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Original articles

Evaluation of bullous pemphigoid 180 ELISA in the diagnosis of bullous pemphigoid patients

Saleh, Marwah A.

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Journal of the Egyptian Women's Dermatologic Society: September 2014 - Volume 11 - Issue 3 - p 197-200
doi: 10.1097/01.EWX.0000443991.69321.52
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Abstract

Introduction

Bullous pemphigoid (BP) is an acquired autoimmune blistering skin disease that has a higher incidence in elderly population 1,2. The autoantibodies in those patients target the hemidesmosomes (dermoepidermal adhesion molecules) – BP antigens I (230) and II (180). The noncollagenous domain 16a (NC16a) of BP180 is considered to be the immunodominant epitope 3. Diagnosis of BP is established by a combination of the presence of tense blisters clinically, the presence of subepidermal blister histopathologically, linear deposits of immunoglobulin G (IgG) and C3 along the dermoepidermal junction in immunofluorescence 4, and the detection of the antibodies targeting BP180 or BP230 antigens by enzyme-linked immunosorbent assay (ELISA) 5 or immunoblot 1.

Bullous pemphigoid disease area index (BPDAI) is a scoring system developed by the international autoimmune bullous disease committee to standardize the description of disease severity among different centers 6. ELISA is an easy and rapid quantitative assay for the diagnosis of autoimmune diseases. The evaluation of BP180 7 as well as BP230 8 ELISAs was carried out in many populations. As BP180 is considered to be the immunodominant epitope and as the sensitivity of BP180 ELISA is higher than BP230 ELISA, we sought to evaluate the usefulness of BP180 ELISA in the diagnosis of Egyptian BP.

Patients and methods

Patients’ data

In this analytic cross-sectional study, 15 patients who fulfilled the typical clinical and pathological criteria of BP were enrolled. Fifteen volunteers served as normal controls. The age and sex of the patients and controls were matched as much as possible. None of the patients received treatment before. The disease severity was assessed by BPDAI. Skin biopsies were obtained from the patients using 4-mm punch for direct immunofluorescence (DIF). The skin was immersed in OCT compound (Tissue-Tek; Sakura, Tokyo, Japan) and rapidly frozen by liquid nitrogen then kept at −30°C until cutting. The skin was cut into 5-μm sections and stained using polyclonal rabbit antihuman IgG-fluorescein isothiocyanate antibody (Dako, Glostrup, Denmark). Sera were collected from patients as well as controls. All sera were collected and stored at −30°C until testing. Verbal consent was obtained after proper orientation of the participants with respect to the objectives of the study. Data confidentiality and the impact of the study were respected and maintained.

All participants were informed according to the Helsinki Declaration of Biomedical Ethics. A consent was obtained after proper orientation of the participants with respect to the objectives of the study.

Bullous pemphigoid 180 ELISA

The reactivity of the sera against human BP180 was measured using BP180 ELISA coated with NC16a recombinant protein (MBL, Nagoya, Japan) and was detected using horseradish peroxidase-conjugated antihuman IgG according to the manufacturer’s instructions. The index value was calculated by the following equation: [(OD−negative control)/(positive control−negative control)]×100. Index greater than 20 was considered positive according to the manufacturer’s instructions. The ELISA index was measured for all patients before treatment. Follow-up was performed for three patients every month for 6 months.

Statistical analysis

The collected data were analyzed using Microsoft excel 2007 and GraphPad prism 5 for Windows (GraphPad Software, San Diego, California, USA). Continuous quantitative data were described in terms of range, mean, and SD when parametric tests are appropriate or median when nonparametric tests are appropriate. Categorical data were described in terms of number and percentage. Data were corrected using logarithmic transformation. The Mann–Whitney test was used for comparing the results of patients and controls. Spearman’s correlation was used for correlating baseline BPDAI and the ELISA index. P value of less than or equal to 0.05 was considered significant.

