Antibodies are incredibly useful in
a number of different diagnostic
assays and actually throughout the
whole of biology, there are uses for
monoclonal antibodies because their
really high degree of specificity makes
them excellent probes to detect the
presence of particular molecules.
One can produce polyclonal antibodies,
that is antibodies produced from
a number of different B-cells by
immunizing an animal with an antigen.
So here we have an example, taking a protein and
immunizing a sheep with this particular protein.
One can then collect blood from the
sheep a period of time later, one gives
the sheep a chance to make some antibodies,
the immune response gets going.
And then a few weeks later, after a couple of
boosters, one can take some blood from the sheep.
And there will be polyclonal antibodies
that are specific for this antigen.
And the proportion of antibodies
because you’ve immunized the sheep, the
proportion of antibodies against this
particular antigen will be increased.
So they won’t all be against this antigen, there’ll
be antibodies against many other antigens as well.
But the percentage of antibodies against the immunizing antigen
will have been increased by the immunization procedure.
So you’ll have different antibodies against different
parts of the particular antigen of interest.
So for example, immunizing with human IgG, you can
develop sheep anti-human IgG as a reagent to use in
immunodiagnostic tests, where you want to see if a patient
has IgG antibodies that bind to a particular antigen.
For example, an autoantigen.
And using these kind of sheep antibodies
and then labeling them with a fluorescent
dye or with an enzyme will allow their
use in immunodiagnostic assays.
So those are polyclonal antibodies,
a mixture of antibodies.
And very often it’s useful to have a mixture
of antibodies rather than a single specificity.
But sometimes you want to have all
of the antibody absolutely identical.
And specific for perhaps one
single epitope on an antigen.
It’s possible to do this using the hybridoma
approach to produce monoclonal antibodies.
In this approach, an animal is
immunized with the antigen of choice.
The spleen is removed from the mouse in this
example, and it’s making-- within the spleen will
be B-cells that are making antibody against the
antigen that the mouse was immunized with.
B-cells don’t survive for
very long outside of the body.
However, B-cells can become
malignant, become tumor cells.
And these tumor cells will have long term
survival, that’s a characteristic of a tumor cell.
It divides very rapidly
and it survives.
By fusing together, the normal B-cells from the
spleen of the immunized mouse, together with
a malignant B-cell in the form of a myeloma,
one can produce what are called hybridomas.
And these hybridomas which is a hybrid of the normal
B-cell and the tumor B-cell, inherit two properties.
They inherit the antigen specificity
of the normal B-cell but they also
inherit the immortality of the tumor
cell, in other words the myeloma cell.
So in this methodology, there is fusion between the
immune spleen cells and the myeloma tumor cells.
These cells, hybrid cells are then cultured
in a selective medium that kills off
the unfused tumor cells, because otherwise
they’d keep growing, they’re immortal.
They’d keep growing and
they’d outgrow the hybrids.
So you want to just have the hybrids, the
normal spleen cells die off naturally.
The myeloma cells that have not fused will
be killed by culture in a selective medium.
So only the few cells
survive after a few days.
And the cells are then diluted into a microtitre
plate, so that there is on average one cell per well.
The cells are then grown in the
individual culture plate wells.
And the culture supernatant, what’s
being secreted by these hybridomas,
can be assayed to see whether they are
producing the antibody of interest.
So the supernatants from the wells containing
the growing hybrid cells are screened for
the presence of the desired antibody using the
enzyme-linked immunosorbent assay, ELISA.
Then the positive wells are grown up to a very large volume
and there is a clone of antibody producing fused cells.
This clone, the hybridoma is an immortal
producer of the desired monoclonal antibody.
So these can be kept growing for
years and years, and years, producing
antibody that is all of identical
specificity, monoclonal antibody.
There is another approach to making monoclonal
antibodies and this is called phage display.
For example, maybe one wants to develop
a monoclonal antibody against a tumor
antigen as a therapeutic agent to treat
patients with a particular tumor.
One can immunize a mouse with this tumor-derived antigen,
take the spleen from these mice and isolate the B-cells.
Messenger RNA is then taken from these
B-cells and converted into cDNA.
PCR, the polymerase chain reaction, is then
used to amplify the immunoglobulin heavy chain
variable region and the immunoglobulin light
chain variable region using specific primers.
These two sequences, the immunoglobulin heavy
chain variable region and the immunoglobulin
light chain variable region are then
linked together using a flexible linker.
And this gene sequence is inserted into
bacteriophages which are viruses that infect bacteria.
Now what you have is a mixture of
phages on the cell surface, or on the
surface of these phages rather, you
will have the antibody present.
And inside the phage, you will have the gene
encoding that particular specificity of antibody.
You can then use a method that is often referred
to as panning; it’s a bit like panning for gold.
But here you’re panning for a phage that has on its surface
an antibody fragment that recognizes the antigen of interest.
So by incubating a mixture of different phages with different
antibodies on their surface, you’re after one particular
antibody with specificity for a particular tumor antigen, by
panning on a plate where there is immobilized tumor antigen.
You can select phages that
have high affinity binding.
They will have inside them the
gene sequence that you want.
And then you can express that in
a particular expression system.