The Immunologic Response of Mice to Specific Antigens Will Be Studied in an Exercise That

THE MOUSE STUDY

The immunologic response of mice to specific antigens will be studied in an exercise that will continue throughout the semester. Students will work in three separate groups. Briefly, mice (12/group) will be immunized to two different antigens by injection of antigens mixed with an adjuvant (see figure 1-1 on the next page) followed by two booster injections of the antigens. Mice will be bled three times (one pre-immune and two post-immune bleeds) to provide whole blood and serum for analyses. The total blood picture (e.g. RBC & WBC counts, hematocrit, differentials, etc.), as described in detail below, must be determined for each bleed. The serum obtained at each bleed will be used for analysis of immune response as described below. Techniques required for assay of the immune responses will be demonstrated and run in the first 10 weeks of the course. The last few lab sessions can be devoted to assays of the immune response of mice to each injected antigen.

The final laboratory period will be devoted to the presentation of results by each research group. Each group member must present a portion of the group's results as part of a well-organized and cohesive presentation. Groups should prepare a complete description of the data and results to be ready at the time of the presentation. The data should be well-organized, using tables, graphs and drawings of all results. Each student must submit an independent written report of the entire mouse experiment. This report may refer to the group data section without including it in the individual write-up. The following areas should be addressed in the presentations and reports:

1. The general health of the mouse throughout the study ‑ weight and any signs of disease or stress.

2. An analysis of the blood picture of the mouse during the study (for each bleed):

a. RBC and WBC counts

b. hematocrits

c. hemoglobin content

d. differentials.

e. Immune cell flow cytometry

3. Immune response (including comparison of pre-immune, first immune, and final immune levels) of mice to each antigen including:

a. antibody titers against both antigens measured by:

i. agglutination reactions

ii. precipitation techniques

iii. ELISA

b. An evaluation of the specificity of the antibodies measured by:

i. gel diffusion

ii. immunoelectrophoresis

iii. western blots

c. An estimation of IgG vs. IgM response from western blots

4. A summation of the results.

IMMUNIZATION PROTOCOL: Each group will be assigned two of the following antigens:

1. bovine serum albumin 6. bovine serum

2. human globulin 7. chicken serum

3. human albumin 8. goat serum

4. Salmonella typhi 9. rabbit serum

5. horse serum 10. Chicken Albumin

Figure 1-1

All 12 mice for each group will be bled and immunized with both antigens combined with a boosting agent known as an adjuvant. See figure 1-2 below for description of many common adjuvants. The following schedule of immunization will be followed:

Week 0 bleed mice (pre‑immune bleed). Immunize mice to antigens in Ribi adjuvant

Week 2 give booster immunizations using antigens in Ribi Adjuvant

Week 3 bleed mice (first immune bleed)

Week 5 give booster immunizations using antigens in Ribi Adjuvant

Week 7 bleed mice (final bleed) 2 weeks after Booster

Figure 1-2

All mice will be anesthetized and bled from the retro-orbital venus plexus by the lab instructor or CLT. All student lab groups should be prepared to run analysis on fresh blood immediately to measure hematocrits, total RBC and WBC counts, hemoglobin levels, and differentials. Blood will then be allowed to clot and the serum isolated for subsequent antibody and complement analyses.

Mice will be immunized to two antigens from the above list. The antigens will be mixed with Ribi adjuvant which is known to boost the antibody response. Mice will be injected subcutaneously in the lower left abdominal region with the antigen:adjuvant mixture in a total volume of 0.1 ml/injection.

Results: A data summary should be prepared listing all experimental results. This should include all raw data and statistical analysis of data (for example, the results of differential counts should be represented as the mean (x) + the standard error of the mean (SEM) for a minimum of three separate counts for each blood sample). All data, including differential slides, gels, and electrophoresis slides must be properly labeled and saved. These along with your data summaries must be turned in before your presentation.

CARE AND HANDLING OF MICE: Mice must be handled in a humane manner. Bleeding may cause significant pain and will only be performed after appropriate anesthesia has been given. Injections cause less pain and can be done without anesthesia. Some animals may have a reaction to the injected material (especially serum) and should be monitored for an acute response for about 30 minutes. It would also be beneficial to vary the injection site slightly for the comfort of the animal so the second immunization will be given in the lower right side.

Materials required for immune assays should be requested in advance of the laboratory period for which they are required. All requests should be written and given to the CLT ‑ allowing sufficient time for preparation.

