Antiplectin autoantibodies in subepidermal blistering diseases.

BACKGROUND: Hemidesmosomal proteins may become targets of autoimmunity in subepidermal blistering diseases. Well-known recognized autoantigens are the intracellular plaque protein BP230, the transmembrane BP180 and its shed ectodomain LAD-1. OBJECTIVES: To establish the prevalence of autoimmunity against plectin, another intracellular plaque protein, and to investigate its antigenic sites. METHODS: Two hundred and eighty-two patients with subepidermal blistering diseases, investigated by routine immunoblot analysis for possible antiplectin antibodies, were included in the study. Epitope mapping was performed using recombinantly produced overlapping plectin domains from the actin-binding domain to the rod domain. The COOH-terminal region of plectin was not included in the study. RESULTS: In 11 of 282 (3.9%) patients an immunoblot staining pattern identical to that of antiplectin monoclonal antibody HD121 was found. Affinity-purified antibodies bound back to normal human skin in a pattern typical for plectin, i.e. to the epidermal basement membrane zone as well as to keratinocytes in the epidermis, and to myocytes. No binding was seen to plectin-deficient skin of a patient with epidermolysis bullosa simplex with muscular dystrophy. Epitope mapping of the plectin molecule showed that the central coiled-coil rod domain is an immunodominant hotspot as 92% of the sera with antiplectin antibodies reacted with it. Most patients with antiplectin antibodies also had antibodies to other pemphigoid antigens. CONCLUSIONS: Plectin is a minor pemphigoid antigen with an immunodominant epitope located on the central rod domain.

Coexistence of IgA antibodies to desmogleins 1 and 3 in pemphigus vulgaris, pemphigus foliaceus and paraneoplastic pemphigus.

BACKGROUND: Pemphigus is a bullous mucocutaneous autoimmune disease characterized by IgG autoantibodies to desmoglein (Dsg) 1 and/or Dsg3. Occasionally direct immunofluorescence of pemphigus skin reveals IgA depositions with an intraepidermal intercellular pattern in addition to the IgG deposition. OBJECTIVES: To investigate if pemphigus patients, in addition to having IgG autoantibodies, also generate IgA antibodies to Dsg1 and/or Dsg3. PATIENTS/METHODS: Sera of 100 pemphigus patients and 36 bullous pemphigoid controls were tested by IgA enzyme-linked immunosorbent assay (ELISA) to the recombinant extracellular domains of Dsg1 and Dsg3. The patients were selected on clinical grounds and positive IgG ELISA index values for Dsg1 and/or Dsg3. They were divided into four groups: patients having IgG to only Dsg1 (n=34), patients having IgG to both Dsg1 and Dsg3 (n=31), patients having IgG to only Dsg3 (n=27) and patients who had paraneoplastic pemphigus (PNP) (n=8). RESULTS: IgA antibodies to Dsg1 were found in 13 (38%) of the patients with IgG to Dsg1, in five (16%) of the patients with IgG to both Dsg1 and Dsg3, in four (15%) of the patients with IgG to Dsg3 and in none of the PNP patients. IgA antibodies to Dsg3 were found in one (3%) of the patients with IgG to Dsg1, in 18 (58%) of the patients with IgG to both Dsg1 and 3, in 18 (67%) of the patients with IgG to Dsg3, and in four (50%) of the PNP patients. Immunofluorescence analysis demonstrated intraepidermal intercellular staining IgA antibodies in serum and intercellular IgA deposits in skin of IgA ELISA-positive patients, although to a lesser extent than by ELISA. CONCLUSIONS: This study shows that in a considerable number of supposedly IgG-mediated pemphigus patients IgA to Dsg1 and Dsg3 is also present. In most cases the antigen specificity of the IgA follows the antigen specificity of the IgG, although in a small number of cases IgA is present against the Dsg not recognized by IgG.

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 degrees C. 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 T1/2 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.

Bullous pemphigoid and linear IgA dermatosis sera recognize a similar 120-kDa keratinocyte collagenous glycoprotein with antigenic cross-reactivity to BP180.

Circulating IgG from a large subset of bullous pemphigoid (BP) patients reacted on immunoblot with a 120-kDa protein in conditioned keratinocyte culture medium and in keratinocyte cell extracts. A protein with a similar molecular weight was recognized by circulating IgA from a subset of patients with linear IgA dermatosis (LAD). Both affinity-purified 120-kDa-specific BP IgG and 120-kDa-specific LAD IgA bound to the roof of salt-split skin. Both proteins recognized are collagenous glycoproteins. Deglycosylation with N-glycosidase F resulted in an identical reduction in molecular weight for both the BP-IgG-recognized protein and the LAD-IgA-recognized protein. Both proteins were equally susceptible to digestion with type VII collagenase. Furthermore, both proteins were absent from conditioned culture medium of keratinocytes from patients with BP180-deficient general atrophic benign epidermolysis bullosa (GABEB). Immunodepletion studies showed that the 120-kDa LAD antigen could be removed from conditioned culture medium by anti-120-kDa BP IgG. Thus these results indicate that these proteins are either highly related or, most probably, identical. A strong antigenic relationship between the 120-kDa protein and the 180-kDa bullous pemphigoid antigen (BP180) was detected by cross-reaction of affinity-purified anti-120-kDa BP patient antibodies to BP180 and cross-reaction of monoclonal anti-180-kDa antibodies to the 120-kDa protein. Notwithstanding this cross-reactivity, the 120-kDa protein also exhibits unique epitopes demonstrated by the nonreactivity of individual anti-120-kDa BP and LAD patient serum with the 180-kDa antigen.

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.