_The immunologic response of mice to specific …maxweber.hunter.cuny.edu/pub/eres/MLS460_JOHNSTON/MAN99.doc · Web viewThe immunologic response of mice to specific antigens will

THE MOUSE STUDYThe 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. hematocritsc. hemoglobin contentd. 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 reactionsii. precipitation techniquesiii. ELISA

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

i. gel diffusion ii. immunoelectrophoresisiii. western blots

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

4. A summation of the results.

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THE MOUSE STUDYIMMUNIZATION PROTOCOL: Each group will be assigned two of the following antigens:

1. bovine serum albumin 6. bovine serum2. human globulin 7. chicken serum3. human albumin 8. goat serum4. Salmonella typhi 9. rabbit serum 5. horse serum 10. Chicken Albumin

Figure 1-1

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THE MOUSE STUDYAll 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 AdjuvantWeek 3 bleed mice (first immune bleed)Week 5 give booster immunizations using antigens in Ribi AdjuvantWeek 7 bleed mice (final bleed) 2 weeks after Booster

Figure 1-2

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THE MOUSE STUDYAll 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 ReactionsAgglutination: 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.

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Agglutination Reactions

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.

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Agglutination Reactions

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.

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Agglutination ReactionsFigure 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,

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Precipitation Reactions

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.

5. To the second et of tubes, add 0.25 ml antigen suspension diluted to equivalence (check your results from step 3). Incubate as before and enter your results on line 3 of the table.

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Precipitin Reactions

Precipitation Reactions in Gels.

These reactions are discussed in depth in your text. The fundamental differences between these types of reactions and precipitation in fluids, other than the media, are that : 1. this test relies on the ability of either an antigen or antibody to diffuse and 2. for diffusable antigens, precipitation in gels allows for the separation and identification of immune reactants in a multispecific system (e.g., the appearance of three precipitation bands indicates that the system is multispecific with antibodies against three specific antigens; one band indicates a monospecific system).

The medium to be used is agarose which is essentially a component of agar which lacks sulfate, sulfhydryl, or carboxyl groups. You must use type I agarose which has a relatively low melting point. It will not allow diffusion of substances significantly larger than 200,000 D: hence, once an antigen-antibody complex is formed, it remains in a fixed position in the gel since it will exceed this molecular weight. It should be obvious that this technique is not suitable for large antigen molecules and macroglobulins such as IgM. In addition, relative nonpolar antigens diffuse poorly and also may be unprecipitable.

The various types of gel diffusion will be discussed during the lab period. We employ the single diffusion in one dimension. (Oudin method), the double diffusion in one dimension (Preer method). and the double diffusion in two dimensions (Ouchterlony method). Generally, toperform the Oudin procedure, antiserum is placed into liquefied agarose at 45-50oC and evenly dispersed prior to solidification of the gel in a test tube.

1. Single diffusion in one dimension. An antigen solution is layered over the solidified antibody-containing gels in a 6 mm x 50 mm test tube. One or more precipitation bands will be formed depending on the number of immune reactants. The bands will appear to move away from the antigen suspension-gel interface as time elapses. Since the gel does not allow for diffusion of molecules or aggregates in excess of 200,000 D, this is a seemingly paradoxical effect. How would you explain this? The antigen/antibody reactions and the actual diffusion process is greatly influenced by temperature and other external factors. To slow down or stop these reactions, refrigerate your sample. You have to allow this to diffuse for up to a week. The refrigeration slows down the reaction somewhat.

2. Double diffusion in one dimension. This is technically more difficult than the Oudin method (#1). Pour 200 ul of antibody suspension in the bottom of a 6 mm x 50 mm tube. Add to this 400 ul of liquid agarose (heated just beyond the melting point). After the agarose solidifies, add 200 ul of the antigen suspension to the top of the column. Follow for 1-2 weeks. Precipitation bonds will form where diffusing antibody reaches equivalence with diffusing antigen. This is the Preer method.

