Production of Nfa1-specific monoclonal antibodies that influences the in vitro cytotoxicity of Naegleria fowleri trophozoites on microglial cells.
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.
Lee
Yang-Jin
YJ
Department of Microbiology, Molecular Science & Technology, Ajou University School of Medicine, Suwon, 443-721, South Korea.
Kim
Jong-Hyun
JH
Jeong
Seok-Ryoul
SR
Song
Kyoung-Ju
KJ
Kim
Kyongmin
K
Park
Sun
S
Park
Moon-Sung
MS
Shin
Ho-Joon
HJ
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*
D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D002860:Chromium Radioisotopes; C460560:Nfa1 protein, Naegleria fowleri; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins