Biology Lesson: Food Forensics: A Case of Mistaken Identity

Abstract
This 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?"

Objectives
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 system.

National Science Education Standard:
Content Standard A – Science as Inquiry

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 investigations.

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
THE CELL
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.

Teacher Background
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 diffusion assay.

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 line.

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 typical allergic symptoms. An extreme response is called an anaphylactic response.

Resources
This lesson is from Access Excellence. Access Excellence is an incredibly rich source of lessons for teachers.
http://www.accessexcellence.org/AE/AEC/AEF/1995/grupe_identity.html

Activity
Before activity:

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.

Day 1
Experiment 1:

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 interest.

2. Highlight that different people are allergic to different and specific substances. This will be explained later by antigen-antibody specificity.

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, before allowing a group to proceed with actual food and reagent.

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 should 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 center well.
vi. Plates can be stored overnight in a flat position at room temperature.

Day 2

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 antibodies.

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 Saga:

Mystery:

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 something else?

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.

Day 3
Experiment 2:

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.

Day 4
Follow up:

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?

Embedded Assessment

1. 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 and allergies?

Extension

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.

Embedded Assessment