title: 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.
authors: Department of Molecular Medicine, Mayo Clinic, Rochester, MN 55905, USA.
authors: Ianko D
authors: ID
authors: Iankov
authors: Alan R
authors: AR
authors: Penheiter
authors: Stephanie K
authors: SK
authors: Carlson
authors: Evanthia
authors: E
authors: Galanis
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D002522:Chlorocebus aethiops; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D018604:Epitope Mapping; D000939:Epitopes / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D016480:Helicobacter pylori / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015151:Immunoblotting; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014709:Vero Cells
chemical_list: D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D000939:Epitopes; D011994:Recombinant Proteins; C092412:neutrophil-activating protein A, Helicobacter pylori; D049452:Green Fluorescent Proteins