Characteristics of 26 kDa Antigen of H. Pylori by Monoclonal Antibody
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...
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Tehran University of Medical Sciences
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doaj-3402a999b637426e83cc7dda5173b9962020-11-25T04:12:39ZengTehran University of Medical SciencesIranian Journal of Allergy, Asthma and Immunology1735-15021735-52492015-02-01141369Characteristics of 26 kDa Antigen of H. Pylori by Monoclonal AntibodyHossein Ghahremani0Shohreh Farshad1Hossein Amini Najafabadi2Susan Kashanian3Mohammad Amin Momeni Moghaddam4Nariman Moradi5Maliheh Paknejad6Department of Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.Alborzi Clinical Microbiology Research Center, Namazi Hospital, Shiraz, Iran.Department of Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.Department of Immunology, School of Medicine, International Campus, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.Department of Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.Department of Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.Department of Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran. 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. https://ijaai.tums.ac.ir/index.php/ijaai/article/view/404Alkyl hydroperoxide reductaseELISAH. pyloriImmunoblottingMonoclonal antibody |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Hossein Ghahremani Shohreh Farshad Hossein Amini Najafabadi Susan Kashanian Mohammad Amin Momeni Moghaddam Nariman Moradi Maliheh Paknejad |
spellingShingle |
Hossein Ghahremani Shohreh Farshad Hossein Amini Najafabadi Susan Kashanian Mohammad Amin Momeni Moghaddam Nariman Moradi Maliheh Paknejad Characteristics of 26 kDa Antigen of H. Pylori by Monoclonal Antibody Iranian Journal of Allergy, Asthma and Immunology Alkyl hydroperoxide reductase ELISA H. pylori Immunoblotting Monoclonal antibody |
author_facet |
Hossein Ghahremani Shohreh Farshad Hossein Amini Najafabadi Susan Kashanian Mohammad Amin Momeni Moghaddam Nariman Moradi Maliheh Paknejad |
author_sort |
Hossein Ghahremani |
title |
Characteristics of 26 kDa Antigen of H. Pylori by Monoclonal Antibody |
title_short |
Characteristics of 26 kDa Antigen of H. Pylori by Monoclonal Antibody |
title_full |
Characteristics of 26 kDa Antigen of H. Pylori by Monoclonal Antibody |
title_fullStr |
Characteristics of 26 kDa Antigen of H. Pylori by Monoclonal Antibody |
title_full_unstemmed |
Characteristics of 26 kDa Antigen of H. Pylori by Monoclonal Antibody |
title_sort |
characteristics of 26 kda antigen of h. pylori by monoclonal antibody |
publisher |
Tehran University of Medical Sciences |
series |
Iranian Journal of Allergy, Asthma and Immunology |
issn |
1735-1502 1735-5249 |
publishDate |
2015-02-01 |
description |
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.
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topic |
Alkyl hydroperoxide reductase ELISA H. pylori Immunoblotting Monoclonal antibody |
url |
https://ijaai.tums.ac.ir/index.php/ijaai/article/view/404 |
work_keys_str_mv |
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