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• W2164239969 abstract "Helicobacter pylori is an important risk factor of duodenal ulcer (DU). Although many virulence factors of H. pylori have been identified, few have been reported to show an association with the pathogenesis of DU. The aims of this study were to identify H. pylori antigens showing a high seropositivity in DU and to develop a platform for rapid and easy diagnosis for DU. Because DU and gastric cancer (GC) are considered clinical divergent gastroduodenal diseases, we compared two-dimensional immunoblots of an acid-glycine extract of an H. pylori strain from a patient with DU probed with serum samples from 10 patients with DU and 10 with GC to identify DU-related antigens. Of the 11 proteins that were strongly recognized by serum IgG from DU patients, translation elongation factor EF-G (FusA), catalase (KatA), and urease α subunit (UreA) were identified as DU-related antigens, showing a higher seropositivity in DU samples (n = 124) than in GC samples (n = 95) (FusA, 70.2 versus 45.3%; KatA, 50.8 versus 41.1%; UreA, 44.4 versus 27.4%). In addition, we found that the use of multiple antigens improved the discrimination between patients with DU and those with GC as the odds ratios increased from 1.82 (95% confidence interval (CI), 0.79–4.21; p = 0.1607) for seropositivity for FusA, KatA, or UreA alone to 4.95 (95% CI, 2.05–12.0; p = 0.0004) for two of the three antigens and to 5.71 (95% CI, 1.86–17.6; p = 0.0024) for all three antigens. Moreover a protein array containing the three DU-related antigens was developed to test the idea of using multiple biomarkers in diagnosis. We conclude that FusA, KatA, and UreA are DU-related antigens of H. pylori, and the combination of these on a protein array provided a rapid and convenient method for detecting serum antibody patterns of DU patients. Helicobacter pylori is an important risk factor of duodenal ulcer (DU). Although many virulence factors of H. pylori have been identified, few have been reported to show an association with the pathogenesis of DU. The aims of this study were to identify H. pylori antigens showing a high seropositivity in DU and to develop a platform for rapid and easy diagnosis for DU. Because DU and gastric cancer (GC) are considered clinical divergent gastroduodenal diseases, we compared two-dimensional immunoblots of an acid-glycine extract of an H. pylori strain from a patient with DU probed with serum samples from 10 patients with DU and 10 with GC to identify DU-related antigens. Of the 11 proteins that were strongly recognized by serum IgG from DU patients, translation elongation factor EF-G (FusA), catalase (KatA), and urease α subunit (UreA) were identified as DU-related antigens, showing a higher seropositivity in DU samples (n = 124) than in GC samples (n = 95) (FusA, 70.2 versus 45.3%; KatA, 50.8 versus 41.1%; UreA, 44.4 versus 27.4%). In addition, we found that the use of multiple antigens improved the discrimination between patients with DU and those with GC as the odds ratios increased from 1.82 (95% confidence interval (CI), 0.79–4.21; p = 0.1607) for seropositivity for FusA, KatA, or UreA alone to 4.95 (95% CI, 2.05–12.0; p = 0.0004) for two of the three antigens and to 5.71 (95% CI, 1.86–17.6; p = 0.0024) for all three antigens. Moreover a protein array containing the three DU-related antigens was developed to test the idea of using multiple biomarkers in diagnosis. We conclude that FusA, KatA, and UreA are DU-related antigens of H. pylori, and the combination of these on a protein array provided a rapid and convenient method for detecting serum antibody patterns of DU patients. Helicobacter pylori is the major factor involved in the pathogenesis of gastritis, gastric ulcer, duodenal ulcer (DU), 1The abbreviations used are: DU, duodenal ulcer; 2D, two-dimensional; CI, confidence interval; GC, gastric cancer; OR, odds ratio. and gastric cancer (GC). It is known that this bacterium infects more than half the world's population (1Blaser M.J. Helicobacter pylori and gastric diseases.BMJ. 1998; 316: 1507-1510Crossref PubMed Scopus (243) Google Scholar). However, the clinical outcomes of H. pylori infection are highly variable and influenced by both bacterial factors and host immune responses. Most infected persons are asymptomatic with no apparent disease, but 6–20% have duodenal ulceration, and a small proportion develop GC (2Miwa H. Go M.F. Sato N. H. pylori and gastric cancer: the Asian enigma.Am. J. Gastroenterol. 2002; 97: 1106-1112Crossref PubMed Google Scholar). DU and GC are considered clinically divergent gastroduodenal diseases in which the pattern of gastritis is regarded as the major factor in the pathogenesis. Antral-predominant gastritis leads to increased acid production and duodenal ulceration, whereas corpus-predominant atrophic gastritis leads to acid reduction and a higher risk of developing GC (3Uemura N. Okamoto S. Yamamoto S. Matsumura N. Yamaguchi S. Yamakido M. Taniyama K. Sasaki N. Schlemper R.J. Helicobacter pylori infection and the development of gastric cancer.N. Engl. J. Med. 2001; 345: 784-789Crossref PubMed Scopus (3712) Google Scholar, 4Blaser M.J. Atherton J.C. Helicobacter pylori persistence: biology and disease.J. Clin. Investig. 2004; 113: 321-333Crossref PubMed Scopus (766) Google Scholar). It has been recognized that the inflammation induced by H. pylori infection is a factor related to the pathogenesis of gastroduodenal diseases. Chronic active gastritis, the hallmark of H. pylori infection, is characterized by infiltration of the mucosa by neutrophils, lymphocytes, and monocytes/mac ro phages (5Dixon M.F. Genta R.M. Yardley J.H. Correa P. Classification and grading of gastritis. The updated Sydney System. International Workshop on the Histopathology of Gastritis, Houston 1994.Am. J. Surg. Pathol. 1996; 20: 1161-1181Crossref PubMed Scopus (4480) Google Scholar). A strong humoral immune response to a variety of H. pylori antigens is also elicited (6Portal-Celhay C. Perez-Perez G.I. Immune responses to Helicobacter pylori colonization: mechanisms and clinical outcomes.Clin. Sci. (Lond.). 2006; 110: 305-314Crossref PubMed Scopus (79) Google Scholar). H. pylori antigens are therefore regarded as potential candidates for biomarkers or vaccines (7Zevering Y. Jacob L. Meyer T.F. Naturally acquired human immune responses against Helicobacter pylori and implications for vaccine development.Gut. 1999; 45: 465-474Crossref PubMed Scopus (49) Google Scholar). Serological tests are noninvasive methods for diagnosing H. pylori infection. Moreover evaluation of the humoral immune responses to H. pylori antigens by immunoblotting appears to be more sensitive than ELISA for detecting low abundance antibodies. Importantly ∼70% of patients with DU are infected with H. pylori (8Ciociola A.A. McSorley D.J. Turner K. Sykes D. Palmer J.B. Helicobacter pylori infection rates in duodenal ulcer patients in the United States may be lower than previously estimated.Am. J. Gastroenterol. 1999; 94: 1834-1840Crossref PubMed Scopus (205) Google Scholar, 9Chu K.M. Kwok K.F. Law S. Wong K.H. Patients with Helicobacter pylori positive and negative duodenal ulcers have distinct clinical characteristics.World J. Gastroenterol. 2005; 11: 3518-3522Crossref PubMed Scopus (25) Google Scholar). The aims of the present prospective study were to identify DU-related antigens by two-dimensional (2D) immunoblotting and mass spectrometry and to develop a platform for detecting antibody patterns for diagnostic use. Protein array technology has been shown to be a useful tool for multiplexed measurements and proteomics studies. Protein arrays can be classified into two types, analytical and functional protein arrays. The most representative analytical protein arrays are antibody arrays. Functional protein arrays have been applied to many fields, including studies of protein-protein, protein-DNA, and protein-drug interactions; biomarker discovery; and clinical diagnosis (10Chen C.S. Zhu H. Protein microarrays.BioTechniques. 2006; 40: 423-429Crossref PubMed Scopus (68) Google Scholar). The binding of humoral antibodies to allergens, autoantigens, cancer-specific antigens, or infectious or ga nisms is the basis of the design of protein arrays for biomarker discovery and clinical diagnosis (11Deinhofer K. Sevcik H. Balic N. Harwanegg C. Hiller R. Rumpold H. Mueller M.W. Spitzauer S. Microarrayed allergens for IgE profiling.Methods. 2004; 32: 249-254Crossref PubMed Scopus (117) Google Scholar, 12Lueking A. Huber O. Wirths C. Schulte K. Stieler K.M. Blume-Peytavi U. Kowald A. Hensel-Wiegel K. Tauber R. Lehrach H. Meyer H.E. Cahill D.J. Profiling of alopecia areata autoantigens based on protein microarray technology.Mol. Cell. Proteomics. 2005; 4: 1382-1390Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 13Wang X. Yu J. Sreekumar A. Varambally S. Shen R. Giacherio D. Mehra R. Montie J.E. Pienta K.J. Sanda M.G. Kantoff P.W. Rubin M.A. Wei J.T. Ghosh D. Chinnaiyan A.M. Autoantibody signatures in prostate cancer.N. Engl. J. Med. 2005; 353: 1224-1235Crossref PubMed Scopus (530) Google Scholar, 14Zhu H. Hu S. Jona G. Zhu X. Kreiswirth N. Willey B.M. Mazzulli T. Liu G. Song Q. Chen P. Cameron M. Tyler A. Wang J. Wen J. Chen W. Compton S. Snyder M. Severe acute respiratory syndrome diagnostics using a coronavirus protein microarray.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 4011-4016Crossref PubMed Scopus (111) Google Scholar). Applying this concept, we wished to develop a DU-related protein array, which can be used for immunodiagnostics in the form of miniaturized and multiplexed assays. In this study, we used a proteomics approach to identify DU-related antigens of H. pylori by comparing the profiles of 2D immunoblots of an acid-glycine extract of H. pylori probed with DU and GC sera. We identified three DU-related antigens, FusA, KatA, and UreA, all of which were more frequently recognized by DU sera than GC sera. Statistical analysis of the data showed that the discrimination power of the three-antigen combination was greater than that of a single antigen. We therefore developed a DU-related protein array that can be used to simultaneously and rapidly detect the reactions of serum antibodies to different DU-related antigens. H. pylori strain HD12 was isolated from endoscopic biopsy samples from the stomach of a patient with DU at the National Taiwan University Hospital. The bacteria were cultured on a BBL™ Stacker™ plate (BD Biosciences) at 37 °C under microaerobic conditions. Serum samples were prospectively collected from individuals who participated in a national project for the investigation of H. pylori and gastroduodenal disorders in Taiwan between December 1999 and December 2001. Our study protocol was approved by both the Institutional Research Board and the Department of Health, Executive Yuan, Taiwan. One hundred and twenty-four patients with DU who received an upper gastrointestinal endoscopic examination were enrolled. Ninety-five patients with GC who underwent curative gastrectomy at our institution were also enrolled. H. pylori status was determined by culture and/or histological examination of gastric biopsy specimens. In addition, 40 subjects with a normal appearance of the gastric mucosa and no evidence of H. pylori infection were selected as controls. Fasting serum samples were collected from all participants, catalogued, aliquoted, and stored at −80 °C. Aliquots were only thawed once prior to analysis. Cell surface proteins were extracted from H. pylori using an acid-glycine extraction procedure as described previously (15Utt M. Nilsson I. Ljungh A. Wadstrom T. Identification of novel immunogenic proteins of Helicobacter pylori by proteome technology.J. Immunol. Methods. 2002; 259: 1-10Crossref PubMed Scopus (49) Google Scholar). Proteins in the H. pylori acid-glycine extract were precipitated using trichloroacetic acid (20%) and separated by 2D electrophoresis as described previously (16Kao S.H. Su S.N. Huang S.W. Tsai J.J. Chow L.P. Sub-proteome analysis of novel IgE-binding proteins from Bermuda grass pollen.Proteomics. 