• 한국식품영양과학회지
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• 제20권6호
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• pp.621-628
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• 1991

Heat-stable enterotoxin(ST) from enterotoxigenic E. coli eKT-53($ST^{+}\;LT^{-}$, transformant from isolate KM-7) that was produced in succinate salts medium. The culture supernatant(crude ST) was purifed by mulitpled steps and investigated some characterization of the ST. The heatstability of purified ST activity was completely lost by treating at $100^{\circ}C$ for 30minutes. ST activity was lost by treatment at pH 1 and 12 conditions, while the activity was not reduced by treatment at pH 2~10, and then the ${\alpha}-amylase$ and pepsin was not decreased activity but disulfide reducing agnets was lost the activity. The molecular weight of the purified ST was approximately 4,200, the isoelectric point was about 4.0.

• 대한미생물학회지
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• 제22권4호
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• pp.377-392
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• 1987

Monoclonal antibody to the Escherichia coli heat-stable enterotoxin(ST) was produced to develop a rapid and convenient diagnostic method to the ST. The toxin was purified from culture supernatant of enterotoxigenic E. coli O148H28($ST^+/LT^+$) and conjugated to bovine serum albumin(BSA). The ST-BSA conjugate was used to immunize BALB/c mice and the immune spleen cells from these mice were fused with $P3{\times}63$ Ag8.V653 plasmacytoma cells. Hybridomas were screened by ELISA and positive hybridomas were cloned by limiting dilution. Finally, one stable clone (AS36) specific to ST was selected for further growth and characterization. Antibody titers of culture supernatant and ascitic fluid from BALB/c mice were 1:1,024 and 1:20,480 respectively in ELISA. The isotype and subclass of monoclonal antibody was IgG1 in sandwich ELISA. To test the neutralizing effect of monoclonal antibody on toxin activity of ST, mixture of ascitic fluid and ST was assayed by infant mouse assay and this monoclonel antibody was proved to be a neutralizing antibody. The titer of ascitic fluid which completely neutralized biological activity of 4 units of ST was 1:4. Purified ST was quantitatively measured by competitive ELISA and minimum amount of ST detectable by this assay was 250pg, which was an amount six-fold smaller than that detectable by infant mouse assay. Four reference strains of enterotoxigenic E. coli from WHO were detected by competitive ELISA and highly specific, sensitive and reproducible result was obtained.

• 대한미생물학회지
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• 제21권1호
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• pp.47-52
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• 1986

A heat-labile enterotoxin and no heat-stable enterotoxin producing($LT^+ST^-$) plasmid (110 kilobases in size) was isolated from an enterotoxigenic Escherichia coli of human strain O15:H11 and used for analysis of the $LT^+$ deoxyrionucleic acid region using recombinant DNA technology. A DNA segment containing the $LT^+$ DNA region which was one restriction endonuclease BamHl fragment(6.2 kb in size) was joind to a small multicopy plasmid, pUC9. E. coli K-12 strain, JM103 harboring the chimeric plasmid produced greater amounts of LT than did the enterotoxigenic E. coli O15:H11 strain. The BamHl fragment was further digested with various restriction endonucleases and contained no HindIll restriction site which is an essential in $LT^+ST^+$ plasmid. The detailed DNA sequencing of this $LT^+ST^-$ plasmid is required.