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.


CONCLUSIONS
Immunoprecipitation was the most sensitive technique for detection of anti-laminin 5 antibodies, while immunoblotting of HK ECM or HK extracts represented practical alternatives.
Lazarova
Zelmira
Z
Department of Dermatology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226-4801, USA. zlazarov@mcw.edu
Sitaru
Cassian
C
Zillikens
Detlef
D
Yancey
Kim B
KB
D001323:Autoantibodies; D015815:Cell Adhesion Molecules / Q000276:immunology*; D006801:Humans; D015151:Immunoblotting; D008856:Microscopy, Fluorescence; D010391:Pemphigoid, Bullous / Q000276:immunology*; D015531:Radioimmunoprecipitation Assay; D000097794:Kalinin
D001323:Autoantibodies; D015815:Cell Adhesion Molecules; D000097794:Kalinin