Studies of the relationship of the 230-kD and 180-kD bullous pemphigoid antigens.
In bullous pemphigoid (BP), autoantibodies from most patients recognize a high molecular weight 230-kD epidermal antigen (Ag) by immunoprecipitation. By Western immunoblotting, 50-70% of sera recognize the high molecular weight Ag, but 30-50% recognize a low molecular weight, 180-kD epidermal Ag. We examined the specificities of affinity-purified antibodies against these Ag. Antibodies specific for the 230- and 180-kD Ag were prepared by immunoaffinity against Ag immobilized on nitrocellulose and released by acid glycine. IgG eluted from the 230-kD Ag band retained its specific binding to the 230-kD Ag by immunoblotting, and bound to the epidermal basement membrane zone (BMZ) by indirect immunofluorescence (IF) and to hemidesmosomes by indirect immunoelectron microscopy (EM). IgG affinity purified by the 180-kD Ag band bound only the 180-kD Ag in immunoblotting, with no cross reaction to the 230-kD Ag, bound the epidermal BMZ by indirect IF, and also bound to hemidesmosomes in immuno-EM. IgG specific for the 230-kD Ag in immunoblotting immunoprecipitated only the 230-kD Ag, with no apparent precipitation of the 180-kD Ag. Surprisingly, IgG specific for the 180-kD Ag precipitated both the 180- and the 230-kD Ag in immunoprecipitation, and the 230-kD Ag band was much more intense than the 180-kD Ag band. This study shows that apparent cross-reactivity between these Ag by BP autoantibodies can only be detected in native conditions by immunoprecipitation, and cannot be demonstrated using denatured Ag in immunoblotting. The two proteins appear to be distinct Ag, closely associated in the epidermal hemidesmosome, but the exact relationship of these Ag to each other may not be clarified until complete structural data become available.
Department of Dermatology, Johns Hopkins University, Baltimore, Maryland 21205.
M A
MA
Robledo
S C
SC
Kim
N J
NJ
Korman
J R
JR
Stanley
R S
RS
Labib
S
S
Futamura
G J
GJ
Anhalt
D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D001324:Autoantigens / Q000276:immunology*; D015153:Blotting, Western; D002352:Carrier Proteins; D003094:Collagen; D003598:Cytoskeletal Proteins; D000071821:Dystonin; D005455:Fluorescent Antibody Technique; D006801:Humans; D007158:Immunologic Techniques; D008854:Microscopy, Electron; D008970:Molecular Weight; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D010391:Pemphigoid, Bullous / Q000276:immunology*; D011233:Precipitin Tests; D012872:Skin Diseases, Vesiculobullous / Q000276:immunology*; D000097805:Collagen Type XVII
D000906:Antibodies; D001324:Autoantigens; D002352:Carrier Proteins; D003094:Collagen; D003598:Cytoskeletal Proteins; D000071821:Dystonin; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D000097805:Collagen Type XVII; C486268:DST protein, human