please help with cell biology lab questions, all data given
EXPERIMENTAL PROTOCOL:
In this laboratory, you will test seven different patients using a direct ELISA. You will test each of them for the presence of ZIKA virus and HIV, as well as perform a pregnancy test, using known antigens/antibodies for those diseases/test. Students will work in pairs. Serum for each patient has been incubated overnight at 4°C in the wells of an ELISA plate.
EQUIPMENT:
• 37°C incubator
• ELISA 96 wells plate.
You will be using the portion of the ELISA plate highlighted in red (A1-A8, B1-B8, C1-C8)
REAGENTS:
• Washing solution 1 : 1x PBS
• Washing solution 2: 1x PBS, 0.05% Tween-20.
• Blocking solution: 5% non-fat dry milk, 0.05% Tween-20 in PBS.
• Conjugates: Anti-EDIII; Anti-hCG; Anti-p24. Each diluted 1:30,000 and conjugated to HRP.
• Substrate: 3,3’,5,5’-tetramethylbenzidine (TMB)
PROCEDURE:
1. Ask for one ELISA plate from your instructor. Each plate should have a lid on it. Plates have been pre-incubated overnight with the serum of each subject in the study as follows:
2. Wash only the used wells in the plate 2 times with 200 µL of PBS each time. Ask your instructor how to dispose of the washes (PBS from each wash can be disposed of in the sink). Dry the wells in the plate after each wash by inverting the plate and tapping it several times on a clean paper towel.
3. Add 200 µL of blocking solution to each well. Place the lid back on the plate, label your plate with your group number, and incubate for 30 minutes at 37°C in the incubator.
4. When the incubation is done, dispose of the blocking solution in the sink and take the plate back to your bench. Invert the plate and tap the wells on a clean paper towel as described in step 2.
5. At this point, you will add the specific antibody for each test you are performing. You have 3 different HRP conjugated antibodies. You will use the antibody against ZIKA in the first row of your plate (from A1 to A8), the antibody for the pregnancy test in the second row (B1 to B8), and the antibody against HIV in the third row of the plate (C1 to C8). You will add 100 µL per well. Pay attention! You must add the proper antibody in the proper wells and add exactly 100 ul/well. A summary of the application of the antibodies is as follows: Cell Biology Laboratory PCB 4023 8
6. Place the lid on and incubate your plate for 30 minutes at 37°C in the incubator.
7. After the incubation is complete, dispose of your plate’s contents in the sink and take it back to your bench.
8. Wash the wells 3 times with 200 µL of PBS-T20 each time. Let each washing solution sit on the plate for 1 min. Invert and dry the plate on a paper towel after each wash.
9. Wash the wells 1 time with 200 µL of PBS.
10. Add 50 µL of the substrate (TMB) for Horseradish peroxidase (HRP) to each well.
11. Cover the plate with the lid and develop at room temperature (your bench) for 15 minutes against a white background. Agitate the plate every 3-4 minutes with a circular motion (with the bottom of the plate always touching the bench).
12. Record your results (+ or -) in the template below and share your results with the class in the table on the board.
RESULTS:
ZIKA (A1-A8): A6, A7, A8 came out positive.
HCG (B1-B8): B3, B4, B7 came out positive.
HIV (C1-C8): C5, C8 came out positive.
QUESTIONS:
1.) What basic principles of antibody-mediated immunity are utilized in an ELISA assay?
2.) What might have caused some positive results to be lighter in color than others?
3.) Why can some viruses not be targeted by antibodies for destruction? Is this a limitation for developing an ELISA-based protocol for detecting the disease?
4.) Postulate a hypothesis on how the patients infected with Zika contracted the disease (in time and space); consider that two of them live nearby and the other visits them frequently.