MAN99

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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.
1
THE MOUSE STUDY
IMMUNIZATION PROTOCOL: Each group will be assigned two of the following antigens:
1.
2.
3.
4.
5.
bovine serum albumin
human globulin
human albumin
Salmonella typhi
horse serum
6. bovine serum
7. chicken serum
8. goat serum
9. rabbit serum
10. Chicken Albumin
2
THE MOUSE STUDY
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
Week 2
Week 3
Week 5
Week 7
bleed mice (pre-immune bleed). Immunize mice to antigens in Ribi
adjuvant
give booster immunizations using antigens in Ribi Adjuvant
bleed mice (first immune bleed)
give booster immunizations using antigens in Ribi Adjuvant
bleed mice (final bleed) 2 weeks after Booster
3
THE MOUSE STUDY
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 reacquaint 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.).
4
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.
II.
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.
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.
5
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:
II.
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.
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
6
Agglutination Reactions
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.
7
Agglutination Reactions
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.
8
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
9
Precipitation Reactions
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.
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.
10
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, to
perform 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
11
Precipitin Reactions
is used clinically to determine the quantity of various serum antigens including different classes and
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.
12
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.
13
Precipitin Reactions
14
Immunoelectrophoresis
This 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.
15
Immunoelectrophoresis
Serum
Antigen 1
5.
o
o
Serum 1
Serum 2
o
Antigen 2
Antigen
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.
16
ELISA ASSAY
The 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
17
ELISA ASSAY
18
ELISA ASSAY
Protocol:
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.
b.
c.
d.
add 0.4 ml of buffer to 7 tubes (label them 2 - 8)
add 0.1 ml of antiserum to tube number 2
mix thoroughly and remove 0.1 ml and add to the next tube
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
19
Cellular Immunity
Measurement 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.
20
Cellular Immunity
21
Cellular Immunity
A 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.
22
Cellular Immunity
B.
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.
23
Cellular Immunity
2.
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
24
Cellular Immunity
25
Cellular Immunity
4.
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:
19.
Tube #
Antibody
1
no antibody (100l of HBSS)
2
anti-CD4 antibody
3
anti-CD8 antibody
Incubate for 30 minutes on ice.
26
Cellular Immunity
20.
Spin down cells and pour off supernatant
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.
27
IMMUNOBLOTTING
Immunoblotting 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
28
IMMUNOBLOTTING
DAY 2
- Formation of stacking gel with sample wells
- 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
29
IMMUNOBLOTTING
standard strip is cut off with a scalpel and allowed to dry. Eight vertical strips are then cut from
each antigen lane.
30
IMMUNOBLOTTING
Std
#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
1
overnight at 4o
Antibody incubation
Wash
5X Tween-20
31
IMMUNOBLOTTING
2
Antibody incubation
1 hour at 37o
Wash
5X Tween-20
3
1 hour at 37o
Antibody incubation
Add substrate and stop when bands are dark
Protocol:
1.
Set up two incubation trays as shown below
Anti IgG
"
"
"
Anti IgM
"
"
"
A
B
C
D
E
F
G
H
Negative Control
Pre-immune Bleed
Second Bleed
Third Bleed
Negative Control
Pre-immune Bleed
Second Bleed
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
B
C
D
E
F
G
H
none (negative control)
pre-immune bleed
2 immune bleed
3 immune bleed
none (negative control)
pre-immune bleed
2 immune bleed
3 immune bleed
32
Anti IgG 2Antibody
Anti IgG 2Antibody
Anti IgG 2Antibody
Anti IgG 2Antibody
Anti IgM 2Antibody
Anti IgM 2Antibody
Anti IgM 2Antibody
Anti IgM 2Antibody
IMMUNOBLOTTING
6.
When blot strips are cut they will be placed in the troughs of Tray A and blocked for 1
hour.
7.
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 antimouse 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.
33
IMMUNOBLOTTING
34
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