| Co-Investigator(Kenkyū-buntansha) |
YAMAKAWA Kiyotaka Kanazawa Univ., Dep. Med., Instructor., 医学部, 助手 (20110629)
KAMIYA Shigeru Kanazawa Univ., Dep., Med., Asst. Prof., 医学部, 講師 (10177587)
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| Research Abstract |
Highly purified toxin A of Clostridium difficicle was obtained by bovine thyroglobulin affinity chromatography followed by two sequential anionーexchange chromatography steps on Q Sepharose FF and Mono Q. The mol. wt of toxin A in nonーdenaturing conditions was estimated to be 520-540 KDa by native PAGE. In contrast, with SDS-PAGE under reducing or nonーreducing conditions, a major band of 240KDa, 10 minor bands of 38-345 KDa and 27 faint bands of 46-440 KDa (nonーreduced conditions), or four minor bands of 38-345 KDa and 31 faint bands of 46-440 KDa (reduced conditions), were detected. In two-dimensional PAGE, the seven minor bands of 255-345 KDa obtained by non-reducing SDS-PAGE migrated to the 240 KDa position after reduction with betaーmercaptoethanol. These findings indicate that toxin A would be composed of subーunits of smaller than 240 KDa.
Nine Monoclonal Antibodies (MAb), 2E15, 3B4, 23C5, 37B5, 45A3, 46A6, 49C4, 61B5 and 112G6, to the purified toxin A were produced. The isotype of 37B5 was IgG2b, kappa, and that of all other 8 MAbs was IgM, kappa. The highest ELISA value of the MAbs was 10^5 for 37B5, and that of other MAbs waS 10^<3-4>. Immunoblot analysis after nonーdenatured PAGE showed that 8 MAbs except for 112G6 gave positive reaction with 540 KDa band of toxin A. The immunoblot analysis after SDS-PAGE showed that 4 MAbs, 2E15, 3B4, 37B5 and 49C4, gave positive reaction with 240 KDa major band of toxin A. In neutralisation test by these MAbs for enterotoxicity, mouse lethality, haemagglutination activity and cytotoxitity, 37B5 was demonstrated to neutralise completely enterotoxicity checked by rabbit ileal loop response test but not any other biological activities, and none of other 8 MAbs showed any neutralising activities. These results suggest that haemagglutination activity would not be necessary for induction of enterotoxicity of toxin A.
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