Name:___________________
Activity 1.1.5: ELISA
Introduction
Given Sue’s diagnosis, all of the patients from the past two days need to be called
back in for immediate testing. School officials are concerned about a possible
outbreak of bacterial meningitis on campus. In order to diagnose bacterial
meningitis, it is necessary to obtain a sample of cerebral spinal fluid using a spinal
tap. Since this procedure is extremely invasive and painful, only those patients
doctors feel are at greatest risk for the disease will be tested. Use the spinal fluid
samples to identify those who are infected with meningitis and to trace how this
disease may have spread amongst the students on campus. Devise a plan to halt
the spread of the disease before it is too late!
In Human Body Systems, you investigated the workings of the immune system and
learned how antibodies, specific proteins produced in response to invading antigens,
circulate to keep us healthy. Antibodies seek out and attach themselves to invaders,
flagging them for destruction by the immune system. These antigens are molecules
foreign to the body and can include bacteria, viruses and fungi. Since antibodies are
extremely specific to the antigens they attack, these proteins can be used in the
laboratory to help identify disease agents. One test, the Enzyme-Linked
Immunosorbant Assay (ELISA), combines targeted tagging with antibodies and an
enzyme reaction that produces a visible color change to test for the presence of
disease antigens or antibodies produced in response to that antigen. The ELISA
assay can even detect disease agents in body fluids before the body has a chance
to mount an immune response and produce antibodies. An ELISA can provide
qualitative results, indicating whether a patient is positive or negative for the
presence of the antigen or antibody, or an ELISA can provide quantitative results,
determining how much of the detected substance is present.
In this lab, you will use ELISA to test simulated cerebral spinal fluid (CSF) samples
taken from patients at Sue’s school for the presence of bacterial meningitis. This
rapid test can be completed in less than one hour and can detect antigens of the
Neisseria meningitidis bacteria. Your job is to determine which college students are
infected with this deadly bacterium and to propose a strategy for halting further
spread. For those who are infected, use quantitative data to propose a chain of
infection. Use data from the ELISA test and from the patient histories to trace how
this disease may have spread from person to person.
Equipment
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Computer with Internet access
Activity 1.1.4 Student Response Sheet
Bio-Rad ELISA Immuno Explorer™ Kit
o Simulated primary and secondary antibody
o Simulated antigen (prepared at various concentrations)
o 12-well microplate strips
o Microcentrifuge tubes
o Wash buffer
Name:___________________
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Micropipettor and disposable tips
Plastic transfer pipets
Paper towels
Marker
Safety goggles
Procedure
Part I: Principles of the ELISA Test
1. Use the Internet to review the relationship between antigens and antibodies.
2. Write a paragraph that explains how antibodies work to keep us well. Underline
each of the following terms in your paragraph:
o
o
o
o
Antigen
Antibody
Immune system
B lymphocytes
3. Note that scientists can use antibodies in the lab to help detect disease antigens.
To learn more about the ELISA test, log on to the Bio-Rad Interactive ELISA
Immuno Explorer site at http://www.bio-rad.com/LifeScience/jobs/2004/040522/04-0522_ELISA.html
4. Choose Antigen Detection ELISA from the main screen.
5. Use the blinking arrow on the left-hand side of the screen to navigate through the
animation. Pay attention to the way in which antibodies are used to identify an
antigen of interest.
6. Use what you have learned in the animation to draw a labeled and colored
sequential diagram that shows how the ELISA test can be used to identify
patients who are infected with bacterial meningitis. Your drawing or model should
include the following labeled components:
o
o
o
o
o
o
Antigen
Primary antibody
Secondary antibody
Enzyme
Substrate
Neisseria meningitidis bacteria
7. Answer Conclusion questions 1-2.
Name:___________________
Part II: Serial Dilutions
Scientists and doctors want to know who is infected with a specific disease, but they
may also want to know quantitative data about the strength of infection. By diluting
samples to be tested, scientists can determine just how much of the disease antigen is
present.
8. Obtain a Student Resource Sheet from your teacher.
9. Read the information presented about Serial Dilutions.
10. Use information from the Resource Sheet to complete practice problems A-C
found below.
If the final concentration in the first tube in Problem C is 200 ng sample/ml, what is the
concentration in the other three tubes? Show your work below.
Name:___________________
11. Compare your answers with a partner. Correct any mistakes.
12. Answer Conclusion question 3.
Part III: Running an ELISA
Use the ELISA test to determine which patients test positive for the presence of the
disease antigen and to determine the relative concentration of the bacteria in the fluid of
those who are affected. Early findings assure adequate treatment to those who are
affected and provide information necessary to prevent the disease from spreading all
over the college. In this lab activity, you will be testing the simulated CSF of two of the
patients presented in Activity 1.1.2 and Activity 1.1.3. These samples have randomly
been assigned by your teacher.
13. Put on your safety goggles.
14. Locate two 12-well strips at your lab station. Place one strip off to the side. You
will use one strip to prepare your concentration standards and one strip to
complete your ELISA test.
15. Use a marker to label the outside wall of each well on one 12-well strip with the
numbers 1-12. This strip will serve as your concentration standards.
16. Use a micropipettor to add to 50µl of Phosphate Buffered Saline (PBS) from the
tube labeled PBS to the wells labeled #2-#12.
17. Add 100µl from the tube labeled 1,000 ng/ml antigen (AG) to the well labeled #1.
18. To prepare the various concentration standards, complete the serial dilutions as
described below.
o Pipet 50µl out of well #1 and add it to well #2. Pipet up and down
gently three times to mix the sample in well #2.
o Using the same pipet tip, transfer 50µl from well #2 to well #3 and
mix the sample in well #3
o Continue this process with each of the remaining wells. Stop when
you reach well #11. Remove 50µl of fluid from well #11 and discard
this fluid into a waste container. Do not add additional liquid to well
#12.
