Transcript bms 1.1.5 elisa

Biomedical Science Activity 1.1.5
ELISA
[email protected]
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PLTW Biomedical Science
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Workshop Overview
 Introduction to Immunology, Antibodies and ELISA
 Set up a serial dilution of our antigen
 Detect antigen in patient samples
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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
H. Memory B cells
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I. Plasma cells
Antibodies are ineffective
Against Zombie Virus
Crash Course in Immunology and ELISA
Antibody Structure
ELISA tests are
based on immune
system antibody
molecules
Heavy
chain
Disulfide
bonds
Light
chain
Antigens
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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.
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Creating standards using serial dilution
*16. Label microtiter plate strip
*Using step numbering from PLTW Activity 1.1.5 manual
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Creating standards using serial dilution
17. Add 50µL PBS to wells 2-12
PBS = Phosphate Buffered Saline, good for solubilizing proteins
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Creating standards using serial dilution
18. Add 100µL of antigen (AG) to well 1
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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
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Trash
Creating standards using serial dilution
20. Draw a diagram of the 12 well plate
in your notebook
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Creating standards using serial dilution
21-23. Calculate the concentration in
each well and annotate your notebook
drawing. Show calcs in notebook
0 ng/ml
0.98 ng/ml
1.95 ng/ml
3.91 ng/ml
7.81 ng/ml
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15.63 ng/ml
31.25 ng/ml
62.5 ng/ml
125 ng/ml
250 ng/ml
500 ng/ml
1,000 ng/ml
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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
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Patient
initials
ELISA Time!
Testing for who is infected
27. Add 50µL “+” control to labeled wells
Patient
initials
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Patient
initials
ELISA Time!
Testing for who is infected
28. Add 50µL “-” control to labeled wells
Patient
initials
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Patient
initials
ELISA Time!
Testing for who is infected
29. Add 50µL patient 1 serum
Patient
initials
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Patient
initials
ELISA Time!
Testing for who is infected
30. Add 50µL patient 2 serum
Patient
initials
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Patient
initials
ELISA Time!
Testing for who is infected
31. Wait 5 minutes
Patient
initials
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Patient
initials
ELISA Time!
Testing for who is infected
Concept
Corner
Patient
initials
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Patient
initials
ELISA Time!
Testing for who is infected
Concept
Corner
Patient
initials
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Patient
initials
ELISA Time!
Testing for who is infected
Concept
Corner
Protein (Antigen)
H
R
H
R
Hydrophobic
interactions
Polystyrene (plate)
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ELISA Time!
Testing for who is infected
32-34. For both microtiter strips tap liquid
out of wells onto papertowels, discard top
papertowel
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ELISA – WASH CYCLE
35. Fill wells with wash buffer
Patient
initials
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Patient
initials
ELISA – WASH CYCLE
36-37. Tap liquid out of wells onto
papertowels, discard top papertowel
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ELISA – WASH CYCLE
38. Repeat Wash Cycle
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ELISA Time!
Testing for who is infected
39. Add 50µL Primary Antibody “PA” to all wells
Patient
initials
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Patient
initials
ELISA Time!
Testing for who is infected
40. Incubate 5 min
Patient
initials
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Patient
initials
ELISA Time!
Testing for who is infected
Concept
Corner
Patient
initials
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Patient
initials
ELISA Time!
Testing for who is infected
Concept
Corner
Patient
initials
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Patient
initials
ELISA Time!
Testing for who is infected
Concept
Corner
Variable
region
Heavy
chain
Light
chain
constant
region
3,368,600 human antibody combos
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ELISA – WASH CYCLE
38. Repeat Wash Cycle 2 times
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ELISA Time!
Testing for who is infected
42. Add 50µL Secondary Antibody “SA” to all wells
Patient
initials
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Patient
initials
ELISA Time!
Testing for who is infected
43. Incubate 5 min
Patient
initials
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Patient
initials
ELISA Time!
Testing for who is infected
Concept
Corner
Patient
initials
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Patient
initials
ELISA Time!
Testing for who is infected
Concept
Corner
Patient
initials
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Patient
initials
ELISA Time!
Testing for who is infected
Concept
Corner
Amplification of signal
Introduce HRP
(Horseradish Peroxidase)
for colormetric detection
Patient
initials
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Patient
initials
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
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ELISA – WASH CYCLE
38. Repeat Wash Cycle 3 times
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ELISA Time!
Testing for who is infected
45. Add 50µL Substrate “Sub” to all wells
Patient
initials
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Patient
initials
ELISA Time!
Testing for who is infected
46. Incubate 5 min – watch for color development
Patient
initials
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Patient
initials
ELISA Time!
Testing for who is infected
Concept
Corner
Patient
initials
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Patient
initials
ELISA Time!
Testing for who is infected
Concept
Corner
Patient
initials
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Patient
initials
ELISA Time!
Testing for who is infected
Concept
Corner
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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
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Patient
initials
How to get quantitative data (real world)
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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.
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What did we use today?
Bio-Rad’s Immuno Explorer Lab
166-2400EDU
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