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

Abstract

Background and Objective: An outer membrane protein (OMP) of Helicobacter pylori namely OipA, is an important vir- ulence 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-Aga- rose 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 deter- mined 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.

Keywords

• 34 KD protein
• Helicobacter pylori
• OipA
• Outer membrane proteins