ELISA Virtual Lab

Directions​: Click through the virtual lab, answer the following questions as you go along. Make
sure to change the font color.

Introduction​:
1. Where are antibodies found?
Antibodies are found in the liquid portion of blood and help protect the body from harm.
2. How can antibodies be used in the laboratory
They can be used to help diagnose disease caused by malfunctions of the immune system or
by infections.

3. What are ELISA assays used for in labs?

It is used in labs to determine whether a particular antibody is present in a patient’s blood
sample.
4. What are the three important limitations of an ELISA? Explain each.
Limitation Explanation

Positive results containing antibodies don’t The body can produce antibodies even
always mean the patient is sick. though the person may have had the disease
previously and already have recovered.

People may be poor producers of antibodies They amount of antibody may be too low to
or have an interfering substance in their measure and is will be undetected.
blood.

Positive results can occur if an unrelated Testing patients and running tests multiple
antibody reacts with the antigen test help reduce false alarms.
nonspecifically.

Background​:
1. What test can be used to determine whether a patient has an infectious or autoimmune
disease?
The interaction of antigen and antibody outside the body in the laboratory can help determine
whether a patient has an infectious or autoimmune disease.
2. What does a positive result indicate?

3. The watery fluid of the blood is called_​serum__​.

4. Detection is possible when_a​ second antibody is added_​__.

5. Once isolated , the secondary antibody can be_​chemically linked to a system that can
produce a detectable signal_.
 6. What is the signaling system?
It consists of an enzyme attached to the second antibody.

7. What happens when the appropriate chemical (substrate) is added?

When the appropiate chemical is added, the enzyme converts it to a colored substance that can
be measured.
8. How is the test quantified?
The test is quantified by how much enzyme is present by the amount of color produced.
9. What does the amount of color reflect?
The amount of color reflects the amount of antigen initally present.
Lab​: Continue through the lab simulation protocol Be sure to read the captions below the
pictures (left) and the information in the lab notebook (right). To begin the lab hit “start over.”

1. What is systemic lupus erythematosus (SLE)?

Systemic lupus erythematosus is a chronic disease.
2. From Figure 1 (click on it), what are the four steps of an ELISA protocol?
a. ​Centrifuge whole blood samples of patients A, B and C for 15 minutes at room
temperature to get the sera
b. Prepare three dilutions using serum from patient A
c. Prepare an ELISA plate with 0.1 ml of the different dilution of patient serum
using an Eppendorf pipette
d. Add to the ELISA plate 0.1ml dilutions for each titer of anti DNA primary antibody
positive and a buffer.
3. In step 1, you centrifuge the samples. What does the centrifuge do?
You must centrifuge to precipitate the blood cells and obtain the serum.
4. What are you preparing in step 2? Why are there three different solutions?
You are preparing three different solutions. There are three different solutions in order to
determine the level of the antibody.
5. In steps 3 and 4, you prepare an ELISA plate. What has the ELISA plate been
pretreated with? Why?
a. What is the positive control? (step 4)
The positive control is the primary antibody.
b. What is a primary antibody?
A primary antibody is to detect the foreign particle.
c. What is the negative control? (step 4)
The negative control is
d. Why is it necessary to have a positive and a negative control? (step 4)
It’s important to have a positive and negative control because pne produces a positve response
if the reagents and conditions are correct whereas the other will never produce the positive
response.
6. Why incubate the plate in step 5?
Incubate the plate to ensure that the antibody present in the sample will interact correctly with
the antigen.
 7. Next, in step 6, the plate is washed. Why was the plate?
The plate is washed because it helps remove any antibody that didn’t react with the SLE antigen
in the well.

8. In step 7, a secondary antibody is added. What is a secondary antibody? Please

define.
A secondary antibody is used in an immunoassay that detects the primary antibody.
a. What is the attached enzyme in this assay? (step 7)
The attached enzyme is the HRP enzyme.
b. What is the specific substrate for HRP? What color does it produce? (step 7)
The specific substrate for HRP is horseradish peroxidase. It produces yellow solution.
9. How can the yellow color be quantitatively measured? At what wavelength? (step 10, in
“why”)
The yellow color can be quantitatively measured in a spectrometer at 414 nanometers.
10. Record your results. Indicate on this page and on the computer which boxes turned
color.
A B C Positive (+) Negative (-)

1:2 Yellow Yellow Yellow

1:10 Yellow Yellow Yellow

1:100 Yellow Yellow

11. Did you complete the ELISA correctly? (Yes/ No)

If yes, go to #12 and #14.
If no, go to #13 and #14. No

12. What do the results indicate about:

a. Patient A:
b. Patient B:
c. Patient C:

13. Explain what you think you did wrong and what you will need to do next time. (For
additional information check the printable summary page.) Did your incorrect procedure
provide you with any results? Explain what went wrong.
I don’t know exactly what I did wrong because I followed the directions and did as I was told to. I
probably accidently pushed something else because my results didn’t seem to all come out the
same.
 14. This virtual lab was testing for lupus. ​ How could this same test be used to test for the
presence of HIV?​ If the results for an HIV test were the same as in this exercise, what
would they indicate about the three patients?
If the results for an HIV test were the same as in this exercise, it would indicate that the two out
of three patients are positive.

Additional information on HIV/AIDS