Blood analysis: You will require your Hematology Laboratory Manual or a Hematology text to re-acquaint yourself with these methods. Three blood smears should be made and stained with Wright's to perform a leukocyte differential. Red and white blood cell counts should be made using the hemacytometer and hematocrit and hemoglobin should be determined. Let the blood stand at room temperature for 1 hour, then rim the top of the clot with an applicator stick to separate it from the tube. Centrifuge at 1500 rpm for 30 minutes. Remove the serum with sterile pipettes and transfer to a sterile tube. Label the tube and freeze for later experimentation (antibody and complement titers, etc.).

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Agglutination Reactions

Agglutination: refer to the text (chapter #2) for supplemental material and a general description of agglutination.

I. Slide Agglutination: This is the method most commonly used for serological study of gram‑negative enteric bacilli and Brucella. The O and H Salmonella antigens will be analyzed separately using this procedure. Antibodies to these antigens are derived from mice that were immunized by the previous immunology class.

1. Use a 0.2 ml pipette to place 0.08, 0.04, 0.02, and 0.01 ml of antiserum on different depressions of an agglutination slide.

2. Add one drop of Ag (standardized) to each and mix well with an applicator stick. The final mixtures are approximately equal to 1:25, 1:50, 1:100 and 1:200. Note: For this and all similar procedures take extreme care to ensure that the antigen pipette tip never comes in physical contact with the antiserum.

3. After mixing, tilt the agglutination slide back and forth slowly for about two minutes.

4. Place the slide at various angles to a light source and observe the results.

II. Tube Agglutination:

1. Make the following dilutions of antisera to Salmonella: 1:10, 1:20, 1:40, 1:80, 1:160 1:320, and 1:640. The 1:10 is made by adding 0.1 ml of serum to 0.9 ml PBS buffer solution. Each successive dilution is made by adding 0.5 ml of the previously diluted antiserum to 0.5 ml of buffer (e.g., to make a 1:20 dilution, add 0.5 ml of the 1:10 to 0.5 ml of buffer, and so on).

2. Include a control tube consisting of 0.5 ml saline.

3. To each tube, add 0.5 ml of Salmonella antigen suspension containing the appropriate (H or O) antigen.

4. Incubate for 3‑4 hours at room temperature and overnight in a refrigerator. Read the results in the morning.

5. Most of the bacteria will have sedimented by the next day so the tubes must be gently tapped on the bottom to send the contents back into the fluid portion. Positive agglutination will be manifested by distinct clumps of varying sizes (a

magnifying glass and/or a mirror might help you to read your tubes). Negative agglutination will be evidenced by a "wisp of smoke" as you tap the bottom of your tubes.

6. The titer of the antiserum corresponds to the last tube in the dilution sequence which shows positive agglutination.

Diagnostic Agglutination Tests:

I. Rheumatoid Factor (RF) Titration (tube agglutination).

RF is an antibody of the IgM class which is titerable in patients with autoimmune diseases such as rheumatoid arthritis and juvenile arthritis. It is not the causative agent in these diseases but rather is coincident with the diseased state. The key feature of RF is that it reacts with human gamma globulin (IgG) and therefore may be quantified. IgG may be adsorbed onto tanned erythrocytes (RBC which are treated with tannic acid which alters the membrane and reveals receptor sites for adsorption of antibody or certain antigens) or poly‑styrene latex particles. The titration of RF should be done with the latter since tanned or otherwise treated red cells display an abnormally high osmotic fragility. Follow the following procedure:

1. dilute the test serum 1:10 (0.1 ml serum to 0.9 ml glycine buffer).

2. label 13 test tubes and place 0.5 ml of buffer into each.

3. add 0.5 ml of the 1:10 dilution into tube #1, mix well with an application stick (1:20 dilution).

4. add 0.5 ml of the 1:20 to the next tube and so on. Carry the dilutions to the 12th tube. The 13th tube should contain only buffer, thereby serving as a control.

5. add one drop of the IgG coated latex particle suspension to each of the 13 tubes.

6. Incubate at 37oC/15 min.

7. centrifuge, check for agglutination, and determine the titer.

