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. Bio-Rad Curriculum and Training Specialist Tamica Stubbs tamica_stubbs@bio-rad.com Damon Tighe damon_tighe@bio-rad.com Sherri Andrews, Ph.D. sherri_andrews@bio-rad.com Leigh Brown 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 • Symptoms • Undead • Hungry for flesh • Treatment • None known

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. 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

10. 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

11. 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

12. ELISA Time! Testing for who is infected

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

14. ELISA Time!Testing for who is infected Add 50 µl “+” (Antigen) Your ID # Your ID #

15. Add 50ul “-” (wash buffer) Your ID # Your ID # Your ID #

16. Add 50ul Bodily Fluid Your ID # Your ID # Your ID #

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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 Antigen = chicken gamma globulin Primary Antibody = anti-chicken polyclonal antibody Secondary Antibody = goat anti-rabbit conjugate to HRP

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

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

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

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

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

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

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