보툴리눔 독소생물활성 분석 및 고감도 진단법 개발 연구

초록

Purpose : Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are divided into seven antigenically distinct proteins designated A through G. In human, types A, B, E and F cause botulism and， in case of Korea， botulism suspected cases have been continuously reported since 2003. A standard method for laboratory diagnostics of botulism in the world is mouse bioassay. However, it takes long time for diagnosis and requires sacrifice of animals, so that the necessity of development of an in vitro diagnostic method was caused. Therefore, in this study， we developed a highly sensitive capture ELISA method for the detection of botulinum neurotoxin type A. Methods : Rabbit anti-BoNT/A IgG and biotinylated monoclonal antibody(MAb) were prepared. Capture ELISA was constructed using rabbit IgG as a capture antibody and biotinylated MAb as a detection antibody, and its detection limit was determined as toxin concentration with optical density(OD) value higher than cut-off，[average of OD values plus 2 standard deviations]. Also，we repetitively conducted the ELISA three times at intra-and inter—day to confirm producibility，and compared toxin detectability with LD_(50) in mice. Results : Detection of this method was possible at 0.031 ng/ml. The OD was in proportion to amount of BoNT/A ranging from 0 to 1.5625 ng/well (R²=0.9955). The intra-and inter-variations were typically 2-12% and the specificity was confirmed by showing no cross reactivity against BoNT/B and /E. Also, the sensitivity was confirmed with four BoNT/As from various origins and was compared with mouse LD_(50) of each BoNT/A. The data showed that sensitivity of this assay was similar to that of mouse bioassay. Conclusions : These results suggest that the capture ELISA could serve as a rapid and sensitive screening tools for detecting BoNT/A and instead of mouse bioassay.

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