Normal human skin is superior to monkey oesophagus substrate for detection of circulating BP 180‐ NC 16A‐specific IgG antibodies in bullous pemphigoid

Abstract

Summary Background Bullous pemphigoid (BP) is the most common autoimmune subepidermal blistering skin disease. Two antigens have been identified as targets of circulating autoantibodies (autoAbs) – BP180 and BP230 – with BP180 being a critical transmembrane adhesion protein of basal keratinocytes of the epidermis. The noncollagenous domain 16A (NC16A) of BP180 is the immunodominant epitope in patients with BP, and anti‐BP180‐NC16A IgG antibodies (Abs) correlate to disease activity. Routine serological testing and follow‐up of BP relies on indirect immunofluorescence (IIF) of serum Abs, commonly performed on monkey oesophagus (ME), and/or enzyme‐linked immunosorbent assay (ELISA) testing on recombinantly produced fragments of BP180 and BP230 (BP180‐NC16A, BP230‐C/N). Objectives To determine if NC16A epitopes are well represented on ME substrate. Methods Sera from different BP cohorts were tested by IIF on ME and normal human skin (NHS). To confirm findings, affinity‐purified anti‐BP180‐NC16A/BP230 polyclonal Abs and recombinant anti‐BP180‐NC16A/BP230 monoclonal antibodies (mAbs) were used. Results For sensitive detection of BP180‐NC16A‐specific IgG Abs, sections of NHS are superior to the widely used ME. Confirmation comes from polyclonal affinity‐purified anti‐BP180‐NC16A/BP230 Abs, and by mAbs cloned from a patient with active BP. Conclusions Use of NHS is preferable over ME in routine IIF testing for BP. These results are of clinical relevance because anti‐BP180‐NC16A IgG titres are correlated to disease activity and detecting them reliably is important for screening, diagnosis and follow‐up of patients with BP.

Lichen Planus Pemphigoides: From Lichenoid Inflammation to Autoantibody-Mediated Blistering

“…In contrast, direct immunofluorescence studies of lichenoid lesions may show typical features of LP: irregular band of fibrinogen at the dermal-epidermal junction and colloid bodies in the papillary dermis (53). Circulating autoantibodies in patient sera that bind to the NC16A or C-terminal domains of COL17 or desmogleins may be detected using routine methods including indirect immunofluorescence on monkey esophagus or human neonatal foreskin (54), ELISA and immunoblotting of human keratinocyte extracts (32, 55). In addition to this, circulating autoantibodies will bind to the roof of the artificial blister in indirect immunofluorescence on 1M NaCl-salt split skin (56).…”

The Diagnosis and Blistering Mechanisms of Mucous Membrane Pemphigoid

“…For the sensitive detection of COL17-NC16A-specific IgG, Emtenani et al reported that normal human skin was superior to monkey esophagus. The monkey esophagus was able to detect only 17% (2/12) of COL17-NC16A antibodies, whereas skin detected 100% (12/12) of the antibodies (50). Together with the low homology of COL17-NC16A between human and monkey esophagus, these pieces of evidence suggest that the expression of COL17 protein differs by anatomical location, such as skin vs. mucosa.…”

Diagnostic Techniques in Autoimmune Blistering Diseases

“…Pre-adsorption of test sera with soluble A/B blood group antigens, as a blocking step, may reduce this non-specificity [50]. Secondly, monkey oesophagus expresses very low levels of BP180 protein, the most common antigen targeted in bullous pemphigoid and, therefore, false negative diagnosis of this disease is possible if monkey oesophagus is the sole substrate used in IIF analysis [51].…”