Identification of New Autoantigens for Primary Biliary Cirrhosis Using Human Proteome Microarrays

Abstract

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease of unknown etiology and is considered to be an autoimmune disease. Autoantibodies are important tools for accurate diagnosis of PBC. Here, we employed serum profiling analysis using a human proteome microarray composed of about 17,000 full-length unique proteins and identified 23 proteins that correlated with PBC. To validate these results, we fabricated a PBC-focused microarray with 21 of these newly identified candidates and nine additional known PBC antigens. By screening the PBC microarrays with additional cohorts of 191 PBC patients and 321 controls (43 autoimmune hepatitis, 55 hepatitis B virus, 31 hepatitis C virus, 48 rheumatoid arthritis, 45 systematic lupus erythematosus, 49 systemic sclerosis, and 50 healthy), six proteins were confirmed as novel PBC autoantigens with high sensitivities and specificities, including hexokinase-1 (isoforms I and II), Kelch-like protein 7, Kelch-like protein 12, zinc finger and BTB domain-containing protein 2, and eukaryotic translation initiation factor 2C, subunit 1. To facilitate clinical diagnosis, we developed ELISA for Kelch-like protein 12 and zinc finger and BTB domain-containing protein 2 and tested large cohorts (297 PBC and 637 control sera) to confirm the sensitivities and specificities observed in the microarray-based assays. In conclusion, our research showed that a strategy using high content protein microarray combined with a smaller but more focused protein microarray can effectively identify and validate novel PBC-specific autoantigens and has the capacity to be translated to clinical diagnosis by means of an ELISAbased method. Molecular & Cellular

Identification of Novel Biomarkers for Behcet Disease Diagnosis Using Human Proteome Microarray Approach

“…Therefore, it is in urgent need to employ an unbiased, high-throughput method to identify novel autoantibodies in BD patients. Protein microarrays, especially human proteome microarrays, are emerging as a highly effective approach for profiling new autoantibodies in numerous autoimmune diseases, as well as cancers (21,30). Here, we employed the HuProt arrays, each comprised of ϳ20,000 human recombinant proteins, to identify BD associated autoantibodies.…”

Systematic Analysis of Intracellular-targeting Antimicrobial Peptides, Bactenecin 7, Hybrid of Pleurocidin and Dermaseptin, Proline–Arginine-rich Peptide, and Lactoferricin B, by Using Escherichia coli Proteome Microarrays

“…In our previous studies, we have shown that proteome microarrays are useful for identifying the intracellular protein targets of LfcinB (15,46). In the present study, we identified the protein targets of Bac7, P-Der, and PR-39, and analyzed the specific and common target proteins of the four AMPs through a bioinformatics analysis.…”

Rnaset2 inhibits melanocyte outgrowth possibly through interacting with shootin1

“…HuProt microarray (CDI Laboratories, Inc., Mayaguez, Puerto Rico), which was composed of 17,225 human full-length proteins, was performed as previously described with minor modifications [14,15]. Briefly, the microarray was blocked with blocking buffer (3% bovine serum albumin in PBS with 0.1% Tween-20, pH = 7.2) at 4 °C for 1 h. In the Rnaset2 group, 500 μl of recombinant full-length Rnaset2 (4 μg/ml) were added and incubated under a glass cover slip at 4 °C for 1.5 h. After rinsing for three times, 500 μl of mouse anti-DDK monoclonal antibody (1:1,000 dilution) was added to the microarray slide and incubated at 37 °C for 1 h. After rinsing again, 500 μl of goat anti-mouse immunoglobulin G-Cy3-conjugated antibody (1:200, Jackson Laboratory, Bar Harbor, ME) was added to the microarray slide and incubated in the dark at 37 °C for 1h.…”