ELISA (enzyme-linked immunosorbent assay) WORKSHEET
The Immunology Virtual Lab
Go tohttps://media.hhmi.org/biointeractive/vlabs/immunology/index.htmlStart the Virtual Lab and maximize the screen if you wish. Answer the following questions in the spaces provided. In order to learn from a virtual interactive lab, it is essential that you read all the information thoroughly and pay attention to what you are doing. Please do not waste your time just clicking through without reading everything and without thoughtful consideration.
Record your answer underneath the questions in a different font and color. Turn in to the assignment folder.
- Where are antibodies found?
- How can they be used in the laboratory?
- What are ELISA assays used for in labs?
- What are the three important limitations of an ELISA? Explain each.
- What does a positive result indicate?
- What is allowed to react with the target antigen?
- Detection is possible when…
- Once isolated, the secondary antibody can be…
- What is the signaling system?
- What happens when the appropriate chemical (substrate) is added?
- How is the test quantified?
- What does the amount of color reflect?
Proceed through the entire lab simulation protocol. Be sure to read the captions below the pictures (left side) and the information in the lab notebook (right side). Be sure to “start over” to begin the lab. You CANNOT skip any steps. Answer the following questions as you proceed.
1.From Figure 1 (click on it), what are the four steps of an ELISA protocol?
- What are you preparing in step 2? Why are there three different solutions?
- In step3, you prepare an ELISA plate.
- What has the ELISA plate been pretreated with?
- In step 4, you prepare an ELISA plate
- What is the positive control?
- What is a primary antibody? Please define.
- What is the negative control?
- Why is it necessary to have a positive and a negative control?
- Why incubate the plate in step 5?
- Next, in step 6, the plate is washed. Why wash the plate?
- In step 7, a secondary antibody is added. What is a secondary antibody? Please define.
- What is the attached enzyme in this assay?
- What is the specific substrate for HRP? What color does it produce?
- Record your results. Indicate on this page and on the computer which boxes turned color.
|Dilution||A||B||C||+ (positive)||– (negative)|
- Did you complete the ELISA correctly? (Yes/No) __________
If yes, proceed to #10.
If no, proceed to #11.
- What do the results indicate about:
- Explain what you did wrong and what you will need to do next time. For more information, check your printable summary page. Did your incorrect procedure provide you any results? Explain what went wrong.
- This virtual lab was testing for lupus. How is this same test used to test for the presence of HIV? What are the dangers of a false negative result?