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Comparative Study
. 2009 Jul 31;346(1-2):18-25.
doi: 10.1016/j.jim.2009.04.013. Epub 2009 May 5.

A Novel ELISA Reveals High Frequencies of BP180-specific IgE Production in Bullous Pemphigoid

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Free PMC article
Comparative Study

A Novel ELISA Reveals High Frequencies of BP180-specific IgE Production in Bullous Pemphigoid

Kelly A N Messingham et al. J Immunol Methods. .
Free PMC article

Abstract

Bullous pemphigoid (BP) is a humoral autoimmune disease directed predominantly against the non-collagenous NC16A domain of the BP180 hemidesmosomal protein. Our laboratory has recently shown, using a mouse xenograft model, that passive transfer of IgE autoantibodies from BP sera induces a skin phenotype that recapitulates the early phases of the disease. Herein, we describe the development of a highly specific and sensitive ELISA to detect circulating IgE autoantibodies that recognize BP180-NC16A. Using this assay, we detected NC16A-specific IgE-class autoantibodies in 77% of BP sera. This frequency, which is significantly higher than reported previously, is comparable to that of anti-NC16A IgG autoantibody production. In 3 BP patients monitored over time, the circulating NC16A-specific levels of both IgE and IgG were associated with clinical disease activity; however, patient sera did not always contain high levels of both isotypes. In conclusion, our ELISA provides a highly sensitive and specific tool for the detection of BP180-specific IgE in patient sera. Furthermore, we report that the majority of BP sera contain both IgE and IgG class autoantibodies specific for NC16A and suggest that screening for both isotypes of autoantibodies may provide a better diagnostic value than IgG alone.

Conflict of interest statement

CONFLICT OF INTEREST

The authors state no conflict of interest.

Figures

Figure 1
Figure 1. Sensitivity and specificity of theNC16A-specific IgE ELISA
The diagnostic properties of the NC16A-specific IgE ELISA are depicted as a receiver operating characteristic curves for bullous pemphigoid (BP) sera. Sera of patients with active BP (n=43) and age/gender-matched normal controls (C; n=55) with no known autoimmune disease were screened for NC16A-GST or GST.
Figure 2
Figure 2. Diagnostic value of the NC16A-specific IgE ELISA
BP and control sera (from patients with systemic lupus erythematosus (SLE); epidermolysis bullosa acquisita (EBA); and normal healthy controls (C)) were incubated with immobilized NC16A-GST fusion protein or recombinant GST. Each serum was tested in duplicate with both proteins and the index value was calculated based on internal positive and negative controls. * = p<0.001 as determined by Kruskal-Wallis test and Dunn’s Multiple Comparsions post-test.
Figure 3
Figure 3. NC16A-specific IgE and IgG are present with similar frequency in BP sera
BP and control (C) sera were assayed by ELISA for IgE (panel A) and IgG (panel B) autoantibodies specific for NC16A. For IgE, reactivity to control GST fusion protein was also assayed and the difference of the means of OD450 with GPP-NC16A-GST and recombinant GST were determined. For both IgE and IgG Index values were calculated based on internal positive and negative controls. Correlation between NC16A-specific IgE and IgG index values (panel C). Each serum was tested in duplicate and data points represent the mean determination for each sample. The numbers in the quadrants indicate the numbers of patients positive or negative for the corresponding Abs. Mann-Whitney test p<0.0001 compared to control (C) for IgG or IgE.
Figure 4
Figure 4. Effect of serum dilution on ELISA sensitivity
Samples consisted of a BP serum with high levels of both IgG and IgE, a BP serum with intermediate (med) levels of both antibody isotypes and a (neg) control. Sera were undiluted or serially diluted 1 to 10, 20, 40, 80 and 100. Index values are shown for the IgG (A) ELISA used as directed (closed symbols) and OD450 is shown for dilutions developed with anti-IgE secondary antibody (open symbols). For comparison, index values over a range of dilutions are shown for the IgE ELISA protocol described in this report (B). Results are representative of two independent experiments and each data point represents the mean of duplicate samples.
Figure 5
Figure 5. NC16A-specific IgE and IgG levels reflect clinical disease activity
Serum IgE and IgG were assayed on serial blood draws obtained from untreated patients at the time of diagnosis and at various time points thereafter. All patients had active disease at the initial draw (time = 0 days) and responded to treatment and had not been previously treated. Disease activity was rate from 1–4: 4 = generalized disease with multiple new blisters and erosions at multiple sites on the body; 3 = localized active disease; 2 = controlled disease with immunosuppressive medications; 1 = no skin lesions in the absence of immunosuppressive therapy (remission).

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