BIO K 061/
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 economical 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 are dying each year of respiratory diseases in the European Community. The cost of these diseases which includes medical treatments, growth delays and mortality is about 450 millions 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 is principally affecting young cattle. Beef cattle is especially vulnerable because of the muscular mass of the animal which is very important compared to the pulmonary volume. Clinical manifestations can be dramatic.
Often signs of severe pneumonia such as polypnea, abdominal breabling and hyperthermia are present.
Reinfections can be observed but most often they remain subclinical. Clinical diagnosis is very difficult and laboratory assistance is required for a precise diagnosis. 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.
II – PRINCIPLE OF THE TEST
The test uses 96-well microtitration plates sensitised by monoclonal antibodies specific to F protein of BRSV virus. This antibody is used to trap a recombinant F protein. The plate’s odd columns (1, 3, 5, 7, 9 and 11) contain the recombinant viral protein, whereas the even columns (2, 4, 6, 8, 10 and 12) contain a control antigen. We thus have a genuine negative control to differentiate the specific anti- viral antibody from the antibodies directed against the control antigen. Using such a control reduces the number of false positives considerably. The test blood sera, plasma or milks are diluted in the dilution buffer. Samples are added to the plate which is then incubated and washed. The conjugate, a peroxidase-labelled anti-bovine IgG1 monoclonal antibody, is added to the wells. The plate is then incubated a second time at 21°C +/- 3°C and washed again and the chromogen (tetramethylbenzidine) is added. This chromogen has the advantage of being more sensitive than the other peroxidase chromogens and not being carcinogenic. If specific BRSV immunoglobulins are present in the test sera the conjugate remains bound to the microwell that contains the viral antigen and the enzyme catalyses the transformation of the colorless chromogen into a pigmented compound. The intensity of the resulting blue colour is proportionate to the titre of specific antibody in the sample. The signal read off the negative control
microwell is subtracted from that of the positive microwell sensitised by the viral antigen. It is possible to
quantify the reactivity of an unknown sample on a scale ranging from 0 to +++++.
III – COMPOSITION OF THE KIT
– Microplates: 96-well microtitration plates. The odd columns (1, 3, 5, 7, 9 and 11) are sensitised by the
recombinant F protein from BRSV and the even columns (2, 4, 6, 8, 10 and 12) by the control antigen.
– 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 so that all the crystals disappear. Mix the solution well and remove the necessary volume. Dilute the buffer 1:20 with distilled or demineralised water.
– Dilution buffer: One bottle of 5x colored, concentrated buffer for diluting blood sera, plasma, milks and
conjugate. The bottle’s content is to be diluted with distilled or demineralised water. If a deposit forms at the bottom of the receptacle filter the solution on Whatman filter paper.
– Conjugate: 1 bottle of anti-bovine immunoglobulin-peroxidase conjugate (horseradish peroxidase-labelled anti-bovine IgG1 monoclonal antibody).
– 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.
– Tracer: One bottle of tracer. Reconstitute this reagent with 0.5 ml distilled or demineralised water. Once reconstituted, the reagent is stored at -20°C. Divide this reagent up into several portions before freezing it to avoid repeated freeze/thaw cycles. If these precautions are taken, the reagent can be kept for several months.
The tracer is a reference sample that can be used to check the intra-laboratory reproducibility of the kit’s batch.
Intra-laboratory reproducibility: Degree of agreement between the results of reiterated tests on the same sample with an identical technical protocol in a given laboratory under variable working conditions.
– Single component TMB: One bottle of the chromogen tetramethylbenzidine (TMB). Store between +2°C and +8°C protected from the light.
– Stop solution: One bottle of the 1 M phosphoric acid stop solution.