3. Single diffusion in two dimensions. This method is also referred to as radial immunodiffusion or the Mancini technique and is described in your text. Briefly, antiserum is suspended in liquefied agarose (45-50oC) and allowed to solidify on a plate or petri dish. Wells are cut in the agarose and filled with different dilutions of antigen and/or control. A precipitation ring will form with the square of the diameter proportional to the concentration of antigen. This method is used clinically to determine the quantity of various serum antigens including different classes and

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Precipitin Reactions

subclasses of immunoglobulins, several complement proteins, lysozyme, and alpha fetoprotein amongst others.

Protocol:

1. The agarose will be preheated for you. Monospecific antibody is generally employed in this test; you will be using antibody to human albumin or globulin. Dilute the antisera approximately 1:10 in saline and mix with agarose to achieve a 2% suspension (if you use 5.0 ml of agarose you will need 100 l of antisera. Mix well and pour into a dish. Note: This must be a thin layer or the test will not work.

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Precipitin Reactions

2. Cut wells in the agar approximately 20 mm apart (check for the proper template before you use it). Use a Pasteur pipette attached to a vacuum hose to lift the agarose plug after you cut it.

3. Fill successive wells with different dilutions of antigen (1:1, 1:2, 1:4, 1:8). Incubate at room temperature for 48-96 hr and determine the diameters of the diffusion bands using a millimeter ruler. If properly done, the diameter of the ring should be proportional to the concentration of antigen.

4. Double diffusion in two dimensions. This technique has the widest practical application. Briefly, it may be used: to determine homogeneity, heterogeneity, and cross reactivity, to provide information concerning the degree of purification of an antigen, and to identify the number of antigen-antibody systems. Agarose (4 – 5 ml) is liquefied as in the Preer method, and poured into a Petri dish, and allowed to solidify. Wells are then cut in the gel using the smaller template in the pattern indicated. Generally, the antibody is placed in the center well and the antigens in the satellite wells. As they diffuse toward each other and reach the proper relative proportions, they will precipitate. Your group will be assigned antigens and antibodies as per the chart provided. Cut the wells with the smallest plug cutter available. Attach a Pasteur pipette to a suction hose and lift the plugs. Follow the pattern exactly and maintain equal spacing between the walls (7-10 mm). Fill each well up to its top but be careful; spillover will necessitate redoing the entire procedure. Incubate at room temperature for two hours and then place in a refrigerator. Check the plates on each weekday following the lab and record your results. These reactions occur slowly and may require as many as ten days for certain antigens.

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Precipitin Reactions

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ImmunoelectrophoresisThis method combines electrophoresis and gel diffusion/precipitation and is described in

your test. This method may be used to quantify IgG, IgM, IgA, transferrin, C3 and C4 as well as various hormones. With immunoelectrophoresis, you can differentiate multiple components (i.e., Ag:Ab systems) with similar electrophoretic mobility or components which are antigenically similar but have different electrophoretic mobilities.

Protocol:

1. Pure agarose is heated to liquid consistency, layered on a glass microscope slide, and allowed to solidify.

2. Use the template to form the wells in the following patterns (do not cut the troughs at this time):

3. Each group should do one of each type.

4. After antigen wells are filled, place slides in the electrophoresis apparatus which is filled with the buffer. Use buffer-dampened filter paper as wicks: one end of the wick is placed on the edge of the slide and the other hangs in the buffer. Adjust the power supply to 260 volts and run for 90 minutes.

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Immunoelectrophoresis

5. Remove the slides from the electrophoresis apparatus and use the templates to cut the troughs.

Antigen Antiserum

whole human serum (WHS) anti-WBS

human albumin (HA) anti-HA

whole bovine serum (WBS) anti-WBS

human globulin (HG) anti-HG

6. Place the slides in a petri dish containing moist gauze and incubate for 48-72 hr in the refrigerator. The slides may be stained with Coomasie blue or Thiazine red R solution and preserved. However, the precipitation bands should be clearly visible with the quantity of Ag and Ab which you used, so this step is not really necessary.

