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Characteristics of 26 Kda Antigen of H.Pylori by Monoclonal Antibody Pubmed



Ghahremani H1 ; Farshad S2 ; Najafabadi HA1 ; Kashanian S3 ; Moghaddam MAM1 ; Moradi N1 ; Paknejad M1
Authors
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Authors Affiliations
  1. 1. Department of Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
  2. 2. Alborzi Clinical Microbiology Research Center, Namazi Hospital, Shiraz, Iran
  3. 3. Department of Immunology, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

Source: Iranian Journal of Allergy# Asthma and Immunology Published:2015


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. Copyright © Winter 2015.