ON PRELIMINARY STUDIES PERFORMED BY ANSES NANCY ON THE BIOPRO RABIES ELISA AB KIT FOR WILDLIFE SAMPLES

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1 1 st September 2011 TECHNICAL REPORT ON PRELIMINARY STUDIES PERFORMED BY ANSES NANCY ON THE BIOPRO RABIES ELISA AB KIT FOR WILDLIFE SAMPLES 1- Background A new ELISA test has been developed and commercialised by O.K. Servis BioPro (Czech Republic) for the detection of antibodies against rabies in domestic and wild carnivores and was validated with fox and dog serum samples (Mojzis, 2008). This test is particularly recommended by the manufacturer for detection of antibodies against rabies in foxes. According to the manufacturer specification, this test is based on blocking ELISA format for antibody detection. It is said to have a specificity of 100% on panel of negative fox samples and a sensitivity of 95.5%, compared with FAVN test on a panel of experimental fox and dog sera. The manufacturer specification presents the kit as robust and routinely used in the laboratory where the kit has been developed for several years. Additionally, it is species independent. Study on dogs and raccoon dogs are currently under process. It is suitable for rather diluted samples due to a high analytical sensitivity. It is easy to use and suitable both for manual or robotic performance. The evaluation of new products available on the market for rabies serological testing is part of the duty of the National Reference Laboratory for rabies. The following analyses were undertaken to evaluate the performances of this kit with serum samples from foxes received in our laboratory in the context of surveillance of oral vaccination campaigns undertaken in France. The samples analysed in this study were collected on foxes during the period. The samples were stored at -20 C. These studies have been conducted from March to July BioPro Rabies ELISA Ab kit - Technical report on wildlife samples - Page 1 / 7

2 The sera had to be titrated with the BioPro Rabies ELISA Ab kit according to the specifications of the kit. The sample and control dilutions were undertaken directly in plate. The conditions of validation described by the manufacturer had to be respected in order to be allowed to valid the results obtained for the different samples. The conditions of validation were the following: The O.D. of the negative control serum must be higher than 1.0. Difference between means of OD of negative and positive control serums must be equal or higher than 0.8. Regarding the panel of control: Percentage of blocking for control serum 1 must be between 50% and 70% Percentage of blocking for control serum 2 must be between 30% and 50% Percentage of blocking for control serum 1 must be lower than 30% If validation criteria were not met, tests results of that specific test plate were invalid and samples have to be retested. The percentage of blocking (PB%) was calculated for each sample according to the specifications of the manufacturer. The performances of BioPro Elisa test have been assessed by using two different tests as references. The first reference test was the FAVN test (Cliquet, 1998) which is a prescribed test for the detection of rabies antibodies by seroneutralisation. The second reference test was a rabies ELISA test developed in our laboratory and published (Cliquet, 2000, Cliquet, 2003) to monitor the efficacy of oral rabies campaigns on foxes. To avoid any confusion between the two ELISA tests, this ELISA will be named Reference ELISA in this report. For result interpretation, the terms sensitivity and specificity are used for comparing the performances of the BioPro ELISA test with those of either the FAVN test or the Reference ELISA test. For FAVN test, positive titre means titre equal or above the threshold of 0.5 IU/mL and negative titre means titre below this threshold. For Reference ELISA test, positive titre means titre equal or above the threshold of 0.05 EU/mL and negative titre means titre below this threshold. For the BIOPRO ELISA test, positive titre for rabies neutralizing antibodies means signal equal or above the percentage of blocking (PB) of % and negative titre means neutralizing antibody levels below this threshold of % as described in the leaflet. BioPro Rabies ELISA Ab kit - Technical report on wildlife samples - Page 2 / 7

3 2- Serological tests 2.1 Reference ELISA test versus BIOPRO ELISA test On fox samples from vaccinated areas ( ) Two hundred eighty-three fox samples received in Anses Nancy from 2000 to 2006 in the frame of the monitoring of oral vaccination campaigns have been tested with the BioPro Rabies ELISA Ab kit. These samples were previously tested with the Reference ELISA test developed in our laboratory and they were stored at -20 C till to be tested by BioPro ELISA test. Results Figure 1: Presentation of the different titres obtained by ELISA test and Reference ELISA test on fox sera received at Anses Nancy laboratory in the frame of monitoring of rabies oral vaccination campaigns ( ) Reference ELISA test VS BioPro ELISA test 100 Titres obtained by Biopro ELISA test ( % of blocking) ,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1 Titres obtained by Reference ELISA test ( EU/mL) On 283 sera titrated with ELISA test, 3.53% (10 sera) of sera had conflicting titres compared to those obtained by Reference ELISA test. On 270 sera having positive titres by Reference ELISA test, 3 sera had negative titres by ELISA test; therefore the diagnostic sensitivity was equal to %. The EU/mL titres obtained with the Reference ELISA test of these 3 sera were 0.066, and BioPro Rabies ELISA Ab kit - Technical report on wildlife samples - Page 3 / 7