19. Draw a diagram of the 12-well plate on your sheet of paper.
20. Under the diagram, calculate the dilution in each well. Think about what you have
added to each well and about the practice serial dilution questions you completed
in Part II.
Name:___________________
21. Note that the concentration of antigen in well #1 is 1000 ng/ml. Calculate the
concentration of antigen in wells 2-12. Show your work on your sheet of paper.
22. Write the concentration of each well under the appropriate well on your drawing.
You will need this diagram for reference in the lab.
23. Carefully place the standard strip off to one side.
24. Obtain the second 12-well strip. This strip will function as your test strip. Label
the first three wells with a “+” for the positive controls, the next three wells with a
“-“ for the negative controls, the next three wells with the name of the first patient
you will be testing, and the last three wells with the name of the second patient
you will be testing.
25. Record the names of the patients you will be testing on your paper.
26. Use a fresh pipet tip to transfer 50µl of the positive control into the three wells
labeled “+.”
27. Use a fresh pipet tip to transfer 50µl of the negative control into the three wells
labeled “-.”
28. Use a fresh pipet tip to transfer 50µl of the simulated serum from your 1st patient
into the three wells labeled with the patient name.
29. Use a fresh pipet tip to transfer 50µl of the simulated serum from your 2nd patient
into the three wells labeled with the patient name.
30. Wait five minutes to allow the proteins in the samples to bind to the plastic wells.
Remember, if the bacterial antigen is present in the samples, it will now be bound
to the walls of the strip.
31. When the five minutes is up, process both the standards strip and your test strip
as described in the following steps.
32. Wash the unbound sample out of the wells by tipping the microplate strip upside
down onto a stack of paper towels. Allow the fluid to drain out and gently tap the
strip a few times to remove any excess drops. Make sure to avoid splashing
sample back into the wells. Keep the two strips separate.
33. Discard the top layer of paper towels.
34. Use a transfer pipet filled with wash buffer to each fill each well of both strips. Be
careful not to spill over into the neighboring wells or let the pipet tip touch the
side of the wells. The same transfer pipet can be used for all washing steps.
35. Tip the microplate strips upside down on the paper towels and allow the wash
buffer to drain out onto the paper. Tap each strip a few times to remove all of the
liquid.
36. Discard the top layer of paper towels.
37. Repeat steps 34-37 to complete a second wash.
38. Use a fresh pipet to transfer 50µl of primary antibody into all 12 wells of each
microplate strip.
Name:___________________
39. Wait five minutes for the primary antibody to bind.
40. Wash the unbound primary antibody out of the wells by repeating the wash steps
in 34-37 two times.
41. Use a fresh pipet tip to transfer 50µl of secondary antibody into all 12 wells of
each microplate strip.
42. Wait five minutes for the secondary antibody to bind.
43. Wash the unbound secondary antibody out of the wells by repeating the wash
steps in 34-37 three times.
44. Use a fresh pipet tip to transfer 50µl of the enzyme substrate into all 12 wells of
each microplate strip.
45. Wait five minutes.
46. Observe your results and record your findings on your sheet of paper. Note: Do
not wait more than ten minutes to observe your results or the results may
be invalid. Find Mrs. Belton to look at your results.
47. Determine which patients test positive for the presence of the antigen.
48. Using your concentration standards, approximate the level of infection for any
patient that tests positive. Refer to your notes for the concentration values in
each well of the standard strip.
49. Share your findings with other lab groups.
50. Record results for all nine patients who are being tested on your paper.
51. For those patients who tested positive, use the concentration data as well as the
patient history information presented in Activity 1.1.2 and Activity 1.1.3 to deduce
a possible path of infection. Lower levels of disease antigen may signal a newer
infection. Refer back to the connections chart or web you started in Activity 1.1.2.
On your sheet of paper, explain who you believe was the first person infected
and describe how the infection might have spread from this person to the rest of
those with the disease. Provide evidence for your theory.
52. Share your theory with the class.
53. View the animation on bacterial meningitis in Merck 3-D animations at
http://www.mercksource.com/ppdocs/us/cns/content/blausen/flash_content
/clientNF.html
54. On a sheet of paper, describe how patients are physically affected by bacterial
meningitis and explain why this is such a serious infection. Explain how the name
of the disease relates to the anatomy of the brain.
55. Research appropriate treatment for bacterial meningitis including those that are
affected AND how to prevent the spread of the disease around campus.
56. Answer the remaining Conclusion questions.
Name:___________________
Activity 1.1.5: ELISA Conclusion Questions
1. Explain why antibodies allow scientists to target and identify specific disease
agents.
2. Why is the secondary antibody used in an ELISA test conjugated with an
enzyme?
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What happens when this enzyme meets up with its substrate?
3. What does it mean if a disease can be detected in samples from one person at a
dilution of 1/5 and in another patient at a dilution of 1/100?
4. Describe a situation that illustrates why it is a good idea to complete the ELISA
assay in triplicate.
Name:___________________
5. Why do you think college students living in dorms are often populations who see
meningitis outbreaks?
6. How did ELISA data allow you to track the path of infection at the college?
7. Explain why in sudden outbreaks, it may be better to test for disease antigens
rather than for antibodies.
Bonus:
8. Home pregnancy tests utilize ELISA technology. When a woman is pregnant, her
body produces a hormone call human chorionic gonadotropin (hCG). Explain
how antibodies can be used to detect this hormone and are linked to a color
change a woman may see on a positive test.