This particular exercise demonstrates that the most profound
discoveries in
immunology are really quite simple techniques. After completion
of this
section the student is expected:
l. To be able to perform the direct Plaque Assay or the Jerne
technique as it is also known and apply it to several different
experiments
2. To describe the principle demonstrated by this technique.
3. To be able to compare the direct and indirect plaque assays
4. To answer the questions at the end of the exercise.
Direct Plaque Assay
McMaster and Houdack observed that lymphatic tissue produces
antibody and
since then researchers have been searching for the antibody
producing cell.
Jerne and Nordin developed the hemolytic plaque technique and
thus were able
to visualize the antibody forming cell directly. A brief
description is
given here while the method is detailed in the
dpaMethodgeneral procedure.
The direct Plaque assay or Jerne technique, allows the
enumeration in agar of
cells producing antibodies to sheep RBC's or other erythrocytes.
A
suspension of spleen cells from a previously injected animal is
mixed with
the test erythrocytes and molten agar. After the agar has
solidified the
mixture is incubated at 37C for one hour, during which time the
antibody
producing cells will release antibody that will bind unto the
antigen (the
sheep RBC's). Subsequent addition of complement will lyse the
antibody
coated erythrocytes, causing the appearance of a clear plaque
with the
antibody producing cell in the middle.
Several specific points should be noted.
Generally, sheep erythrocytes are used as an antigenic stimulus
but almost
any type of cell can be substituted for the antigen, including
tissue culture
cells and bacteria. The technique can be further modified to
detect cells
producing antibodies to soluble antigens. Erythrocytes are coated
with the
soluble immunizing antigen before they are mixed with the spleen
cells and
the agar. The secreted antibody then binds unto the coated
erythrocytes which
are destroyed upon addition of complement.
Indirect Plaque Assay
Erythroctyes will lyse in the presence of complement if the
antibody is of
the lgM class Those cells coated with antibody of the lgG class
will not
lyse because the antibodies are too far apart to bind complement.
However,
the system may be modified to detect lgG producing cells. The
DPAdirect plaque
assay technique isDPAMethodperformed and the
plaques are counted and marked. Anti-lgG is
added and the plate is re-incubated with more complement. Any new
plaques
are the result of the IgG producing cells.
Direct Plaque
Assay: Method
l. Put 2.0 ml of Hanks balanced salt solution (HBSS) in a small
mortar and
pestle and cool it in an ice bath.
2. Kill the mouse with an overdose of ether by placing the mouse
in a small
jar with an ether soaked cotton swab and replacing the lid.
3. Remove the dead mouse from the jar, put it on a paper towel,
swab the
abdomen with 70% ethanol and cut open the abdomen. Cut out the
spleen and
make sure that excess fat and tissue is removed. The location of
the spleen
was described in the exercise on the anatomy of the immune
system.
4. Put the spleen in the 2.0 ml of cold HBSS and cut it into
small pieces.
Grind these small pieces with the pestle until an even cell
suspension is
formed.
5. Filter the suspension through a cheesecloth that has been
placed in a
small funnel. This will remove any large clumps of cells. Flush
the few
remaining trapped cells from the cloth with 5.0 ml of cold HBSS.
6. Perform a ten-fold dilution series of the spleen suspension in
the
following manner: Label 4 test tubes - l, l:l0, l:l00, l:l000 and
put 5.0 ml
of the spleen cell suspension in tube l. Put 9.0 ml of cold HBSS
in each of
the other three tubes. Mix l.0 ml of the cell suspension from
tube l with
9.0 ml of the HBSS to form a l:l0 dilution. Then mix l.0 ml of
the l:l0
dilution with 9.0 ml of the HBSS to give a l:l00 dilution. Repeat
with the
last tube to obtain a l:l000 dilution.
7. Remove 4 test tubes of agar from the 50`C water bath and place
them in a
beaker filled with warm water. Label these test tubes l, l:l0,
l:l00 and
l:l000 as before. Add 0.5 ml of the appropriate dilution of the
spleen
suspension to the agar and mix the contents.
