Abstract |
Neutrophil-activating protein (NAP) is a major virulence factor expressed by Helicobacter pylori isolates associated with severe chronic gastroduodenal inflammation and peptic ulcers. NAP is one of the main protective antigens and a target for vaccine development against Helicobacter infection. In addition, NAP is a potent immune stimulator with potential application as a general vaccine adjuvant and in treatment of allergic diseases and cancer immunotherapy. NAP-specific immunoassays are needed for both H. pylori diagnostics and characterization of NAP-based vaccines and immunomodulatory preparations. We generated a panel of NAP-specific monoclonal antibodies (MAbs) by immunization of BALB/c mice with synthetic NAP peptides. The antibody reactivity against recombinant or native NAP antigen was characterized by enzyme-linked immunosorbent assay (ELISA), immunoblotting and immunofluorescence. A sensitive capture ELISA was developed using MAbs 23C8 and 16F4 (directed against different NAP epitopes) for detection of native or measles virus (MV) vector-expressed recombinant NAP in a concentration range of 4 ng/ml to 2000 ng/ml. MAb 23C8 antigen-binding depends on Tyr101 in a variable amino acid sequence of the NAP molecule, indicating the existence of antigenic variants among H. pylori strains. MAb 16F4 reacted with NAP from different H. pylori strains and was a sensitive tool for detection of small amounts of isolated NAP antigen or whole bacteria by immunoblotting or immunofluorescence. In conclusion, MAb-based immunoassays are highly specific and sensitive for detection of native NAP antigen and recombinant NAP immunostimulatory transgenes expressed by replication competent virus vectors.
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Authors | Ianko D Iankov, Alan R Penheiter, Stephanie K Carlson, Evanthia Galanis |
Journal | Journal of immunological methods
(J Immunol Methods)
Vol. 384
Issue 1-2
Pg. 1-9
(Oct 31 2012)
ISSN: 1872-7905 [Electronic] Netherlands |
PMID | 22750540
(Publication Type: Journal Article)
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Copyright | Copyright © 2012 Elsevier B.V. All rights reserved. |
Chemical References |
- Antibodies, Monoclonal
- Antigens, Bacterial
- Bacterial Proteins
- Epitopes
- Recombinant Proteins
- neutrophil-activating protein A, Helicobacter pylori
- Green Fluorescent Proteins
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Topics |
- Amino Acid Sequence
- Animals
- Antibodies, Monoclonal
(immunology, isolation & purification, metabolism)
- Antigens, Bacterial
(genetics, immunology, metabolism)
- Bacterial Proteins
(genetics, immunology, metabolism)
- Chlorocebus aethiops
- Enzyme-Linked Immunosorbent Assay
(methods)
- Epitope Mapping
- Epitopes
(genetics, immunology, metabolism)
- Female
- Fluorescent Antibody Technique, Indirect
- Green Fluorescent Proteins
(genetics, metabolism)
- Helicobacter pylori
(genetics, immunology, metabolism)
- Immunoblotting
- Mice
- Mice, Inbred BALB C
- Microscopy, Fluorescence
- Molecular Sequence Data
- Recombinant Proteins
(immunology, metabolism)
- Sequence Homology, Amino Acid
- Vero Cells
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