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Development of monoclonal antibody-based immunoassays for detection of Helicobacter pylori neutrophil-activating protein.

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.
AuthorsIanko D Iankov, Alan R Penheiter, Stephanie K Carlson, Evanthia Galanis
JournalJournal of immunological methods (J Immunol Methods) Vol. 384 Issue 1-2 Pg. 1-9 (Oct 31 2012) ISSN: 1872-7905 [Electronic] Netherlands
PMID22750540 (Publication Type: Journal Article)
CopyrightCopyright © 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
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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