8. Add 0.l ml of sheep RBC's to each tube and mix again.
9. Quickly pour the mix contents into a petri dish that already
has a bottom
layer of agar. Swirl the petri dishes in a figure eight to obtain
an even
top layer; allow that agar to harden and then incubate the plates
at 37`C for
one hour. Make sure that the petri dishes are labelled with your
name and
the dilution used.
10. Add 2.0 ml of diluted guinea pig complement to each petri
dish, swirl
the dish to distribute the solution evenly and re-incubate at
37`C for 30
min.
ll. Remove the plates from the incubator and store them at room
temperature
for 30-60 min. After which pour off the complement, rinse the
plates twice
with saline and store the plates in the cold room overnight.
l2. Stain the plates the next morning by adding 8.0 ml of freshly
prepared
benzidine reagent to each plate and allowing it to develop for l0
min.
l3. Pour off the stain and rinse the plates twice with saline
before
counting the plaques which appear as light areas on a dark blue
background.
l4. Calculate the number of plaques/ml of spleen suspension.
Remember that
only 0.5 ml of each dilution was used. Calculate the number of
antibody
producing cells per spleen.
Direct Plaque
Assay: Experiments
Establish how the number of antibody producing cells increases
with time
Mice are injected intraperitoneally, with sheep RBC's as the
antigen. Some
mice are injected 24 hours before the experiment, others 48, 72,
96 and l20
hours earlier. A control group of mice are not injected. All the
animals
are sacrificed and the number of antibody producing cells in each
spleen is
determined by the Direct plaque assay ( or immunocytoadherence).
Plot this
value against the time of injection.
An antibody producing cell forms antibody against a single
antigen.
Mice are injected intraperioneally with a mixture of two
different antigens
that could be identified easily; sheep RBC's which have no
nucleus and
chicken RBC's which are nucleated. Sacrifice these mice after
five days;
remove the spleen and perform the Direct Plaque Assay with the
following
modification. Add 0.l ml of sheep RBC's and 0.l ml of chicken
RBC's to the
spleen suspension as described in step eight (8) of the general
procedure. Incubate and stain as described but look at the
plaques under low
power magnification.
If an antibody producing cell formed antibodies against both
sheep RBC's and
chicken RBC's, a zone of complete lysis would occur around this
cell. If the
cell produced antibody to only one species of erythrocyte (i.e.,
one antigen)
the plaques would appear hazy because of the presence of the
other type of
erythrocyte (antigen).
Direct Plaque
Assay: Questions
l. Are there any antibody producing cells in the uninoculated
animals? If
so, how many? Does this observation favour the selective theory
or the
instructive theory of antibody formation? Why?
2. The lag phase is that time before the number of antibody
forming cells
increases. What event occurs during this phase?
3. How many cells are produced by the end of the log phase of the
curve?
4. How many generations of cells are involved? How long does it
take for a
cell to divide after the antigen has been processed.
5. Can a single cell produce antibodies against more than one
antigen?
6. Does this favour Ehrlich's selective theory or the clonal
selective
theory?
7. Does the mouse produce some antibody forming cells directed
against sheep
RBC's and at the same time does it produce antibody forming cells
directed
against chicken RBC's or is only one antigen recognized by the
animal at a
time?
8. The Jerne technique is capable of detecting directly only
antibodies of
the lgM class yet lgG will fix complement also. Why are
antibodies of the
lgG class not detected directly?
9. How would you modify the experiment to measure the number of
antibody
producing cells that release lgG?
l0. How would you detect cells that release lgD, lgE or lgA since
these
classes of immunoglobulins do not fix complement?
ll. What would be the effect of increasing the incubation time in
step 9
before you add the complement?
l2. What would be the effect of increasing and decreasing the
concentration
of complement
l3. What does benzidine do?
