react with cell-derived and recombinant forms of this autoantigen.3,4,7,9,20,28 In addition, earlier studies mapping the epitopes recognized by autoantibodies and T cells from pemphigoid gestationis patients demonstrated a tight clustering to the 16th noncollagenous region of bullous pemphigoid antigen 180.7–10,29 These previous observations and our own results indicate that immunoblotting using a recombinant form of the immunodominant region of bullous pemphigoid antigen 180 is a highly sensitive assay and can be used to confirm the clinical diagnosis of pemphigoid gestationis. Complement-fixing autoantibodies were detected in 2 patients who tested negative by immunoblotting and ELISA with recombinant protein. In addition, autoantibodies in these 2 patients were shown to recognize the full-length form of cell-derived bullous pemphigoid antigen 180 in extracts of cultured keratinocytes by immunoblotting.10 In contrast to the majority of patients with pemphigoid gestationis, autoantibodies in these 2 patients may bind to epitopes outside the immunodominant region of bullous pemphigoid antigen 180. Compared with Western blotting, ELISA tests can be performed in a shorter period and allow one to obtain quantitative results. In addition, for a referral laboratory, ELISA is more cost-effective than immunoblotting and immunofluorescence microscopy. When we analyzed pemphigoid gestationis sera with a previously reported ELISA using a recombinant form of the 16th noncollagenous A domain of bullous pemphigoid antigen 180,21 we found autoantibodies in 86.3% of the patients. There was a strong correlation between the results using our ELISA and those determined using another ELISA that has recently become commercially available.24 Interestingly, immunoblotting was somewhat more sensitive than ELISA for the detection of autoantibodies to bullous pemphigoid antigen 180. The reason for this discrepancy remains obscure; however, it is conceivable that autoantibodies of some patients preferentially react with denaturation-dependent epitope(s).
In bullous pemphigoid, several studies demonstrated that serum levels of autoantibodies to bullous pemphigoid antigen 180, as measured by ELISA, parallel disease activity.13,24,30,31 In pemphigus gestationis, however, the possible correlation of levels of bullous pemphigoid antigen 180–specific autoantibody with disease activity had not yet been studied. When we prospectively followed up some of our pemphigoid gestationis patients, we found that levels of autoantibodies to the immunodominant region of bullous pemphigoid antigen 180 parallel disease activity. It is likely that immunosuppressive treatment with corticosteroids in our patients affected serum levels of antibodies to bullous pemphigoid antigen 180 and thus our ELISA readings. The ELISA for the detection of autoantibodies to this autoantigen may not only be a helpful tool to diagnose the disease, but also to guide treatment decisions; especially in those patients who are free of skin lesions yet still receive treatment.
In conclusion, our data indicate that ELISA using recombinant bullous pemphigoid antigen 180 is a sensitive tool for the detection and monitoring of levels of autoantibodies to this hemidesmosomal protein in patients with pemphigoid gestationis. In addition, our data substantiate the pathogenic relevance of autoantibodies to bullous pemphigoid antigen 180 in this disease.
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© 2004 The American College of Obstetricians and Gynecologists
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