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Antigen-Antibody Testing

Lesson Plan

Learning objectives:

This exercise demonstrates the biological phenomenon of the formation of a precipitate when an antigen reacts with an antibody. The exercise can be used to illustrate the specificity of antigen-antibody reactions, showing that a precipitation reaction only occurs when an antibody reacts with the antigen that was used to induce the formation of the antibody. The exercise is also a general demonstration of diffusion.

Exercise description:

Molten solutions of agar or agarose are poured into petri plates and allowed to harden. Wells are cut into the solid gel layer using a plastic pipette. Solutions of simulated antigen and antibody preparations are placed in the wells. The antigens and antibodies diffuse into the gel layer and react to form a precipitate.

Time frame:

Solutions of the simulated antigens and antibodies will need to be prepared the first time that the exercise is performed. This will require about 30 minutes. The solutions are stable, however, and will only need to be replenished when they are depleted. Gels will need to be prepared each time that the exercise is performed. Depending on the number of gels prepared and the method used to heat and dissolve the agar or agarose, this will require 30 to 45 minutes. The number of students involved, the availability of equipment and supplies, and the manual dexterity of the students will influence the amount of time needed to set up each gel in the classroom, but they can often be prepared within 5 to 15 minutes. The gels need to stand for 45 min to one hour for the precipitin lines to appear. The lines will become more apparent the longer that they stand.

Study questions:

1. What is diffusion?
   
   
2. What is an antibody?
   
   
3. What is an antigen?
   
   
4. When silver nitrate (simulated Antibody A) reacts with sodium chloride (simulated Antigen a), what is the precipitate that forms? When lanthanum nitrate (simulated Antibody B) reacts with potassium fluoride (simulated Antigen b), what is the precipitate that forms?
   
   
5. In the single diffusion test (where sodium chloride is incorporated into the gel and silver nitrate solutions of differing concentrations are placed into the wells), why are some rings larger than others?
   
   
6. In a double diffusion test with silver nitrate in the center well and sodium chloride solutions of differing concentrations in the outer wells, why do some lines form earlier than others? Which lines form first? Why? Why do the lines of precipitate not cross? Which lines are closer to the center well? Why?
   
   
7. In double diffusion tests (with silver nitrate and lanthanum nitrate together in the center well and sodium chloride and potassium fluoride separately in outer wells), which precipitate lines form first? Why? Why do the precipitate lines cross?

Answers to Study Questions:

1. What is diffusion?

Diffusion is the movement of suspended or dissolved molecules (or ions or particles) from an area of high concentration to an area of low concentration. It is the net movement of such molecules (or ions or particles) down their concentration gradient. In the absence of other forces, molecular motion and random spontaneous collisions cause the net outward movement of molecules (or ions or particles) from one region into a neighboring region where they are less concentrated. Because collisions are more frequent where the molecules are most crowded together, the movement is from an area of high concentration to an area of low concentration.

2. What is an antibody?

An antibody is a globular protein molecule that has binding sites complementary to and specific for combining with antigen molecules. Antibodies are produced only by B-lymphocyte cells in the blood of vertebrate animals. The antibodies are positioned either on the surface of the B-cells or are secreted into the blood plasma. Antibodies are sometimes called immunoglobulins. They are part of a higher animal's defense mechanism against disease. After their formation, antibodies will combine only with antigens identical or very similar to the one that induced their formation.

3. What is an antigen?

An antigen is an antibody generator. It is any molecule that is recognized as foreign by the body of a vertebrate animal and which triggers an immune response, inducing the formation of antibodies. Most natural antigens that elicit antibody responses in animals are protein molecules.

4. When silver nitrate (simulated Antibody A) reacts with sodium chloride (simulated Antigen a), what is the precipitate that forms? When lanthanum nitrate (simulated Antibody B) reacts with potassium fluoride (simulated Antigen b), what is the precipitate that forms?

The reaction of silver nitrate (Ag⁺ = simulated Antibody A) with sodium chloride (Cl^- = simulated Antigen a) forms silver chloride (AgCl) as a precipitate. The reaction of lanthanum nitrate (La⁺⁺⁺ = simulated Antibody B) with potassium fluoride (F^- = simulated Antigen b) produces lanthanum fluoride (LaF₃) as a precipitate.

5. In the single diffusion test (where sodium chloride is incorporated into the gel and silver nitrate solutions of differing concentrations are placed into the wells), why are some rings larger than others?

The diameters of rings around some wells are larger than others because the concentration of silver nitrate in those wells is higher. The diameter of the ring around a well is proportional to the concentration of silver nitrate that is in the well.

6. In a double diffusion test with silver nitrate in the center well and sodium chloride solutions of differing concentrations in the outer wells, why do some lines form earlier than others? Which lines form first? Why? Why do the lines of precipitate not cross? Which lines are closer to the center well? Why?

Some lines form earlier because the concentration of sodium chloride in the wells is higher than in other wells. The higher the concentration, the more rapidly the diffusion will occur; thus, molecules coming from wells with higher concentrations will encounter one another sooner and form the visible precipitate sooner. The lines that form first are the ones with the highest concentration. The lines of precipitate do not cross because the antibodies react with the antigens as they encounter them; thus, no antibody molecules diffuse beyond the line of precipitation and the lines do not cross.

7. In double diffusion tests (with silver nitrate and lanthanum nitrate together in the center well and sodium chloride and potassium fluoride separately in outer wells), which precipitate lines form first? Why? Why do the precipitate lines cross?

If the concentrations of all components are equal, the silver chloride precipitate line will generally form first, because the silver and chloride ions diffuse more readily into the gel than the lanthanum and fluoride ions. The precipitate lines cross because of the specificity of the reactions involved. Lanthanum will not react with chloride, so lanthanum diffuses through the silver chloride precipitate line and reacts only with fluoride. Likewise, the silver does not react with fluoride, so silver diffuses through the lanthanum fluoride precipitate..

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