Improved purification process for cholera toxin and its application to the quantification of residual toxin in cholera vaccines.
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-microm 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-microm permeate was then concentrated 3-fold and diafiltered against 10 mM phosphate buffer, pH 7.6, 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/microg of toxin. The purified toxin was used to prepare antiserum against whole toxin, which was used in a G(M1) ganglioside-binding ELISA to determine residual levels of toxin in an oral inactivated whole-cell cholera vaccine. The G(M1) ganglioside-binding ELISA was shown to be very sensitive and capable of detecting as little as 1 ng/ml of cholera toxin.
Jang
Hyun
H
Vaccine Development Section, Laboratory Sciences Division, International Vaccine Institute, Seoul, Korea.
Kim
Hyo Seung
HS
Kim
Jeong Ah
JA
Seo
Jin Ho
JH
Carbis
Rodney
R
D000992:Antitoxins; D019149:Bioreactors; D002411:Cation Exchange Resins; D002771:Cholera / Q000517:prevention & control; D002772:Cholera Toxin / Q000096:biosynthesis / Q000302:isolation & purification*; D022121:Cholera Vaccines / Q000592:standards*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D008851:Micropore Filters; D012680:Sensitivity and Specificity; D014462:Ultrafiltration / Q000379:methods; D014734:Vibrio cholerae / Q000378:metabolism
D000992:Antitoxins; D002411:Cation Exchange Resins; D022121:Cholera Vaccines; D002772:Cholera Toxin