Overview of ELISA Experiments
These ELISA experiments differ in an important way from the
previous assignments you've had in this course. Previously,
you've been given a recipe to follow, and the most challenging
parts may have been to understand the principles, the rationale
for the method's recipe, and to interpret your results. In ELISA,
we're adding an additional challenge. This makes it more like a
real-life experiment -- more like what most of you will need to
learn how to do in your future employment.
The additional challenge is to design and select the individual
tests that make up each experiment. Each ELISA experiment will
consist of several dozen individual tests, all done at once in a
single plate (capable of holding up to 96 test wells). Your lab
manual gives a recipe for conducting such tests, but doesn't
specify in detail exactly which tests are needed. You'll have to
make enough decisions to answer the questions at the bottom of page
108. Perhaps most importantly, you'll have to figure out what
kinds of information you need to get from control tests in order
to interpret your tests on unknown samples. You have to decide
what control tests to do, and then do them. If your decisions are
ill-founded, you'll simply have to redesign the experiment and
then do it over. (That's real life!)
Each student should do her or his own experiments individually. It is
not necessary to work in pairs for the ELISA experiments. Of course you
should discuss your plans and help each other freely.
We have allotted four classes to ELISA. Generally
we suggest that you use your time this way:
- Day 1 (mostly dry; only COAT available)
- Get a good understanding of our ELISA methods
and the principles behind them.
- Plan your first experiment (standard curve
only), including the layout of test wells in your plate (use the
form on page 112).
Be sure to check your plan with a TA.
- Coat 96 wells (see "Coating Wells" on page 109). Store
the coated wells dry between classes.
- Write a clear procedure that will enable you to do
this first experiment on Day 2 efficiently and without making
mistakes. This includes details for all dilutions you'll need to
make, including how much to make of each one.
- Day 2 (all ELISA reagents available)
Perform your first ELISA experiment.
- Graph and interpret the results.
- Day 3 (all ELISA reagents available):
- Design your second experiment. If your first was inconclusive,
you should focus the second experiment on getting an
optimal standard curve.
If your first standard curve was solid, add some unknowns in
your second experiment. ("Unknowns" are the samples from various
stages of your IgG purification.)
- If you have time, perform your second ELISA experiment. If not,
do it on Day 4.
- Store unused coated wells for use on Day 3.
Coat additional wells only if you expect to need more than you have.
- Day 4 (all ELISA reagents available):
- Continue planning and performing ELISA experiments to get optimal
standard curves, and to analyze
as many unknowns as time permits.
- Day 4 or at home:
- Complete graphing and interpreting your results.
- Organize your records and results neatly in your lab book.
- Summarize your major results in the running summary in the
front of your lab book.
- There is no lab report for ELISA. Your lab book will be graded
for clear and correct
presentation of your ELISA results, and the quality of