Science in Motion
Ursinus College
Name_____________________________
Protein Identification Through Immunoassay
“THE CASE OF THINGS THAT GO BUMP IN THE NIGHT”
Standards:
3.2.7 Apply process knowledge to make and interpret observations
3.6.10 Apply technologies that relate to propagating, growing, adapting,
treating or converting
3.2.12 Evaluate experimental information for appropriateness
Introduction and Background:
Immunoassay is one method of determining the presence of biochemicals in a chemical
mixture or biological sample. This technique identifies chemicals by unique features of
the chemical’s shape and surface charges. The assay uses antibodies to detect the
presence of compounds in a sample. The chemical’s presence is known when it bonds to
an antibody and forms a detectable clump known as a precipitation band.
Blood typing is one of the earliest forms of immunoassay. It was developed by an
Austrian scientist, Carl Landsteiner.
To design an immunoprecipitation test, researchers must first isolate the chemical or
antigen that they interested in detecting. They inject the antigens into a laboratory
animal. In response to the injection, the lab animal produces antibodies specifically
designed to adhere to that antigen. The researcher then collects the animal’s blood. The
blood is then purified in a manner that yields a supply of that particular antibody.
Immunoprecipitation assays are regularly used by physicians, biologists, and forensic
scientists. They are used to determine the presence of disease, evolutionary relationships,
and the origin of biological samples found at a crime scene. It is also used by the U.S.
Department of Agriculture and the Food and Drug Administration to identify the
components in food. They run tests on a variety of proteins to make sure the food is
labeled correctly and there are no impurities. For example, sometimes soybean and pork
proteins end up in hamburgers that are supposed to be 100% beef.
Dried antibodies can be attached to paper or plastic to make a “trap” for chemicals. Then
the trapped chemical can be analyzed using other tests. This is the basis of the enzymelinked immunosorbent assay (ELISA) used in medicine and agriculture to look for
disease causing organisms. Home pregnancy tests use this principle to find the presence
of a pregnancy hormone in a urine sample. This test also can be used to look for the
presence of hazardous chemicals in soil and drinking water. Recently, immunological
testing has been used to find particular sequences of DNA.
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Guiding questions:
1. If you were allergic to peanuts, how could a scientist find out if there was
peanut protein in a certain food product?
2. What are your body’s defenses against disease?
3. What are antibodies and antigens and what are their roles?
Important definitions:
Antibodies: Proteins produced by white blood cells (B-cells) in response to a
foreign chemical or disease organism in the body. Antibodies bond to the foreign
substance, aiding the body’s ability to recognize and remove the foreign material.
Antigen: A term for a foreign material in the body. Antigens could be
carbohydrates, lipids, nucleic acids, or proteins. They stimulate an immune response in
animals with an immune system.
Antiserum: Antiserum is serum containing antibodies
Assay: A term used for chemical testing.
Immunoprecipitation: The binding of an antibody to a specific antigen which
comes out of solution and forms a clump.
Serum: The liquid portion of the blood.
Diffusion: The natural spreading out of molecules due to molecular motion.
Materials:
Petri Dishes
Droppers of serum and antiserum
Sharpie Fine Point Markers
Straws
Safety
Safety glasses should be worn but there are no unusual safety concerns in this lab.
Avoid contact with the solutions and the skin or mucous membranes because they may
cause irritation. Have students wash hands before and at the conclusion of the lab.
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To begin the lab, read the short story below and then follow the procedure,
THE CASE OF THINGS THAT GO BUMP IN THE NIGHT
Officer Rick Williams noticed a 2000 Porshe doing 75mph in a 35mph zone. He stopped
the driver, Bill Bonta, for speeding. When the officer making the stop was walking to the
car, he noticed what appeared to be blood on the bumper. There recently had been a hit
and run in the area involving an injury to a pedestrian. A witness identified the car
involved as some type of sports car. The blood made Officer Williams suspicious. When
questioned, Bill said that the blood came from some small animal that he accidentally hit
that evening. Blood was taken from the bumper for identification. It is your job to
determine the source of the blood- human or animal.
