Purification of Clostridium Perfringens Epsilon Toxin Type D from Culture Supernatant of the Microorganism
Published: March 14, 2013
By: Dr. Esteban Guerra
Clostridium perfringens type D is responsible for producing microorganism enterotoxemia in sheep. The main toxins produced by the organism are the alpha and epsilon (E). E toxin being responsible for the pathogenesis produce in sheep and goats, since it increases the capillary permeability which favors its absorption through the intestinal wall into the blood reaching critical levels and the installation of a frame enterotoxemia.
The objective of this work is the purification of the toxin of C. E perfringens type D for use in serum neutralization test in vaccine potency tests. For this purpose a commercial strain (ATCC) Cl perfringens type D was cultured in culture medium 1Lt Cooked Meat (Oxoid) for 72 hours.
The medium supernatant was centrifuged at 10000 rpm for 30 minutes at 4 ° C and then filtered by filters in successive steps of 1 um, 0.45 um and 0.22 um. Purification of the protoxin from the supernatant was carried out in two stages, first by salting and second by anion exchange.
The saline precipitation was carried out with saturated ammonium sulfate (35% v / v) by adding small volumes and stir at room temperature. Subsequently, the culture medium was centrifuged at 3500 rpm for 10 minutes. The supernatant was discarded and the pellet containing the protoxin was suspended in 1/10 the original volume with distilled water and dialyzed for 24hours against phosphate buffered saline (PBS) pH 7.2. Following dialysis the protoxin was purified by anion exchange chromatography (DEAE-Sepharose).
The material thus obtained was tested biological controls by challenge of mice with the purified fraction activated with 0.2% trypsin and biochemical controls by SDS-PAGE. 72 hours after the intravenous inoculation of mice with different dilutions of toxin were evaluated to be positive deaths dilution 1/10000. The electrophoresis of the purified toxin was stained with Coomassie revealing a single fraction PM 34 Kda. Thus we have purified toxin E Clperfringens type D with a high purity that allows its use for immunological assays for both serum neutralization test in vaccine potency testing and for determination of antibody titer in immunized animals, also from this reagent polyclonal antibodies.
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Authors:
Esteban Guerra
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