A quantitative in vitro assay for detecting biological activity of endotoxin using rabbit peripheral blood.
The pyrogen test or the endotoxin test has been playing a crucial role in detecting endotoxin in parenteral drugs. The current test methods, however, have disadvantages such as requiring a relatively high number of animals or an inadequacy in direct evaluation of in vivo activity. We made an attempt to establish a new in vitro assay method that can overcome the shortcomings of the current assay methods. We standardized the system of tumor necrosis factor-alpha (TNF-alpha) induction from the peripheral blood of rabbits for assaying endotoxin activity. This in vitro assay showed a linear dose-response regression between 0.1 and 5.0 endotoxin units per milliliter of endotoxin and a definite homogeneity of variance by logarithmically transforming the endotoxin and TNF-alpha concentrations in the reaction mixtures at 5 h of incubation at 37 degrees C. The assay showed a definite correlation with the pyrogen test but not with the endotoxin test when endotoxins from various bacteria were tested.
Ochiai
M
M
Department of Safety Research on Biologics, National Institute of Infectious Diseases, Tokyo 208-0011, Japan. masakio@nih.go.jp
Kataoka
M
M
Toyoizumi
H
H
Yamamoto
A
A
Horiuchi
Y
Y
D000818:Animals; D001431:Bacteriological Techniques / Q000379:methods* / Q000592:standards; D001769:Blood; D019963:Clinical Chemistry Tests / Q000379:methods* / Q000592:standards; D004731:Endotoxins / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D011817:Rabbits; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis / Q000096:biosynthesis
D004731:Endotoxins; D014409:Tumor Necrosis Factor-alpha