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Materials and Methods: Antigen-Antibody Testing

Materials needed are:

• Agar or agarose
plates with wells
2 g of agar or agarose in 100 ml distilled water will
make  10 plates 60 mm in diameter)
• La+++ solution
(lanthanum nitrate, 1 M)
(43.3 g La(NO3)3.6H2O/ 100 ml solution)
• F- solution
(potassium fluoride, 1 M)
(9.4 g KF.2H2O/ 100 ml solution)
• Ag+ solution
(silver nitrate, 0.2 M)
(3.4 g AgNO3 / 100 ml solution)
• Cl- solution
(sodium chloride 0.4 M)
(2.3 g NaCl/ 100 ml solution)
• Cl- diluted solution
(sodium chloride 0.1 M)
(dilute the above solution 1:4 with distilled water)
(The silver nitrate solution should be stored in an opaque bottle out of the light.)


All the solutions are stable indefinitely at room temperature. The chemicals are available from several supply companies, but are often in stock in academic chemistry departments, so don't buy any without checking with your chemistry teacher first.

60 mm plastic petri plates
Disposable Graduated Small or Large Bulb 1 ml plastic pipettes, with the first and second gradations cut off (used for cutting the wells)
Pasteur pipettes (or other narrow tipped pipettes; used for filling the wells)

The petri plates and pipettes are available from several scientific supply companies. They can all be cleaned and reused; they last indefinitely.

This figure shows the pipettes, a petri plate and one of the solutions needed for this exercise.
Figure 1.
Click image for a larger view.


Make the plates.

The easiest way to prepare large numbers of gel plates is to use a microwave oven. Suspend 2 grams of agar or agarose powder in 100 ml of distilled water in a 250- or 300-ml flask. Swirl to suspend the powder thoroughly. Heat on high power in the microwave for 1 min. Carefully swirl to re-suspend the powder and repeat the heating until the powder is dissolved. Caution should be used when swirling because the solution may become superheated and boil vigorously when it is mixed.

If a microwave oven is not available, agar can be dissolved by suspending the powder in water and heating with a bunsen burner or hot plate. Be sure to stir the suspension continuously while it is heating to prevent the agar from burning and sticking to the bottom of the container.

Agarose is easier to dissolve than agar. Suspend the powder as described above, and heat it with swirling in a boiling water bath or in a microwave oven. Students can prepare the gel for their plates individually by placing 0.2 g of agarose in 10 ml of distilled water in a small beaker and heating it with swirling in a boiling water bath.

If a large volume of gel has been prepared, it can be dispensed into the petri plates using a 10-ml pipette equipped with a pipetting device. A volume of 10 ml of gel per plate is sufficient for excellent results. If you plan to reuse the pipette, rinse it with warm water immediately after use to prevent residual gel inside it from solidifying. If individual 10 ml volumes of the gel solution have been prepared, they can be transferred by pouring directly into the petri plates.

(Note: You may want to test several concentrations of agar or agarose for use in your class. 2% agar or agarose generally works well. At lower concentrations, the agar is mushy, and students have a hard time cutting good wells. Agar is much less expensive than agarose. If unwanted bands appear on the plates, they may be due to contaminants in the agar. Suspending the agar in distilled water and collecting it by centrifugation two to three times before dissolving it by heating will remove the contaminants. Agarose dissolves more easily than agar, and convenience may be a factor in choosing which one to use.)

Depending on how hot the gel solution is, it will solidify in 5 to 15 minutes after being poured into the petri plates. The lids should be left off of the plates so that water does not condense on them while the agar is cooling. The plates can be prepared ahead and stored in sealed containers (plastic bags work well) in the refrigerator for several weeks.

Cut the wells.

To cut the wells, use a plastic disposable graduated 1-ml pipette that has had the smallest two gradations cut off. Students can easily cut their own wells. A piece of paper is marked with a hexagonal array of dots. (See Figure 2, which can be used to make working copies. The dots should be 17 mm apart. They can be labeled and are visible through the gel which helps in setting up the experiments as described below). The petri plate is positioned over the markings on the paper (Figure 3).

To cut the wells:

  1. Compress the bulb of the pipette as completely as conveniently possible.
  2. Press the tip of the pipette into the gel, going all the way to the bottom of the gel layer.
  3. Release the compression on the bulb. This should pull the gel plug out of the well and into the pipette.

Several wells can be cut before cleaning the pipette. To clean the pipette, draw water into it and forcefully expel the water with the gel pieces suspended in it (Figure 4).

Figure 2: Template of hexagonal array to guide the cutting of wells in the gel layer; the dots should be 2 mm in diameter and are 17 mm apart, on center. (Courtesy D. Schadler)
Figure 2.
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Figure 3: Petri plate placed of the hexagonal array template. (Courtesy D. Schadler)
Figure 3.
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Figure 4: Petri plate with wells cut in the gel layer. (Courtesy D. Schadler)
Figure 4.
Click image for a larger view.
Fill the wells.

For a typical experiment, label the silver nitrate (Ag+) solution and the lanthanum nitrate (La+++) solution as Antibody A and Antibody B, respectively. The sodium chloride (Cl-) and the potassium fluoride (F-) solutions are designated Antigen a and Antigen b, respectively. Standard laboratory safety procedures should be followed when handing the solutions.

