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FMDv NSP Ab Rapid test kit
- 1. Passion, Integrity, Professional, and Ethics
- 2. Costs of eradicating 1997 outbreak estimated to be US$ 378.9 million, Potential export losses of approximately US$ 1.2 billion (Data analysis Yang et al. (1999)) Foot & Mouth Disease (FMD) – A big economic Threat
- 3. Sentinel® FMDV Non Structural Protein Antibody Rapid Test Sentinel® FMDV Non Structural Protein Antibody ELISA kit
- 4. What is NSP? 4 Foot and mouth disease virus Viral structural proteins, SP Viral non structural proteins (NSPs)
- 5. Vaccine and viral protein Viral structural proteins Viral non structural proteins (NSPs) Early vaccine New vaccine Not present in FMDV structure, Not in New vaccine! Could be present Only appeared in viral replication process
- 6. Removing NSP in vaccines Distinguish between animals that have been vaccinated and animals that are infected! FMD Vaccine (NSP removed) inoculation blood Antibody : Only Anti-structural protein infection FMDV Antibody : Anti-structural protein & Anti-nonstructural protein blood
- 7. Results serology FMDV NSP test Vaccinated Infected + Vaccinated Not infected - Not vaccinated Infected + Not vaccinated Not infected -
- 8. To prove absence of infection or virus circulation To certify individual animal free of FMD prior to move or trade To confirm suspected case To distinguish from infected and vaccinated animal (DIVA) NSPAb testing for FMD are performed in support for 4 main purposes
- 9. Vaccinated or infected animals Extremely Easy High specificity and sensitivity Monitor FMD Virus activity Time saving Sentinel® FMDV NSPAb Raipid test
- 10. ELIS A Sentinal® FMDV NSP Ab Rapid Test 16 hours 60 mins 20 mins Read by ELISA ReaderStop reactionWash Add Secondary Antibody Develop with Substrate Wash Add Sample and Control 30 mins Add Running buffer Interpret by eyesAdd Sample Over 17 hours!!
- 11. Protocol
- 12. Validation test and Field test
- 13. • Swine sera provided by Animal Health Research Institute (AHRI). • Total 62 positive swine samples from pigs experimentally infected with FMDV O/Taiwan/1997 strain at 14 days post-infection(dpi) were tested, and results are as follows: Sensitivity :98.39 % (61/62) Lab tests-Sensitivity
- 14. Lab tests-Specificity •Animal Health Research Institute (AHRI) provided swine sera and analyzed. • Total 412 FMD negative swine sera from vaccinated pigs, pre- outbreak pigs and SPF pigs were tested. Specificity :92.96 % (383/412)
- 15. •A serum panel collected sequentially from 32 secondary SPF pigs experimentally challenged by the FMDV O/Taiwan/1997 strain was tested. The sera in the panel were sampled at different days post infection(dpi). •Results were compared with PrioCHECK, UBI and Chekit ELISA kit. testing limited Testpositive% Lab tests-early testing
- 16. The validation test was executed by the Animal Technology Institute Taiwan (ATIT). 169 swine sera were blind tested. Then compared results with PrioCHECK® FMDV NS (Ceditest) and UBI® FMDV NS ELISA(swine), respectively. The data had showed that the Kappa value of Sentinel were more than 0.8, it indicate the data of Sentinel® were almost the same result with PrioCHECK® and UBI®. Observed agreement Kappa value Sensitivity Specificity PrioCHECK 0.95 0.87 90.2 % 96.9 % UBI 0.95 0.86 86.4 % 97.6 % Field tests-comparison with other kit
- 17. Kappa（K coefficient of agreement；K） P = (observed agreement)： P (chance agreement)：= ───── P -Po Pc c-1 K= o c
- 18. Kappa（K coefficient of agreement；K） 0.81~1 (almost perfect) 0.61~0.80 (substantial) 0.41~0.60 (moderate) 0.21~0.40 (fair) 0.0~0.20 (slight) K value is between -1 and 1. If the raters are in complete agreement then κ = 1. If there is no agreement among the raters other than what would be expected by chance
- 19. Sentinel ® FMDV NSPAb ELISA kit
- 20. Sentinel ® FMDV NSPAb ELISA kit
- 21. Reliability
- 22. Two internal controls (IC-SPC , IC-PC )* were run in 2 replicates on 5 different plates in the same lot. (The data presented here are CV) within run precision PI Value Plate-A Plate-B Plate-C Plate-D Plate-E SPC 0.27% 0.24% 0.00% 0.07% 0.62% PC 0.31% 0.69% 1.46% 1.31% 2.32% between run precision Two internal controls (IC-SPC , IC-PC )* were run 10 replicates in the same lot. *Strong positive control-SPC *positive control-PC PI Value AVE. STD CV SPC 96.5 0.31 0.33% PC 89.0 1.26 1.42%
