RESUMO
The objective of this study is to investigate whether the avidity of proteinase-3-anti-neutrophil cytoplasmic antibody (PR3-ANCA) changes during follow-up in different subgroups of patients with granulomatosis with polyangiitis (GPA). We selected 10 patients with renal relapsing GPA, 10 patients with renal non-relapsing GPA and 10 patients with non-renal relapsing GPA. In all patients, an ANCA rise occurred during remission. The avidity was measured using a chaotropic approach at the time of an ANCA rise and at the time of a relapse in relapsing patients or time-matched during remission in non-relapsing patients. No difference was observed in the avidity at the ANCA rise between renal relapsing patients [26·2% (15·5-47·5)], renal patients without a relapse [39·6% (21·2-63·4)] and non-renal relapsing patients [34·2% (21·6-59·5)]. In renal relapsing patients, the avidity increased significantly from the moment of the ANCA rise to the relapse [difference 6·4% (0·0-17·1), P = 0·0273]. The avidity did not increase after an ANCA rise in renal non-relapsing patients [difference 3·5 (-6·0 to 10·1), P = 0·6250] or in non-renal relapsing patients [difference -3·1% (-8·0 to 5·0), P = 0·5703]. The avidity of PR3-ANCA increases after an ANCA rise during follow-up in renal relapsing patients, but not after an ANCA rise in renal patients who remain in remission or in non-renal relapsing patients.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/imunologia , Afinidade de Anticorpos , Granulomatose com Poliangiite/imunologia , Nefropatias/imunologia , Mieloblastina/imunologia , Adulto , Idoso , Anticorpos Anticitoplasma de Neutrófilos/sangue , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Mieloblastina/sangue , RecidivaAssuntos
Autoanticorpos/sangue , Laminina/imunologia , Lúpus Eritematoso Cutâneo/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Biomarcadores/sangue , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Humanos , Lúpus Eritematoso Cutâneo/sangue , Lúpus Eritematoso Sistêmico/sangue , Estrutura Terciária de ProteínaRESUMO
BACKGROUND: Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease characterized by circulating autoantibodies against BP180 and BP230. For BP180, the NC16A domain has previously been identified as the main antigenic target in BP, while data about the diagnostic value of epitopes on BP230 were inconclusive. OBJECTIVES: To identify the most appropriate epitopes on BP230 to be applied in a simple, sensitive, and highly specific enzyme-linked immunosorbent assay (ELISA) for routine detection of serum autoantibodies. METHODS: Ten overlapping linear fragments covering the whole length of BP230 were expressed in Escherichia coli. Based on Western blot analysis with sera from patients with BP (n = 49) and healthy controls (n = 94), the diagnostic performance of the fragments was compared by receiver operating characteristics curve analysis. The BP230-C3 fragment comprising the C-terminal portion (amino acids 2326-2649) was subsequently applied in a novel ELISA. The operating characteristics of this ELISA were analysed by probing sera from patients with BP (n = 118), pemphigus vulgaris (n = 50), rheumatoid arthritis and other inflammatory arthritides (n = 170), and systemic lupus erythematosus (n = 56), and from healthy blood donors (n = 483). RESULTS: Among all the fragments, BP230-C3 provided the best efficiency in serologically diagnosing BP by Western blot. An ELISA employing BP230-C3 revealed a diagnostic sensitivity of 56·8% and specificity of 97·6%. Its diagnostic added value amounted to 4·2% compared with the anti-BP180-NC16A-4X ELISA alone. CONCLUSIONS: Recombinant BP230-C3 is a suitable target antigen for the detection of serum autoantibodies against BP230.
Assuntos
Autoanticorpos/metabolismo , Mapeamento de Epitopos/métodos , Glicoproteínas de Membrana/metabolismo , Penfigoide Bolhoso/imunologia , Autoanticorpos/imunologia , Western Blotting , Proteínas de Transporte , Estudos de Casos e Controles , Proteínas do Citoesqueleto , Distonina , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Proteínas do Tecido Nervoso , Penfigoide Bolhoso/diagnóstico , Curva ROC , Proteínas RecombinantesRESUMO
BACKGROUND: Antineutrophil cytoplasmic antibodies (ANCA) with a C-ANCA or P-ANCA pattern are detected in ANCA-associated vasculitis (AAV). While in most patients with AAV a C-ANCA pattern is due to reactivity with proteinase-3 (PR3)-ANCA, some C-ANCA-positive sera do not react with PR3. OBJECTIVE: The development and evaluation of a direct enzyme-linked immunosorbent assay (ELISA) for PR3-ANCA with increased sensitivity. METHODS: A mixture of human native (hn) and human recombinant (hr) PR3 was used as antigen coating. The resulting ELISA (anti-PR3-hn-hr) was compared with ELISAs using directly coated hn-PR3 or hr-PR3, as well as with a hn-PR3 capture ELISA. Assay characteristics were determined in patients with AAV (n = 248), with special attention for those patients with C-ANCA (n = 132), as well as disease controls (n = 585) and healthy controls (n = 429). Additionally, for prediction of relapses serial samples of 46 patients with PR3-AAV were analysed. RESULTS: At a predefined specificity of 99% both ELISAs containing hr-PR3 revealed a substantial increase in sensitivity. For the prediction of relapses by rises in PR3-ANCA titres the capture ELISA was most optimal (odds ratio 12.5). With an odds ratio of 8.9 the novel anti-PR3-hn-hr ELISA was second best. CONCLUSIONS: Owing to the very high sensitivity of the novel anti-PR3-hn-hr ELISA for the detection of PR3-ANCA in C-ANCA-positive samples of patients with AAV this assay has an excellent diagnostic performance. This feature is combined with a good predictability of clinical relapses in patients with PR3-AAV. These characteristics challenge the dogma that, for detection of PR3-ANCA, capture ELISAs are superior for diagnosis and follow-up.