BIO K 336/1
I – INTRODUCTION
In Europe, BRSV is the most important etiological agent responsible for respiratory affections in young cattle. In cattle as in children, respiratory syncytial viruses can cause a very deep attack of the respiratory tree. The affection is often provoking very severe injuries, which are responsible for important economic losses. As a matter of fact, in Europe, 7 million calves suffer from infections diseases yearly, 60% of which are caused by respiratory pathogens. One million calves dye of respiratory diseases in the European Community each year. The cost of these diseases, including medical treatments, growth delays and mortality, is about 450 million EURO per year for calves under one year. For dairy cattle, the cost of BRSV has been evaluated at around 25 EURO per animal. BRSV principally affects young cattle. Beef cattle are especially vulnerable because of the high proportion of muscle mass compared with the pulmonary volume in such animals. The clinical manifestations can be dramatic. Often signs of severe pneumonia such as polypnoea, abdominal breathling and hyperthermia are present. Reinfections may be observed but most often they remain subclinical. Clinical diagnosis is very difficult and laboratory assistance is required for a precise diagnosis. The virus can be detected in lung tissue by fluorescein labelled specific antibodies. Diagnosis can also be achieved by measuring a virus specific seroconversion. To do so, a first sample will be collected during the acute phase of the disease and a second sample will be collected 2 or 3 weeks later. These two samples will be evaluated for their content in specific antibodies against BRSV by ELISA. The BRSV kit can be used to obtain a diagnosis from a minced lung tissue sample taken from a corpse.
II – PRINCIPLE OF THE TEST
The test uses 96-well microtitration plates sensitised by specific antibodies for the BRSV. These antibodies allow specific capture of the corresponding pathogens which are present in the samples (minced lung tissue or culture medium). Rows A, C, E, G have been sensitised with these antibodies and rows B, D, F, H contain nonspecific antibodies. These control rows allow the differentiation between specific immunological reactions and non-specific binding so as to eliminate false positives. Samples are diluted in lysis solution 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-BRSV 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 BRSV is present in the sample, 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 color is proportionate to the titer of BRSV in the sample. The enzymatic reaction can be stopped by acidification and the resulting optical density at 450 nm recorded using a photometer. The signals recorded 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
– Microplate: One 96-well microtitration plate. Rows A, C, E, G are sensitised by anti-BRSV specific antibodies, while rows B, D, F, H are sensitised by the non-specific antibodies (control antibody). – 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. – Lysis buffer: One 100-ml bottle. The reagent is ready to use. Store the solution between +2°C and +8°C. – Conjugate: One 12-ml vial of coloured conjugate. This solution is ready to use. – Positive Control: 1 vial of 2 ml. The reagent is ready to use. – Single component TMB One 12-ml 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 6-ml bottle of the 1 M phosphoric acid stop solution.