BIO K 317
I – INTRODUCTION
Enterotoxaemia is a fatal enteric disease that affects all species of domestic animals and is attributable to a toxigenic type of Clostridium perfringens. The latter is an anaerobic, strongly gram-positive bacterium that has the ability to form heat-resistant endospores. This bacterium is grouped into five types (types A, B, C, D and E) according to the four major lethal toxins, alpha, beta, epsilon, and iota ( ) produced. C.perfringens has been shown to be a cause of human diseases such as gas gangrene (clostridial myonecrosis), food poisoning, necrotising enterocolitis of infants, and enteritis necroticans (pigbel). It is also the causative agent of lamb dysentery, ovine enterotoxaemia (struck) and pulpy kidney disease of sheep, and other enterotoxaemic diseases of lambs and calves. Large amounts of toxin in addition to large numbers of C.perfringens cells can usually be detected in the intestinal fluid of the diseased or dead animals. As C. perfringens is a natural commensal of human and animal intestines, identifying of the bacterium is not enough. Toxinotyping and quantifying of the isolated strains are essential. The BIO K 317 test is designed to monitor the animal’s serological response after immunisation by a vaccine or natural contact with Clostridium perfringens. As it is a blocking test, it can be used in all animal species.
II – PRINCIPLE OF THE TEST
The 96-well microplate has been sensitised by a monoclonal antibody specific for C. perfringens beta toxin. C. perfringens beta toxin was then added to these microplates. The kit’s user deposits the previously diluted test sera and plasma in the microplate’s wells. After 2 hours’ incubation and a rinse step, the operator adds the conjugate, which is a specific monoclonal antibody against C. perfringens beta toxin coupled to peroxidase. After incubating and washing the preparation, the operator adds the chromogen tetramethylbenzidine (TMB). This chromogen has the advantages of being more sensitive than the other peroxidase chromogens and not being carcinogenic. The intensity of the colour is inversely proportionate to the sample’s serum titre. Positive and negative sera are provided with the kit to be able to validate the test results.
III – COMPOSITION OF THE KIT
– Microplates: 96-well microtitration plates. The entire surface of each microplate has been sensitised with C. perfringens beta toxin. – Washing solution: One bottle of 20x concentrated washing solution. The solution crystallises spontaneously when cold. If only part of the solution is to be used, bring the bottle to 21°C +/- 3°C until disappearance of all crystals. Mix the solution well and remove the necessary volume. Dilute the buffer 1:20 with distilled or demineralised water. Store the diluted solution between +2°C and +8°C. – Dilution buffer: One bottle of colored buffer for diluting samples and conjugate. The dilution buffer is ready to use. Store the solution between + 2°C and + 8°C. If a deposit forms at the bottom of the container filter the solution on Whatman filter paper. – Conjugate: 1 vial of anti – C. perfringens beta toxin peroxidase conjugate (horseradish peroxidase-labelled anti- C. perfringens beta toxin monoclonal antibody). The reagent must be diluted 1:20 with dilution buffer. – Positive reference: One bottle of positive serum. Store this reagent between +2°C and +8°C. – Negative reference: One bottle of negative serum. Store this reagent between +2°C and +8°C. – Single component TMB: One bottle of the chromogen tetramethylbenzidine (TMB). Store between +2°C and +8°C protected from light. This solution is ready to use. – Stopping solution: One bottle of the 1 M phosphoric acid stop solution.