Identification of helicobacter pylori antigenic proteins from isolates of patients with gastric pathologies
Loading...
Date
2011
Authors
Chun Wei, Lee
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Helicobacter pylori is a gram negative, spiral-shaped, microaerophilic bacterium, which
colonizes the apical side of human gastric epithelial cells and mucous layer. H pylori
infections are associated with various gastric diseases with wide spectrum of clinical
outcomes, ranging from gastric cancer to ulceration of the gastrointestinal tract. The
pathogenicity of the infection depends on the strain virulence, host susceptibility and
environmental co-factors. Thus it is very important to understand the pathogenesis of the
disease. Identification of the antigenic proteins of H pylori, may eventually lead to the
development of diagnostic test for this pathogen.
Several media were evaluated for isolation and propagation of H pylori. Three
clinical isolates were used and ATCC 700824 strain were employed as reference. For
liquid medium, Brain Heart Infusion broth and Tryptic Soy broth with 10% fetal bovine
serum were tested and the former was found to give higher microbial growth (cfu).
Meanwhile comparisons between serum or blood supplemented solid media i.e. Tryptic
Soy Agar (TSA), Columbia Agar, Eugon Agar and Brain Heart Infusion Agar (BHIA),
showed that the media with blood supplement gave higher cfu. The conventional agar
medium, TSA with 5% defibrinated sheep blood, produced the best growth among the
solid media.
In this study, stomach biopsy samples were from the patients who underwent
Esophageal-Gastro-Duodenal scope. Out of 564 biopsy samples, 50 isolates of H pylori
were successfully isolated from patients with various gastric pathologies. PCR screening
showed that all isolates were positive for Ure C gene, whereas only 42 isolates were
positive for Cag A gene.
An in-house anti-H pylori ESA-IgG ELISA was developed and compared with a
commercially available EIAgen H pylori IgG kit (Adaltis, Italy) and the results showed
similar sensitivities and specificities. Four groups of serum samples were used, they were
categorised according to the results of biopsy culture and the in-house IgG-ELISA:
Group 1: culture positive and seropositive; Group 2: culture negative and seronegative;
Group 3: healthy and seronegative, and Group 4: patients with other diseases and
seronegative. Three different isolates which originated from different gastric pathologies
were used for protein preparation, namely Sll002 from gastroduodenitis, Sll016 from
gastric ulcer, and Sll017 from duodenal ulcer. Two different protein preparations were
used i.e. Excretory Secretory Antigen (ESA) and Surface Associated Antigen (SAA).
ESA and SAA were then subjected to SDS-PAGE and Western blot analysis using the
above serum samples.
A series of antigenic bands were identified as proteins with potential diagnostic value.
For ESA, three bands were identified from isolate Sll002 namely 17 kDa, 29 kDa and
57 kDa. From isolate Sll016, four bands were identified namely 13 kDa, 17 kDa, 29
kDa and 41 kDa; and from isolate SIJ017 three bands were identified namely 13 kDa,
17 kDa and 50 kDa. Using SAA three antigenic bands were identified from isolates
Sll002 & Sll016 i.e 13 kDa, 29 kDa and 57 kDa. From isolate SIJ017, four bands
were identified i.e 13 kDa, 17 kDa, 29 kDa and 57 kDa. As can be observed there
were some common bands between the antigen preparations and among the isolates.
Thus this study has successfully identified a panel of antigenic bands that are
potentially very useful for future development of diagnostic test for H pylori infection.
Since these isolates were from local patients, the antigens derived from them would be
very suitable to be used for diagnosis and screening of Malaysian Patients.
Description
Keywords
Helicobacter pylori , Gastric Pathologies