The (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) colorimetric assay for the quantitation of Actinobacillus pleuropneumoniae cytotoxin.
Using swine neutrophils as target cells, two MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) colorimetric assay systems, one with and one without phorbol 12-myristate 13-acetate (PMA) stimulation were established for the quantitation of Actinobacillus pleuropneumoniae cytotoxin. The MTT assays were optimized for the number of neutrophils, incubation time, and PMA concentration by a series of experiments. The optimal conditions were 25 x 10(4) cells/well incubated for four hours for the assay system without PMA stimulation, and 12.5 x 10(4) cells/well incubated for two hours for the assay system with PMA stimulation. One culture supernatant of a toxigenic Pasteurella multocida strain and five A. pleuropneumoniae cytotoxin preparations produced from three A. pleuropneumoniae strains were used to test assay reproducibility. Results showed both assays were reproducible with a coefficient of variation ranging from 7.8 to 18% for the assay system without PMA stimulation and from 10.7 to 18.2% for the assay system with PMA stimulation. The PMA-stimulated assay had 40 to 60-fold higher sensitivity than the nonstimulated MTT assay. The MTT assay also was applied to the measurement of neutralizing antibody titers against A. pleuropneumoniae cytotoxin.
Chung
W B
WB
Department of Medical Science, University of Wisconsin-School of Veterinary Medicine, Madison 53706.
Bäckström
L R
LR
McDonald
J
J
Collins
M T
MT
D016977:Actinobacillus pleuropneumoniae / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D003124:Colorimetry; D004396:Coloring Agents; D003603:Cytotoxins / Q000032:analysis* / Q000276:immunology / Q000633:toxicity; D007106:Immune Sera / Q000276:immunology; D009504:Neutrophils / Q000187:drug effects; D012044:Regression Analysis; D015203:Reproducibility of Results; D013552:Swine; D013755:Tetradecanoylphorbol Acetate; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism
D004396:Coloring Agents; D003603:Cytotoxins; D007106:Immune Sera; D013778:Tetrazolium Salts; D013844:Thiazoles; C022616:thiazolyl blue; D013755:Tetradecanoylphorbol Acetate