2005; 5: 3805-3813Crossref PubMed Scopus (36) Google Scholar). Briefly the protein extract was incubated with 2D sample buffer (8 m urea, 2% Pharmalyte, pH 3–10, 60 mm DTT, 4% CHAPS, bromphenol blue), the first dimension of the 2D gel was run on IPG strips (Immobiline DryStrip, pH 3–10, 11 cm, GE Healthcare), and the second dimension was run on 12.5% SDS-poly ac ryl am ide gels. For immunodetection, the proteins on the gel were transferred to a PVDF membrane (Millipore, Bedford, MA). Then the membrane was blocked by incubation for 1 h at room temperature in blocking buffer (26 mm Tris-HCl, 150 mm NaCl, pH 7.5, 1% skimmed milk) and incubated overnight at 4 °C with serum samples from DU patients or GC patients or pooled normal sera (1:1000 in blocking buffer containing 0.05% Tween 20) and then for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-human IgG (Chemicon, Temecula, CA). Then bound antibody was detected using 3-amino-9-ethylcarbazole (Sigma) as substrate. The individual Coomassie Blue-stained protein spots were excised and subjected separately to in-gel tryptic digestion. Briefly the spots were destained using 50 mm NH4HCO3 in 50% acetonitrile and dried in a SpeedVac concentrator. The protein was then digested by incubation overnight at 37 °C with sequencing grade trypsin (Promega, Madison, WI) in 50 mm NH4HCO3, pH 7.8. The resulting peptides were extracted sequentially with 1% TFA and 0.1% TFA, 60% acetonitrile, and the combined extracts were lyophilized and analyzed using a QSTAR™ XL Q-TOF mass spectrometer (Applied Biosystems, Framingham, MA) coupled to an UltiMate™ nano-LC system (Dionex/LC Packings, Amsterdam, Netherlands). Peak lists of MS/MS spectra were created using Mascot Search version 1.6b4 in Analyst® QS 1.1 (Applied Biosystems) and uploaded to the Mascot MS/MS Ions Search program (Mascot version 2.0) on the Matrix Science public website, and protein identification was performed against the National Center for Biotechnology Information non-redundant (NCBInr) database (containing 3,957,439 protein entries at the time searched). Up to one missed cleavage was allowed. Cysteine carbamidomethylation, glutamine/asparagine deamidation, and methionine oxidation were set as possible modifications. The error windows for peptide and MS/MS fragment ion mass values were 0.3 and 0.5 Da, respectively. MH22+ and MH33+ were selected as the precursor peptide charge states in the search. Ion scores greater than 54 indicate a significant match; the individual score for the MS/MS spectrum of each peptide was more than 20. From the hit lists, the protein names and locus_tag for H. pylori strain 26695 were selected and are listed in Table I and Supplemental Table 1.Table IFrequency of seropositivity for the indicated H. pylori antigens in the DU and GC groups identified by nano-LC-MS/MS analysisSpot no.ProteinLocus_tagTheoretical pI/molecular mass (Da)Sequence coverageScoreSeropositivityDU (n = 10)GC (n = 10)%%1Translation elongation factor EF-G (FusA)HP11955.25/76,97256870402Flagellar hook proteinHP08705.04/76,160362921001003Flagellar hook-associated protein (FliD)HP07525.15/74,04334183100804Chaperone protein DnaK (Actinobacillus actinomycetemcomitans)4.88/68,3522843690805Molecular chaperone DnaKHP01095.04/67,01136330100706Urease β subunit (UreB)HP00725.71/61,6765278890807Chaperonin GroELHP00105.44/58,2365250070808Flagellin B (FlaB)HP01155.95/53,923291531001009Flagellin A (FlaA)HP06015.58/53,2354357510010010Catalase (KatA)HP08758.57/58,599402591005011Flagellin A (FlaA)HP06015.58/53,23557210010012Serine protease (HtrA)HP10199.18/51,593564431008013Urease α subunit (UreA)HP00738.47/26,53746144402014Urease α subunit (UreA)HP00738.47/26,53766288802015Urease α subunit (UreA)HP00738.47/26,537613138020 Open table in a new tab H. pylori was lysed, the lysate was subjected to RNase treatment, and then genomic DNA was extracted using phenol-chloroform and precipitated with 70% ethanol. To amplify the DNA fragments containing the H. pylori fusA gene, katA gene, ureA gene, and flaA gene (control) by PCR, the primer pairs used were: fusA: sense, 5′-GGT ACC ATG GCT AGA AAA ACC CCA-3′; antisense, 5′-CTG CAG TCA GCC TTT GCG TTT TTC-3′; katA: sense, 5′-GAA TTC GAT GGT TAA TAA AGA TGT GAA-3′; antisense, 5′-CTC GAG CTT TTT CTT TTT TGT GTG GT-3′; ureA: sense, 5′-GGA TCC ATG AAA CTC ACC CCA-3′; antisense, 5′-GGA TCC TTA CTC CTT AAT TGT T-3′; and flaA: sense, 5′-GGA TCC ATG GCT TTT CAG GTC AAT-3′; antisense, 5′-GGT ACC CTA AGT TAA AAG CCT TAA G-3′. PCR was performed using 30 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 2 min followed by a final extension at 72 °C for 15 min. The fusA, ureA, and flaA gene fragments