J Korean Med Sci.  2017 Jul;32(7):1139-1146. 10.3346/jkms.2017.32.7.1139.

Helicobacter pylori Antigens Inducing Early Immune Response in Infants

• Seo JH
• Youn JH
• Kim EA
• Jun JS
• Park JS
• Yeom JS
• Lim JY
• Woo HO
• Youn HS
• Ko GH
• Park JS
• Baik SC
• Lee WK
• Cho MJ
• Rhee KH

• KMID: 2379609
• DOI: http://doi.org/10.3346/jkms.2017.32.7.1139

Abstract

To identify the Helicobacter pylori antigens operating during early infection in sera from infected infants using proteomics and immunoblot analysis. Two-dimensional (2D) large and small gel electrophoresis was performed using H. pylori strain 51. We performed 2D immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin M (IgM) antibody immunoblotting using small gels on sera collected at the Gyeongsang National University Hospital from 4-11-month-old infants confirmed with H. pylori infection by pre-embedding immunoelectron microscopy. Immunoblot spots appearing to represent early infection markers in infant sera were compared to those of the large 2D gel for H. pylori strain 51. Corresponding spots were analyzed by matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS). The peptide fingerprints obtained were searched in the National Center for Biotechnology Information (NCBI) database. Eight infant patients were confirmed with H. pylori infection based on urease tests, histopathologic examinations, and pre-embedding immunoelectron microscopy. One infant showed a 2D IgM immunoblot pattern that seemed to represent early infection. Immunoblot spots were compared with those from whole-cell extracts of H. pylori strain 51 and 18 spots were excised, digested in gel, and analyzed by MALDI-TOF-MS. Of the 10 peptide fingerprints obtained, the H. pylori proteins flagellin A (FlaA), urease β subunit (UreB), pyruvate ferredoxin oxidoreductase (POR), and translation elongation factor Ts (EF-Ts) were identified and appeared to be active during the early infection periods. These results might aid identification of serological markers for the serodiagnosis of early H. pylori infection in infants.

Keyword

Helicobacter pylori Infection; IgM Antibody; Infant; Immunoblot

MeSH Terms

Biotechnology
Electrophoresis
Flagellin
Gels
Helicobacter pylori*
Helicobacter*
Humans
Immunoblotting
Immunoglobulin A
Immunoglobulin G
Immunoglobulin M
Infant*
Microscopy, Immunoelectron
Peptide Elongation Factors
Peptide Mapping
Proteomics
Pyruvate Synthase
Serologic Tests
Spectrum Analysis
Urease
Flagellin
Gels
Immunoglobulin A
Immunoglobulin G
Immunoglobulin M
Peptide Elongation Factors
Pyruvate Synthase
Urease

Figure

• 

  Fig. 1 Histopathologic findings of the gastric antral mucosa in infant 4. (A) H-E-stained gastric mucosa (×400) showed a mild degree of chronic gastritis. (B) Immunohistochemistry with rabbit anti-H. pylori polyclonal antibody and DAB as a chromogen showed brown-colored gastric epithelial lining. (C) Immunoelectromicroscopic examination (× 12,000). Electron microscopic view of the immunohistochemical-stained area revealed several H. pylori. DAB = 3,3′-diaminobenzidine-tetrahydrochloride-dihydrate

• 

  Fig. 2 Western blot of IgG, IgA, and IgM antibodies to whole-cell proteins of H. pylori strain 51 showed several weak IgG-reactive bands and several strong IgA- and IgM-reactive bands. IgG = immunoglobulin G, IgA = immunoglobulin A, IgM = immunoglobulin M.

• 

  Fig. 3 2D immunoblotting of 8 infant serum samples. (A) With IgM. Similar positive spots were observed in each membrane (circles). (B) With IgA. Similar and week positive spots were observed in each membrane (circles). 2D = two-dimensional, IgM = immunoglobulin M, IgA = immunoglobulin A.

• 

  Fig. 4 Eighteen numbered IgM-reactive spots for the 3-month-old infant 2. IgM = immunoglobulin M.

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  Fig. 5 2D PAGE of whole-cell lysates of H. pylori strain 51. 2D = two-dimensional, PAGE = polyacrylamide gel electrophoresis.

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