Keratinocyte footprint assay discriminates antilaminin‐332 pemphigoid from all other forms of pemphigoid diseases

BACKGROUND: Antilaminin‐332 mucous membrane pemphigoid is a chronic severe pemphigoid disease characterized by autoantibodies to laminin‐332. At present no commercial assay is available to demonstrate antilaminin‐332 antibodies, and diagnosis relies on in‐house techniques with limited sensitivities. OBJECTIVES: In order to move, keratinocytes cultured in vitro secrete laminin‐332 to attach to the culture dish. In that way, they leave behind a unique footprint trail of laminin‐332. We aimed to develop a sensitive and specific laboratory assay to determine antilaminin‐332 autoantibodies in patient serum based on binding of patient IgG to these unique footprints. METHODS: Normal human keratinocytes were grown on glass coverslips and incubated with patient or control serum for 1 h. The binding of IgG was then investigated by immunofluorescence. After validating the test for its ability to identify antilaminin‐332 autoantibodies it was converted into a daily available test based on binding of IgG to dried coverslips that can be stored frozen. The staining patterns of sera from patients with antilaminin‐332 pemphigoid were then compared with those of sera from patients with other autoimmune bullous diseases and normal human sera. RESULTS: IgG of all antilaminin‐332 pemphigoid sera (n = 16) bound to laminin‐332 footprints, while all normal human controls (n = 55) were negative. From the sera of patients with other diseases (n = 72) four sera tested positive. The footprint assay was also positive for sera that were negative by salt‐split skin analysis, demonstrating that it is a very sensitive technique. CONCLUSIONS: The keratinocyte footprint assay is a fast and specific assay to confirm or rule out the presence of antilaminin‐332 autoantibodies. What's already known about this topic? Antilaminin‐332 mucous membrane pemphigoid is a severe form of pemphigoid, and patients may have an increased risk of malignancies. The diagnosis of antilaminin‐332 mucous membrane pemphigoid is complicated by the lack of specific commercial tests for antilaminin‐332 antibodies and can be confirmed only in specialized laboratories. Keratinocytes in culture need laminin‐332 for adhesion and migration and therefore deposit it on the bottom of the culture dish. What does this study add? The keratinocyte footprint assay detects antilaminin‐332 autoantibodies in patient serum using the native laminin‐332 produced by cultured keratinocytes. What is the translational message? The keratinocyte footprint assay is a fast and specific assay to confirm or rule out the presence of antilaminin‐332 autoantibodies.