Results

Ten patients (66.6%) were women and 5 (33.3%) were men. The age ranged from 40 to 76 years. The mean age was 54.5±10.8 years. The data are summarized in Table 1. All patients showed linear IgG deposits along the dermoepidermal junction using DIF (Fig. 1). The baseline BP180 ELISA index of patients ranged from 0.5 to 146. The median BP180 ELISA index of patients was 24. Of the 15 patients, 6 (40%) were positive using ELISA, whereas 9 (90%) were negative (Fig. 2a). All controls were negative using ELISA. The results showed that the patients’ ELISA results were significantly higher than the controls (P<0.0001). The baseline BPDAI of patients ranged from 3 to 30. The median BPDAI was 5. The baseline BPDAI positively correlated with the baseline BP180 ELISA index of patients (Fig. 2b). The correlation coefficient was 0.790, P value was 0.0005. The disease time course of three patients was followed for 6 months. Figure 2c showed an example of the disease time course of a BP patient. The disease severity ran parallel to the BP180 ELISA index throughout the disease time course of three BP patients.

Table 1
Table 1:
Summary of the results of DIF and BP180 ELISA
Figure 1
Figure 1:
Direct immunofluorescence of a bullous pemphigoid patient showing linear IgG deposits along the dermoepidermal junction. Staining was performed using polyclonal rabbit antihuman IgG-fluorescein isothiocyanate antibody. Magnification ×20. IgG, immunoglobulin G.
Figure 2
Figure 2:
(a) Scatter plot diagram of the indices of BP patients and the normal controls using BP180 ELISA. Six patients were positive using ELISA, whereas nine patients were negative. (b) Correlation between the baseline BPDAI and the baseline BP180 ELISA index. The baseline BPDAI positively correlated with the baseline BP180 ELISA indices of the patients. The correlation coefficient was 0.790,P value was 0.0005. (c) The disease time course of a BP patient. The disease severity ran parallel to the ELISA index throughout the disease time course of the BP patient. BP, bullous pemphigoid; BPDAI, bullous pemphigoid disease area index; ELISA, enzyme-linked immunosorbent assay.

Discussion

Despite BP being the commonest autoimmune bullous disease in many European countries 2, it is a rare disease in the Middle east 9,10. BP180 is a 180 kDa transmembrane protein that is considered to be the immunodominant epitope of BP 2. BP180 ELISA was evaluated for the diagnosis of BP in several populations 11–13. However, to the best of our knowledge, it was not evaluated in the diagnosis of Egyptian BP patients. Therefore, the aim of this study was to evaluate the use of BP180 ELISA for the diagnosis of Egyptian BP patients.

Conventional assays used for the diagnosis of autoimmune bullous diseases include DIF and ELISA 1. DIF is considered to be the standard method of diagnosis. However, it needs high technical skills. In addition to that, it is not available in all hospitals in Egypt. The results showed that DIF is the most sensitive test for the diagnosis of BP. This goes along with the results of several studies showing that the sensitivity of DIF is more than 90% 14.

However, ELISA is an easy, rapid quantitative technique for the diagnosis of autoimmune diseases. The results showed that 6/15 (40%) of the patients reacted to the BP180 antigen. The sensitivity of ELISA was less than that reported by Kobayashi et al.7, probably because of the lower number of patients in this study. Nine sera were negative using ELISA. The first explanation could be because of the low sensitivity of ELISA. A second reason of negativity of those sera might be that the autoantibodies in those patients might target the BP230 antigen 15. A third explanation is that those antibodies might recognize epitopes outside the NC16a domain. As ELISA is coated by only the recombinant NC16a domain, those antibodies might target epitopes in the BP180; however, outside this domain that is why it was not detected by ELISA 16,17. A fourth reason could be that those antibodies target the NC16a domain; however, they might be of the IgE 17 not IgG class, and therefore were not detected using the horseradish peroxidase-conjugated antihuman IgG antibody. Further research is needed to screen the BP patients using BP230 ELISA. Moreover, further research is needed to characterize the BP180 ELISA-negative sera.

There was a wide range of ELISA indices among patients (0.4–146), which was because of the different disease severities (3–30). The results showed that the baseline BPDAI correlated with the baseline BP180 ELISA index. In addition to that, the BPDAI correlated with the BP180 ELISA index during the disease time course of three patients. This suggests that BP180 ELISA is useful in the follow-up of some BP patients.

Conclusion

BP180 ELISA can be useful in the diagnosis and follow-up of some BP patients; yet, DIF remains the standard method for diagnosis of this disease. Further research is needed to characterize the autoantibodies in the Egyptian BP patients.

Acknowledgements

Conflicts of interest

There are no conflicts of interest.

References

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Keywords:

adhesion molecules; autoantibodies; bullous pemphigoid; ELISA; hemidesmosome

© 2014 Egyptian Women's Dermatologic Society