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KMID : 0545120090190010108
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Journal of Microbiology and Biotechnology
2009 Volume.19 No. 1 p.108 ~ p.112
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Improved Purification Process for Cholera Toxin and its Application to the Quantification of Residual Toxin in Cholera Vaccines
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Hyun Jang
Kim Hyo-Seung
Kim Jeong-Ah
Seo Jin-Ho
Carbis Rodney
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Abstract
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A simplified method for the purification of cholera toxin was developed. The 569B strain of Vibrio cholerae, a recognized hyper-producer of cholera toxin, was propagated in a bioreactor under conditions that promote the production of the toxin. The toxin was separated from the bacterial cells using 0.2-μm crossflow microfiltration, the clarified toxin was passed through the membrane into the permeate, and the bacterial cells were retained in the retentate. The 0.2-μm permeate was then concentrated 3-fold and diafiltered against 10 mM phosphate buffer, pH7.6, using 30-kDa crossflow ultrafiltration. The concentrated toxin was loaded onto a cation exchange column, the toxin was bound to the column, and most of the impurities were passed unimpeded through the column. The toxin was eluted with a salt gradient of phosphate buffer, pH7.0, containing 1.0M NaCl. The peak containing the toxin was assayed for cholera toxin and protein and the purity was determined to be 92%. The toxin peak had a low endotoxin level of 3.1 EU/μg of toxin. The purified toxin was used to prepare antiserum against whole toxin, which was used in a GM1 ganglioside-binding ELISA to determine residual levels of toxin in an oral inactivated whole-cell cholera vaccine. The GM1 ganglioside-binding ELISA was shown to be very sensitive and capable of detecting as little as 1 ng/ml of cholera toxin.
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KEYWORD
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Cholera toxin, purification, GM1 ganglioside, ELISA, vaccine
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