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Food Forensics: A Case of Mistaken Identity

By Michael Grupe



ABSTRACT

This lesson is a summary of a mini-unit put together by myself and five other St. Louis area teachers during a summer internship in immunology. The entire packet with complete preparation instructions and student sheets can be obtained from the St. Louis Mathematics and Science Education Center, 8001 Natural Bridge Road, St. Louis, MO 63121.

This 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 like these: "Why am I allergic to some things but not to others?" or "Why does clotting occur when incompatible blood types are mixed?"


BACKGROUND INFORMATION

Notes for the Teacher:

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 around 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 leads to typical allergic symptoms. An extreme response is called an anaphylactic response.

Materials for experiments 1 and 2 (per class and per team of students):

  • Anti-chicken egg albumin (Sigma Chemical Co.)
  • 2 - 1.5% agar plates
  • 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: I clean out leftovers from my 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 supernatent. Sterile technique is not necessary and some inaccuracy in dilutions is allowable.)
  • fine-tipped dropping pipettes (plastic)
  • test tubes or flasks for food solutions

Preparation:

Most of these 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. Total prep time is about 2 hours.

Class Time Needed:

This lesson is designed for four 50-minute periods. Activities can be reorganized to fit your schedule.

Day 1:

  • Introduction and exploration
  • Class discussion of allergies
  • Exploratory activity

Day 2:

  • Gather/discuss data
  • Observe, record, discuss results of experiment 1
  • Concepts presented
  • Mystery read by students

Day 3:

  • Design experiment to solve mystery and set-up of experiment 2

Day 4:

  • Gather/discuss data
  • Follow-up
  • Observe, record, discuss results of experiment
  • Form conclusion (solve mystery)
  • Return to day 1 questions
  • Solve application problems


LESSON

Day 1:

A quick discussion during which students are questioned about their allergies and symptoms and how their allergies compare to the allergies of family members or friends. This discussion is not intended to result in answers, but rather to stimulate interest. Highlight that different people are allergic to different and specific substances. This will be explained later by antigen-antibody specificity.

Experiment 1:

Fooling with Food is a chance for students to explore the interaction between various foods (some negative and some positive) and Reagent A (the antibody). 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.

  1. Cut wells in agar using a template (teacher prepared) under the plate as a guide.
  2. Remove plugs and label wells and plate.
  3. Students 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.
  4. Reagent A is placed in the center well. (I suggest teacher does this because of expense.)
  5. Plates can be stored overnight in a flat position at room temperature.

(graphic)

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 all are egg-containing.
  3. Teacher can now discuss specific interaction between antibody (reagent A) and antigen (albumin in egg). Terms can be presented at this time. Basic (forked) structure of antibodies that allows for cross-linkage and formation of precipitate can also be discussed.

Mystery:

Stan's Salad Saga
(Let students read this or assign)

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:

Students (or teacher) can design a test to answer the question posed in the mystery. 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).

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. Again, store plates at room temperature until the following day.

Day 4:

Follow-up:

Results are observed and compared to predictions. A conclusion is formed about Stan's Salad Saga (of course, Stan is innocent).

Now you can refer back to your original discussion about allergies and answer some of the unanswered questions from day 1. Other uses of specificity can also be discussed at this time. 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.

Challenge Problems:

I have not included these but they are reviews and extensions of the terms and concepts presented in this lesson. They are part of the entire packet, should you decide to decide to get the activity packet from the Mathematics and Science Education Center of St. Louis.


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