egg albumin (Sigma Chemical Co.) , 2 x - 1.5% agar
plates , 3 x 6 mm diameter soda straw, toothpick
Glass marking pen
Small quantities of:
Raw egg white (diluted 1:625)
Uncooked egg-enriched pasta (1:40)
Uncooked egg-free pasta (1:40)
Samples of various foods:
some positives (egg-containing) like mayonnaise (1:10), custard (1:10), pasta
(1:40), baked items (1:10), egg white
some negatives (without egg) like sugar, salt, milk, beef broth, molasses, etc.
The dilution is not critical on negatives.
(Note: the author suggests leftovers from refrigerator)
(For dilutions use 1gm of solid foods or 1 ml of
liquids in .85% saline. For egg white dilution, start
with a 1:25 dilution and dilute first dilution again
1:25. For dilutions of solids, use only the supernatant.
Sterile technique is not necessary and some inaccuracy
in dilutions is allowable.)
fine-tipped dropping pipettes (plastic)
test tubes or flasks for food solutions
Salad Saga -
lesson is from the Access Excellence website, but is part
of a larger set of lessons that is available from the St.
Louis Mathematics and Science Education Center, 8001 Natural
Bridge Road, St. Louis, MO 63121. The lesson is designed
to serve as an introduction to the immune system. It can
stand alone or it can lead into further studies of the
immune system. The primary focus of this inquiry-based
lesson is antigen-antibody specificity. After focusing
the students' attention on allergic reactions, two hands-on
experiments allow students to explore the specific reaction
between an antigen and the antibody that recognizes it.
Students carry out an exploratory experiment leading to
the concept of specificity. A second experiment allows
students to apply the techniques and concepts learned in
the first activity and subsequent discussion to solve a
mystery. Follow-up discussion and problems apply the concept
of specificity to related topics.
This lesson can be used with high school students and can
be adapted to any level (biology or advanced biology) in
association with the study of biochemistry, cell biology,
health, or physiology. It can be used in association with
topics such as allergies, food safety, or antigen-antibody
precipitation. As a result of completing this lesson, students
will be able to answer questions such as: "Why am
I allergic to some things but not to others?" or "Why
does clotting occur when incompatible blood types are mixed?"
Students will be able to:
1. Develop an experiment to test a hypothesis.
2. Use their understanding of antigen-antibody specificity
to develop a hypothesis.
3. Articulate that the antibody-antigen specificity is
one part of the immune system.
4. Develop questions around the components of the immune
Standard A – Science
ABILITIES NECESSARY TO DO SCIENTIFIC INQUIRY
IDENTIFY QUESTIONS AND CONCEPTS THAT GUIDE SCIENTIFIC INVESTIGATIONS.
Students should formulate a testable hypothesis and demonstrate the
logical connections between the scientific concepts guiding a hypothesis
and the design of an experiment. They should demonstrate appropriate
procedures, a knowledge base, and conceptual understanding of scientific
DESIGN AND CONDUCT SCIENTIFIC INVESTIGATIONS.
Designing and conducting
a scientific investigation requires introduction to the major concepts
in the area being investigated, proper equipment, safety precautions,
assistance with methodological problems, recommendations for use
of technologies, clarification of ideas that guide the inquiry, and
scientific knowledge obtained from sources other than the actual
investigation. The investigation may also require student clarification
of the question, method, controls, and variables; student organization
and display of data; student revision of methods and explanations;
and a public presentation of the results with a critical response
from peers. Regardless of the scientific investigation performed,
students must use evidence, apply logic, and construct an argument
for their proposed explanations.
Content Standard C – Life Science
Cells have particular structures that underlie their functions. Every
cell is surrounded by a membrane that separates it from the outside
world. Inside the cell is a concentrated mixture of thousands of
different molecules which form a variety of specialized structures
that carry out such cell functions as energy production, transport
of molecules, waste disposal, synthesis of new molecules, and the
storage of genetic material.
Cell functions are regulated. Regulation occurs both through changes
in the activity of the functions performed by proteins and through
the selective expression of individual genes. This regulation allows
cells to respond to their environment and to control and coordinate
cell growth and division.
The major concept of this lesson is the specificity of
the reaction between an antibody and an antigen. Antibodies
are proteins produced by cells of the immune system in
response to the exposure of an individual to a foreign
substance (an antigen). This concept will be illustrated
through the use of an experimental procedure called a double
This assay is based on the formation of a precipitate (precipitin
line) when an antibody reacts with its specific antigen.
