Warlord’s Revenge - Can You Dig It?

Developed by: Becky Aulenbacher, Loretta Fortner, Thomas Kazanecki,
Judy Nicholson, and Andrew Shaw
© American Association of Immunologists 1990

Focus
This activity allows students to understand how immunologists use antigen-antibody interactions to study diseases. The concepts of self/non-self recognition and the specificity of antigen-antibody reactions are essential to understanding this lesson.

Description
This activity involves three laboratory procedures. Antibodies can be precipitated from the serum by addition of an inorganic salt. In the first experiment, antibodies are precipitated by the addition of ammonium sulfate. A simple explanation for this phenomenon is that the salt ions bind to the surface of the antibodies. When the antibody-salt complex reaches a particular density, it comes out of solution and settles to the bottom of the liquid. This test represents a check to determine whether or not the immune system is functioning.

In the second experiment, the reaction of an antibody with its specific antigen results in the clumping of red blood cells. This clumping is called agglutination. The reaction is dependent upon the antigen being bound to a red blood cell or other insoluble particle. This test represents a check for IgM immunoglobulins.

The third test is the ELISA: Enzyme Linked Immunosorbant Assay. It is the most specific of all the tests, and is used to detect the presence of an antibody. When the antibody is present, a color change will be produced: the greater the color change, the greater the amount of the antibody. A quantitative measurement of the antibody can be made by carefully measuring the optical density. Each antigen stimulates the production of a specific antibody. We use this specificity to study the immune reactions. This test represents a check for the presence and amount of IgG immunoglobulins.

Curricular Context
This lesson can be used with units dealing with the topics of immunology, diseases, blood and circulation, or personal and community health.

Prior Knowledge
Students need basic laboratory skills and prior knowledge about antigens, antibodies, precipitation and agglutination reactions.

Objectives
Upon completion of this activity the student should be able to:

1. demonstrate the ability to follow specific instructions in a protocol and obtain correct results;
2. apply scientific methods to solve a problem;
3. work in a team setting where his/her success depends upon cooperation;
4. follow safety precautions inherent in immunologic research;
5. define the terms "antigen" and "antibody" in his/her own words;
6. illustrate by means of diagram a simple precipitation reaction (e.g. antibody/salt reaction);
7. state the relationship between the presence of an antibody and a color change in an ELISA;
8. explain the relationship between the exposure to a given antigen and the presence of a specific antibody;
9. propose and test hypotheses about the presence of antigens and antibodies;
10. explain how agglutination can be used to test for the presence of an antibody;
11. form a conclusion using the results from assays and laboratory tests;
12. describe careers in immunology and related areas of research.

Suggested Time
Preparation time: two hours
Instructional time: three class periods (45-50 minutes each)

Day One: Introduction to Warlords Revenge/pre-lab discussions

1. Read student packet
2. Discuss purpose and procedure for each of the 3 laboratory tests
3. Demonstrate precipitation, agglutination and serial dilution
4. Answer introduction questions

Day Two: Laboratory Activity

1. Perform lab and fill out data table
2. Answer procedure questions
3. Complete conclusion

Day Three: Interpretation of Class Data

1. Complete discussion questions and for further research in small groups
2. Complete student evaluation of lab experience individually
3. Collect student packets for grading

Materials
The following equipment will be needed for each student team (2-3 students are recommended per team):

• 1 microcentrifuge (1.5 ml) test tube
• 1 regular test tube
• 3 pipettes
• 5 paper towels
• 1 clean microscope slide
• 1 toothpick
• 1 clean white spot plate (with 10-12 depressions)
• 2 dropping bottles (for ammonium sulfate and SUBSTRATE solutions)

The following materials will be needed for 25 teams (2-3 students are recommended per team):

• 110 g ammonium sulfate
• 200 ml distilled water
• 2 ml diluted alkaline phosphate (the ENZYME)
• 100 ml glycine buffer
• 25 (5 mg) tablets phosphatase substrate (the SUBSTRATE)
• 50 ml bovine serum
• 2 ml anti-sheep red blood cells (the ANTIBODY)
• 25 ml sheep blood
• 0.75 g salt (for saline solution)

To receive a copy of this curriculum, please contact: The American Association of Immunologists, telephone: (301) 634-7178, fax: (301) 634-7887, email infoaai@aai.org.