title: Characteristics of 26 kDa antigen of H. Pylori by Monoclonal Antibody.
abstract: Alkylhydroperoxide reductase (AhpC, the 26 kDa antigen) is one of the abundant antioxidant enzymes in Helicobacter pylori and seems to have a good potential for use in development of immunoassays to detect H. pylori infection in clinical specimens. This study aimed to investigate some properties of this antigen by the produced monoclonal antibodies. Five established hybridoma cell lines secreting monoclonal antibodies (MAbs) against 26 kDa antigen of H. pylori were cultivated and MAbs were purified by affinity chromatography. Subsequently, MAbs were conjugated with biotin, and different combinations of capture and tracer antibodies used in sandwich ELISA. Immunoblotting of bacterial extracts were performed to estimate aggregation status of the antigen. Release of antigen from the cultivated bacteria on solid media was examined by sandwich ELISA, and also, existence of interference in fecal extract was investigated by immunoblotting and sandwich ELISA. Our findings showed that the MAbs against 26 kDa antigen of H. pylori could recognize three bands of nearly 25 kDa, 50 kDa, and 75 kDa in immunoblotting. This study also indicated presence of more antigens in the culture medium around the bacteria than the bacterial extract itself. The results of sandwich ELISA and immunoblotting on fecal extracts suggest the presence of interfering agents that prevent detection of antigen by antibody in ELISA but not in immunoblotting. In this study the oligomerization of the 26 kDa antigen, presence of interfering agents in stool matrix, and release of antigen to outside of bacteria, were demonstrated.
authors.affiliation: Department of Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran. paknejadma@tums.ac.ir.
authors.forename: Hossein
authors.initials: H
authors.lastname: Ghahremani
authors.affiliation: Alborzi Clinical Microbiology Research Center, Namazi Hospital, Shiraz, Iran. paknejadma@tums.ac.ir.
authors.forename: Shohreh
authors.initials: S
authors.lastname: Farshad
authors.affiliation: Department of Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran. paknejadma@tums.ac.ir.
authors.forename: Hossein
authors.initials: H
authors.lastname: Amini Najafabadi
authors.affiliation: Department of Immunology, School of Medicine, International Campus, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. paknejadma@tums.ac.ir.
authors.forename: Susan
authors.initials: S
authors.lastname: Kashanian
authors.affiliation: Department of Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran. paknejadma@tums.ac.ir.
authors.forename: Mohammad Amin
authors.initials: MA
authors.lastname: Momeni Moghaddam
authors.affiliation: Department of Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran. paknejadma@tums.ac.ir.
authors.forename: Nariman
authors.initials: N
authors.lastname: Moradi
authors.affiliation: Department of Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran. paknejadma@tums.ac.ir.
authors.forename: Maliheh
authors.initials: M
authors.lastname: Paknejad
mesh_terms: D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay; D005243:Feces / Q000382:microbiology; D016481:Helicobacter Infections / Q000175:diagnosis*; D006801:Humans; D010544:Peroxidases / Q000302:isolation & purification*
chemical_list: D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D010544:Peroxidases; C508531:ahpC protein, Helicobacter pylori