Production of Mouse Monoclonal Antibody to Extracellular Keratinase of Microsporum canis.

Tazawa Mari, Higuchi Dousei, Takiuchi Iwao, Tsuchida Kazuo, Ishidate Shuzo, Adachi Kenji

doi:10.3314/jjmm.34.35

Male BALB/c mice were immunized with extracellular keratinase (eKase) fromMicrosporum canis(KK-1203-91 strain). Their splenocytes were hybridized with mouse myeloma cells.<BR>After screening by EIA, a cell line secreting a monoclonal antibody (mAb) against eKase was obtained. After eKase was incubated with mAb at 37°C for 15min, a substantial decrease in caseinolytic activity was observed. When eKase was applied to CNBr-activated Sepharose 4B gel which had been coupled with the mAb, eKase activity was detected with the gel using guinea pig hair as a substrate. When Western blot analysis was performed using eKase ofM. canisand tested with the mAb, two antigens could be identified. The molecular weight of the main band and minor band was estimated to be 48KD and 34KD, respectively. However, when SDSPAGE was performed using eKase repeatedly isolated from another strain ofM. canis(Kdog-0114-92), polypeptides of 34 and 31.5KD or those of 48, 34 and 31.5KD were stained in the gel. In immunoblotting experiments, the mAb reacted with latter three polypeptides respectively. Furthermore, enzymographic studies by overlying gelatin containing gel on these SDS-PAGE gels revealed gelatinolytic activity. These results suggest that eKase molecules might have two or three isozymes.

Production of Mouse Monoclonal Antibody to Extracellular Keratinase of Microsporum canis.

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