Isolation and purification of a pemphigus vulgaris antigen from human epidermis.

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Abstract

Pemphigus vulgaris is an autoimmune disease associated with an autoantibody directed against a keratinocyte membrane antigen. The purpose of this study was to purify the human pemphigus vulgaris antigen, to produce an antibody to this antigen, and to use the antibody to induce pemphigus in newborn mice. Various techniques to extract the membrane-rich pellet from human epidermal homogenate were compared; 1% sodium dodecyl sulfate (SDS) and 1% dimethylsulfoxide proved to be superior to extract the pemphigus vulgaris antigen. This antigen was identified by transfer blotting to nitrocellulose paper, incubated with pemphigus vulgaris serum, or 20 control sera, and detected with fluorescein labeled antisera to human IgG. Since concanavalin A inhibits the binding of pemphigus vulgaris antibody to tissue sections, we studied the binding of the extracted proteins to concanavalin A covalently coupled to Sepharose. Pemphigus vulgaris antigen bound to the concanavalin A column and was released by 0.02 M methyl alpha-D-mannopyranoside. The proteins thus recovered were subjected to AcA 54 gel permeation chromatography, and the pemphigus antigen was detected by the transfer blot assay. The antigen corresponded to a discrete peak at 66,000 D by gel permeation and gave one homogeneous band at 33,000 D in urea-SDS-polyacrylamide gel electrophoresis. Monospecific antibody to the antigen raised in rabbits stained human epidermis in the same manner as the pemphigus vulgaris autoantibody and induced pemphigus vulgaris in newborn mice when injected intraperitoneally. A pemphigus vulgaris antigen has been purified from adult human epidermis. It is a 66,000-D membrane glycoprotein that is composed of two apparently identical subunits of 33,000 D each.

Authors

L L Peterson, K D Wuepper