A Solid-Phase Protein Assay:Quantitation of Protein in the Nanogram Range

A solid-phase protein assay (SPA) for the determination of protein concentrations in the nanogram range is described. The technique is based on biotinylation of immobilized protein on the solid phase of a microtiter plate and quantitation of the protein-biotin complexes by peroxidase-coupled avidin. Compared to the routinely used Bradford and Lowry protein detection techniques, the described SPA assay is (depending upon the protein assayed) 1000 to 10,000 times more sensitive. Moreover, the SPA assay can be suitable for protein quantitation of samples containing agents which commonly interfere with the routinely used detection techniques. The SPA assay is a valuable addition to the Bradford and the Lowry techniques. Used in combination with an antigen-specific ELISA it gives a reliable ratio of specific versus total protein

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

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

Type XVII collagen (BP180) and LAD-1 are present as separate trimeric complexes

This study characterized the high molecular mass BP180 complex that is observed when unheated sodium dodecyl sulfate extracts of human skin or keratinocytes are subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. In heated extracts BP180 is present as a monomer with a molecular weight of 180 kDa, in unheated extracts BP180 runs at a molecular weight position over 500 kDa, By preincubating the unheated extracts at temperatures between 31 degrees C and 40 degrees C, the high molecular weight complex could be "melted" down to monomeric BP180. Under the conditions employed the T-1/2 Of the dissociation process was between 35 degrees C and 36 degrees C. The temperature resistance of the high molecular weight complex was used to analyze its molecular composition by performing two-dimensional electrophoresis with a "low-temperature" first dimension step and a "high-temperature" second dimension step. Silver staining and immunoblotting of the two-dimensional gels revealed the high molecular weight complex to be composed of solely BP180, indicating that the complex is the nondissociated homotrimeric form of BP180. The 120 kDa linear IgA dermatosis antigen (LAD-1) is an collagenous anchoring filament protein with homology to the extracellular collagenous part of BP180, Two-dimensional immunoblotting showed that LAD-1, as BP180, is also present as a high molecular mass complex and does not form mixed complexes with BP180