1. Biomedical Science Activity 1.1.5 ELISA

2. PLTW Biomedical Science
From Activity we know about the spread of bacterial meningitis at the school. Using the ELISA we want to figure out who else is infected and determine who spread the disease to who

3. Workshop Overview Introduction to Immunology, Antibodies and ELISA
Set up a serial dilution of our antigen
Detect antigen in patient samples

4. Crash Course in Immunology and ELISA
Immune Response
C. Macrophage
A. Pathogen
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

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

6. ELISA Enzyme-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.

7. Creating standards using serial dilution
*16. Label microtiter plate strip
*Using step numbering from PLTW Activity manual

8. Creating standards using serial dilution
17. Add 50µL PBS to wells 2-12
PBS = Phosphate Buffered Saline, good for solubilizing proteins

9. Creating standards using serial dilution
18. Add 100µL of antigen (AG) to well 1

10. Creating standards using serial dilution
19. Create a serial dilution by moving 50µL from well 1 to next well, mix, move 50µL, repeat until well 11, remove 50µL add to trash
50µL
Trash

11. Creating standards using serial dilution
20. Draw a diagram of the 12 well plate in your notebook

12. Creating standards using serial dilution
Calculate the concentration in each well and annotate your notebook drawing. Show calcs in notebook
1,000 ng/ml
500 ng/ml
250 ng/ml
125 ng/ml
62.5 ng/ml
31.25 ng/ml
15.63 ng/ml
7.81 ng/ml
3.91 ng/ml
1.95 ng/ml
0.98 ng/ml
0 ng/ml

13. ELISA Time! Testing for who is infected
24. Place standards strip off to the side
25. Label other microtiter strip as below
26. Record patient names in notebook
Patient initials
Patient initials

14. ELISA Time! Testing for who is infected
27. Add 50µL “+” control to labeled wells
Patient initials
Patient initials

15. ELISA Time! Testing for who is infected
28. Add 50µL “-” control to labeled wells
Patient initials
Patient initials

16. ELISA Time! Testing for who is infected
29. Add 50µL patient 1 serum
Patient initials
Patient initials

17. ELISA Time! Testing for who is infected
30. Add 50µL patient 2 serum
Patient initials
Patient initials

18. ELISA Time! Testing for who is infected
31. Wait 5 minutes
Patient initials
Patient initials

19. ELISA Time! Testing for who is infected
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Protein in solution start sticking to the polystyrene of the microtiter plate
Patient initials
Patient initials

20. ELISA Time! Testing for who is infected
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Patient initials

21. ELISA Time! Testing for who is infected
Polystyrene (plate)
Protein (Antigen)
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Hydrophobic
interactions
Proteins stick to the polystyrene because of the interaction of the protein’s hydrophobic residues with the benzene rings of the polystyrene

22. ELISA Time! Testing for who is infected
For both microtiter strips tap liquid out of wells onto papertowels, discard top papertowel

23. 35. Fill wells with wash buffer
ELISA – WASH CYCLE
35. Fill wells with wash buffer
Patient initials

24. ELISA – WASH CYCLE
Tap liquid out of wells onto papertowels, discard top papertowel

25. ELISA – WASH CYCLE
38. Repeat Wash Cycle

26. ELISA Time! Testing for who is infected
39. Add 50µL Primary Antibody “PA” to all wells
Patient initials
Patient initials

27. ELISA Time! Testing for who is infected
40. Incubate 5 min
Patient initials

28. ELISA Time! Testing for who is infected
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Patient initials
The waiting period is so that the primary antibody can find and stick to any antigens stuck to the microtiter plate. Antibodies due this with a very high specificity.

29. ELISA Time! Testing for who is infected
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Patient initials

30. ELISA Time! Testing for who is infected
Variable region
Light chain
Heavy chain
constant region
3,368,600 human antibody combos
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The human genome has 25,000 or less genes, but we are able to make almost 3.5 million types of antibodies and this all comes down to a very cool translationaly trick where 3 different regions can be combined in different patterns to generate all of those different antibodies. The variable region at the far end of the “Y” is where the antibodies protein residue interface with the antigen, so most changes in translation effect this area. The constant regions are what allow us to make antibodies against other antibodies as we will see later.

31. 38. Repeat Wash Cycle 2 times
ELISA – WASH CYCLE
38. Repeat Wash Cycle 2 times

32. ELISA Time! Testing for who is infected
42. Add 50µL Secondary Antibody “SA” to all wells
Patient initials
Patient initials
Patient initials
Patient initials

33. ELISA Time! Testing for who is infected
43. Incubate 5 min
Patient initials

34. ELISA Time! Testing for who is infected
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Patient initials

35. ELISA Time! Testing for who is infected
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Patient initials
The secondary antibody has an Enzyme Linked to the constant region hence Enzyme Linked ImmunoSorbent Assay. This enzyme will ultimately be used on a substrate that will give us a chromogenic response.

36. ELISA Time! Testing for who is infected
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Amplification of signal
Introduce HRP (Horseradish Peroxidase) for colormetric detection
Patient initials
The secondary antibody does two things – it amplifies the signal in that it can have multiple antibodies bind to 1 primary antibody and from a manufacturing point of view its hard to make the enzyme linked antibody, so if you can just make one of those that attacks all antibodies from a given organism you can produce the primary antibodies for much cheaper and make the entire processes less expensive.

37. ELISA Time! Testing for who is infected
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Antigen = chicken gamma globulin
Primary Antibody = anti-chicken polyclonal antibody
Secondary Antibody = goat anti-rabbit conjugate to HRP
The antigen that is used in this kit is chicken Gamma Globulin. Polyclonal antibodies are made in a rabbit model against the chicken Gamma Globulin. The rabbit antibodies are put in goats in such a way as to develop sensitivity to the constant region of the rabbit antibodies and this goat antibody has the enzyme linked to it after they are harvested.

38. 38. Repeat Wash Cycle 3 times
ELISA – WASH CYCLE
38. Repeat Wash Cycle 3 times

39. ELISA Time! Testing for who is infected
45. Add 50µL Substrate “Sub” to all wells
Patient initials
Patient initials
Patient initials
Patient initials

40. ELISA Time! Testing for who is infected
46. Incubate 5 min – watch for color development
Patient initials

41. ELISA Time! Testing for who is infected
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Patient initials
Upon addition of the substrate the horseradish peroxidase enzyme that has been linked to the secondary antibody can begin to modify the substrate TMB which is usually colorless into its blue colored product. The blue development tells you that an antigen was present in the original body fluid sample

42. ELISA Time! Testing for who is infected
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Patient initials

43. ELISA Time! Testing for who is infected
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44. ELISA Time! Testing for who is infected
47. Record results in your notebook
48. Note which patients tested positive
49. Using your standards determine the level of infection your patients have (ng/ml)
Qualitative => estimate “biospec”
Quantitative => spectrophotometry
Patient initials

45. How to get quantitative data (real world)
The iMark micropalte reader can read the absorbance of all the samples in 30 seconds and you can have your students spend time actually doing data analysis.

46. How did the disease spread?
50. Share your results and gather the other groups results. Based upon antigen concentrations in patients propose the order by which the patients were infected, starting from Sue.

47. Bio-Rad’s Immuno Explorer Lab
What did we use today?
Bio-Rad’s Immuno Explorer Lab
EDU