1. The ELISA Enzyme-Linked ImmunoSorbent Assay A highly specific and sensitive procedure allowing evaluation of multiple agents for a single serum dilution Compiled from a presentation by Dr. Lamichhane

2. ELISA Advantages  Relatively easy to perform  Can be run in a short time  Can be performed against a variety of agents at the same time.  Can be setup in any laboratory  Not strain specific

3. Steps of the Indirect ELISA Step 1  Antigen coated to well  Performed at Synbiotics  Antigen is inactivated.  Very stable

4. Steps of the Indirect ELISA Step 2 ä Addition of serum sample ä Specific reaction between antigen and chicken antibody ä Plate is washed

5. Steps of the Indirect ELISA Step 3 ä Addition of anti- chicken conjugate ä Conjugate and chicken antibody bind ä Plate is washed

6. Steps of the Indirect ELISA Step 4 ä Addition of substrate ä Amount of color directly proportional to amount of chicken antibody ä Reaction stopped

7. Time Sequence in the Elisa Assay Start assay Add serum Wash Plate Add Conjugate Add Substrate Stop Reaction 1 2 3 4 5 6

8. Test High Quality Serum  High Quality Serum = High Quality Results  Light hemolysis, fatty serum causes no problems  Heavy hemolysis, bacterially contaminated samples should be avoided  Store serum at 4 C for up to 1 week, store at -20 C for longer periods.

9. Sample Size (n)  Sample size is critical in order to obtain a representative estimate of mean flock titers  Higher titer variation = larger sample size  Collect more samples than planning to test  Suggested Sample Sizes Broilers - 18-20 samples Broilers - 18-20 samples Breeders/Layers - 30-45 samples Breeders/Layers - 30-45 samples

10. Sample Size Cont.  Variation from the mean titer increases as the number of samples tested decreases  Reducing sample sizes to save money, can decrease the reliability of serological results. Results can be misleading or confusing.

11. Random Samples  Tends to average out effects of uncontrollable variation within the flock  Every bird should have and equal chance of being selected for testing. Tests birds throughout the house!

12. The Serum Plate Samples from three different flocks

13. From Color to ELISA Results Optical Density (O.D.) SP Ratio Titer Value Flock Mean Geometric Mean Titer Groups BaselinesHigh-Normal-Low

14. ProFILE Reports  SP Ratio  Titer  The Mean Titer of the samples  The Geometric Mean Titer  The Coefficient of Variation  Titer Groups ProFILE reports provide the following statistics concerning the flocks tested.

15. ProFILE Reports SP Ratio ä Compares O.D. of sample to O.D. of Positive Control serum ä Removes test “background” Sample O.D. - Avg. Neg. Control Sample O.D. - Avg. Neg. Control Avg. Pos. Control - Avg. Neg. Control SP Ratio =

16. ProFILE Reports Sample Titer ä Converts SP ratio into a linear scale ä Linear regression formula used ä Different formula used for different tests (a x log 10 SP) + b log 10 Titer =

17. ProFILE Reports The Mean Titer:  The mean titer provides information about the titer from an average bird within the flock  The larger the sample size the more accurate the mean. S 1 + S 2 +…S N S 1 + S 2 +…S N n Mean =

18. ProFILE Reports The GMT: ä The GMT provides information about the central “tendency” of the flock. ä Best used when flock has high variability S 1 x S 2 x…S N GMT = n

19. Titer Value Titer Value  Arithmetic Mean Titer (Mean) Good when flock is normally distributed  Geometric Mean Titer (GMT) Better when flock is not normally distributed - bimodal or polymodal

20. ProFILE Reports The Coefficient of Variation ä Measures “uniformity” within a flock ä Calculates the variation of the average individual, expressed as % deviation from the mean titer. Utilizes SP values. Standard Deviation Mean titer Mean titer %CV = x 100

21. Interpreting ELISA Results Ê Uniformity (%CV) Ë Titer Values Ì Flock History

22. Coefficient of Variation (% CV) ResultInterpretation

23. Preferred Population Distribution Flock Age ELISA Titer Protection Threshold Flock A Low %CV

24. Population Distribution Flock Age ELISA Titer Protection Threshold Flock B High %CV

25. Flock Profile Frequency Chart Number or Percentage of Samples in Group

26. New AI Surveillance Method B-ELISA Ab Test  Advantages  1. Screen large sample sizes  2. Can be Automated  3. Detects group specific antibody  4. Can be applied for multi- species (waterfowl and other avian species)  5. Excellent sensitivity, specificity and consistency   Disadvantages  1. Group specific test  2. Need more volume of serum  4. ELISA reader and computer

27. Indirect ELISA AIV rNP Antigen Antibody Conjugate Goat Anti-chicken IgG Polyclonal Goat Anti-chicken IgG Polyclonal antibody/peroxidase antibody/peroxidase Substrate Uncolored/Colored substrate Uncolored/Colored substrate PositiveNegative

28. Blocking ELISA AIV rNP antigen Antibody Conjugate Mouse Anti-AIV NP Monoclonal Mouse Anti-AIV NP Monoclonal antibody/peroxidase antibody/peroxidase Substrate Uncolored/Colored substrate Uncolored/Colored substrate Positive Negative