BI 106 A&P-2 HYBRID LAB ASSIGNMENT and answer to #2 on Lab Midterm
1. Go to http://www.hhmi.org/biointeractive/vlabs/
2. Scroll down to “The Immunology Lab” (you will have to use a computer that has Shockwave) and Click on it.
3. Then read both the “Diagnostic Section” and the “Background Section” before you proceed.
4. Then click on “Notebook” to start the Virtual ELISA Lab.
5. Then answer the following questions (this is a group effort paper):
a. Define ELISA and the DRAW the generalized ELISA protocol for detecting a target antigen and include the separate steps of primary antibody binding and then secondary antibody binding. (You can find it under “Fig. 1” in the “Background” section).
b. The ELISA test quantifies how much enzyme is present by the amount of color produced. How is the color produced?
c. What are some potential experimental problems and what are some limitations of the test?
d. What is SLE?
e. In ‘Lab Notebook’ Step 1, why do you centrifuge the sera?
f. In ‘Lab Notebook’ Step 2, why do you do dilutions?
g. In ‘Lab Notebook’ Step 3, why do you pre-treat the ELISA plate?
h. Why do you use a new pipette tip for each dilution?
i. In ‘Lab Notebook’ Step 4, why do you add the primary antibody as well as buffer (control)?
j. In ‘Lab Notebook’ Step 5, why do you incubate the ELISA plate at 370C for 15 minutes?
k. In ‘Lab Notebook’ Step 6, why do you wash the wells?
l. In ‘Lab Notebook’ Step 7, you add a 2o antibody – this antibody is made in a rabbit and has an attached enzyme that will interact with the substrate in the next step. Why?
m. Next (Step 9) – Remove the fluid from each well with the pipette and wash with 0.1 ml of PBS –WHY?
n. Next (Step 10) – Add 0.1 ml of buffered solution containing the chemical substrate (HRP-substrate). If there are human antibodies present, the clear substrate will turn yellow – WHY?
o. Step 11 – Set timer for 15 minutes – WHY?
p. What were the correct results? Who tested positive for the auto-immune disease, Systemic Lupus Erythematosus (SLE)?
BI 106 A&P-2 HYBRID LAB ASSIGNMENT and answer to #3 on Lab Midterm
Google “Respiratory or Pulmonary Function Tests” and choose 1 to describe in detail how the test is done and what the results can tell the respiratory therapist.