Faculty Collaboration Database

Comparative analysis of methods for detection of anti-laminin 5 autoantibodies in patients with anti-epiligrin cicatricial pemphigoid. J Am Acad Dermatol 2004 Dec;51(6):886-92

Date

12/08/2004

Pubmed ID

15583578

DOI

10.1016/j.jaad.2004.06.004

Scopus ID

2-s2.0-9944261428 (requires institutional sign-in at Scopus site)   46 Citations

Abstract

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.

Author List

Lazarova Z, Sitaru C, Zillikens D, Yancey KB

MESH terms used to index this publication - Major topics in bold

Autoantibodies
Cell Adhesion Molecules
Humans
Immunoblotting
Microscopy, Fluorescence
Pemphigoid, Bullous
Radioimmunoprecipitation Assay