Appendix 30
Modification and further validation by different laboratories of a FMDV type O specific ELISA for detection of antibodies in all susceptible species L. Jacobs 1) ,G. Chènard2) , T. Jelsma 1) and K. de Clercq 3). 1)
Cedi-Diagnostics B.V. The Netherlands ID-Lelystad, the Netherlands 3) CODA, Belgium 2)
Summary A quick and simple Mab based ELISA for the detection of antibodies directed against type O of FMDV was developed by ID-Lelystad. The ELISA was specially developed to be used on a semi-automatic pipetting system. Specificity and sensitivity of the ELISA was previously described by G. Chènard et al. (2001). To decrease the variation between test results when used manually and to increase the specificity some slight modifications were applied. Additionally the modified Ceditest® FMDV type O ELISA was taken in production and send to different laboratories for validation. The modified Ceditest® FMDV type O was compared with the original test using the same sera, if available. Additionally the test was validated during a workshop organised by the FAO-EUFMD commission in Sofia, Bulgaria, by the CODA-CERVA-VAR in Belgium and by the world reference laboratory of FMDV in Pirbright, England. Also the Ceditest type O ELISA was used by the BFAV Tűbingen/Riems, Germany and by the State Veterinary Institute, Lindholm in Denmark. Test procedure and modifications Original test Plates coated with antigen Add 40 μl buffer + 10μl serum and 10 μl serum Incubate 1 hour at room temperature Add 50 μl conjugate Incubate 1 hour at room temperature Wash the plates Add 100μl TMB Stop the reaction after 15 minutes
Modified test Plates coated with antigen Add 90 μl buffer (+5% horse serum ) Incubate 1 hour at room temperature Wash the plates Add 100μl conjugate Incubate 1 hour at room temperature Wash the plates Add 100μl TMB Stop the reaction after 15 minutes
Incubation time of the modified Ceditest® FMDV type O is the same, only one extra washing step is included to increase the specificity and to decrease test variation. Calculation and interpretation of the results of the modified Ceditest® FMDV type O %inhibition = 100-((OD monster-OD blank)/OD max-OD blank)*100) Positive >= 50% Negative < 50% 242