Lab:
Immunocytoadherence
This particular exercise demonstrates that the most profound
discoveries in
immunology are really quite simple techniques. After completion
of this
section the student is expected:
1. to be able to perform the immunocytoadherence technique for
the
detection of rosette forming cells.
2. to distinguish between T-cells and B-cells in the immunocyto-
adherence technique.
IMMUNOCYTOADHERENCE
When lympocytes are incubated in suspension with sheep
erythrocytes, those
lymphocytes with appropriate receptors (antibodies) on their
surfaces will
bind the erythrocytes to form a rosette. These cells designated
rosette
forming cells (RFC) may be either T-cells or B-cells. T-cells
have the
ability to form rosettes in non-immunized animals and the rosette
is only one
layer deep. It is not understood why these rosettes are formed
but their
formation is inhibited by anti-lymphocyte serum, anti T-cell
serum (anti )
and the numbers are lower in people with T-cell deficiency
syndromes.
B-cell rosettes occur if the animal has had a prior antigenic
exposure to the
erythrocyte and on prolonged incubation. B-cell rosettes are
several layers
deep because the B-cell has had time to secrete antibody.
Mice immunized against sheep RBC's are sacrificed and a
suspension of spleen
cells obtained. This suspension is mixed directly with the test
erythrocytes
in solution. The antibody producing cell will appear in the
centre of a
cluster of erythrocytes which are bound unto its surface. The
number of
antibody producing cells can be determined by counting the number
of clusters
or rosettes in a haemocytometer.
Gudat and co-workers examined the rosette forming cells during
the primary
response and found that 84-98% of the cells were lymphocytes, and
the rest
were plasma cells.
The amount of antibody required for immunocytoadherence is less
than that
required for plaque formation.
IMMUNOCYTOADHERENCE:
Method
l. Repeat steps l-6 of the General Procedure of the Jerne
technique (use the
remaining sample).
2. Mix 0.5 ml of the spleen suspension with 0.5 ml of the test
erythrocytes
and incubate the mixture for 30 min at 37`C.
3. Fill a haemocytometer with the mixture and count the number of
rosettes
that occur in the nine large grid squares.
4. Count the number of cells in the spleen suspension alone and
calculate
the number of rosette forming cells/l000 spleen cells.
Immunocytoadherence:
Experiments
EXERCISE C
The objective of this exercise is to establish how the number of
antibody
producing cells increases with time. Mice have been injected
intraperitoneally, with sheep RBC's as the antigen. Some mice
were injected
24 hours before the experiment, others 48, 72, 96 and l20 hours
earlier. A
control group of mice were not injected. Sacrifice all the
animals and
determine the number of rosette forming cells in each spleen.
Plot this
value against the time of injection.
EXERCISE D
A mouse had been injected with a mixture of sheep and chickens
RBC's 5 days
previously. Sacrifice the animal and determine the number of
rosette forming
cells in the spleen. Use a mixture of sheep and chicken
erythrocytes for the
test. Do you observe any rosettes that are composed of a mixture
of chicken
and sheep RBC's? Do you find some rosettes made up of spleen
cells and sheep
RBC's and other rosettes composed of spleen cells and chicken
RBC's or is
only one class present?
REFERENCES
Sterzl, J. and Riha, I. l965. A localized hemolysis in gel method
for the
detection of cells producing 7S antibody. Nature 208: 858-859.
Dresser, D.W. and Wortiss. l965. Use of an antiglobulin serum to
detect
cells producing antibody with low haemolytic efficiency. Nature
208:
859-86l.
Jerne, N.K. and Nordin, A.A. l963. Plague formation in agar by
single
antibody-producing cells. Science l40: 405.
Ingraham, J. and Bussard, A.J. l964. J. Exp. Med. ll9: 667.
Mercants, B and Araba, T. l966. Lymphoid cells producing
antibodies
against simple haptens detection and enumeration. Science l52:
l378-
l379.