Edvotech Instructional Videos
The enzyme-linked
immunosorbent assay or ELISA.
For this experiment, you will need a
microtiter plate, phosphate-buffered saline,
antibody and antigen samples,
a color-changing substrate solution,
a 37-degree incubator,
an adjustable micropipette and tips,
and transfer pipettes.
Step 1: Label the microtiter plate
and transfer pipettes as noted
in the product literature.
Step 2: Add 100 microliters of the
antigen solution to all of the wells.
Step 3: Incubate for five
minutes at room temperature.
At this time, the antigens will
nonspecifically adhere to the plastic
through hydrophobic and
electrostatic interactions.
Step 4: Remove all the liquid from
the wells using a transfer pipette.
Step 5: Wash the wells by adding approximately
eight drops of PBS buffer to each well.
Remove all the PBS from each
of the wells taking care to prevent
the buffer from spilling
over into adjacent wells.
Traditionally following this step,
the wells are blocked with a protein
containing buffer to prevent
nonspecific interactions between
the antibody and the plastic wells.
We have optimized this experiment
to eliminate this step.
Step 6: Add reagents as outlined
in the product literature.
Remember to use a clean
micropipette tip for each reagent.
Step 7: Incubate the plate for
15 minutes at 37 degrees Celsius.
Step 8: Remove the liquid from
the wells using the appropriately
labeled transfer pipette.
Step 9: Wash each well with fresh PBS.
Remove the liquid using the transfer
pipette designated for each sample.
Step 10: Add 100 microliters of the
secondary antibody to each of the wells.
Step 11: Incubate for 15
minutes at 37 degrees Celsius.
Step 12: While the samples are incubating,
prepare the detection substrate as specified
in the product literature.
Step 13: Remove the secondary antibody
solution from the wells using the transfer
pipette designated for each sample.
Wash each well once with fresh PBS buffer.
Remove the liquid using the transfer
pipette designated for each sample.
Step 14: Add 100 microliters of the
substrate solution to each weld.
Step 15: Incubate at 37 degrees
Celsius for five minutes.
Step 16: Examine your results.
Differences between negative and
positive samples will be obvious
with most positive samples
turning brown in color.
If color is not fully developed after
five minutes, incubate at 37 degrees
Celsius for a longer period of time.