7. Evaluate the slides for cross reactivity of antibodies, differential mobility of antigens, etc.

oo o

Serum Serum 1 Serum 2

Antigen 1 Antigen 2 Antigen

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ELISA ASSAYThe Enzyme-Linked ImmunoSorbent Assay (ELISA) is among the most sensitive

methods for detection of antibody:antigen interactions. In its simplest form antigen is attached to the bottom of wells of a 96 well (microtiter) plate by incubation in carbonate buffer. Areas where antigen failed to attached must be covered up (blocked) by incubation with blockers such as milk, BSA, or Tween 20. Antisera is then added to the wells and incubated. Only antibodies that recognize the antigen will bind while all others should not. Unattached antibodies are removed by thorough washing with blocker. A secondary antibody (in our case goat anti-mouse Ig would be appropriate) is added to the wells and incubated. The secondary antibody contains an attached enzyme such as alkaline phosphatase or a peroxidase. If a primary antibody has bound to the antigen then the secondary antibody will bind to the primary. Unattached secondary antibody is removed by extensive washing with blocker. The enzyme substrate is then added to all wells and color development is monitored. The absorbance of each well can be read in an ELISA reader to obtain quantitative measures of antibody levels in the original antisera.

The figures from the text describe a normal ELISA assay and a sandwich assay that is used for small proteins especially cytokines. Note that the left hand figure has an error. We can't use round bottom wells because the absorbance is red directly in the wells and the round bottoms would cause distortion.

In today’s laboratory we will only demonstrate the normal ELISA for proteins. Because the ELISA method is so sensitive we often see strong absorbances for all sera, even if the antibody titers are different. On way to see the differences in antibody titers is to run serial dilutions of the sera. If this is done then the endpoint titers can be measured. The endpoint titer will be the highest dilution that still gives a significant absorbance above background. Three different antigen:antibody systems will be used in today's demonstration:

I. Goat serum:anti-goat serum

II. Chicken serum:anti-chicken serum

III. Bovine albumin:anti-bovine albumin

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ELISA ASSAY

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ELISA ASSAYProtocol:

1. Plates containing the appropriate antigen and already blocked will be available

2. Set up serial (1:5) dilutions (in blocking buffer) of your antiserum as follows:

a. add 0.4 ml of buffer to 7 tubes (label them 2 - 8)b. add 0.1 ml of antiserum to tube number 2c. mix thoroughly and remove 0.1 ml and add to the next tubed. repeat step c until all dilutions have been made

3. Keep 0.4 ml of the original antisera as an undiluted sample and label it tube #1

4. transfer 100 l from each tube (including the undiluted sample) to triplicate wells of the plate. Tube #1 in row A, tube #2 in row B, etc. Group 1 will use columns 1 - 3, #2 - columns 5 - 7, and group 3 - columns 9 - 11.

5. Add buffer only to columns 4, 8, and 12 as negative controls

6. Incubate at 37o for 1 hour

7. Gently wash 4 times by filling wells with 0.05% tween 20

8. Add 100 l of Protein-A:alkaline phosphatase to all wells including the negative control columns

9. Incubate for 1 hour at 37o

10. Wash thoroughly as in 7 above

11. Add 100 l of substrate solution to all wells

12. After strong yellow color is apparent in undiluted samples read absorbances in the ELISA reader

13. The last row that has an average reading at least 0.1 Absorbance units above background will be the end-point titer to your antiserum

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Cellular ImmunityMeasurement of Cellular Immune Functions

A. Isolation Methods:

Several methods may be used to isolate immune cells from human peripheral blood or from animal organs such as the mouse spleen. The simplest and oldest is the Ficoll-Hypaque method that isolates cells by density as shown below. Cells are carefully layered onto a dense solution of Ficoll-Hypaque then centrifuged. Dense cells like RBC's and granulocytes will spin down through the Ficoll-Hypaque but most lymphocytes will stay right on top of the dense layer. These cells are carefully pipetted off.

