BIO K 268/2
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. Clostridium 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 Clostridium perfringens cells can usually be detected in the intestinal fluid of the diseased or dead animals. As Clostridium 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 kit works with culture supernatants as well as biological probes such as liquid intestinal contents and pericardial- or peritoneal fluid.
II – PRINCIPLE OF THE TEST
The test uses 96-well microtitration plates sensitised by specific monoclonal antibodies for the Epsilon-toxin. These antibodies allow a specific capture of the corresponding antigen which is present in the samples. Rows A, C, E, G have been sensitized with these antibodies and rows B, D, F, H are containing non specific antibodies. These control rows allow the differenciation between specific immunological reaction and non specific bindings. Biological samples (for example: contents of the small intestine, peritoneal fluid….) are diluted in dilution buffer and incubated on the microplate for 1 hour at 21°C +/- 3°C. Culture supernatants are used without dilution. After this first incubation step, the plate is washed and incubated for 1 hour with the conjugate – a peroxidase labelled anti-Epsilon-toxin specific polyclonal antibody. After this second incubation, the plate is washed again and the chromogen (tetramethylbenzidine) is added. This chromogen has the advantages of being more sensitive than the other peroxidase chromogens and not being carcinogenic. If Epsilon-toxin is present in the tested samples, the conjugate remains bound to the corresponding microwells and the enzyme catalyses the transformation of the colourless chromogen into a pigmented compound. The Bio-X Diagnostics S.A. – 38, Rue de la Calestienne (PAE) – 5580 Rochefort – Belgique Tél : 0032(0)18.104.22.168 – Fax : 0032(0)22.214.171.124 – E-mail : firstname.lastname@example.org (01/06/16) 2 intensity of the resulting blue colour is proportionate to the titre of Epsilon-toxin in the sample. Enzymatic reaction can be stopped by acidification and resulting optical density at 450 nm can be recorded using a photometer. The signals recorded for the negative control microwells are substracted from the corresponding positive microwells. There is a positive antigen supplied with the kit.
III – COMPOSITION OF THE KIT
– Microplates: 96-well microtitration plate (12 Strips x 8 wells). Rows A, C, E, G are sensitised by antiEpsilon-toxin specific monoclonal antibodies, while rows B, D, F, H are sensitized by the non specific antibodies. – Washing solution: 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. – Dilution buffer: bottle of 5x colored, concentrated buffer for diluting samples. Dilute this concentrated dilution buffer 1:5 with distilled or demineralised water. If a deposit forms at the bottom of the container filter the solution on Whatman filter paper. – Conjugate: vial of anti-Epsilon-toxin-peroxidase coloured conjugate. This solution is ready to use. – Control antigen bottle containing control antigen. This reagent is ready to use. – Single component TMB: bottle of the chromogen tetramethylbenzidine (TMB). Store at + 2°C and + 8°C protected from light. This solution is ready to use. – Stopping solution bottle of the 1 M phosphoric acid stop solution.