In this test, often called the Ouchterlony test, antibody
and possible antigens are placed in wells in agar plates
and allowed to diffuse toward one another. The antibody
is placed in a center well and antigens (specific or nonspecific)
are placed in surrounding wells. When an antibody and its
specific antigen meet one another and are at the proper
concentrations, the precipitate will form a visible white
line between the two wells. This line is called a precipitin
In the diagram below, a precipitin line can be seen
between the center well and wells 2 and 3. The fact
that the line
is continuous indicates that both wells contain the same
antigen. Antibodies and antigens that are not complementary
will diffuse past one another in the agar and will not
form a precipitate.
The scenario for this lesson is centered on hypersensitivity
to environmental antigens that are generally not particularly
harmful (e.g., pollen, dust mite excrement, mold, drugs,
food, etc.). In these situations the immune system reacts
to these antigens by producing a type of antibody known
as immunoglobulin E (IgE). IgE antibodies trigger the
release of histamine by mast cells which then lead to
symptoms. An extreme response is called an anaphylactic
This lesson is from Access Excellence. Access Excellence
is an incredibly rich source of lessons for teachers.
Most of the materials are cheap and easily obtainable.
Dilutions can be made up days in advance and stored until
needed. Plates should be made several days prior to use
to allow proper drying. Antibody is the biggest expense
but a little bit goes a long way. (2 ml supplies 50 teams
of 2) Out-of-date antibody would be cheaper and would
still work for these experiments. Antibody (Anti-Chicken
Egg Albumin) from Sigma Chemical Co., P.O. Box 14508,
St. Louis, MO 63178 Stock # C-6534 2 ml is about $50.
1. Start by asking students whether they have allergies.
What are their symptoms like? Do their friends and families
have similar allergies? Different allergies? This discussion
is not intended to result in answers, but rather to stimulate
2. Highlight that different people are allergic to different
and specific substances. This will be explained later by
3. Introduce the Fooling with Food activity. Explain to the
students that this is a chance for them to explore the interaction
between various foods (some negative and some positive) and
Reagent A (the antibody). At this time students have not
explored formal definitions of antibody or antigen, this
is purely exploratory. The figure below shows the relative
position of wells to be cut in the agar plates with the straws.
Toothpicks are good for removing the plugs from the wells.
Extra plates and colored water can be used first for students
to practice loading the wells. Only 1-2 drops with a fine-tipped
pipettes is needed per well.
4. Provide them with the following instructions and
model for them how to cut wells and load the wells.
i. Cut wells in agar using a template under the plate
as a guide.
ii. Remove plugs (toothpicks are helpful for this)
and label wells and plate.
iii. Practice loading wells using colored water.
You may want to set up a check system to make sure
that everybody is able to load the wells successfully,
allowing a group to proceed with actual food
iv. Select six different foods to load in the
six outer wells.
Give students about 10 foods to choose
from so there is variation in
selections. Different pipettes
be used for each food and foods
should not spill over edge of wells.
v. Go to the teacher to have
Reagent A is placed in the
vi. Plates can be stored
overnight in a flat position
at room temperature.
1. Let students find precipitin lines. Tell them only that
they may have to hold plates up to a light or toward
a window. Faint white lines will be seen by someone.
Then others will see.
2. Compile a list of positive foods and students will quickly
see that they all contain egg.
3. This is the time at which to introduce the terms antibody
and antigen. Explain to students that antibodies are produced
by the body as part of the complex of interacting immune
system components. Antibodies are a type of protein called
an immunoglobulin that can interact to effectively neutralize
or destroy antigen action in a number of ways. Ask students
if they know how the body is able to tell if something
is a foreign substance or an antigen. Ask also if all foreign
substances are bad. Are there times when you might want
to be able to allow a foreign substance in? How can the
body tell self from non self?
4. Explain that antigens are markers on the cells of a
foreign substance that trigger the body to recognize the
invading cells as non self and launch an attack. Antibodies
are part of the attack stimulated by the presence of foreign
antigens. Antibodies bind to antigens and initiate defensive
measures. Some students may have been exposed to this concept
in middle school. Express to the students that they will
be investigating further how antibodies and antigens interact
and other aspects of the immune system in a later lesson.
5. Explain what has happened in the test that they performed.
When the antibody is placed in the center well and antigen
is placed in the surrounding wells they each diffuse through
the agarose in all directions. Where the antibodies and
a specific antigen interact a precipitin line appears.
The Reagent A represented a specific antibody that binds
to antigens associated with egg. Multiple antibody molecules
can bind to each antigen molecule, which can form very
large complexes. These large complexes are so large they
form a visible precipitate. This Ouchterlony test can be
used to test a patient’s blood for the presence of
6. Ask students to explain the relationship between antibody
and antigen. Make sure that the class has a firm grasp
of the relationship to the extent that they have gone over
it. Check also that they understand the purpose of the
Ouchterlony test. Then explain to them that they will be
using this test to solve the mystery of Stan’s Salad
Stan's Salad Saga
(Let students read this)
As Stan lay in his hospital bed, red, swollen and gasping for breath,
he agonized over the cause of the near life-threatening reaction he had
suffered. All of his adult life he had known of his allergy to eggs.