Pasteur pipettes are used to add the solutions to the wells. Only 1 to 2 drops of solution are needed to fill the well. Instead of holding the pipette above the well and attempting to drop the solution into it, put the tip of the pipette into contact with the bottom of the well and then slowly release a small amount of solution. It helps if the student steadies the hand that is used for pipetting by placing their elbow on the table. In double diffusion tests, always put the simulated antibodies in the center well.

Single Diffusion:
Ag+ solutions (simulated Antibody A) of several concentrations are placed separately into wells of a gel plate containing dilute (0.2 mM) Cl- (simulated Antigen a) along with the agar or agarose. Rings of precipitate form around the wells within 30 minutes to one hour.

To prepare gels containing 0.2 mM Cl-:

  1. Add 0.5 ml of 0.4 M NaCl to 99.5 ml of distilled water.
  2. Add 2 g of agar or agarose and heat as described above.

0.2, 0.02 and 0.002 M AgNO3 solutions are recommended
0.02 M and 0.002 M AgNO3 can be prepared by adding 1 and 0.1 ml of the 0.2 M stock solution to 9 ml and 9.9 ml of distilled water, respectively.

Q.  What is the precipitate?


Q.  Why do some rings have larger diameters than others?


Double Diffusion - One Reaction Type:
Place the Ag+ solution (simulated Antibody A) in the center of a hexagonal array of wells. Place two different dilutions of Cl- solution (0.1 M and 0.4 M NaCl) (simulated Antigen a) in separate, adjacent wells around the hexagon. Lines of precipitate will appear within 30 minutes to one hour.

Q.  Why does a line of precipitate form?


Q.  Which line forms first? Why?


Q.  The lines fuse and do not cross one another. Why?


Q.  Which precipitate is closer to the Cl- well? Why?


Double Diffusion - Two Reaction Types:
The most instructive results are obtained in two configurations of double diffusion - experiments in which two reaction types occur:

Experiment 1:

  1. Alternate the simulated Antigens a and b (Cl- and F-) in the 6 outer wells.
    Number the wells consecutively around the hexagon.
    Wells 1, 3 and 5 receive simulated Antigen a.
    Wells 2, 4 and 6 receive simulated Antigen b.
  2. The class is then divided into three groups:
    Group 1 uses only simulated Antibody A (Ag+) in the center well.
    Group 2 uses only simulated Antibody B (La+++) the center well.
    Group 3 uses both simulated Antibody A and simulated Antibody B (one drop of each) in the center well.
    (Figure 5 shows these configurations as labeled wells.)

Experiment 2:

  1. All students use three neighboring outer wells (1, 2, and 3) for simulated Antigen a (Cl-) and three neighboring outer wells (4, 5, and 6) for simulated Antigen b (F-).
  2. (2) The class is then divided into three groups:
    Group 1 uses only simulated Antibody A (Ag+) in the center well.
    Group 2 uses only simulated Antibody B (La+++) the center well.
    Group 3 uses both simulated Antibody A and simulated Antibody B (one drop of each) in the center well.
    (Figure 5 shows these configurations as labeled wells.)

Visible lines of precipitate will appear within 30 minutes to one hour.

Q.  Which precipitate lines form first? Why?


Q.  Where do precipitate line cross? Where do they not cross?


Q.  Why do the precipitate lines cross or not cross?


Figure 5. This figure shows six different configurations for double diffusion - two reaction type demonstrations. (Courtesy D. Schadler)
Figure 5.
Click image for a larger view.


In all cases, the precipitation reactions are usually visible within one hour and are fully developed in two hours. The precipitin bands are stable for at least 24 hours, but they widen with time.

Single diffusion test:
The reaction of silver nitrate (Ag+ = simulated Antibody A) with sodium chloride ( Cl- = simulated Antigen a) forms silver chloride (AgCl) as a precipitate. The diameters of the rings that form around the wells should be proportional to the concentration of Ag+ (simulated Antibody A) in the wells. As the concentration of Ag+ increases, the diameter of the ring should increase.

Double diffusion- one reaction type test:
Clear lines of precipitate should form where the simulated antibody meets the simulated antigen. The lines form earliest around the wells where the simulated Antigen a (Cl-) concentration is highest. The order of appearance of the precipitation lines should be correlated with the concentration of the simulated Antigen a in the wells.

The lines of precipitate should not cross, which is a more informative observation than the time sequence of their appearance. The lines of precipitate do not cross because the simulated antibodies react with the simulated antigens as they encounter them; thus, no simulated antibody molecules diffuse beyond the line of precipitation, and the lines do not cross.

The line of precipitate forms closest to the Cl- well with the highest concentration of Cl- because the concentration of Cl- that has diffused into the gel is sufficiently high to cause the reaction to occur.

Double diffusion - two reaction type test:
Clear lines of precipitate should form. The lines around wells containing like antigens will not cross, for the reason noted above (Figure 6). Around wells containing unlike antigens, however, the lines will cross due to the specificity of the reactions involved. Simulated Antibody B will not react with simulated Antigen a. Simulated Antibody B diffuses through the Antibody A/Antigen a precipitate line to react with the simulated Antigen b beyond the line. Likewise, simulated Antibody A does not react with simulated Antigen b and diffuses through the Antibody B/Antigen b precipitate line to react with Antigen a beyond the line (Figure 7).

Typical results for a double diffusion - two reaction type test.
Figure 6.
Click image for a larger view.

Figure 7: Typical results for a double diffusion - two reaction type test. Simulated antigens are placed in alternate wells. Both simulated antibodies are placed in the center well. (Courtesy D. Schadler)
Figure 7.
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