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-X Duo-Entero ELISA Kit can detect the toxin of Clostridium perfringens and reveal the multiplication of the bacterium. The kit works with culture fluid as well as biological probes such as liquid intestinal contents and pericardial- or peritoneal fluid, faeces.
II – PRINCIPLE OF THE TEST
Rows A and E are sensitised by specific polyclonal antibody produced against toxin of Clostridium perfringens and rows C and G by monoclonal antibody specific for a structural protein of this bacterium. These antibodies allow specifically the capture of the toxin or bacteria that may be present in the samples (intestinal fluid, culture fluids, body fluids, etc.). Rows B, D, F and H are coated with aspecific antibodies as controls. All samples except culture supernatants are diluted in dilution buffer and incubated on the microplate for 1 hour at 21°C +/- 3°C. After this first incubation step, the plate is washed, then conjugates (peroxidase- labelled anti- toxin monoclonal or anti- C. perfringens monoclonal) are added to the wells. The plate is then re-incubated for 1 hour at 21°C +/- 3°C. After this second incubation step, the plate is washed again and the chromogen (tetramethylbenzidine TMB) is added. This chromogen has the advantage of being more sensitive than the other peroxidase chromogens and not being carcinogenic. If toxins and/or C. perfringens are present in the tested samples, conjugates remain bound to the corresponding microwells and the enzyme catalyses the transformation of the colourless chromogen into a pigmented compound. The intensity of the resulting blue colour is proportionate to the content of toxin or pathogenic agent of the sample. The enzymatic reaction can be stopped by acidification and the resulting optical density at 450 nm can be read using a photometer. The signals read for the negative control microwells are subtracted from the corresponding positive microwells. Positive control is provided with the kit so as to validate the test results.
III – COMPOSITION OF THE KIT
– Microplates: Two 96-well microtitration plates. The rows A, C, E, G are sensitised by specific antibodies, the rows B, D, F, H by non specific antibodies.
Row A : anti-Alpha toxin
Row B : control
Row C : anti-C.perfringens
Row D: control Row E : anti-Alpha toxin
Row F: control
Row G : anti-C.perfringens
Row H: control
– Washing solution: One 100 ml 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 50 ml bottle of 5x colored and concentrated buffer. Dilute this concentrated dilution buffer 1:5 with distilled or demineralised water. Store the diluted solution between +2°C. and +8°C. If a deposit forms at the bottom of the container filter the solution on Whatman filter paper.
– Conjugates: Two 12 ml vial of colored conjugates. These solutions are ready to use. Anti-Alpha (red), Anti-C. perfringens (green).
– Positive reference: 1 vial of 4 ml. The reagent is ready to use.
– Single component TMB: One 25-ml bottle of the chromogen tetramethylbenzidine (TMB). This solution is ready to use. Store between + 2°C. and +8°C. protected from light.
– Stop solution: One 15 ml bottle of the 1 M phosphoric acid stop solution.