sodium-dodecyl-sulfate and Pemphigoid--Bullous

There has been controversy concerning the nature of the bullous pemphigoid (BP) antigen: immunoprecipitation identified BP antigen as a single, unique 230-kDa protein, whereas immunoblot analysis showed multiple antigen molecules, mainly 230- and 170-kDa proteins. In this study, to further characterize the 170-kDa protein, we have examined whether the 170-kDa protein is detected by immunoprecipitation. Extracts of human squamous cell carcinoma cells revealed the 170-kDa protein with immunoblot analysis. Although the conventional immunoprecipitation detected only the 230-kDa protein, some BP sera that detected the 170-kDa protein with immunoblotting also precipitated the 170-kDa protein with our modified immunoprecipitation, in which the cells were extracted with 1% sodium dodecylsulfate (SDS) buffer and reacted with the sera under reduced SDS concentration. The 170-kDa protein-specific BP sera clearly showed hemidesmosomal plaque staining with immunofluorescence of cultured cells. These results indicate that the 170-kDa protein is indeed one of the BP antigens and that the 230- and 170-kDa BP antigens are integrated in different ways in hemidesmosomes.

Topics: Autoantigens; Carrier Proteins; Collagen; Collagen Type XVII; Cytoskeletal Proteins; Dystonin; Humans; Immunoblotting; Nerve Tissue Proteins; Non-Fibrillar Collagens; Pemphigoid, Bullous; Precipitin Tests; Sodium Dodecyl Sulfate; Tumor Cells, Cultured

Ninety-seven patients suffering from a pruritic dermatosis were screened for the detection of bullous pemphigoid (BP) antibodies (ab) using the Western immunoblot (WB) analysis technique.. Twenty-four patients (25%) reacted at least twice with the BP antigen on WB analysis at a 1/10 dilution: seven had typical BP, four had papular BP, 10 had pruritus and prurigo of unknown origin, two had eczema, and one had lichen planus. This corresponds to 13% of BP-type ab in patients who did not fulfill criteria for BP. Therefore, we did the following: (1) tested a control group of 24 subjects; (2) assessed the reproducibility of the WB method by retesting the same serum samples on different epidermal extracts; and (3) estimated the BP ab titer. None of the 24 control subjects had detectable BP ab, and reproducible results were obtained in all groups when serum samples were retested at the 1/10 dilution. Although 86% (6/7) of patients with BP had BP ab titers of 1/100 or greater, only 60% (6/10) of the group with pruritus and prurigo and 33% (1/3) of the group with eczema reached such titers.. These results indicate that due to its sensitivity, the WB method can detect low titers of BP ab in patients with pruritic dermatoses who did not fulfill criteria for BP, and therefore we question the specificity of this method.

Topics: Adult; Aged; Aged, 80 and over; Antibodies; Blotting, Western; Eczema; Electrophoresis, Polyacrylamide Gel; Epitopes; Female; Fluorescent Antibody Technique; Follow-Up Studies; Humans; Keratinocytes; Lichen Planus; Male; Middle Aged; Pemphigoid, Bullous; Prurigo; Pruritus; Reproducibility of Results; Skin; Sodium Dodecyl Sulfate