Isolation of specific cell types may be obtained by using cell-specific monoclonal antibodies. One method to achieve this is to combine the monoclonal antibodies with magnetic beads as shown below. For example, CD4 cells can be isolated by using an anti-CD4 monoclonal bound to magnetic (iron) bead. After incubation with the beads the cell suspension is put through a column surrounded by a strong magnet. The beads with attached CD4 cells will bind while all other cells are washed out. The CD4 cells can then be eluted by changing the ionic strength of the buffer and washing out the desired cells.

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Cellular Immunity

21

Cellular ImmunityA second method for specific cell isolation is by panning (see below). In this method the monoclonal antibody (e.g. anti-CD4) is attached to a petri dish. Cells are incubated in the dish and CD4 cells bind to antibody. Other unbound cells are washed out, then the bound cells eluted by increasing ionic strength.

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Cellular ImmunityB. Cellular Assays:

1. Proliferation Assays: Lymphocytes from antigen-primed mice divide rapidly when exposed to antigen in vitro. A common assay is to incubate isolated cells (lymph node or splenocytes) with antigen for 3 - 6 days and measure cell growth as seen below. Cell proliferation can be measured by incorporation of 3H-thymidine into DNA, increase in cell numbers, or dye incorporation such as the MTT dye assay.

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Cellular Immunity2. Cytotoxicity: CD8 cell function is determined by the ability of cells to kill target

cells (known as cellular cytotoxicity). The simplest technique is to load the target cells with a metabolically inert substance such as chromium as shown below. In this case the chromium compound is made with a radioactive Chromium isotope 51Cr. If the target cells are attacked by the CD8 cells the chromium will be released into the culture supernatant (typically the incubation times are 4 hour or 24 hour releases). After incubation cells are spun out and the radioactivity in the supernatant is measured. Some newer methods have been developed that avoid the avoid the use of the high energy 51Cr emitter.

3. Cytokine profiles: CD4 subset (TH1 vs. TH2) cellular activity can be determined by the set of cytokines produced. Typically isolated lymphocytes are incubated with target cells or antigens for 24 - 48 hours. After incubation the cells are spun down and the supernatants collected. Specific cytokines are measured by sandwich ELISA assays. If TH1 cells are activated the profile should include IL-2, IL-12, and Interferon . TH2 cells will produce IL-4, IL-5, and IL-10.

C. Analysis of Immune Cells:

Immune status can be estimated by the number of immune cells (WBC counts) and also by the relative numbers of CD4 and CD8 cells. In healthy individuals the CD4/CD8 ratio should be close to 2:1 as shown below. Relative immune cell numbers can be measured by flow cytometry if monoclonal antibodies to surface markers are available. In today's lab you will isolate splenocytes to measure CD4/CD8 ratios using flow cytometry as shown in the figure below.

Protocol:

1. All mice will be anesthetized and bled out for the final bleeding.

2. After sacrificing, the spleen of one mouse/group will be removed for assay.

3. Place the spleen into a 35 mm petri dish

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Cellular Immunity

25

Cellular Immunity4. Crush the spleen with a glass stopper and grind for 15 seconds.

5. Place a 1-inch square nylon mesh screen on to of a 15 ml centrifuge tube

6. Gently rinse the dish with 5 ml of HBSS (be careful!! The dish barely holds 5 ml) and break up clumps.

7. Collect the suspended cells using the same pipet

8. Slowly drop the cell suspension onto the nylon mesh. The cells should pass through the mesh into the tube while large structures such as the membrane surrounding the spleen remain on top of the mesh.