- 23. different lot precision Two internal controls (IC-SPC , IC-PC ) were run in PD601631 and PD601648, 2 different lots. PI Value Ave. STD. CV SPC 96.4 0.14 0.15% PC 86.8 3.11 3.58% When tested the same sample in different lot, the result were high consistency
- 24. Lab tests- Sensitivity and Specificity Animal Health Research Institute (AHRI) provided swine and goat sera than analyzed. 1. Swine sera 30 swine sera infected with O type FMD virus 50 FMDV-free swine sera Sensitivity：96.7% (29/30) Specificity：100% (50/50) 2. Goat sera 30 goat sera infected with FMD virus 50 FMDV-free goat sera Sensitivity：100% (30/30) Specificity：100% (50/50)
- 25. Field data
- 26. PrioCHECK® Total+ - Sentinel® + 47 5 52 - 4 57 61 Total 51 62 113 113 pigs sera were collected from field farm, Sentinel ® NSP Ab ELISA test kit and PrioCHECK ® NS ELISA were compared • Observed agreement = 0.92 • Kappa value= 0.83 • Sensitivity※ = 92.20% • Specificity※ = 91.90% • (※ PrioCHECK ® as standard) Sentinel ® FMDV NSP Ab ELISA test kit compared with PrioCHECK ® FMDV NS ELISA kit
- 27. PrioCHECK® Total + - Sentinel® + 17 2 19 - 3 159 162 Total 20 161 181 181 cattle sera were collected from field farm, Sentinel ® NSP Ab ELISA test kit and PrioCHECK ® NS ELISA were compared • Observed agreement = 0.97 • Kappa value= 0.86 • Sensitivity※ = 85% • Specificity※ = 98.1% • (※ PrioCHECK ® as standard) Sentinel ® FMDV NSP Ab ELISA test kit compared with PrioCHECK ® FMDV NS ELISA kit
- 28. PrioCHECK® Total + - Sentinel® + 3 1 4 - 1 127 128 Total 4 128 132 132 goats sera were collected from field farm, Sentinel ® NSP Ab ELISA test kit and PrioCHECK ® NS ELISA were compared • Observed agreement = 0.98 • Kappa value= 0.74 • Sensitivity※ = 75% • Specificity※ = 99.2% • (※ PrioCHECK ® as standard) Sentinel ® FMDV NSP Ab ELISA test kit compared with PrioCHECK ® FMDV NS ELISA kit
- 29. Field tests-validation：The agreement of analytical performance compare to commercial ELISA kit (PrioCHECK) Species Sample No. Observed agreement Kappa value Swine 113 0.92 0.83 Cow 181 0.97 0.86 Goat 132 0.98 0.74
- 30. The performance of our FMDV NSP Ab Testing kits • Sensitivity • Specificity • Reliability
- 31. THANK YOU FOR YOUR ATTENTION Passion, Integrity, Professional, and Ethics
Editor's Notes
- Good afternoon everyone. My name is Derek, the specialist of EBS. In following presentation I’ll introduce you our 2 FMDV NSP Ab testing products to you.
- As we know foot & mouth disease has high transmission rate and mild death rate, It’s an important infectious animal disease in the world, and it’s also a big Economic threat in livestock industry. For example, In Taiwan cost 378.9 M USD for eradicating FMD, and FMD also cause export losses about 1.2 B USD. And that is one of the reason why we develop FMD testing kit.
- We have 2 FMDV NSP Ab testing kit, one is FMDV NSP Ab rapid test, the other is Sentinel® FMDV Non Structural Protein Antibody ELISA kit. Rapid test is a useful tool for disease screening on-site, and ELISA is LAB using.
- As we know FMD is cause by FMD virus, This virus contain 2 part protein. They are structural proteins, and Viral non structural proteins (NSPs) Viral non structural proteins only appeared in viral replication process
- In Early vaccine Could be present SP and NSP, But now according to OIE request, the new FMD vaccine need to purified and NSP should not be present in FMDV new vaccine.
- So FMD vaccinated animal only produce Anti-structural protein antibody FMDV infected animal produce both Anti-structural protein antibody and Anti-nonstructural protein antibody. That’s why the FMDV NSP Ab test can distinguish between animals that have been vaccinated and animals that are infected!
- This table show how this kit work Only sample from infected animal will show the positive result.
- NSP Ab testing for FMD are performed in support for 4 main purposes. To prove absence of infection or virus circulation. To certify individual animal free of FMD prior to move or trade. To confirm suspected case. To distinguish from infected and vaccinated animal
- This slide show the benefit of our rapid test kit. Rapid test can distinguish Vaccinated or infected animals. It extremely Easy to use. Time saving. High specificity and sensitivity . Monitor FMD Virus activity.
