title: Production of Nfa1-specific monoclonal antibodies that influences the in vitro cytotoxicity of Naegleria fowleri trophozoites on microglial cells.
abstract: Naegleria fowleri, agent of fatal primary amoebic meningoencephalitis, appears to induce cytotoxicity mechanically through its contact with the cell. The nfa1 gene cloned from a cDNA library of pathogenic N. fowleri by immunoscreening consists of 360 bp and expresses a 13.1-kDa recombinant protein (rNfa1) that demonstrated localization in the pseudopodia when examined using immunocytochemistry. To study the mechanisms involved in N. fowleri cytotoxicity, we developed a large volume of rNfa1-specific monoclonal antibody (McAb) against a 17-kDa His-tag fusion rNfa1 protein using a cell fusion technique. We established eight McAb-producing hybridoma cells. The antibodies were all immunoglobulin G2b and reacted strongly with a 17-kDa band representing the rNfa1 fusion protein in Western blotting, demonstrating immunoreactivity to the Nfa1 protein in pseudopodia (especially in the food cups) of N. fowleri trophozoites. A 51Cr-release assay indicated N. fowleri cytotoxicity by demonstrating that it eliminated 37.8, 60.6, and 98.8% of the target (microglial) cells 6, 12, and 24 h after co-incubation, respectively. When an anti-Nfa1 McAb was added to the coculture system, N. fowleri cytotoxicity decreased to 29.8, 44.1, and 66.3%, respectively.
authors.affiliation: Department of Microbiology, Molecular Science & Technology, Ajou University School of Medicine, Suwon, 443-721, South Korea.
authors.forename: Yang-Jin
authors.initials: YJ
authors.lastname: Lee
authors.forename: Jong-Hyun
authors.initials: JH
authors.lastname: Kim
authors.forename: Seok-Ryoul
authors.initials: SR
authors.lastname: Jeong
authors.forename: Kyoung-Ju
authors.initials: KJ
authors.lastname: Song
authors.forename: Kyongmin
authors.initials: K
authors.lastname: Kim
authors.forename: Sun
authors.initials: S
authors.lastname: Park
authors.forename: Moon-Sung
authors.initials: MS
authors.lastname: Park
authors.forename: Ho-Joon
authors.initials: HJ
authors.lastname: Shin
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000913:Antibodies, Protozoan / Q000276:immunology*; D000953:Antigens, Protozoan / Q000235:genetics / Q000276:immunology*; D002478:Cells, Cultured; D002860:Chromium Radioisotopes / Q000378:metabolism; D017628:Microglia / Q000469:parasitology*; D016848:Naegleria fowleri / Q000276:immunology / Q000472:pathogenicity*; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D053058:Trophozoites / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D002860:Chromium Radioisotopes; C460560:Nfa1 protein, Naegleria fowleri; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins