Peptide Epitopes of NC16A BP180 in the Diagnostics of Bullous Pemphigoid

Abstract

Bullous pemphigoid is a severe autoimmune blistering skin disorder causing significant morbidity and mortality among the elderly. Improved diagnostic strategies rely on the identification of autoantigen epitopes recognized by pathogenic autoantibodies. Continuous (linear) peptide epitopes, unlike conformational epitopes in recombinant proteins, offer advantages such as chemical stability, reproducibility, and lower production costs, making them attractive for diagnostic assay development. To identify relevant linear epitopes, a microarray of overlapping peptides spanning the NC16A domain of BP180 and the C-terminus of BP230 autoantigens was screened with sera of 13 patients with bullous pemphigoid and 2 control sera. A 25-mer peptide of NC16A, termed Pep7-NC16A, was evaluated by ELISA in 2 independent patient cohorts (n = 35 and 26 bullous pemphigoid cases, n = 48 and 53 controls). Pep7-NC16A ELISA showed sensitivity of 71% and 62% and specificity of 92% and 98%, with area under the curve values of 0.92 and 0.754 (P < .001). Reactivity strongly correlated with commercial BP180 NC16A ELISA (r = 0.72, P < .0001). In this study, we demonstrate the diagnostic potential of synthetic peptide in bullous pemphigoid. The identified epitope could also serve as a potential biomarker for BP180-associated conditions or future peptide-based immunotherapies.

Keywords: Autoimmunity; Bullous pemphigoid; Peptide epitopes.

Figure 1

Peptide array fluorescent intensity map generated by patient sera reactivities. The 15mer peptides with 14 amino acid overlap derived from sequences of BP180 NC16A, BP180 C-terminus, and BP230 C-terminus were printed in duplicates on to PepPerPrint microarray. Sera from 13 patients with BP from site 1 (SAL1, SAL2, SAL3, TOS1, TOS2, TOS3, TOS4, TOS5, TOS7, KL1, KL2, and KL3) were compared with 2 nonblistering autoimmune disease sera (scleroderma with anti-Scl70 and anti-CENPB autoantibodies). (a) Fluorescence reactivities of human IgG (red), with internal positive controls represented by wells containing viral peptides along the periphery of the array, alternating between anti-influenza (red) and anti-polio (green). (b) Semiquantification of the relative fluorescence intensities of each serum corresponding to the putative peptide epitopes. a.u., arbitrary unit; BP, bullous pemphigoid.

Figure 2

Diagnostic performance of BP180 NC16A peptide 7 ELISA.(a) ROC analysis of 35 BP cases versus 48 ANA-negative geriatric controls (solid line) showed AUC = 0.92, P < .0001. (b) IgG ELISA reactivities of NC16A-Pep7 relative to the cutoff >0.19 OD_{450–620 nm} (dotted line) determined from ROC analysis. Controls’ mean OD_{450–620 nm} is 0.107 (95% CI = 0.08–0.128). ∗∗∗P < .0001 (unpaired t-test). (c) A second analysis of 26 BP cases versus 53 dermatitis herpetiformis controls yielded AUC = 0.754, P < .001, NC16A-pep7 (solid line) and AUC = 0.787 MBL BP1180 NC16A (dotted line). (d) Passing–Bablok method comparison of MBL BP180 NC16A ELISA versus NC16A-Pep7 ELISA. Spearman’s rank correlation coefficient r = 0.73 (95% CI = 0.6–0.816), P < .0001. neg denotes the number of patients with negative values, pep7 denotes peptide-7 of NC16A, Ctrl denotes control, and pos denotes number of patients with positive values. ANA, antinuclear antibody; AUC, area under the curve; BP, bullous pemphigoid; CI, confidence interval; ctr, control; OD, optical density; ROC, receiver operator curve.

Figure 3

Imprecision testing of NC16A-Pep7 ELISA. Twenty-six BP cases of site 3 were evaluated in 6 repeated runs on NC16APep7 ELISA. (a) The box-whisker plots show the median and range and upper and lower limits of ODs of 2 intraday runs on separate microtiter plates of 2 lots of batch-synthesized Pep7-NC16A NC16A-Pep7 manufactured by PepPerPrint (lot A) and Intavis (lot B), 2 hours after peptide coating. Five interday runs were performed within 2 hours (1B), 4 months (2B, 3B), 1 week (4B), or 1 month (5B) after coating. The mean OD_{450–620 nm} of the DH controls is 0.062 (95% CI = 0.05–0.08). ∗∗∗P < .0001 unpaired t-test difference between mean OD values of BP (run 4) and of DH controls (run 4B). The mean intraday CV% of OD_{450–620 nm} values in the range >0.19 to 0.4 (dark gray zone) and in the range >0.4 (light gray zone) were 11 (95% CI = 3.5–19) and 5 (95% CI = 1.8–8.5), respectively. The mean interday CV% of OD_{450–620 nm} values of 5 runs were 24 (95% CI = 18–30) and 22 (95% CI = 18–27) within the 2 ranges. (b) Color-coded intensities of the ODs (OD_{450 nm–620 nm}) of BP samples in each of the 6 runs (1A, 1B, 2B, 3B, 4B, and 5B). Red OD > 2.4, pink OD > 1.8 < 2.4, orange OD > 1.3 < 1.8, light orange OD > 0.7 t < 1.8, yellow OD > 0.37 < 0.7, light green OD > 0.1 < 0.37, and green OD < 0.1. BP, bullous pemphigoid; CI, confidence interval; CV, coefficient of variation; DH, dermatitis herpetiformis.

Figure 4

Inhibition of the autoantibody binding activity in BP180 recombinant protein MBL. ELISA was by Pep7-NC16A. Sera of 5 patients with BP having anti-BP180 IgG autoantibodies were incubated for 1 hour at room temperature with Pep7-NC16A at 0, 3, 10, 33, and 100 μg/ml and then assessed for binding activity in the MBL BP180 ELISA. Pep7-NC16A concentrations between >10 μg and 100 μg/ml resulted in a 10–50% decrease in autoantibody binding activity (U/ml), depending on the individual patients with BP (sal3-kl, sal7-kl, sal15-kl, and sal2-kl). BP, bullous pemphigoid.