BIO 327 Immunology Term 7, 04/05

Precipitation Reactions:
Ouchterlony Test
From: Animal Blood Sera for Immunologic Reactions in Gel,

Carolina Biological Supply Company

Another reaction that involves the binding of antigen by antibody with the formation of a visible aggregate is called precipitation. The antigen in precipitation reactions is soluble and much smaller than the particulate antigen detected in agglutination reactions. Contrary to particulate antigens detected in agglutination reactions, smaller soluble antigens must combine with more antibody to form a visible clumps. Soluble antigens can be attached to particulate material serving as carriers that can be detected using the more sensitive agglutination technique. This is the basis for numerous commercial agglutination tests such as pregnancy tests and tests for diagnosis of infections like mononucleosis and strept throat.

The precipitation reaction is subject to inhibition if either antigen or antibody is present in excess. Cross-linking will not occur if excess antigen is present or if excess antibody is present. (See figure below). Cross linking and lattice formation will only occur when antigen and antibody concentrations are optimal. In the Ouchterlony double gel diffusion test antigen and antibody diffuse toward each other in a semisolid medium to a point in the medium where optimum concentration of each is reached. A band of precipitation occurs at this point. The qualitative Ouchterlony Test can simultaneously monitor multiple Ab-Ag system and can be used to identify particular antigens in a preparation.

The technique involves cutting cylindrical wells into a purified preparation of agar solidified in a Petri dish. The wells are filled with antibody or antigen and the dish is allowed to incubate. When homologous antigen and antibody diffuse toward each other from the individual wells, a precipitin line will form somewhere between the two wells. Non-specific reactants diffuse past each other, forming no precipitate. You have available three anti-albumin antisera from three different mammals: bovine, horse and swine. Each antibody was produced in goats injected with the protein albumin obtained from ox, horse, or swine blood. Albumin is therefore the antigen. Antibodies contained in the antisera are polyclonal but specific for the albumin component of whole blood. The class will be divided into four groups. Group 1 will test for the purity of ground meat; group 2 will test for the effect of antigen concentration. Group 3 will demonstrate identity and non-identity reactions, and group 4 will play detectives in a crime.

General Directions:

Ouchterlony plates have been prepared for you. Cut 2.5 to 3 mm diameter wells in the agar using a glass medicine dropper. Place the plate over the template as a guide to cutting the wells. This template has a central well surrounded by six equally spaced wells numbered for identification and record keeping. Number the wells on the bottom of the diffusion plate. The wells must be carefully prepared; nicks at the edges of the well can cause distortions in the precipitin ring.

Template for cutting wells.

Group 1: Is Your "Big Mac" What It Claims to Be?
1. Prepare two diffusion plates, one for the test and one for control; cut a center well and wells #1, #3, and #5 in each plate.

2. Extract "blood" from a golf-ball-sized portion of the raw meat. To do this, thoroughly mix 2 ml of water into the raw meat, wrap the meat in cheese cloth, and squeeze out several drops of meat extract into a small test tube.

3. In another tube, mix 1 drop bovine serum + 1 drop horse serum + 1 drop swine serum + 2 drops saline. This produces a 20% concentration of each animal serum.

4. Fill the wells carefully as follows:

5. Cover the plates and set them in a moist chamber at room temperature until the precipitin lines appear (18 to 72 hours).

6. The results are best viewed with diffuse back-lighting while holding the opened dish vertically. Precipitin lines vary in shape and intensity depending upon the reactants' concentration and rates of diffusion. The lines will form crescents which bend away from the center well, indicating that the antigen (albumin) molecules diffuse through the gel faster than the antibodies.

7. Compare the control and test plates. Is your "hamburger" pure beef? Caution: secondary precipitin lines may form near the center well by the horse reactants and swine reactants. Such lines result from secondary antigens which are "seen" by the antibodies. These are cross reactions between the antiserum which contains mixtures of related molecules and antigens similar to the primary one.

