Antigen specificities of antibasement membrane zone antibodies: immunofluorescence and Western immunoblotting studies

Abstract

To clarify the antigen specificities of autoantibodies in sera and blister fluids from patients diagnosed as bullous pemphigoid (BP) by routine histology and immunofluorescence (IF) methods, indirect IF studies using the salt split-skin technique were performed. In addition, to detect the BP antigen(s) in human epidermal extracts, Western immunoblotting analyses were carried out. Of 41 sera, 39 (95%) showed a linear pattern of fluorescence along the epidermal side of the separation. Two (5%) sera showed a linear pattern of fluorescence along the dermal side. Blister fluids produced IF staining patterns identical with those of serum samples. These fluorescence patterns were not related to the BP antigen expression of the skin used as substrates. In Western immunoblotting analyses, selected sera showing an epidermal pattern on separated skin primarily reacted with 240 kD, 220 kD, 180 kD, and 150 kD proteins extracted from normal human epidermis. Two sera showing a dermal pattern on separated skin revealed no specific bands. The protein bands recognized by blister fluids were identical with those of serum samples. These results indicated that blister fluids are also available in immunological analysis, and that BP antibodies have more than one antigenic specificity. Moreover, it is suggested that differential diagnosis between BP and other bullous diseases may be more important than previously recognized, particularly in patients with epidermolysis bullosa acquisita (EBA).