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

Ianko D. Iankov, Alan R. Penheiter, Stephanie K. Carlson, Evanthia Galanis

Research output: Contribution to journalArticle

11 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)1-9
Number of pages9
JournalJournal of Immunological Methods
Volume384
Issue number1-2
DOIs
StatePublished - Oct 31 2012

Keywords

  • Helicobacter pylori
  • Immunoassays
  • Monoclonal antibody
  • NAP

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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