ELN Lab 12 ELISA FINAL 6-9.docx

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University of Florida
Microbiology and Cell Science
MCB 3020L

ELISA STD Purpose The purpose of this experiment is to learn the methods and principles behind the three types of ELISA tests: Direct ELISA, Indirect ELISA, and Sandwich ELISA. We will then use the indirect ELISA test in order to determine if our “bodily fluids” have been infected after direct contact with someone else. Hypothesis If the ELISA test is performed correctly, and our “bodily fluids” have been mixed with someone who is “infected”, the test should indicate that we are too infected. Based on the fact that STD’s tend to spread rapidly if not cared for, regardless of how many people begin infected, a majority will end up infected as well. Procedure Workflow of the ELISA procedure 1. Apply a sample of known antigen to a surface, often in the well of a microtiter plate. The antigen is fixed to the surface to render it immobile. 2. The plate wells or other surface are then coated with blocking buffer. 3. Detecting antibody, usually diluted in blocking buffer, is applied to the plate for binding to the antigen coated on the plate. 4. The plate is washed, so that unbound antibody is removed. After this wash, only the antibody-antigen complexes remain attached to the well. 5. The second antibodies, which will bind to any antigen-antibody complexes, are added to the wells. These second antibodies are coupled to the substrate-modifying enzyme. 6. Wash the plate, so that excess unbound antibodies are removed. 7. Apply a substrate which is converted by the enzyme to elicit a chromogenic or fluorescent signal. 8. View/quantify the result using a spectrophotometer or other optical device. Protocol for Direct ELISA 1. Label an Eppendorf tube with “bodily fluid” from the rack with your initials. 2. Take a 8 well microplate strip and share it with your partner (wells 1-4 for student 1 and the second set of wells 1-4 for student 2) 3. Take 50 l out of the “bodily fluid” tube and transfer it to your well #1 = no sharing partner = control. Were you infected to start with? 4. Pair up with another random student in the class. Write down that student’s name. = Sharing Partner #1 5. Using a transfer pipette, mix your bodily fluid with sharing partner #1 by transferring all of your “bodily fluids” (~500-750 l) into Sharing Partner #1’s Eppendorf tube. Mix gently. 6. Transfer half of that mixture (~500-750 μl) back to your empty original sample tube. 7. Take 50 l out of your tube and transfer it to your well #2 = sharing partner 1. Was your first partner infected? 8. Pair up with a second student and repeat steps 5-7 = sharing partner #2. 9. After bodily fluid exchange, take 50 l out of the tube and transfer it to your well #3 = sharing partner 2. Was your second partner infected? 10. Pair up with a third student and repeat steps 5-7 = sharing partner #3. 11. After bodily fluid exchange, take 50 l out of the tube and transfer it to your well 4 = sharing partner 3. Was your third partner infected? 12. Negative control and Positive control, will be performed by your TAs 13. Continue with the protocol with your original lab partner and perform the ELISA procedure to determine if the “HIV antigen” is present in your bodily fluid samples. 14. Incubate your protein antigens = disease markers at room temperature for 5 minutes to allow the protein samples to bind to the well. 15. Wash a. Tip the strip over onto paper towels and gently tap the strip on them to remove the liquid. Make sure that no liquid splashes back up into the wells. b. Using a new transfer pipet, add wash buffer to each of the wells. c. Tip the strip over onto clean paper towels and tap gently. d. Throw away the wet paper towels. 16. Repeat the wash one more time. 17. The wells should be empty after the second wash. 18. Add 50 μl of the Primary antibody PA to each of the 8 wells. This antibody binds specifically to the disease marker 19. Incubate at room temperature for 5 minutes to allow the antibodies to bind to the antigen. 20. Wash away the unbound antibodies by performing the wash two times. 21. The wells should be empty after the second wash. 22. Add 50 μl of the Secondary antibody SA into each of the 8 wells. The second antibody is bound (conjugated) to the enzyme horseradish
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