ELN Lab 11 ELISA STD
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
direct 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 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 antibodyantigen
complexes remain attached to the well.
5. The second antibodies, which will bind to any antigenantibody complexes, are added to the wells.
These second antibodies are coupled to the substratemodifying 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
2. Take a 8 well microplate
strip and share it with
your partner (wells 14
for student 1 and the
second set of wells 14
for student 2)
3. Take 50 m 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
5. Using a transfer pipette, mix your bodily fluid with sharing partner #1 by transferring all
of your “bodily fluids” (~500750 m l) into Sharing Partner #1’s Eppendorf tube. Mix
6. Transfer half of that mixture (~500750 μl) back to your empty original sample tube. 7. Take 50 m 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 57 = sharing partner #2.
9. After bodily fluid exchange, take 50 m 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 57 = sharing partner #3.
11. After bodily fluid exchange, take 50 m 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.
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
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 min