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2. PP Percent Positivity Values (PP) All Corrected OD Values for the test samples as well as the Negative Control (Neg C) are related to the corrected OD value of the positive control as follows: = Test Sample or Neg C (ODCorr) x 100 Positive Control (ODCorr) Infectious Bovine Rhinotracheitis (IBR-Ab) OD Positive control > 0,5 PP Negative control < 7% ELISA test for the detection of IBR antibodies in serum and milk Interpretation of test sample results Serum Interpretation of the results Criteria for test validity To ensure validity, the duplicate of the OD values should not differ more than 25% from each other. Additionally, the control values should fall within the following limits SVANOVIR® Should any of these criteria’s not be fulfilled, the test is invalid. For invalid tests, technique may be suspect and the assay should be repeated. Milk PP Interpretation < 7 8-12 > 12 Negative (-) Doubtful/Re-test Positive <7 >7 Negative Positive General information Infectious Bovine Rhinotracheitis (IBR) is a severe respiratory herpesvirus infection in cattle characterized by tracheitis, rhinitis and fever. The virus may also cause conjunctivitis, infectious pustular vulvovaginitis (IPV), balanoposthitis, abortions, and in rare cases encephalitis. IBR is transmitted horizontally by contact with respiratory, ocular, and reproductive secretions. IBR also acts as an immunosupressive, predisposing individuals to secondary bacterial infections. The clinical manifestations of the disease affect feed efficiency, milk yield, and reproduction1,2. Another aspect is the transition from a primary manifest infection to a latent state of persistency forming the source of spread following virus reactivation3,4. Economical losses can be high due to these factors and therefore herds should be monitored for the presence of infection. In Switzerland, an indirect Enzyme Linked Immunosorbent Assay (ELISA) of serum and milk samples has been introduced4, resulting in a successful country-wide control by culling the infected cattle. If samples are retested as doubtful, it is recommended to test a second sample from the animal, obtained after a period of at least three weeks. References Principle Manufacturer Svanova Biotech AB Uppsala Science Park SE-751 83 Uppsala, Sweden Phone +46 18 65 49 00 Fax +46 18 65 49 99 [email protected] www.svanova.com Customer Service Phone +46 18 65 49 15 Fax +46 18 65 49 99 [email protected] Manualnumber: 19-2100-00/04 1. Gibbs, E.P.J. and Riveyemamu, M.M. (1977). Bovine herpesviruses, Part 1. Vet. Bul. 47, pp. 317-343. 2. Kahrs, R.F. (1981). Infectious bovine rhinotracheitis. In Viral Diseases of Cattle. The Iowa State University Press, Ames Iowa, pp. 135-156. 3. Homan, E.-J. and Easterday, B.C. (1980). Isolation of bovine herpesvirus-1 from trigeminal ganglia of clinically healthy cattle. Am. J. Vet. Res. 41, 1212-1213. 4. Bommeli, W. and Kihm, U. (1982). ELISA- the nucleus of the IBR/IPV control programme in Switzerland. Current Topics in Veterinar y Medicine and Animal Science 22, 242-251. 5. Juntti, N., Larsson, B., and Fossum, C.(1987) The use of monoclonal antibodies in enzyme linked immunosorbent assays for detection of antibodies to bovine viral diarrhoea virus. J. Vet. Med. B 34, 356-363. 6. Niskanen, R., Alenius, S., Larsson, B., and Juntti, N. (1989) Evaluation of an enzyme- linked immunosorbent assay for the detection of antibodies to bovine virus diarrhoea virus in milk. J. Vet. Med. B 36, 113-118. The Infectious Bovine Rhinotracheitis IBR-Ab ELISA Kit is designed to detect IBR specific antibodies in serum or milk samples. The kit procedure is based on a solid phase indirect Enzyme Linked Immunosorbent Assay This manual covers the following IBR-Ab ELISA kits: Article number 10-2100-02 and 10-2100-10 (Indirect ELISA). In this procedure, samples are exposed to noninfectious IBR antigen coated wells on microtitre plates or strips. IBR antibodies (if present in the test sample) bind to the antigens in the well. The HRP conjugate added subsequently forms a complex with these IBR antibodies. Unbound material is removed by rinsing before the addition of a substrate solution. Subsequently a blue colour develops which is due to the conversion of the substrate by the conjugate. A positive result is indicated by development of the blue colour. The reaction is stopped by addition of stop solution; the colour changes to yellow. The result can be read visually or by a microplate photometer, where the optical density (OD) is measured at 450 nm. Contents • IBR viral and control antigen coated microtitre plates/strips, odd columns coated with viral antigen and even columns with control antigen (sealed and stored dry) • Lyophilized HRP Conjugate (horseradish peroxidase conjugated anti bovine IgG monoclonal antibodies) • PBS-Tween Solution 20 x concentrate • Substrate Solution – (tetramethylbenzidine in substrate buffer containing H2O2) – STORE IN THE DARK ! • Stop Solution – Contains sulphuric acid – CORROSIVE! A. Positive Control Serum – 0.05% merthiolate B. Negative Control Serum – 0.05% merthiolate C. Positive Control Milk – 0.05% merthiolate D. Negative Control Milk – 0.05% merthiolate Materials needed but not provided 1. 