BIO K 344/2
I – INTRODUCTION
Diarrhoea is a major cause of mortality in young cattle under one month. Bovine neonatal gastroenteritis is a multifactorial disease. It can be caused by viruses: coronavirus or rotavirus, by bacteria : Salmonella or E.coli F5, or by protozoan microorganisms such as Cryptosporidium. Coronavirus and rotavirus are often associated with episodes of neonatal diarrhoea. The diagnosis of the etiological agent of diarrhoea can only be performed in the laboratory because clinical signs do not allow to differentiate between these different microorganisms. It is possible to identify these agents by means of different techniques including culture, staining, electron microscopy and floating techniques. However, these techniques are labor intensive, unpracticle and time consuming. These classical techniques have rapidly been replaced by the ELISA technology because of its simplicity, and the limited requirements in laboratory equipment. The sensitivity and specificity of the ELISA technique for the detection of these pathogens is at least as good as that of the more classical techniques; results are very similar. The ELISA technique is rapid and reliable and is particularly suited to the analysis of important numbers of samples.
II – PRINCIPLE OF THE TEST
The test uses 96-well microtitration plates sensitised by specific antibodies for the Coronavirus. These antibodies allow a specific capture of the corresponding pathogens which are present in the faeces 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 differentiation between specific immunological reaction and non specific bindings so as to eliminate false positives. Faeces 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 and incubated for 1 hour with the conjugate, a peroxidase labelled anti-coronavirus specific monoclonal 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 coronavirus is present in the tested faeces, the conjugate remains 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 titre of coronavirus 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. 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. Rows A, C, E, G are sensitised by anti-coronavirus specific antibodies, while rows B, D, F, H are sensitized by the non specific antibodies. – 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. – Dilution buffer: One 50-ml bottle of 5x colored and 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: One 25 ml vial of coloured conjugate. This solution is ready to use. – Positive Control: 1 vial of 4 ml. The reagent is ready to use. – Single component TMB: One 25-ml bottle of the chromogen tetramethylbenzidine (TMB). Store between +2°C and +8°C protected from light. This solution is ready to use. – Stop solution: One 15-ml bottle of the 1 M phosphoric acid stop solution.