Identify Patient Zero of a Zombie Apocalypse With the Power of an ELISA!

1. Identify Patient Zero of a Zombie Apocalypse With the Power of an ELISA!

2. Instructors - Bio-Rad Curriculum and Training Specialists • Sherri Andrews, Ph.D. sherri_andrews@bio-rad.com • Damon Tighe damon_tighe@bio-rad.com • Leigh Brown, M.A. leigh_brown@bio-rad.com

3. TV or real life? The Walking Dead show Feb 11, 2014 Atlanta

4. Zombiefication • Method of infection • Bodily fluid contact • Airborne • Foodborne • Other? • Symptoms • Undead • Hungry for flesh • Treatment • Under investigation

5. Introduction to Immunology, Antibodies and ELISA • Fluid swapping and disease spreading! • Indirect ELISA to narrow down who were the initial carriers of the disease

6. Crash Course in Immunology and ELISA Immune Response C. Macrophage A. Pathogen Zombie virus D. Macrophage B. B cells F. T cell E. Macrophage G. B cell J. Antibodies attach to pathogen Antibodies are ineffective Against Zombie Virus H. Memory B cells I. Plasma cells

7. Heavy chain Disulfide bonds Light chain Crash Course in Immunology and ELISA Antibody Structure ELISA tests are based on immune system antibody molecules Antigens

8. ELISAEnzyme-Linked Immunosorbant Assay (indirect) Add the purified antigen to all the wells. Incubate for 5 min. Rinse Add serum antibodies (student samples) to the appropriate wells. Incubate for 5 min. Rinse Add the enzyme-linked antibody to all wells. Incubate for 5 min. Rinse Add enzyme substrate to all wells. Incubate for 5 min.

9. Time to Swap Fluids and disease!

10. Swapping Fluids and Disease Swap with 1 person at your table (record who) Add your 750 µl together, mix and each take 750 µl back

11. Swapping Fluids and Disease Swap with 1 person at the closest table (record who) Add your 750 µl together, mix and each take 750 µl back

12. Swapping Fluids and Disease Swap with 1 person at the another table (record who) Add your 750 µl together, mix and each take 750 µl back

13. ELISA Time! Testing for who is infected

14. ELISA Time!Testing for who is infected Label Tubes Your ID # Your ID #

15. ELISA Time!Testing for who is infected Incubate: 5 minutes Your ID # Your ID #

16. Your ID # Your ID # ELISA Time!Testing for who is infected Tap out liquid

17. ELISA Time!Testing for who is infected Wash with Buffer (contains Tween20) Your ID # Your ID #

18. Your ID # Your ID # ELISA Time!Testing for who is infected Tap out liquid Repeat

19. ELISA Time!Testing for who is infected Add 50 µl + Control Add 50ul – Control Add 50ul of Bodily Fluid Your ID # Your ID #

20. ELISA Time!Testing for who is infected Incubate: 5 minutes Your ID # Your ID #

21. ELISA Time!Testing for who is infected Concept Corner Your ID # Your ID #

22. ELISA Time!Testing for who is infected Concept Corner Your ID # Your ID #

23. ELISA Time!Testing for who is infected Concept Corner Protein (Antigen) H H R R Hydrophobic interactions Polystyrene (plate)

24. Your ID # Your ID # ELISA Time!Testing for who is infected Tap out liquid

25. ELISA Time!Testing for who is infected Wash with Buffer (contains Tween20) Your ID # Your ID #

26. Your ID # Your ID # ELISA Time!Testing for who is infected Tap out liquid Repeat

27. ELISA Time!Testing for who is infected Add 50 µl “PA” (Primary Antibody) Your ID # Your ID #

28. ELISA Time!Testing for who is infected Incubate: 5 minutes Your ID # Your ID #

29. ELISA Time!Testing for who is infected Concept Corner Your ID # Your ID #

30. ELISA Time!Testing for who is infected Concept Corner Your ID # Your ID #

31. ELISA Time!Testing for who is infected Variable region Concept Corner Heavy chain Light chain constant region 3,368,600 antibody combos

32. ELISA Time!Testing for who is infected Concept Corner Antigen = chicken gamma globulin Primary Antibody = anti-chicken polyclonal antibody Secondary Antibody = goat anti-rabbit conjugate to HRP

33. Your ID # Your ID # ELISA Time!Testing for who is infected Tap out liquid

34. ELISA Time!Testing for who is infected Wash with Buffer Your ID # Your ID #

35. Your ID # Your ID # ELISA Time!Testing for who is infected Tap out liquid Repeat

36. ELISA Time!Testing for who is infected Add 50ul “SA” (Secondary Antibody) Your ID # Your ID #

37. ELISA Time!Testing for who is infected Incubate: 5 minutes Your ID # Your ID #

38. ELISA Time!Testing for who is infected Concept Corner Your ID # Your ID #

39. ELISA Time!Testing for who is infected Concept Corner Your ID # Your ID #

40. ELISA Time!Testing for who is infected Concept Corner Amplification of signal Introduce HRP (Horseradish Peroxidase) for colormetric detection Your ID # Your ID #

41. ELISA Time!Testing for who is infected Concept Corner

42. Your ID # Your ID # ELISA Time!Testing for who is infected Tap out liquid

43. ELISA Time!Testing for who is infected Wash with Buffer Your ID # Your ID #

44. Your ID # Your ID # ELISA Time!Testing for who is infected Tap out liquid Repeat x2

45. ELISA Time!Testing for who is infected Add 50 µl “Sub” (HRP substrate) Your ID # Your ID #

46. ELISA Time!Testing for who is infected Incubate: 5 minutes Watch for color change! Your ID # Your ID #

47. ELISA Time!Testing for who is infected Concept Corner Your ID # Your ID #

48. ELISA Time!Testing for who is infected Concept Corner Your ID # Your ID #

49. Deducing who was originally infected

50. Deducing who was originally infected Everyone Stand UP! If your ELISA was negative sit down