2. 3. 4. 5. 6. Precision pipets (range from 4 to 200 µl) Disposable pipet tips Distilled water Wash bottle 1 container: 1 to 2 litres for PBS-Tween Microplate photometer, 450 nm filter Specimen information Individual serum or pools up to 10 samples: 4 µl of blood serum or plasma is needed for each sample well. Fresh, refrigerated, or previously frozen serum or plasma may be tested. Individual or pooled milk samples up to 50 animals: 100 µl of skim milk is required for each sample well. Milk samples must be centrifuged for 15 minutes at 2000 x g to remove the lipid layer. Preparation of reagents PBS-Tween Buffer: Dilute the PBS-Tween Solution 20 x concentrate 1/20 in distilled water. Prepare 500 ml per plate by adding 25 ml PBST solution to 475 ml distilled water and mix thoroughly. N.B. Please check that there is no crystal precipitation in the bottle. If crystals are seen, please warm and shake well. Anti-Bovine IgG Conjugate: • Reconstitute the lyophilized HRP Conjugate with 11.5 ml PBS-Tween Buffer. Add • the buffer carefully to the bottle. Leave the solution one minute and mix thoroughly. Prepare immediately before use. The remaining reconstituted conjugate can be stored at -20°C and thawed and refrozen up to 3 times. Precautions 1. Carefully read and follow all instructions. 2. Store the kit and all reagents at +2 to +8°C (35 to 45°F) 3. All reagents should equilibrate to room temperature 18 to 25°C (64 to 77°F) before use. 4. Handle all materials according to the Good Laboratory Practice. 5. Do not mix components or instruction booklets from different test kits batches. 6. Care should be taken to prevent contamination of kit components. 7. Do not use test kit beyond date of expire. 8. Do not eat, drink, or smoke where specimens or kit reagents are handled. 9. Use a separate pipet tip for each sample. 10. Do not pipet by mouth. 11. Include positive and negative serum and /or milk controls on each plate or test strip series. 12. Use only distilled water for preparation of reagents. 13. The Stop Solution contains sulphuric acid which is corrosive. 14. All unused biological materials should be disposed according to the local, regional and national regulations. Recommendations! For article 10-2100-02, the volume of the reagents is sufficient for at least 8 separate test occasions. For article 10-2100-02, strips with broken seal can be stored at +2 to +8°C for up to 4 weeks. Reconstituted conjugate may not be stored in refrigerator. Procedure 1. 2. All reagents should equilibrate to room temperature 18 to 25°C (64 to 77°F) before use. Label each strip with a number. Serum Samples A. Add 100 µl of PBS-Tween Buffer to each well that will be used for serum samples and serum controls. B. Add 4 µl of Positive Control Serum (Reagent A) and 4 µl of Negative Control Serum (Reagent B) respectively to selected wells coated with IBR viral antigen and to corresponding wells coated with control antigen. For confirmation purposes it is recommended to run the control sera in duplicates. C. Add 4 µl of serum sample to a selected well coated with IBR viral antigen and to a corresponding well coated with control antigen. For confirmation purposes it is recommended to run the samples in duplicates. 3. Milk Samples A. Add 100 µl of Positive Control Milk (Reagent C) and 100 µl of Negative Control Milk (Reagent D) respectively, to selected wells coated with IBR viral antigen and to corresponding wells coated with control antigen. For confirmation purposes it is recommended to run the control milk in duplicates. B. Add 100 µl of skim milk sample to a selected well coated with IBR viral antigen and to a corresponding well coated with control antigen. For confirmation purposes it is recommended to run the samples in duplicates. 4. Shake the plate thoroughly. Seal the plate/strips and incubate at 37°C (98.6°F) for 1 hour. 5. Rinse the plates/strips 3 times with PBSTween Buffer: at each rinse cycle fill up the wells, empty the plate and tap hard to remove all remains of fluid. 6. Add 100 µl of HRP Conjugate to each well and incubate at 37°C (98.6°F) for 1 hour. 7. Repeat step #5. 8. Add 100 µl Substrate Solution to each well. Incubate for 10 minutes at room temperature (18 to 25°C). Begin timing when the first well is filled. 9. Stop the reaction by adding 50 µl of Stop Solution to each well and mix thoroughly. Add the Stop Solution in the same order as the Substrate Solution in step #8. 10. Measure the optical density (OD) of the controls and samples at 450 nm in a microplate photometer (use air as blank). Measure the OD within 15 minutes after the addition of Stop Solution in order to prevent fluctuation in OD values. Calculations Calculation of results are done in two steps as described below. 1. Corrected OD Values (ODCORR) The optical density (OD) values in wells coated with IBR viral antigen are corrected by subtracting the OD values of the corresponding wells containing the control antigen. ODIBR -ODCONTROL= ODCORR