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Immunological detection of 34 KDa outer membrane protein as a functional form of OipA in clinical isolates of Helicobacter pylori.

Landarani Z, Falsafi T, Mahboubi M, Lameh-Rad B - Iran J Microbiol (2014)

Bottom Line: Screening of various strains by Dot blot method for its presence showed that its expression was more frequent in strains isolated from the patients with more severe pathology.High titer obtained for pAbs antibody, suggested the high immunogenicity of this protein in experimental animals.Detection of 34 KDa OMP in strains isolated from the patients with more severe pathology proposes the possible application of this pAbs in detecting more virulent strains of H. pylori.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Alzahra University, Tehran, Iran ; Department of Biochemistry, Payam-Nour University, Tehran, Iran.

ABSTRACT

Background and objective: An outer membrane protein (OMP) of Helicobacter pylori namely OipA, is an important virulence factor associated with peptic ulcer and gastric cancer risks. The purpose of this study was to isolate the 34 KDa OMP of H. pylori and evaluate its immunogenicity in experimental animals for rapid detection of more virulent H. pylori isolates.

Material and methods: Sarcosine insoluble fraction of membrane proteins (OMPs) were prepared from 15 clinical isolates of H. pylori and their profiles were analyzed by SDS-PAGE. Two out of 15 isolates which demonstrated higher expression for apparent 34 KDa proteins were selected. Under optimal conditions, 34 KDa protein was recovered from 5% SDS-Agarose gel, purified and injected into the New Zealand white rabbits with Fruend's adjuvant in multiple stages with two weeks intervals. Collected antiserum was purified through affinity chromatography with Sepharose column and its titer was determined by ELISA. Specific immune response was demonstrated by Dot blot and western blotting methods.

Results: The titer of antibody was determined about 1/3000 and western blotting demonstrated a 34 KD-protein. Screening of various strains by Dot blot method for its presence showed that its expression was more frequent in strains isolated from the patients with more severe pathology.

Conclusion: High titer obtained for pAbs antibody, suggested the high immunogenicity of this protein in experimental animals. Detection of 34 KDa OMP in strains isolated from the patients with more severe pathology proposes the possible application of this pAbs in detecting more virulent strains of H. pylori.

No MeSH data available.


Related in: MedlinePlus

Western blot analysis of H. pylori strains using specific anti-33 KDa pAb.1: S13 (strain investigated for pAb production), 2: S15, 3: MW standard, 4: positive control: a recombinant OipA protein (21), 5: negative control
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Figure 3: Western blot analysis of H. pylori strains using specific anti-33 KDa pAb.1: S13 (strain investigated for pAb production), 2: S15, 3: MW standard, 4: positive control: a recombinant OipA protein (21), 5: negative control

Mentions: To understand that the OMP investigated in this work corresponded to OipA, we detected a recombinant OipA protein by our pAb in the same experimental conditions (Fig. 3). The importance of OipA, a 34 KDa outer membrane protein on gastroduodenal pathogenesis is apparently increasing (16, 22-25). In the initial stage of infection, binding via OipA would be more important since its expression explains it’s switched “on” status which can initiate the inflammatory cascade (18, 26). By PCR-related methods, it will be difficult to predict the presence of a functional OipA protein. However, its detection by sensitive and specific immunoblot would provide a simple and accurate method for detecting expression of oipA as an important virulence factor in H. pylori. An easier method for primary screening of H. pylori isolates for presence of its functional protein would be Dot blotting since it requires very smaller amount of antigen without performing SDS-PAGE and transfer.


Immunological detection of 34 KDa outer membrane protein as a functional form of OipA in clinical isolates of Helicobacter pylori.

Landarani Z, Falsafi T, Mahboubi M, Lameh-Rad B - Iran J Microbiol (2014)

Western blot analysis of H. pylori strains using specific anti-33 KDa pAb.1: S13 (strain investigated for pAb production), 2: S15, 3: MW standard, 4: positive control: a recombinant OipA protein (21), 5: negative control
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4385572&req=5

Figure 3: Western blot analysis of H. pylori strains using specific anti-33 KDa pAb.1: S13 (strain investigated for pAb production), 2: S15, 3: MW standard, 4: positive control: a recombinant OipA protein (21), 5: negative control
Mentions: To understand that the OMP investigated in this work corresponded to OipA, we detected a recombinant OipA protein by our pAb in the same experimental conditions (Fig. 3). The importance of OipA, a 34 KDa outer membrane protein on gastroduodenal pathogenesis is apparently increasing (16, 22-25). In the initial stage of infection, binding via OipA would be more important since its expression explains it’s switched “on” status which can initiate the inflammatory cascade (18, 26). By PCR-related methods, it will be difficult to predict the presence of a functional OipA protein. However, its detection by sensitive and specific immunoblot would provide a simple and accurate method for detecting expression of oipA as an important virulence factor in H. pylori. An easier method for primary screening of H. pylori isolates for presence of its functional protein would be Dot blotting since it requires very smaller amount of antigen without performing SDS-PAGE and transfer.

Bottom Line: Screening of various strains by Dot blot method for its presence showed that its expression was more frequent in strains isolated from the patients with more severe pathology.High titer obtained for pAbs antibody, suggested the high immunogenicity of this protein in experimental animals.Detection of 34 KDa OMP in strains isolated from the patients with more severe pathology proposes the possible application of this pAbs in detecting more virulent strains of H. pylori.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Alzahra University, Tehran, Iran ; Department of Biochemistry, Payam-Nour University, Tehran, Iran.

ABSTRACT

Background and objective: An outer membrane protein (OMP) of Helicobacter pylori namely OipA, is an important virulence factor associated with peptic ulcer and gastric cancer risks. The purpose of this study was to isolate the 34 KDa OMP of H. pylori and evaluate its immunogenicity in experimental animals for rapid detection of more virulent H. pylori isolates.

Material and methods: Sarcosine insoluble fraction of membrane proteins (OMPs) were prepared from 15 clinical isolates of H. pylori and their profiles were analyzed by SDS-PAGE. Two out of 15 isolates which demonstrated higher expression for apparent 34 KDa proteins were selected. Under optimal conditions, 34 KDa protein was recovered from 5% SDS-Agarose gel, purified and injected into the New Zealand white rabbits with Fruend's adjuvant in multiple stages with two weeks intervals. Collected antiserum was purified through affinity chromatography with Sepharose column and its titer was determined by ELISA. Specific immune response was demonstrated by Dot blot and western blotting methods.

Results: The titer of antibody was determined about 1/3000 and western blotting demonstrated a 34 KD-protein. Screening of various strains by Dot blot method for its presence showed that its expression was more frequent in strains isolated from the patients with more severe pathology.

Conclusion: High titer obtained for pAbs antibody, suggested the high immunogenicity of this protein in experimental animals. Detection of 34 KDa OMP in strains isolated from the patients with more severe pathology proposes the possible application of this pAbs in detecting more virulent strains of H. pylori.

No MeSH data available.


Related in: MedlinePlus