Procedure
Preparing the wells
Obtain a Petri dish with agar.
1. Turn the dish upside down so the agar is on the top.
2. Make a template of Figure 2b (see next page) on the bottom of the Petri dish. The
labels will be reversed so you will be able to read them when the dish is turned
over.
3. Turn the dish over and remove the lid.
4. The template should show clearly through the agar and look like Figure 2a.
5. Carefully press the end of a straw into the circle labeled Y in Section A. The
straw should pass through the agar and hit the Petri dish bottom.
6. Turn the straw while in the agar and then tilt to the side.
7. Slowly withdraw the straw which should leave a hole or well in the agar.
8. Remove the plug from the straw by squeezing the straw behind the plug and place
the plug in the lid of the Petri dish.
9. Continue making the rest of the wells.
Conducting the analysis
1. Add blue food dye to well B in section A one drop at a time until the well is full.
Caution: Be careful not to overfill the well and do not let the tip of the pipette
touch the bottom of the Petri dish as this may force the liquid under the agar.
2. Add yellow to well Y.
3. Now move to section B. Add Human Serum (HS) to the center well and Human
Antiserum (HA) to the left well and Chicken Antiserum (CA) to the right well.
4. Set up the wells in section C. Carefully add Unknown Serum (?S) to the center
well. Add the antisera in the following order:
a. Human Antiserum (HA) to the upper left well
b. Chicken Antiserum (CA) to the lower left well
c. Cat Antiserum (CatA) to the upper right
d. Dog Antiserum (DA) to the lower right
5. Place the plates in a cool place where they will not be disturbed.
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Observing the results
The results should be fully visible in 45 minutes. If the plate cannot be observed
at 45 minutes, it can be observed the next day.
Finishing up
At the completion of the lab the Petri Dishes may be disposed of in the trash.
Questions
1. What is the difference between antibodies and antigens?
2. How does immunological testing work?
3. What is the main goal of this particular lab?
4. What happened in section A of the Petri dish? What did this section demonstrate?
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5. What happened in section B of the Petri dish? What was this section meant to
show?
6. What happened in section C of the Petri dish? What was the purpose of this
section?
7. Did Bill hit the pedestrian? Explain your answer.
8. Describe three possible uses of immunological testing.
Adapted from “Biotechnology on a Shoestring”
Kingwood College
Template developer – Brian Shmaefsky,
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Teacher Notes
Science in Motion
Ursinus College
Protein Identification Through Immunoassay
“THE CASE OF THINGS THAT GO BUMP IN THE NIGHT”
Time needed to complete lab: 30-45 minutes one day to introduce the material and set
up Petri dishes- some time needed the next day to observe
results
Targeted grade level: 10th-12th
Objectives:
At the completion of this lab students will be able to:
 Describe the principles and purpose of immunoassay testing
 Explain the importance of antibody-antigen interactions to the assay
 List three uses of this form of testing
 Explain the limits of this test
Major Concepts
This is an easy lab but many students don’t understand what they are actually doing.
Living things have chemical markers that allow our bodies to recognize what is foreign in
our bodies from what is not. Foreign substances are usually referred to as antigens. They
are often proteins but they do not have to be. Antibodies are made by your body (by Bcells) to help destroy these invaders. Antibodies are specific. A particular antibody will
only attack one antigen. In this lab, they are using specially produced antibodies to find
specific compounds (antigens) The models are to show that when an antibody attaches
to an antigen (both of which usually have a high molecular weight) they will bond and
when they do, they make a complex that is even larger and will precipitate out of
solution. There is another antibody model that will not bond and therefore shows that
antibodies must be specific for an antigen.
