False-negative results in immunoblot assay of serum IgA antibodies reactive with the 180-kDa bullous pemphigoid antigen: the importance of primary incubation temperature

Background Different subepidermal autoimmune blistering skin disorders are characterized by linear deposition of IgA, sometimes accompanied by linear IgG, along the epidermal basement membrane zone. Identification of the targeted autoantigen is usually attempted by immunoblotting. Although immunoblotting works well for human IgG, the method is less successful for IgA and often no or only faint signals are obtained.

Objectives To improve the method of immunoblotting for diagnosis of IgA-mediated bullous dermatoses.

Methods Eleven sera, selected from patients with linear deposition of IgA along the epidermal basement membrane zone in vivo, were tested by immunoblotting for antigen specificity using different primary incubation temperatures.

Results No reliable information regarding IgA antigen specificity was obtained when the primary incubation was undertaken at room temperature. In 10 of 11 sera, IgA bound to the 180-kDa bullous pemphigoid antigen (BP180) when the primary incubation temperature was increased to 37 T.

Conclusions Primary incubation at room temperature may result in false-negative results in the IgA-BP180 immunoblot assay.