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Lab 18: Immunology 18.1 The ELISA: Activity 1: Using An ELISA To Diagnose Lupus To Begin

Human Physiology Lab 18

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319 views6 pages

Lab 18: Immunology 18.1 The ELISA: Activity 1: Using An ELISA To Diagnose Lupus To Begin

Human Physiology Lab 18

Uploaded by

Valerie Okakpu
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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Lab 18: Immunology

18.1 The ELISA


Our immune system is responsible for recognizing foreign invaders and eliminating them
from our body. This is a multi-step process which requires an army of cells, including a type of
leukocyte called a B lymphocyte. These cells have the capacity to recognize foreign antigens
and respond to them by creating antibodies. Antibodies are a powerful tool against disease;
they are custom created to fit perfectly with antigens like a lock and key and are able to
effectively neutralize pathogens. In our class we talk about the usefulness of antibodies in the
humoral immune response, but there is an additional use for antibodies: in the lab.
Antibodies are an important part of a lab tool called an Enzyme-Linked Immunosorbent
Assay (ELISA). In this procedure, an antibody, which has been tagged with an enzyme and is
specific for a given molecule, is used to determine the presence of that molecule. As part of the
reaction, the enzyme causes a change in the color of the test solution. The results can be
viewed with the naked eye or by using a spectrophotometer. Home pregnancy tests utilize this
technique. In the test, antibodies bound to the hormone human chorionic gonadotropin (hCG
the hormone of pregnancy) create a reaction wherein there is a color change. This color
change is viewed on the pregnancy test as a positive result. ELISAs are also utilized to diagnose
certain infectious diseases.
In todays lab, you will be completing a virtual lab activity where an ELISA is used to
diagnose patients with Systemic Lupus Erythematosus (often referred to as just Lupus, or SLE).
Lupus is an interesting disease because it is an autoimmune disease, meaning that the patients
immune system is attacking itself. In this case, there are no foreign antigens; the antigens that
the antibodies bind to are the patients own body proteins. Normally the immune system
recognizes these body proteins as self proteins; but with Lupus the immune system thinks
they are foreign and attacks them. This assault by the immune system causes the symptoms
associated with the disease. Today you will be testing the blood of three patients to see if they
have the disease.
Activity 1: Using an ELISA to diagnose Lupus
To begin:
When you open the program, you will be on the Diagnosis tab of the module. Read through
the information.
When finished, select the Background tab of the module. Read through the information.
To begin the procedure, click on the Notebook tab.
Notice the animation on the upper left of the screen. Directly below it you will find the
directions for completing the activity.

Copyright 2015 Kelly Neary.


Procedures and questions adapted from Howard Hughes Medical Institute Biointeractive Virtual Lab
For exclusive use by Mission College Physiology students.
Lab 18 page 1

Introduction:
1. How many patients will you be testing today?
Step 1: Centrifugation: Centrifuge your sample for 15 minutes at room temperature.
2. What type of sample are you given?
3. Why do you centrifuge your samples?
4. What is serum?

Step 2: Dilution: Prepare three dilutions of your patient samples: 1:2, 1:10 and 1:100. This
process determines the concentration of the sample or the sensitivity of the assay.
5. What buffer do you add? Aside from diluting the sample, what is the purpose of that
buffer?

Step 3: Filling your ELISA plate with the patient samples. Add 0.1 ml of each dilution of each
of the patients samples to the appropriate well on the ELISA plate.
6. Your ELISA plate has different wells. What chemical is coating the wells? Why?
You will be recording your results in Figure 1. This is similar to the ELISA plate that is shown in
step 3. Label the dilution factors and samples on Figure 1, as shown in the program.
Your program will skip back to step 2 and repeat the procedures in steps 2 and 3 with the
other blood samples.
Step 4: Completing the ELISA plate with controls. Add positive and negative controls to the
remaining wells.
7. What chemical is used as your positive control? What makes it a positive control?
8. What chemical is used as your negative control? What makes it a negative control?

Copyright 2015 Kelly Neary.


Procedures and questions adapted from Howard Hughes Medical Institute Biointeractive Virtual Lab
For exclusive use by Mission College Physiology students.
Lab 18 page 2

Step 5: Incubation #1: Place your ELISA plate in an incubator.


9. For how long do you incubate your ELISA plate?
10. At what temperature do you incubate your plate? Why this temperature?

11. Why do you incubate your sample?

Step 6: Washing #1: Remove fluid from each well and wash with 0.1ml of solution. Remove
fluid.
12. Why do you change the pipette tip each time you remove fluid from the wells?
13. What chemical do you use to wash the wells?

14. Why do you wash the wells?

Step 7: Add the secondary antibody: Add 0.1ml of enzyme-linked antibody to each well.
15. What animal is the source of the anti-human antibodies that you are adding to your wells?
16. What enzyme is tagged to the secondary antibody (give the whole name, not the
abbreviation)?
17. With what is the secondary antibody reacting: the SLE antigen or the anti-SLE antibody?
18. Why dont you change the pipette tip after each time you fill a well?

Copyright 2015 Kelly Neary.


Procedures and questions adapted from Howard Hughes Medical Institute Biointeractive Virtual Lab
For exclusive use by Mission College Physiology students.
Lab 18 page 3

Step 8: Incubation #2: Place the ELISA plate in the incubator.


19. For how long do you incubate your ELISA plate?
20. At what temperature do you incubate your plate?

21. Why do you incubate your sample?

Step 9: Washing #2: Remove fluid from each well and wash with 0.1 of solution. Remove fluid.
22. What chemical do you use to wash the wells?

Step 10: Add HRP-substrate


23. What is the name of the substrate (its OK to use the abbreviation here) that reacts with the
enzyme?
24. To what color does the substrate change when it reacts with the enzyme?

Step 11: Enzymatic reaction


25. How much time do you allow for the enzymatic reaction to proceed?
View your results in the animation, and fill them into Figure 1.

Figure 1: ELISA plate. Color in the wells to indicate a positive (+) reaction.
Copyright 2015 Kelly Neary.
Procedures and questions adapted from Howard Hughes Medical Institute Biointeractive Virtual Lab
For exclusive use by Mission College Physiology students.
Lab 18 page 4

Results:
Does Patient A have SLE?
Does Patient B have SLE?
Does Patient C have SLE?
Q1: What, specifically, are we testing for in the patients sample?

Q2: Normally, chemicals like the one in question 1 are created as a result of infection. Describe
the physiological process whereby that type of chemical would be present after infection.

Q3: Lupus is not the result of an infection. Why would the chemical, specified in question 1, be
present if the patient is sick?

Q4: Explain why a positive diagnosis is indicated by a color change in the ELISA plate wells. In
other words, explain how this ELISA worked.

Copyright 2015 Kelly Neary.


Procedures and questions adapted from Howard Hughes Medical Institute Biointeractive Virtual Lab
For exclusive use by Mission College Physiology students.
Lab 18 page 5

Q5: On the figure below, label the antigen, anti-SLE antibody, anti-human antibody, HRP, and
HRP substrate.

Copyright 2015 Kelly Neary.


Procedures and questions adapted from Howard Hughes Medical Institute Biointeractive Virtual Lab
For exclusive use by Mission College Physiology students.
Lab 18 page 6

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