When blood sits for a long time or is centrifuged, it separates into plasma, white blood
cells, and red blood cells. In the plasma are the clotting factors. If the plasma is agitated,
a clot will form. If the clot is removed, what is left is called serum. If this serum came
from a human it would be called human serum and would have markers (antigens) that
would identify it as human. If this serum was injected into a rabbit, the rabbit would
make antibodies because this material is foreign and the resulting solution would be
called human antiserum. Human antiserum could be used to find human blood because it
contains antibodies that will attack human serum (antigens). Antibodies could be
produced that could identify almost anything. All that basically needs to be done is to
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inject the substance you want to find into an organism and use the organisms natural
ability to make antibodies. The antibodies produced can be used to test for your
compound.
Preparation
All materials for this lab are delivered and just need to be distributed to lab tables. If you
do not plan to do this lab the next day after delivery, the Petri dishes should be put in the
refrigerator to prevent drying out.
Typical Results
Quadrant A should show two expanding circles of color.
Quadrant B should have a white precipitation line between the human serum and the
human antiserum. No line will form between the human serum and the chicken
antiserum.
Quadrant C should have a white precipitation line between the human antiserum and the
unknown serum.
Answers to questions
1. Antigens are chemicals that your body can identify. They allow us to distinguish
foreign matter that might try to enter our bodies. Antibodies are special proteins that are
produced by white blood cells that attach to foreign antigens and cause them to clump
together. This clumping makes disposal by other white cells (phagocytes) easier.
2. To perform this test, the chemical that needs to be tested for is injected into some
animal. The animal will make antibodies for the substance. These antibodies are
removed from the blood serum making an antiserum. When this antiserum is exposed to
the original chemical, it will bond with it and form a precipitate.
3. The main goal of this lab is to demonstrate an important technique in biotechnologyimmunoassay. Using this technique, scientists can develop a fairly simple test for almost
any chemical compound.
4. In this lab, quadrant A is just to show diffusion. Since the other solutions are
transparent, this quadrant allows you to see the material diffusing through the agar which
is a semisolid (colloid).
5. Quadrant B shows that human serum and human antiserum will form a precipitate line
because corresponding antigens and antibodies get together but that human serum and
chicken antiserum will not.
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6. In quadrant C, there will be a precipitate line between the unknown serum and the
human antiserum. This demonstrates that the blood on the bumper is human.
7. The precipitation line indicates that the blood was human but it does not prove,
however, that the blood was from the pedestrian. A DNA analysis would need to be done
to prove that. It could be blood from any human even the driver of the car.
8. Immunoassay can be used to test for many things. The USDA uses it to identify the
components of food. The EPA is using it to test for specific pollutants in water samples.
Blood testing for the major blood types (A,B,AB&O) is an immunoassay. Home
pregnancy tests and test for specific disease organisms are done by immunoassay. This
test will even find specific sections of DNA.
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Science in Motion
Ursinus College
Immunoassay Prequiz
1. The main purpose of an immunoassay is to
A. demonstrate a person’s ability to defend against disease
B. demonstrate that a particular antibody is present
C. test for the presence of a chemical in a sample
D. make vaccines for diseases
2. What is the important component in an antiserum
A. antibodies
B. antigens
C. precipitate
D. both antibodies and antigens
KEY
Y- yellow dye
B- blue dye
HA- human antiserum
HS- human serum
CA- chicken antiserum
?S- unknown serum
CatA-cat antiserum
DogA- dog antiserum
- precipitate line
3. What does the data from figure 2a, quadrant C indicate
A. the unknown was dog blood
B. human antiserum reacted with human serum
C. the unknown serum was from a cat
D. the unknown serum could either be cat or dog
4. In quadrant B in the above diagram,
A. the line between HA and HS would form but not between HS and CA
B. the line between HS and CA would form but not between HA and HS
C. both lines are possible
D. neither line should form
5. Material moves through the agar in this test because of
A. gravity
B. diffusion
C. attraction of opposite charges
D. osmosis
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