9. Spin down the cells then pour off the supernatant.

10. Resuspend cells in 5.0 ml of ice-cold 0.87% ammonium chloride to lyse the RBC's.

11. Incubate for 5 minutes gently mixing every minute.

12. After 5 minutes add 5.0 ml of HBSS then spin down cells.

13. Remove the supernatant and resuspend cells in 10.0 ml of HBSS. Take an aliquot (50 l) out and count cells.

14. While counting spin down cells and pour off the supernatant.

15. Resuspend cells in HBSS to give 2 x 106 cells/ml

16. Transfer 0.5 ml aliquots to three separate microfuge tubes (labeled with group # and tube numbers 1-4)

17. Spin down cells and pour off supernatant.

18. Add ice-cold primary monoclonal antibodies (rat IgG's) in 100 l of HBSS as follows:

Tube # Antibody

1 no antibody (100l of HBSS)

2 anti-CD4 antibody

3 anti-CD8 antibody

19. Incubate for 30 minutes on ice.

20. Spin down cells and pour off supernatant

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Cellular Immunity

21. Wash 3 x 1.0 ml ice-cold 2% BSA in HBSS

22. Add ice-cold FITC-secondary antibody (goat anti-rat IgG) in 100 l of HBSS

23. Incubate for 30 minutes on ice in the dark.

24. Spin down and wash cell 3 X as above.

25. Resuspend cells in 400 l of fixing buffer and transfer to labeled tubes.

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IMMUNOBLOTTINGImmunoblotting combines the technique of electrophoresis and a sensitive immunodetection system such as an enzyme-linked antibody. In general, antigens are separated by electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) which separates according to molecular weight. Because the polyacrylamide gel does not allow most secondary antibody conjugates to diffuse in, the separated antigens must be removed from the gel. This is accomplished by electrophoresing them horizontally onto a thin sheet of nitrocellulose paper. The pattern of components separated by the SDS-PAGE is now immobilized on the nitrocellulose paper. The antigens can now be detected in a manner very similar to the ELISA assay. The paper is incubated with primary antibody, washed several times with blocker, then incubated with an enzyme-linked secondary antibody. However in immunoblotting the enzyme is usually a peroxidase because the colored product is not soluble in solution and precipitates directly onto the nitrocellulose paper. Thus, wherever the antigens are separated by electrophoresis they will be blotted onto the nitrocellulose and bind the primary antibody. The secondary antibody will bind and the enzyme reaction will produce a dark band at the site of the separated antigens. Immunoblotting is a very powerful technique for detection of antigens that are difficult to isolate form complex mixtures such as cell lysates. Because of the enzyme-linked detection system it is extremely sensitive and can detect reactions which could not be seen by precipitations in gels.

Western blots typically take three days as follows:

DAY 1 - Formation of slab gel

DAY 2 - Formation of stacking gel with sample wells

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IMMUNOBLOTTING

- PAGE separation of antigens

- Blotting onto nitrocellulose

- Cutting individual strips of nitrocellulose

- Blocking of strips

- Addition of antisera to individual strips and overnight incubation

DAY 3 - Addition of biotinylated secondary antibody to strips and 1 hour incubation

- Addition of avidin:peroxidase to strips and 1 hour incubation

- Addition of substrate

The electrophoresis and blotting steps typically take 5 hours. We can run two minigels simultaneously. Each lab section will have a single gel and blot. These will be subdivided into three sections so that a complex antigen for each group can be run. The gels will be pre-cast on Friday and the stacking gel formed an hour before use on Monday. The stacking gels will be formed as shown below with a small well for the molecular weight standards and larger wells for three antigens. Complex antigens (using 100 - 200 g of protein) will be mixed with an extraction buffer containing SDS and a blue tracking dye (bromphenol blue) and put into the individual wells. A standard mixture of proteins of known molecular weights is added to the control well. The gel antigen loading for both sections will be done first thing in the morning lab using the template below.

