Jpn. J. Infect. Dis., 54, 131-136, 2001

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Original Article

A Quantitative In Vitro Assay for Detecting Biological Activity of Endotoxin Using Rabbit Peripheral Blood

Masaki Ochiai*, Michiyo Kataoka, Hiromi Toyoizumi, Akihiko Yamamoto and Yoshinobu Horiuchi

Department of Safety Research on Biologics, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashimurayama, Tokyo 208-0011, Japan

(Received July 4, 2001. Accepted August 31, 2001)

SUMMARY: 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-a) 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-a concentrations in the reaction mixtures at 5 h of incubation at 37℃. The assay showed a definite correlation with the pyrogen test but not with the endotoxin test when endotoxins from various bacteria were tested.

*Corresponding author: Tel: +81-42-561-0771, Fax: +81-42-565-3315, E-mail: masakio@nih.go.jp

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