Novel Autoimmune Hepatitis-Specific Autoantigens Identified Using Protein Microarray Technology

Abstract

Autoimmune hepatitis (AIH) is a chronic necroinflammatory disease of the liver with a poorly understood etiology. Detection of non-organ-specific and liver-related autoantibodies using immunoserological approaches has been widely used for diagnosis and prognosis. However, unambiguous and accurate detection of the disease requires the identification and characterization of disease-specific autoantigens. In the present study, we have profiled the autoantigen repertoire of patients with AIH versus those with other liver diseases, identifying and validating three novel and highly specific biomarkers for AIH. In Phase I we fabricated a human protein chip of 5,011 non-redundant proteins and used it to quickly identify 11 candidate autoantigens with relative small serum collection. In Phase II we fabricated an AIH-specific protein chip and obtained autoimmunogenic profiles of serum samples from 44 AIH patients, 50 healthy controls, and 184 additional patients suffering from hepatitis B, hepatitis C, systemic lupus erythematosus, primary Sjögren’s syndrome, rheumatoid arthritis, or primary biliary cirrhosis. Using this two-phase approach, we identified three new antigens, RPS20, Alba-like, and dUTPase, as highly AIH-specific biomarkers, with sensitivities of 47.5% (RPS20), 45.5% (Alba-like), and 22.7% (dUTPase). These potential biomarkers were further validated with additional AIH samples in a double-blind design. Finally, we demonstrated that these new biomarkers could be readily applied to ELISA-based assays for use in clinical diagnosis/prognosis.

Characterization of monoclonal antibody's binding kinetics using oblique-incidence reflectivity difference approach

“…[15][16][17][18] With advances in proteome microarray technology, such as the E. coli, yeast, and HuProt TM arrays, specificity of an antibody can now be surveyed against the entirety or majority of an organism's proteome. 9,[19][20][21] Mono-specificity of an antibody reagent is thus established only when it recognizes a single antigen on a proteome microarray.…”

Antibody Profiling of Bipolar Disorder Using Escherichia coli Proteome Microarrays

“…4200 E. coli proteins in a microarray format. The two-phase strategy for identification and validation protein hits (30) was used in this study. Although the antigens on the microarray may not be directly associated with BD, this microarray provided hundreds of thousands of epitopes for analyzing antibody profiles of plasma samples in a high throughput fashion.…”

Immunoproteomics technologies in the discovery of autoantigens in autoimmune diseases

“…As a relatively new technology, autoantigen microarrays have been successfully used to detect and characterize autoantibody profiles for several autoimmune diseases (68)(69)(70)(71)(72)(73)(74)(75). These protein/peptide chips have been generated with as few as 14 proteins to as many as ~17 000 proteins (72) that can be used to screen patient sera for corresponding autoantibodies.…”