4 On 13 negative sera obtained by Reference ELISA test, 7 sera had positive titres in BIOPRO ELISA test. These 7 sera had titres ranging from to EU/mL by Reference ELISA test. 2.2 FAVN test versus BIOPRO ELISA test On naïve fox samples from the field (2011) Fifty-eight samples received in Anses Nancy in 2011 from unvaccinated foxes in the field have been tested in parallel with the BioPro Rabies ELISA Ab kit and by FAVN test. The whole of samples had a negative titre by using both methods. In conclusion, the specificity of the BioPro ELISA was 100% for naïve fox samples On fox samples from vaccinated areas ( ) Two hundred sixty-six fox samples, received in Anses Nancy from 2000 to 2006 in the frame of monitoring of rabies oral vaccination campaigns and stored at -20 C, have been tested in parallel with the BioPro Rabies ELISA Ab kit and by FAVN test. Results Figure 2: Presentation of the different titres obtained by ELISA test and FAVN test on fox sera received at Anses Nancy laboratory in the frame of monitoring of rabies oral vaccination campaigns ( ) and tested in parallel with the two techniques. FAVN test VS BioPro ELISA test 100 Titres obtained by Biopro ELISA test ( % of blocking) Titres obtained by FAVN test ( IU/mL) BioPro Rabies ELISA Ab kit - Technical report on wildlife samples - Page 4 / 7

5 On 266 sera titrated with ELISA test, 12.03% of sera had conflicting titres compared to those obtained by FAVN test. Twenty conflicting sera had FAVN test titres ranging from 0.29 to 0.38 IU/mL which are values close to the 0.5 IU/mL threshold. On 229 sera having positive titres in FAVN test, 3 sera had negative titres by ELISA test, so the diagnostic sensitivity was equal to %. The IU/mL titres of these 3 sera were 0.5, 0.66 and On 37 negative sera obtained by FAVN test, 29 sera had positive titres in ELISA test. Among them, 20 samples had titres between 0.29 and 0.38 IU/mL which were very close to the threshold and therefore could be considered as positive samples in FAVN test especially considering the bad quality of samples. In this case only 9/17 sera were considered as negative and their titres were ranging from 0.10 to 0.22 IU/mL. BioPro Rabies ELISA Ab kit - Technical report on wildlife samples - Page 5 / 7

6 3- Conclusions 3.1 Diagnostic specificity criterion Fifty-eight samples from unvaccinated foxes taken on the field have a negative titre in FAVN test and in BioPro ELISA test. Therefore the diagnostic specificity is equal to 100%. 3.2 Diagnostic sensitivity criterion Two hundred and seventy samples of foxes from vaccinated areas have a positive titre in Reference ELISA test. Among them, 3 have negative titres in BioPro ELISA test. Therefore the diagnostic sensitivity is equal to 98.89%. Two hundred and twenty-nine samples of foxes from vaccinated areas have a positive titre in FAVN test. Among them, 3 have negative titres in BioPro ELISA test. Therefore the diagnostic sensitivity is equal to 98.69%. 3.3 Coefficient of concordance We obtained a coefficient of concordance equal to 96.46% with the Reference ELISA test. We obtained a coefficient of concordance equal to 87.97% with the FAVN test. We obtained a coefficient of concordance equal to 95.48% with the FAVN test if we consider the 20 samples with FAVN test titres IU/mL as possibly positives. Authors Technical Assistance M. Wasniewski, F. Cliquet L. Tribout, J.L. Schereffer, A. Labbé and J. Rieder BioPro Rabies ELISA Ab kit - Technical report on wildlife samples - Page 6 / 7

7 Bibliography Mojzis M, Korytar P, Jerg S. Development and validation of ELISA test for detection of rabies antiglycoprotein antibodies. International conference on rabies in the Americas (RITA XIX). Atlanta, 2008: Cliquet F, Aubert M, Sagne L. Development of a fluorescent antibody virus neutralisation test (FAVN test) for the quantitation of rabies-neutralising antibody. J Immunol Methods 1998;212(1): Cliquet F, Sagne L, Schereffer JL, Aubert MF. ELISA test for rabies antibody titration in orally vaccinated foxes sampled in the fields. Vaccine 2000;18(28): Cliquet F, Müller T, Mutinelli F, Geronutti S, Brochier B, Selhorst T, Schereffer JL, Krafft N, Burow J, Schameitat A, Schlüter H, Aubert M. Standardisation and establishment of a rabies ELISA test in European laboratories for assessing the efficacy of oral fox vaccination campaigns. Vaccine Jun 20;21(21-22): BioPro Rabies ELISA Ab kit - Technical report on wildlife samples - Page 7 / 7