Autoantigen microarrays for multiplex characterization of autoantibody responses.

We constructed miniaturized autoantigen arrays to perform large-scale multiplex characterization of autoantibody responses directed against structurally diverse autoantigens, using submicroliter quantities of clinical samples. Autoantigen microarrays were produced by attaching hundreds of proteins, peptides and other biomolecules to the surface of derivatized glass slides using a robotic arrayer. Arrays were incubated with patient serum, and spectrally resolvable fluorescent labels were used to detect autoantibody binding to specific autoantigens on the array. We describe and characterize arrays containing the major autoantigens in eight distinct human autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis. This represents the first report of application of such technology to multiple human disease sera, and will enable validated detection of antibodies recognizing autoantigens including proteins, peptides, enzyme complexes, ribonucleoprotein complexes, DNA and post-translationally modified antigens. Autoantigen microarrays represent a powerful tool to study the specificity and pathogenesis of autoantibody responses, and to identify and define relevant autoantigens in human autoimmune diseases.

Development and evaluation of a standardized ELISA for the determination of autoantibodies against cN-1A (Mup44, NT5C1A) in sporadic inclusion body myositis.

PURPOSE: Sporadic inclusion body myositis (sIBM) is an autoimmune degenerative disease of the muscle, with inflammatory infiltrates and inclusion vacuoles. Its pathogenesis is not fully understood and the diagnosis is hampered by its imprecise characteristics, at times indistinguishable from other idiopathic inflammatory myopathies such as polymyositis and dermatomyositis. The diagnosis may be assisted by the detection of autoantibodies targeting Mup44, a skeletal muscle antigen identified as cytosolic 5'-nucleotidase 1A (cN-1A, NT5C1A). A novel standardized anti-cN-1A IgG ELISA was developed and its diagnostic performance was evaluated by two reference laboratories. METHODS: Recombinant human full-length cN-1A was expressed and purified, and subsequently utilized to set up a standardized ELISA. To evaluate the novel assay, laboratory A examined sera from North American patients with clinically and pathologically diagnosed definite sIBM (n = 17), suspected sIBM (n = 14), myositis controls (n = 110), non-myositis autoimmune controls (n = 93) and healthy subjects (n = 52). Laboratory B analyzed a Dutch cohort of definite sIBM patients (n = 51) and healthy controls (n = 202). RESULTS: Anti-cN-1A reactivity was most frequent in definite sIBM (39.2-47.1%), but absent in biopsy-proven classic polymyositis or dermatomyositis. Overall diagnostic sensitivity and specificity amounted to 35.5 and 96.1% (laboratory A) and 39.2 and 96.5% (laboratory B). CONCLUSIONS: Anti-cN-1A autoantibodies were detected by ELISA with moderate sensitivity, but high specificity for sIBM and may therefore help diagnose this infrequent and difficult-to-diagnose myopathy. The novel anti-cN-1A IgG ELISA can improve and accelerate the diagnosis of sIBM using sera where muscle biopsy is delayed or unfeasible.

Significance of the detection of HIV-1 gag- and/or pol-CD8/A2 T-lymphocytes in HIV-patients.

UNLABELLED: Cytotoxic T-lymphocytes (CTL) play an important role in the immune system's defense against human immunodeficiency virus (HIV) infection. The functional status of CTL closely relates to the progression of HIV disease. We have validated the characteristics of the assay for HIV-1 gag- and pol-specific-CD8/HLA-A2 T-cells from peripheral blood by flow cytometry. Sixty-nine healthy individuals and 38 HIV-patients with HLA-A2 antigen-positive subjects were included in the study. Neither HIV-1 gag- nor pol-specific-CD8/HLA-A2 T-cells were determined in these healthy subjects. HIV-1 gag- and pol-specific-CD8/HLA-A2 T-cells could be detected in HIV-patients. The frequency of specific CTL was 58% (22/38) in the patient group. There was a significantly inverse correlation (p < 0.05) between HIV-1 gag- and pol-specific-CD8/HLA-A2 T-cells and HIV plasma viremia in the patients. CONCLUSION: The HIV-1 gag- or pol-specific-CD8/HLA-A2 T-cells assay is sensitive and specific, being able to detect at the single T-cell level. This assay may provide a versatile tool for structured HIV treatment and for monitoring vaccination efficacy.

Acute hemorrhagic myositis in inflammatory myopathy and review of the literature.

We describe two patients with dermatomyositis that presented with interstitial lung disease, positive V and Shawl sign who developed acute spontaneous abdominal/retroperitoneal bleed. Both patients expired despite aggressive treatment and resuscitation. Hemorrhagic myositis in these two patients with inflammatory myopathy is a very rare complication. The association of anti-Ro52 with this potentially very serious complication remains unclear. This potential relationship should be further evaluated in future studies.

Computer-assisted pattern recognition of autoantibody results.

Immunoassay-based anti-nuclear antibody (ANA) screens are increasingly used in the initial evaluation of autoimmune disorders, but these tests offer no "pattern information" comparable to the information from indirect fluorescence assay-based screens. Thus, there is no indication of "next steps" when a positive result is obtained. To improve the utility of immunoassay-based ANA screening, we evaluated a new method that combines a multiplex immunoassay with a k nearest neighbor (kNN) algorithm for computer-assisted pattern recognition. We assembled a training set, consisting of 1,152 sera from patients with various rheumatic diseases and non-diseased patients. The clinical sensitivity and specificity of the multiplex method and algorithm were evaluated with a test set that consisted of 173 sera collected at a rheumatology clinic from patients diagnosed by using standard criteria, as well as 152 age- and sex-matched sera from presumably healthy individuals (sera collected at a blood bank). The test set was also evaluated with a HEp-2 cell-based enzyme-linked immunosorbent assay (ELISA). Both the ELISA and multiplex immunoassay results were positive for 94% of the systemic lupus erythematosus (SLE) patients. The kNN algorithm correctly proposed an SLE pattern for 84% of the antibody-positive SLE patients. For patients with no connective tissue disease, the multiplex method found fewer positive results than the ELISA screen, and no disease was proposed by the kNN algorithm for most of these patients. In conclusion, the automated algorithm could identify SLE patterns and may be useful in the identification of patients who would benefit from early referral to a specialist, as well as patients who do not require further evaluation.