- This slide show that when we perform a ELISA kit, we will need a well trained, A lot of equipment like pipette, and ELISA Reader, and you also need to spend at least 5 hour Then you can get the result. But when you perform our rapid test you just need a men with hand, and a pipette or a dropper, you can get The result in 15-30 min without using a EXPENSIVE ELISA reader.
- The slide show the operation protocol of Sentinel FMDV NSP Ab rapid test. 1. Using a dropper to get 10ul whole blood or using a pipette to get 5ul serum. 2. Add into sample area. 3.Add 200ul buffer into sample area. 4. Read the result in 15-30 minutes. Two lines appear in test window indicate the Positive result. Only control line appear in test window indicate the Negative result
- Now I show some data from Validation and Field tests.
- Lab test for Sensitivity. This experiment is done by Authorities in Taiwan, Animal Health Research Institute (AHRI). 62 pigs experimentally infected with FMDV. And sampled at 14 days-post-infection(dpi) And our kit can identify 61 out of 62 samples are NSP positive, that is indicated the Sensitivity is 98.39 %.
- This experiment is also done by AHRI in Taiwan. 412 FMD negative swine sera from vaccinated pigs, pre-outbreak pigs and SPF pigs. And our kit can identify 383 out of 412 samples are NSP negative, that is indicated the Specificity is 92.96 %.
- This slide shows the testing limited of 4 commercial FMDV NSP Ab testing kit. 32 secondary SPF pigs experimentally challenged by the FMDV, and sampled at different days-post-infection(dpi). Y-axis is percentage of test positive and X-axis is different days after infection. Different colors means different kits. And purple color is Sentinel FMDV NSP Ab rapid test At 8dpi, sentinel NSP Ab rapid test can indicate almost 90% positive samples were tested positive.
- As we know the result of field tests are affected by a lot of factor. And the real performance of a testing kit should be exam by field test. So we used the same field sample, then perform our kit and other 2 most accepted kits, and compared their performance. Table show that our sentinel FMDV NSP Ab rapid test has high kappa value when compare with PrioCHECK and UBI.
- This is the formula of kappa.
- Kappa value is used to compare 2 testing methods. In general, high Kappa value means more similarity, low kappa value means less similarity, and when kappa value higher than 0.81 it means the 2 methods are almost the same.
- In this part of presentation, I will introduce you our ELISA Kit.
- This slide is the principle of Sentinel FMDV NSP Ab ELISA kit. Our ELISA is a solid phase blocking ELISA. It means when the sample contain the target antibody, this Ab will block the binding site of conjugate, then after a serial reaction the less conjugate produce less color. On the other side, the negative sample does not contain target antibody, it means more conjugate will binding to their binding site, And it can produce deeper color.
- How to chose a suitable testing kit? In my opine, first we need consider what’s our main purpose. And we also need consider the sensitivity, specificity and reliability. All of our kit are manufacturing follow the ISO quality system, and before selling. We also done a lot of test to make sure the quality of our kits.
- First, we used 2 different internal controls and then run in same plate from the same lot. Second, we used 2 different internal controls then run 10 replicates in the same lot. Final we record the CV.
- We also used 2 different internal controls then run in different lot. Final we record the CV. And the result show that our kits were high consistency.
- This slide is the performance of our ELISA kit. This experiment is also done by AHRI. 30 swine sera infected with O type FMD virus And 50 FMDV-free swine sera were provided by AHRI. The Sensitivity of our ELISA kit is 96.7%. The Specificity of our ELISA kit is 100%. Similar, 30 FMDV NSP Ab positive serum and 50 FMDV-free goat sera were tested use our ELISA kit. The Sensitivity of our ELISA kit is 100%. The Specificity of our ELISA kit is 100%.
- We collected the pig, cattle and goat samples from field perform our FMD NSP Ab ELISA kit and PrioCHECK FMD NS ELISA kit. WE compared these 2 kit.
- 113 pigs sera were collected from field farm. PrioCHECK ® as standard. Sensitivity of Sentinel is 92.20% Specificity of Sentinel is 91.90% Kappa value is 0.83
- 181 cattle sera were collected from field farm. PrioCHECK ® as standard. Sensitivity of Sentinel is 85%. Specificity of Sentinel is 98.1%. Kappa value is 0.86.
- 132 goats sera were collected from field farm. PrioCHECK ® as standard. Sensitivity of Sentinel is 75%. Specificity of Sentinel is 99.2%. Kappa value is 0.74. And we can find that the sample size of positive group is too small, so that is affect the sensitive. We need to do further experiment or to collect more samples for this test.
- This slide show the overview of last 3 slides. Our kit compared with other commercial ELISA kit. It means the performance of our kit is as good as the most acceptable kit.
- Sentinel ® FMDV NSP Ab Rapid Test and FMDV NSP Ab ELISA Test kit provide a powerful tool for FMDV surveillance from farm to Laboratory.