II. Monospot Test for Infectious Mononucleosis (IM).

To confirm a physical diagnosis of IM (lassitude, low grade fever, pharyngeal irritation) a routine smear (abnormal shape, PAS+, inclusions) and an Esptein‑Barr virus antibody titer (heterophile antibody titer) may be performed. A heterophile antibody is produced in infected humans, presumably against the causative Epstein‑Barr virus or against the products of a lysed cell. This antibody is capable of reacting with erythrocytes of different species (horse red cells are most commonly used). Because of this it is referred to as a heterophile antibody. Briefly, the ability of test serum to agglutinate a 20% suspension of horse erythrocytes compared to several controls is a positive reaction for IM. Generally, a positive Monospot test must be verified by a determination of heterophile antibody titer.

III. Agglutination‑inhibition test for pregnancy.

The human embryo manufactures a trophic substance, chorionic gonadotrophin (CG). CG is adsorbed onto latex beads for this test. Briefly, anti‑CG of commercial origin is added to an equal volume of test serum. After 5‑30 minutes of incubation, the latex bead‑CG suspension is added. If the test serum is positive, the CG will bind to the anti‑CG and neutralize it. When the latex‑CG suspension is added, no reaction will occur. Conversely, in a negative response, test serum which contains no CG cannot neutralize the anti‑CG. Therefore, when the latex bead‑CG is added, the antibody bridges the antigen‑latex and a macroscopic agglutination is observed. Positive agglutination tests are also available using latex beads. These are coated with anti‑human CG. Test serum/urine is incubated with the latex‑anti‑CG. In this test, agglutination

indicates a positive reaction.

Figure 2.7 (from Text) Hemagglutination is used to type blood groups and match compatible donors and recipients for blood transfusion. Common gut bacteria bear antigens that are similar or identical to blood group antigens, and these stimulate the formation of antibodies to these antigens in individuals who do not bear the corresponding antigen on their own red blood cells (left column); thus, type O individuals, who lack A and B, have both anti-A and anti-B antibodies, while type AB individuals have neither. The pattern of agglutination of the red blood cells of a transfusion donor or recipient with anti-A and anti-B antibodies reveals the individual's ABO blood group. Before transfusion, the serum of the recipient is also tested for antibodies that agglutinate the red blood cells of the donor, and vice versa, a procedure called a cross-match, which may detect potentially harmful antibodies to other blood groups that are not part of the ABO system.

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Precipitation Reactions

Precipitation.

See your text for a more in‑depth description of this reaction. Basically, precipitation is similar to agglutination but differs in one respect: precipitation (or precipitin) antigens are not cells or large particles but are in solution (proteins, polysaccharides). Visible precipitation occurs after lattice formation reaches a certain point, called equivalence (see figure below).

Figure 2.8 (from Text) Antibody can precipitate soluble antigen. Analysis of the precipitate can generate a precipitin curve. Different amounts of antigen are added to a fixed amount of antibody, and precipitates form by antibody crosslinking of antigen molecules. The precipitate is recovered and the amount of precipitated antibody measured; the supernatant is tested for residual antigen or antibody. This defines zones of antibody excess, equivalence, and antigen excess. At equivalence, the largest antigen:antibody complexes form. In the zone of antigen excess, some of the immune complexes are too small to precipitate. These soluble immune complexes can cause pathological damage to small blood vessels when they form in vivo.

Equivalence is dependent on the relative ratios of antigen and antibody. You will be performing an alpha precipitation procedure in which antigen is titered against a fixed amount of antibody. Some circumstances may dictate the use of the beta precipitation procedure,

where antibody is titered against a fixed amount of antigen.

Protocol:

1. A series of 12 tubes is set up and serial dilutions of the antigen are performed (the stock suspension should be diluted 1:4) using 0.5 ml aliquots.

2. The antisera is diluted 1:5 with saline and 0.5 ml is added to each of the 12 tubes. Mix via angular rotation of the tubes.

3. Incubate at 37oC for 60 minutes. Check for precipitation at 30 minutes and record the antigen dilution. Grade the degree of response from 0 (no reaction) to ++++ (positive reaction) on the first line of the chart. Compare the response at 30 minutes to that observed at 60 minutes. The greatest degree of precipitation occurs at equivalence.

4. Centrifuge to remove particulate matter from the supernatant portion of the tubes and pipette the supernatant in 0.25 ml of aliquots into two sets of properly labeled tubes. 5. To one of these tubes, add 0.25 ml of antisera and mix well. Incubate as in "3" and record your results on line 2 of the table.