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.
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
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
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
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
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
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
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
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
20. Wash away the unbound antibodies by
performing the wash two times.
21. The wells should be empty after the second
22. Add 50 μl of the Secondary antibody SA
into each of the 8 wells. The second
antibody is bound (conjugated) to the