Enzyme‐linked immunosorbent assay using multimers of the 16th non‐collagenous domain of the BP180 antigen for sensitive and specific detection of pemphigoid autoantibodies

Abstract

Bullous pemphigoid (BP) and pemphigoid gestationis (PG) are acquired autoimmune subepidermal blistering diseases characterized by autoantibodies against the hemidesmosomal proteins BP180 ⁄ type XVII collagen and BP230. In the vast majority of BP and PG patients, these autoantibodies bind to epitopes clustered within the 16th non-collagenous domain of BP180. An ELISA system for the detection of these autoantibodies was developed and evaluated using 16th non-collagenous domain (NC16A) tetramers instead of monomers. In contrast to antigens fused to large proteins used in the past for the detection of autoantibodies against type XVII collagen, tetrameric antigen fragments bearing a small hexahistidine tag allow for high expression levels without the need to cleave off the fusion partner. Using tetrameric BP180 NC16A, positive reactions were found in 106 (89.8%) of 118 randomly selected BP sera and in all of 20 (100%) randomly selected PG sera, whereas only 2.2% of a large cohort of control subjects were positive in this assay, including patients with rheumatoid arthritis (two of 107), progressive systemic sclerosis (two of 50), systemic lupus erythematosus (one of 72), and healthy blood donors (10 of 494). Thus, the sensitivity and specificity of the new anti-tetrameric NC16A ELISA were 89.9% and 97.8% respectively. Levels of circulating autoantibodies against BP180 paralleled disease activity in the pemphigoid patients. In conclusion, the use of tetrameric NC16A in ELISA results in a sensitive and specific tool for diagnosis and monitoring of BP and PG.Key words: autoantigen -autoimmunity -BPAG2 -collagen -ELISA -tetramer Please cite this paper as: Enzyme-linked immunosorbent assay using multimers of the 16th non-collagenous domain of the BP180 antigen for sensitive and specific detection of pemphigoid autoantibodies. Experimental Dermatology 2007; 16: 770-777.

Epitope-Specific Evolution of Human B Cell Responses to Borrelia burgdorferi VlsE Protein from Early to Late Stages of Lyme Disease

“…The constructs were confirmed by DNA sequencing. His-C3b-VlsE(D1-21, D45-335) was expressed and purified under denaturing conditions as described for another protein (28). Identity and purity of the final protein preparation, referred to thereafter as VlsE mp , were assessed by mass spectrometryassisted peptide mass mapping (29) and SDS-PAGE.…”

Anti-Skin Specific Autoantibodies Detected by a New Immunofluorescence Multiplex Biochip Method in Patients with Autoimmune Bullous Diseases

“…BP180, also known as type XVII collagen or BP antigen 2, is a transmembrane glycoprotein of about 1,500 amino acids that ultrastructurally spans the lamina lucida before kinking back from the lamina densa into the lamina lucida [14]. The extracellular portion of the 16th noncollageneous domain (NC16A) has been identified as the immunodominant region of BP180 [14] and the recombinant NC16A domain has been employed in highly sensitive and specific ELISA systems to detect anti-BP180 autoantibodies [15,16,17,18]. …”

Membranous glomerulonephropathy in a patient with bullous pemphigoid

“…Although anti-Dsg1 and 3 antibodies are a feature of patients with pemphigus, anti-BP180 and 230 antibodies are elevated in those with BP. In particular, anti-BP180 antibody reactivity is closely associated with BP, as it is detectable with absolute accuracy in almost all patients with BP [5][6][7]. Moreover, serum levels of anti-BP180 antibody correlate with disease activity [8].…”