Development of a simple enzyme-linked immunosorbent assay for the detection of autoantibodies in anti-p200 pemphigoid.
BACKGROUND
Anti-p200 pemphigoid is a subepidermal blistering skin disease characterized by autoantibodies against a 200-kDa protein (p200) of the dermal-epidermal junction. The laminin γ1 chain has recently been identified as target antigen in this disease and the C-terminus was described as an immunodominant region of laminin γ1. Diagnosis of anti-p200 pemphigoid requires detection of serum IgG at the dermal side of 1 mol L(-1) salt-split skin by indirect immunofluorescence microscopy and labelling of a 200-kDa protein by Western blotting of dermal extract. However, preparation of dermal extract is not widely available, limiting the possibility of diagnosing this disease to a few laboratories.


OBJECTIVE
To develop a simple, sensitive and specific diagnostic tool for anti-p200 pemphigoid.


METHODS
Sera from patients with anti-p200 pemphigoid (n = 35), bullous pemphigoid (BP, n = 101), epidermolysis bullosa acquisita (EBA, n = 10), antilaminin 332 mucous membrane pemphigoid (MMP, n = 14), pemphigus vulgaris (PV, n = 51) and healthy volunteers (HV, n = 131) were tested by a novel enzyme-linked immunosorbent assay (ELISA) that employed a recombinant monomeric C-terminal fragment of human laminin γ1 (hLAMC1-cterm) expressed in Escherichia coli.


RESULTS
Serum reactivity with hLAMC1-cterm was detected in sera from 24 of 35 (69%) patients with anti-p200 pemphigoid, two of 101 (2%) with BP, 0 of 10 with EBA, two of 14 (14%) with anti-laminin 332 MMP, 0 of 51 with PV, and 0 of 131 HV.


CONCLUSIONS
This novel ELISA will facilitate the diagnosis of anti-p200 pemphigoid.
Groth
S
S
Department of Dermatology, University of Lübeck, 23538 Lübeck, Germany.
Recke
A
A
Vafia
K
K
Ludwig
R J
RJ
Hashimoto
T
T
Zillikens
D
D
Schmidt
E
E
D000429:Alcohol Oxidoreductases / Q000276:immunology; D019540:Area Under Curve; D001323:Autoantibodies / Q000097:blood / Q000276:immunology*; D001324:Autoantigens / Q000097:blood / Q000276:immunology; D015153:Blotting, Western; D004268:DNA-Binding Proteins / Q000276:immunology; D015180:Electrophoresis, Gel, Two-Dimensional; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards; D016107:Epidermolysis Bullosa Acquisita / Q000175:diagnosis / Q000276:immunology; D006801:Humans; D007797:Laminin / Q000276:immunology*; D010391:Pemphigoid, Bullous / Q000175:diagnosis* / Q000276:immunology; D012680:Sensitivity and Specificity; D012872:Skin Diseases, Vesiculobullous / Q000175:diagnosis / Q000276:immunology
D001323:Autoantibodies; D001324:Autoantigens; D004268:DNA-Binding Proteins; D007797:Laminin; C089141:laminin gamma 1; D000429:Alcohol Oxidoreductases; C096794:C-terminal binding protein