Intro
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| Pregnancy Test Stick |
In this lab, we will be testing and learning how to use the method of ELISA, or Enzyme Linked Immunosorbent Assay, to determine if we have been exposeded to a certain disease. ELISA works by placing antigens into a container. Then primary antibodies are injected and they bind to the antigens. After the primary antibodies have binded, secondary antibodies with an enzyme are also injected, and the secondary antibodies bind to the primary. Then a colorless substrate is added and it reacts with the enzyme. If the disease is present, then the color will change in the liquid.
Procedure
1. Label a yellow tube and a plastic transfer pipet with your initials.
2. Use the pipet to transfer all your "bodily fluid" sample into the tube of another student. Gently mix the samples, then take back half of the shared sample (750 microliters) to your own tube. Write down the name of the student next to "Sharing Partner #1".
3. When instructed to do so, repeat the sharing protocol two more times. Discard this transfer pipet after this step.
4. Label your 12-well strip. On each strip label the first 3 wells with a "+" for the positive controls and the next wells with a "-" for the negative controls. Label the remaining wells with your and your lab partner's initials. (3 wells each).
5. Use a fresh pipet tip to transfer 50 µl of the positive control (+) into the three “+” wells.
6. Use a fresh pipet tip to transfer 50 µl of the negative control (–) into the three “–” wells.
7. Transfer 50 µl of each of your team’s samples from step 3 into the appropriately initialed three wells, using a fresh pipet tip for each sample.
8. Wait 5 minutes while all the proteins in the samples bind to the plastic wells.
9. WASH:
a. Tip the microplate strip upside down onto the paper towels, and gently tap the strip a few times upside down. Make sure to avoid samples splashing back into wells.
b. Discard the top paper towel.
c. Use a fresh transfer pipet to fill each well with wash buffer, taking care not to spill over into wells. Note: the same transfer pipet is used for all washing steps.
d. Tip the microplate strip upside down onto the paper towels and tap.
e. Discard the top 2–3 paper towels.
10. Repeat wash step 9.
11. Use a fresh pipet tip to transfer 50 µl of primary antibody (PA) into all 12 wells of the microplate strip.
12. Wait 5 minutes for the antibodies to bind to their targets.
13. Wash the unbound primary antibody out of the wells by repeating all of wash step 9 two times.
14. Use a fresh pipet tip to transfer 50 µl of secondary antibody (SA) into all 12 wells of the microplate strip.
15. Wait 5 minutes for the antibodies to bind to their targets.
16. Wash the unbound secondary antibody out of the wells by repeating wash step 9 three times.
17. Use a fresh pipet tip to transfer 50 µl of enzyme substrate (SUB) into all 12 wells of the microplate strip.
18. Wait 5 minutes. Observe and record the results.