were cloned into the expression vector pQE30 (Qiagen, Hilden, Germany) and transformed into Esch e richia coli strain M15, whereas the katA gene fragment was cloned into the pET-22b(+) vector (Novagen, Madison, WI) and transformed into E. coli strain BL21. For expression of recombinant proteins, cells were grown to an A600 value of 0.6, induced with 1 mm isopropyl β-d-thiogalactoside, and harvested after 6 h at 25 °C (for UreA), 2 h at 37 °C (for FlaA), or 4 h at 37 °C (for FusA and KatA). The recombinant proteins were dissolved in binding buffer (20 mm Tris-HCl, 0.5 m NaCl, 5 mm imidazole, pH 7.9) containing 8 m urea and purified on a Ni2+-chelating Sepharose column (GE Healthcare). An H. pylori acid-glycine extract and recombinant FusA, KatA, UreA, and FlaA were electrophoresed on 12.5% SDS-poly ac ryl am ide gels, transferred to the PVDF membrane, and then subjected to immunoblotting using serum samples from patients with DU or GC or from normal controls diluted 1:1000 as described above. Statistical analysis was performed using SAS version 9.1 (SAS Institute Inc., Cary, NC). The odds ratio (OR) and 95% confidence interval (CI) were estimated by multiple logistic regression analysis. Comparisons between tests of serum reactions on protein arrays were made using Student's t test. A p value of <0.05 was considered statistically significant. Poly(l-lysine)-coated slides were placed in an OmniGrid arrayer (GeneMachines, San Carlos, CA) and four SMP3 printing pins (TeleChem, Sunnyvale, CA) with a tip diameter of 100 μm were used to generate the protein arrays. All proteins were spotted in quadruplicate at three different concentrations (100, 300, and 600 μg/ml) diluted with 0.14–2 m urea in PBS. Each protein array contained the control spots, human IgG in three dilutions (one normal serum was diluted with PBS to 1:1500, 1:1000, and 1:500). The resulting protein chips were stored in a desiccator. All reactions were performed at room temperature. The chips were blocked for 2 h with 2% BSA in TBST (20 mm Tris, 137 mm NaCl, 0.1% Tween 20, pH 7.6) in a humidified atmosphere and then incubated with the test sera (1:100 in 2% BSA in TBST) for 1 h. Following three TBST washes, the chips were incubated for 1 h in the dark with Cy3-conjugated anti-human IgG antibody (1:500 in 2% BSA in TBST; Abcam, Cambridge, UK) and then washed three times in TBST and twice in distilled water. All incubation steps were performed in a volume of 50 μl underneath a cover slide. Following air drying, the protein chips were imaged using a GenePix™ 4000B scanner (Axon Instruments, Union City, CA), and image analysis was performed using GenePix Pro 6.0 (Axon Instruments). To identify the DU-related antigens of H. pylori, we first examined the one-dimensional immunoblot profiles of the acid-glycine-extracted bacterial proteins probed with sera from H. pylori-infected patients with DU. The molecular masses of the immunoreactive proteins ranged from 23 to 97 kDa (Fig. 1A). The binding pattern of each serum sample was distinct and unique. Moreover several proteins were recognized by sera from normal individuals (Fig. 1B). We then performed 2D electrophoresis on the bacterial proteins and examined the patterns of the 2D immunoblots probed with sera from 10 patients with DU in the active stage. Fig. 2A shows the complex 2D profile of the H. pylori acid-glycine-extracted proteins after silver staining. The representative 2D immunoblot probed with one DU serum is shown in Fig. 2B. Numerous protein spots recognized by the DU sera were observed. The majority of the recognized antigens had molecular masses greater than 30 kDa under reducing conditions, and those showing the strongest reaction had molecular masses greater than 50 kDa. To determine which antigens were DU-related and non-DU-related, we compared the frequency of recognition of these by sera from patients with DU and GC (n = 10) and identified the proteins by nano-LC-MS/MS analysis (Table I and Supplemental Table 1). The representative 2D immunoblot probed with one GC serum is shown in Fig. 2C. Several proteins recognized at a high frequency by both DU and GC sera were flagellar hook protein, flagellar hook-associated protein (FliD), molecular chaperone DnaK, urease β subunit, flagellin A, flagellin B, and serine protease (HtrA). Importantly comparison of DU and GC prevalence data showed that translation elongation factor EF-G (FusA; DU versus GC, 70 versus 40%), catalase (KatA; 100 versus 50%) and urease α subunit (UreA; 40 and 80% versus 20%) were potential candidates for DU-related antigens. Fig. 3 shows the reactivity of FusA, KatA, and UreA in 2D immunoblots probed with 10 DU sera. The location of FusA was close to a spot with strong immunoreactivity. The different isoforms of KatA were recognized equally well by DU sera; in contrast, 40% of DU sera recognized two of three UreA isoforms, and 40% of DU sera recognized all UreA isoforms.Fig. 22D profiles of immunoreactive proteins in the H. pylori sample. The acid-glycine extract of cell surface proteins from H. pylori was separated by 2D electrophoresis using a linear pH 3–10 gradient in the first dimension and 12.5% SDS-PAGE in the second dimension. The separated proteins were detected by silver staining (A) or were transferred to a PVDF membrane and probed with serum from an H. pylori-infected patient with DU (B) or GC (C).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3The patterns of DU-related antigens on 2D immunoblots. The acid-glycine extract of cell surface proteins from H. pylori was separated by 2D electrophoresis and transferred to the PVDF membrane; the portions of the silver-stained gel and the immunoblots containing FusA, KatA and UreA protein spots are shown (indicated by arrows). The 2D immunoblots were analyzed by blotting with sera from 10 DU patients. WB, Western blot.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further examine the clinical significance of FusA, KatA, and UreA as potential candidates for DU-related antigens, we expressed the recombinant His-tagged fusion proteins in E. coli. Recombinant FusA, KatA, and UreA with apparent molecular masses of 80, 63, and 28 kDa, respectively, were successfully expressed and purified (Fig. 4, lanes 1–3). The identity of the purified proteins was confirmed by nano-LC-MS/MS, and their antigenicity was examined by immunoblot analysis using pooled sera from DU patients (Fig. 4, lanes 4–6). They were then used in an immunoblot assay using a series of clinical samples including sera from H. pylori-infected patients with DU (n = 124) or GC (n = 95) or non-H. pylori-infected normal individuals (control, n = 40). As shown in Table II, the prevalence of FusA seropositivity was significantly higher in DU patients than in GC patients (70.2 versus 45.3%; OR = 2.68; 95% CI, 1.52–4.72; p = 0.0007) or controls (70.2 versus 20%; OR = 8.42; 95% CI, 3.23–22.0; p < 0.0001). The corresponding values for KatA seropositivity in patients with DU, GC, and control were 50.8, 41.1, and 5%. KatA seropositivity in DU patients was significantly higher than in controls (OR = 17.7; 95% CI, 3.83–81.8; p = 0.0002) but not significantly different from that in GC patients (OR = 1.45; 95% CI, 0.82–2.56; p = 0.1976). UreA seropositivity in DU patients was significantly higher than in GC patients (44.4 versus 27.4%; OR = 2.05; 95% CI, 1.13–3.71; p = 0.0183) or controls (44.4 versus 15%; OR = 4.43; 95% CI, 1.51–13.1; p = 0.0069). Moreover the seropositivity of each protein in DU patients was significantly different from that in the GC patients and normal individuals combined (FusA: OR = 3.37; 95% CI, 1.97–5.76; p < 0.0001; KatA: OR = 2.16; 95% CI, 1.25–3.73; p = 0.0055; UreA: OR = 2.38; 95% CI, 1.35–4.20; p = 0.0029). As a result, H. pylori FusA, KatA, and UreA were antigens related to DU disease.Table IIFrequency of seroreactivity with a single recombinant antigen in patients with gastroduodenal diseases or controls and the odds ratioAntigensPositive no. (%)OR (95% CI), p valueaThe OR, 95% CI, and p value were obtained by multiple logistic regression analysis.DU (n = 124)GC (n = 95)Control (n = 40)DU vs. GCDU vs. controlDU vs. (GC + control)FusA87 (70.2)43 (45.3)8 (20)2.68 (1.52–4.72), 0.00078.42 (3.23–22.0), <0.00013.37 (1.97–5.76), <0.0001KatA63 (50.8)39 (41.1)2 (5)1.45 (0.82–2.56), 0.197617.7 (3.83–81.8), 0.00022.16 (1.25–3.73), 0.0055UreA55 (44.4)26 (27.4)6 (15)2.05 (1.13–3.71), 0.01834.43 (1.51–13.1), 0.00692.38 (1.35–4.20), 0.0029a The OR, 95% CI, and p value were obtained by multiple logistic regression analysis. Open table in a new tab To evaluate whether a combination of multiple antigens increased the ability to distinguish patients with DU from those with GC and normal individuals, we compared the frequency of seroreactivity with a single antigen (FusA, KatA, or UreA) and with combinations of these antigens in gastroduodenal diseases (Table III). Comparing the results for the DU samples with those for the GC samples, the odds ratios increased with the number of recognized antigens, going from 1.82 (95% CI, 0.79–4.21; p = 0.1607) for a single recognized antigen to 4.95 (95% CI, 2.05–12.0; p = 0.0004) for two recognized antigens and to 5.71 (95% CI, 1.86–17.6; p = 0.0024) for three recognized antigens. Similarly when the results for the DU samples were compared with those for the GC samples and normal samples combined, the odds ratios again increased with the number of recognized antigens: 3.14 (95% CI, 1.46–6.77; p = 0.0035) for a single antigen, 9.87 (95% CI, 4.35–22.4; p < 0.0001) for two antigens, and 12.5 (95% CI, 4.23–36.8; p < 0.0001) for three antigens. Using combinations of two of the three antigens, each pair had a significantly higher odds ratio than the single antigen when DU samples were compared with GC samples alone or the population of GC and normal individuals combined. Importantly the combination of KatA and UreA led to the best discrimination of DU disease from GC (OR = 16.0; 95% CI, 1.77–144; p = 0.0136) or a population of GC and normal individuals (OR = 34.9; 95% CI, 3.95–308; p = 0.0014). According to the statistical data, we can use two-antigen and three-antigen combinations of FusA, KatA, and UreA as biomarkers of DU.Table IIIEffect of an increased number of recognized antigens on the odds ratioRecognized antigensPositive no. (%)OR (95% CI), p valueaThe OR, 95% CI, and p value were obtained by multiple logistic regression analysis.No.AntigensDU (n = 124)GC (n = 95)GC + control (n = 135)DU vs. GCDU vs. (GC + control)0None11 (8.9)22 (23.2)48 (35.6)1.01.01Any one antigen41 (33.1)45 (47.4)57 (42.2)1.82 (0.79–4.21), 0.16073.14 (1.46–6.77), 0.00352Two of the three antigens52 (41.9)21 (22.1)23 (17.0)4.95 (2.05–12.0), 0.00049.87 (4.35–22.4), <0.0001 FusA and KatA26 (21.0)12 (12.6)12 (8.9)4.33 (1.60–11.7), 0.00399.45 (3.67–24.4), <0.0001 KatA and UreA8 (6.5)1 (1.1)1 (0.7)16.0 (1.77–144), 0.013634.9 (3.95–308), 0.0014 FusA and UreA18 (14.5)8 (8.4)10 (7.4)4.50 (1.49–13.6), 0.00757.85 (2.85–21.6), <0.00013All three antigens20 (16.1)7 (7.4)7 (5.2)5.71 (1.86–17.6), 0.002412.5 (4.23–36.8), <0.0001a The OR, 95% CI, and p value were obtained by multiple logistic regression analysis. Open table in a new tab To improve the efficiency and convenience of diagnostic usage, we designed a multiple protein array including DU-related antigens and a non-disease-related antigen in a poly(l-lysine)-coated slide. The DU-related antigens were FusA, KatA, and UreA, whereas FlaA was selected as a positive control antigen to monitor the experimental performance. We found that FlaA was recognized by sera from H. pylori-infected persons and non-infected persons. All antigens were spotted at three different concentrations (100, 300, and 600 μg/ml) to show the trend of antigen-antibody recognition. Human IgG was also included at three serial dilutions for interchip comparison. The chips were incubated with sera from six DU patients and seven normal individuals. Based on the immunoblot analysis, six DU patients whose serum IgG recognized all the DU-related antigens were selected. In contrast, none of the DU-related antigens were recognized by serum IgG from the seven normal individuals. The results of chip reactions with six DU sera and four normal sera are shown in Fig. 5A. The analysis of human IgG showed a low coefficient of variation (26.4, 28.9, and 3.6% for 1:1500, 1:1000, and 1:500, respectively), indicating good reproducibility between chip-serum incubations. When comparing DU sera with normal sera using the average intensity of fluorescence, obvious" @default.
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