Bullous pemphigoid (BP) is the most frequent autoimmune blistering skin disease, primarily affecting the elderly. Direct immunofluorescence (DIF) testing of a perilesional skin biopsy is considered to be the gold standard in diagnosing autoimmune blistering skin diseases such BP. By performing staining against human IgG linear deposits of IgG antibodies can be visualized as bound to the basement membrane zone (BMZ), and most cases also demonstrate linear C3 fixation at the BMZ. The BMZ antigens targeted in BP are well characterized as BPAG1 (BP230) and BPAG2 (BP180, Collagen XVII), and circulating serum antibodies against the NC16A epitope of BP180 correlate with disease activity.

Previous immunofluorescence data suggested that non-complement-fixing IgG4 antibodies are predominantly bound to the BMZ when compared to complement-fixing IgG1 antibodies. However, it is unclear which exact antibody reactivities (BPAG1 or BPAG2) contribute most to the tissue-bound IgG4 or IgG1 antibodies detected by immunofluorescence, and if the ratios of antigen-specific IgG4:IgG1 bound to the skin change over time.

To address these questions we already established a sophisticated protocol for eluting antibodies from patient skin biopsies, affinity-purifying these skin-eluted antibodies specifically for BP180-NC16A and BP230-C reactivity, followed by LC-MS/MS analyses. This highly sensitive proteomic approach allows to relatively quantify the IgG4:IgG1 ratio for autoantibodies of a given single reactivity, e.g. anti-BP180-NC16A only, without requiring immunofluorescence techniques.