Electrophoresis of the gels will take about 1 & 1/2 hours. It is complete when the tracking dye reaches the bottom of the gel. The gels will be removed, washed in buffer, and the stacking gels removed. The separated proteins will then be blotted from the gels onto nitrocellulose paper. Both gels will be done simultaneously. A sandwich will be made containing both gels and nitrocellulose papers separated by sponges and filter paper. This is a delicate procedure to be certain that gels tightly fit against the nitrocellulose without any air bubbles and in the proper order. The sandwich is then placed into the gel apparatus and the blotting takes place. The actual transfer of proteins occurs because they are electrophoresed onto the nitrocellulose. This electrophoresis continues for 2.8 hours.

After blotting the sandwich is removed and the top and bottom of the gels are marked on the nitrocellulose by drawing lines using a pen. The gel can then be removed and discarded (sometimes the gel is stained for protein to make sure that all were blotted onto the nitrocellulose. The nitrocellulose is immediately placed into PBS; it can not be allowed to dry. Each sheet must be done separately as follows: The sheets are placed on a glass plate and the three separate antigen lanes identified and marked on top and bottom. The molecular weight standard strip is cut off with a scalpel and allowed to dry. Eight vertical strips are then cut from each antigen lane.

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IMMUNOBLOTTINGStd #1 #2 #3

The top part of each strip should be labeled A - H. Each strip goes into the appropriate trough of an incubation tray.

The remainder of the assay is similar to the ELISA method. Except the final substrate used is one that forms an insoluble precipitate on the blot instead of soluble color in a well. The general steps will be:

Block 5% milk/PBS for 1 hour

1Antibody incubation overnight at 4o

Wash 5X Tween-202Antibody incubation 1 hour at 37o

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Wash 5X Tween-20

3Antibody incubation 1 hour at 37o

Add substrate and stop when bands are dark

Protocol:

1. Set up two incubation trays as shown below

Anti IgG A Negative Control" B Pre-immune Bleed" C Second Bleed" D Third Bleed

Anti IgM E Negative Control" F Pre-immune Bleed" G Second Bleed" H Third Bleed

2. Clearly label each tray with Group # and Tray A or B

3. Add 2.0 ml of blocker to all troughs of Tray A

4. Add 1.0 ml of blocker to all troughs of Tray B

5. Add 10 l of your mouse sera to the following troughs

A none (negative control) Anti IgG 2AntibodyB pre-immune bleed Anti IgG 2AntibodyC 2 immune bleed Anti IgG 2AntibodyD 3 immune bleed Anti IgG 2AntibodyE none (negative control) Anti IgM 2AntibodyF pre-immune bleed Anti IgM 2AntibodyG 2 immune bleed Anti IgM 2AntibodyH 3 immune bleed Anti IgM 2Antibody

6. When blot strips are cut they will be placed in the troughs of Tray A and blocked for 1 hour.

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IMMUNOBLOTTING7. The strips will then be transferred to Tray B for overnight incubation with antibody

8. The assay will be completed tomorrow

9. Strips will be washed extensively with 0.05% tween-20/PBS

10. Incubate strips with biotinylated 2Antibody (A - D:goat anti-mouse IgG, E - H goat anti-mouse IgM)

11. Strips will be washed extensively with 0.05% tween-20/PBS

12. Incubate strips with avidin:peroxidase complex

13. Strips will be washed extensively with 0.05% tween-20/PBS

14. Add substrate and develop

15. Stop reaction by washing with distilled water

16. Dry strips overnight

17. Align strips in proper order adjacent to molecular weight markers and mount strips on filter paper

18. Relative antibody titers to each antigen can be determined by examining the intensity of each band. This may be eye-balled or can be quantified by scanning the lanes into an appropriate analysis program.

19. The molecular weights of each band can likewise be determined. They may be estimated by eye by comparison to the molecular weight standards. They may be precisely calculated by comparison to a plot of the standard molecular weights vs. migration distance

Below is an example of western detection of antibodies to HIV proteins GP24, 40 and 120.

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