Proteomic characterization of Helicobacter pylori CagA antigen recognized by child serum antibodies and its epitope mapping by peptide array.

Serum antibodies against pathogenic bacteria play immunologically protective roles, and can be utilized as diagnostic markers of infection. This study focused on Japanese child serum antibodies against Helicobacter pylori, a chronically-infected gastric bacterium which causes gastric cancer in adults. Serological diagnosis for H. pylori infection is well established for adults, but it needs to be improved for children. Serum samples from 24 children, 22 H. pylori (Hp)-positive and 2 Hp-negative children, were used to catalogue antigenic proteins of a Japanese strain CPY2052 by two-dimensional electrophoresis followed by immunoblot and LC-MS/MS analysis. In total, 24 proteins were identified as candidate antigen proteins. Among these, the major virulence factor, cytotoxin-associated gene A protein (CagA) was the most reactive antigen recognized by all the Hp-positive sera even from children under the age of 3 years. The major antigenic part of CagA was identified in the middle region, and two peptides containing CagA epitopes were identified using a newly developed peptide/protein-combined array chip method, modified from our previous protein chip method. Each of the epitopes was found to contain amino acid residue(s) unique to East Asian CagA. Epitope analysis of CagA indicated importance of the regional CagA antigens for serodiagnosis of H. pylori infection in children.

A new type of protein chip to detect hepatocellular carcinoma-related autoimmune antibodies in the sera of hepatitis C virus-positive patients.

BACKGROUND: We report here a new type of protein chip to detect antibodies in sera. This chip method was used to a prototype created to detect hepatocellular carcinoma (HCC) -related autoantibodies in the sera of hepatitis C virus (HCV) infected individuals. RESULTS: Five cysteine-tagged (Cys-tag) and green fluorescent protein (GFP)-fused recombinant heat shock protein 70 (HSP70), superoxide dismutase 2 (SOD2), and peroxiredoxin 6 (PRDX6), were spotted and immobilized on maleimide-incorporated diamond-like carbon (DLC) substrates. The antibodies in diluted sera were trapped by these proteins at each spot on the chip, and visualized by a fluorescence-conjugated anti-human IgG. The total immobilized protein level of each spot was detected with anti-GFP mouse IgG and a fluorescence-conjugated secondary anti-mouse IgG. The ratio between the two fluorescence intensities was used to quantify autoantibody levels in each serum sample. Heat treatment of the chip in a solution of denaturing and reducing agents, before serum-incubation, improved autoantibody detection. We tested serum samples from healthy individuals and HCC patients using the chips. The HSP70 autoantibodies were found at high levels in sera from HCV-positive HCC patients, but not in HCV-negative sera. CONCLUSION: This protein chip system may have useful properties to capture a specific set of antibodies for predicting the onset of particular cancers such as HCC in HCV-infected individuals.

A novel approach of protein immobilization for protein chips using an oligo-cysteine tag.

Protein chip technology is essential for high-throughput functional proteomics. We developed a novel protein tag consisting of five tandem cysteine repeats (Cys-tag) at termini of proteins. The Cys-tag was designed to allow covalent attachment of proteins to the surface of a maleimide-modified, diamond-like, carbon-coated silicon substrate. As model proteins, we created an enhanced green fluorescent protein (EGFP) and an EGFP-stathmin fusion protein, both of which contained a Cys-tag. We also included an oligo-histidine tag to allow its purification by the use of Ni beads, and we expressed the protein in Escherichia coli. The purified Cys-tagged EGFP could be captured on the maleimide-coated substrate efficiently so that 50 pg of the fusion protein was detected by fluorescence, and as little as 5 pg was immunodetected by combination with enhanced chemiluminescence. This highly sensitive immunodetection may be due to the strong covalent binding of the Cys-tag to the substrate combined with efficient exposure of the protein to the surrounding solution. Thus, the Cys-tag should be useful for developing a novel protein printing method for protein chips that requires very low amounts of protein and can be used for high-performance analysis of protein-ligand interactions.