Comparative analysis of methods for detection of anti-laminin 5 autoantibodies in patients with anti-epiligrin cicatricial pemphigoid

Background: Anti-epiligrin cicatricial pemphigoid (AECP) is a subepidermal blistering disease characterized by circulating anti-basement membrane autoantibodies to laminin 5. Objective: To evaluate the relative sensitivity of immunoblotting and immunoprecipitation techniques for the detection of anti-laminin 5 antibodies, comparative studies using reference laminin 5 antiserum as well as sera from patients with AECP, other immunobullous diseases, and normal volunteers were performed. Methods: Equivalent amounts of protein from five different Substrates were studied by immunoblotting; immunoprecipitation experiments examined biosynthetically radiolabeled human keratinocyte (HK) extracts. Results: HK extracellular matrix (ECM) was the most sensitive substrate for detection of antibodies to laminin 5; extracts of HKs, A-431 cells and HaCat cells represented alternative test substrates (though the later required higher amounts of protein input). Sera from patients with AECP immunoblotted laminin 5 in HK ECM at end titers exceeding those identified in indirect immunofluorescence microscopy studies of 1 M NaCl split skin. Immunoprecipitation studies found that a 10,000-fold dilution of reference laminin 5 antiserum retained the ability to identify laminin 5. Maximal dilutions of sera from AECP patients retaining the ability to immunoprecipitate laminin 5 ranged from 500 to 5,000. Conclusion: Immunoprecipitation was the most sensitive technique for detection of anti-laminin 5 antibodies, while immunoblotting of HK ECM or HK extracts represented practical alternatives.