His physician had made abundantly clear to him the severity of the reaction
that he could expect if he included eggs in his diet. Now he was suffering
from the very symptoms that had been predicted. He wasn't allergic to
lots of different things. Eggs were the only substance that could have
brought him to this extreme condition. Now he faced a multi-thousand
dollar hospital bill and his insurance agent was placing the blame on
him. The company would refuse to pay if Stan was shown to have been negligent.
He had been far too careful to have made a mistake on his own. He had
to somehow convince his agent that he was not at fault. Someone else
was responsible for his being here! For the benefit of both his insurance
agent, Carl, and his allergist, Judy, he recapped the activities prior
to this onset of anaphylactic shock.
It had been a typical day with the exception of his departure time for
work. Running late, he had not had time to eat breakfast or make his
lunch. He grabbed an apple on his way out the door. When the lunch hour
came, he went to the nearest branch of a local grocery chain to get a
salad bar. The pasta salad looked particularly appealing that day. Conscientiously,
Stan asked the salad technician whether any eggs were used in the salad.
He was assured that the salad was egg-free. Stan's decision was made.
His wife would be pleased that he was avoiding his usual high cholesterol
diet. Stan had walked to the park to eat his lunch and that was when
the crisis began. After eating only three or four bites of lunch, he
began to experience a burning sensation in his ears and had trouble breathing.
A police officer who happened to be nearby noticed his difficulty and
made a 911 emergency call. That is how Stan ended up in the hospital.
Knowing that Stan was not allergic to anything else that he had eaten,
the contents of the pasta salad became the immediate focus of the allergist's
attention. A sample had been brought into the hospital by an alert paramedic.
In addition to the pasta, it had contained tomatoes, onions, black olives
and an oil and vinegar dressing. Since all the other ingredients clearly
did not contain egg, the only possible source of egg was the pasta itself.
The salad technician had told Stan there was no egg in the salad. Had
a mistake been made? Had egg-enriched pasta been used? Or had Stan eaten
You are the lab technician asked to test for the presence of egg in the
pasta. Your evidence might place responsibility on the grocery store,
in which case the insurance company will pay Stan's medical bills. Or
you will show no evidence of egg in the pasta and Stan will be handed
the blame and will be forced to pay for his negligence.
1. Following the mystery presented yesterday students should
design a test to answer the question posed in the mystery.
Have them write up a hypothesis for the experiment using
an ‘if..then..’ statement. Let them know that
they should make sure both positive and negative controls
are included. With six wells you could test the unknown
(egg-enriched pasta), egg-enriched pasta (+ control), egg-free
pasta (- control), egg white - 1:625 dilution and 1:3125
dilution (+ controls that show a range of concentrations
that will result in formation of a precipitin line), saline
solution (- control that is used for all dilutions). Students
should create a data collection chart and identify within
the chart what are the positive and negative controls.
2. Experiment 2 is also a double-diffusion test as was
experiment 1. Now, however, students know that they are
putting different antigens in the outer wells and antibody
in the center well. They know why there will be positive
results and why there will be negative results so they
can predict which wells will have precipitin lines. Store
plates at room temperature until the following day.
3. Students should observe results and compare to predictions.
They should form a written conclusion about Stan's Salad
Saga (of course, Stan is innocent) incorporating the concept
of antibody and antigen into the conclusion.
4. Now you can refer back to your original discussion about
allergies and answer or further develop some of the unanswered
questions from day 1.
5. Can students think of other uses of specificity? Many
home pregnancy tests use an antibody to detect the presence
of human chorionic gonadotropin (HCG) that is present in
a woman's urine during pregnancy. The test for HIV also
involves formation of an antibody-antigen complex.
6. Inform the students that they will be looking in more
depth at antibody-antigen interactions and also addressing
other components of the immune system. Do they have questions
about the immune system?
Students’ ability to develop an ‘if…then’ hypothesis
and the subsequent conclusion that addresses this hypothesis
can be assessed by the students’ written pieces.
2. In the written hypothesis the ability to incorporate
a new concept, such as antibody-antigen interactions can
also be assessed.
3. During class discussion can students develop questions
about immunity and about the relationship between immunity
The Ouchterlony test is rather dated and has some limitations.
As an extension students can research the Ouchterlony test,
its predecessors, similar tests and those used now. Have
them place the invention along a time line and write a
paragraph about how improvements in technology affect our
understanding of the human body and disease.