Group 2: Testing the Effect of Antigen Concentration

1. Prepare three diffusion plates; cut a center well and wells #1 to #6 in each plate.

2. Label one plate "B" for bovine, another "H" for horse, and the last "S" for swine.

3. Prepare 20% and 5% dilutions of each animal serum in separate test tubes. To make a 20% dilution, combine once drop stock (100%) serum with four drops saline. To make a 5% dilution, combine one drop stock serum with 19 drops saline. Label the tubes. Stir each dilution with a different transfer pipette and use the same pipette to fill the corresponding well in the diffusion plate.

4. Fill the wells as follows:

5. Cover the plates and set them in a moist chamber at room temperature until the precipitin lines appear (18 to 72 hours).

6. Unlike the results for group 1 (see #6), the precipitin lines will bend toward the center well since you have antibody in the center well. To determine the effect of antigen concentration, observe the precipitin lines appearing between the center well and wells 1, 3, and 5. compare intensity, length and width, and distance from the center well. An "ideal" line produced by a well-balanced system is narrow, dense, has sharp margins, and is positioned away from the antibody well. Which antigen concentration forms the best precipitin lines? Are any secondary reaction lines present (see #7, Group 1)? What is the role of wells 2,4, and 6?

Group 3: Demonstrating Identity and Non-Identity Reactions (see website).

There are three characteristic precipitin reactions which may occur in the Ouchterlony analysis: identity, non-identity, and partial identity. The demonstration of these reactions requires that the test wells be positioned closely enough to allow precipitin lines to fuse or cross.

Examine the figure. When the two antigens are different, yet both react with the antibody, a pattern of non-identity results. Precipitin lines are seen to cross at their ends. Such a pattern would form if antibody against whole human serum were tested with human albumin and human immunoglobulin. These two proteins are thus distinct in antigenic determinants. Note that antibody molecules specific for one antigen must diffuse through the precipitate of the other antigen (and conversely) to produce an overlapping pattern. A partial identity reaction occurs when the two antigens involved have common antigenic determinants, but one antigen also has a unique determinant. Antigens which are closely related chemically often produce a pattern of partial identity when diffused against antibody homologous to one. Anti-human serum reacted with human serum and rhesus monkey serum is an example. Indications of partial identity have proven useful for systematists interested in determining evolutionary relationships between organisms.

1. Prepare a diffusion plate with a center well and wells #1 to #6 cut in the gel according to the template.

2. Combine two drops anti-bovine albumin and two drops anti-horse albumin in a test tube.

3. Prepare 10% dilutions of bovine serum and horse serum in separate tubes (one drop 100% stock with 9 drops saline).

4. Stir the dilutions with a different pipette and use the same pipette to fill the corresponding well in the diffusion plate. Be careful; do not contaminate the pipettes.

5. Fill the wells as follows:

6. Cover the plates and set them in a moist chamber at room temperature until the precipitin lines appear (18 to 72 hours).

7. A pattern of identity is demonstrated by the precipitin bands formed between the center well and each pair of wells which contained the same antigen, namely, wells 1 to 2, and 3 to 4. A non-identity reaction occurs between the center well and wells 2 and 3 where the diffusion of bovine and horse sera have crossed. What would you expect to see from wells 5 and 6?

Group 4: A Hint of Crime

Read the explanations of each of the previous groups. You will be given a "blood-stained" cloth. You need to determine whether the blood is of human or animal origin using the Ouchterlony test.

1. Cut out the portion of the cloth stained with blood and place the sample in a beaker containing 5 ml of saline.

2. Let the cloth soak for a few minutes, then rinse out as much of the blood as possible into the saline.

3. Prepare one Ouchterlony plate for each antiserum type to be tested. Contrary to the other groups, you also have anti-human sera and human albumin. You will need 4 plates.

4. Prepare 1% saline solutions of serum albumin from each species to be tested. Keep the solutions separate. Mix each solution with a different pipette that will also be used to fill the wells.

5. Fill the center well of each plate with one of the various samples of anti-species antisera using the following formula. The wells should be filled but not overfilled.

6. Cover the plates and set them in a moist chamber at room temperature until the precipitin lines appear (18 to 72 hours).

7. Look for lines of precipitation to determine the identity of the blood sample.