Chapter 6:

IMMUNOTHERAPY

• Nanobodies
• A Library of
Antibodies
• ELISA
 I don’t want you to think that all of
biotechnology is serendipitous,
but…an observation research group
working with specific species of
camels and llamas has discovered that
those creatures possess a very
strange form of antibody called
nanobodies.

• These entities appear to be small parts of

antibodies. Some forms are heavy chains
without light chains, but with the antigen affinity
of light chains. Alternatively, other forms are
light chains with the ability to commandeer
other parts of the immune system, a function
usually associated with the heavy chain. 2
• If a system can be devised to
“humanize” the antibodies – that is, to
engineer a large enough portion of the
protein to fool the human immune
system into accepting it – use of
nanobodies may overcome some of
the obstacles that compromise
monoclonal antibody therapy.
Nanobodies are much smaller than
other types of monoclonal
antibodies and penetrate into
tissues much more easily.
They would be much more effective,
for example, in delivering toxins that
you can chemically link the antibodies 3

to cancer tissues.
• A large variety of monoclonal
antibodies has been and is being
developed, as the specific genes that
code for specific antibodies are
deciphered. A strategy to maintain this
large collection of genetic information in
one place would be invaluable. To this
end, the genes for the light chain of a
number of monoclonal antibodies have
been introduced into living bacterial
phages. 4
• Bacterial phages are viruses that infect bacteria. The
phages are then maintained as a kind of library of this
information, a living library that contains the genetic
information that you want to keep, just like you would
keep a book collection. Such living libraries are known
as combination libraries.
Bacterial phages
• Theoretically, you can go to the antibody library and
order an antibody. The specific phage that has housed
the genetic information for your antibody will be
provided to you. You can introduce this genetic
sequence into bacteria by allowing the phage to invade
your bacteria. If you do this correct, the infected
bacteria will produce the antibody light chain that you
want. This must be later hooked to a heavy chain to
produce the Fab segment of interest. See Figure 6-3. 5

Bacterial phages under an electron microscope

 “A” gene introduced
Gene for the mAb
into living bacteria
“A” isolated
phages

Need for mAb “A” phages grow and

“A” identified live in culture

Bacteria produce “A” phages induced

mAb “A” to infect bacteria 6
 The development of monoclonal
antibodies vastly improved our ability
to diagnosis a number of human
diseases. Using antibodies against a
very specific target, we can detect low
levels of antigens or of antibodies that
are present in the human body under
disease conditions.
• The presence of the antibodies is a very
sensitive indicator that the antigen is
present. The antigen might be
associated with bacteria, a virus, or an
element of the person’s own body,
against which the immune system is
inappropriately mounting a defense 7

(autoimmune disease).
 • A very widely used monoclonal
antibody technique is called ELISA
(Enzyme-Linked Immunosorbent
Assay). ELISA is a method of detecting
the formation of an immunocomplex,
Consider this challenge. the linkage of antibodies and of
You as a diagnostician antigens. You will use the following:
want to know if your
patient has a certain • An antibody to the antigen in question,
antigen in his/her system. attached to a substrate
You might suspect a
streptococcus infection • The patient’s blood, containing the
and want a quick screen. antigen
You know that a patient • A free antibody to the antigen in
with a strep infection
would have strep
question; this one with a linked enzyme
antigens. that will react with a colored substrate
8
• In ELISA, the antibody is fixed to a plastic
well and picks up whatever antigen is
present in the patient’s blood, as shown in
Figure 6-4. Then the material is treated
with another copy of the antibody, this one
enzyme linked. The bound antigen will
form a complex with the new antibody by
binding the new antibody at a site other
than the site binding the antibody fixed to
the well. The enzyme-linked antibody that
has not been bound is washed away. The
remaining molecules form a sandwich
consisting of layers of
antibody/antigen/enzyme-linked antibody. 9
• This sandwich is treated with a substrate that the
enzyme will digest. The digestion results in a
change in color in the substrate. Voila! The antigen
is present if the substrate changes color.
• This technology is the basis for the quick screen
for strep infections and for the over-the-counter HIV
pregnancy-test kits.
• There are other conditions in which the antigen
may not be circulating or may be present in the
body in very small quantities. An example is HIV
infection (or other viruses) where, during a
substantial period of the infection, the antigen is
sequestered inside the victim’s own cells. Another
example of low-level antigen presence is the need
10
to detect antigens for victims of autoimmune ELISA Immuno Explorer Kit
diseases.
• You might have a child patient at risk
for diabetes and need to determine if
the child has antibodies that indicate
he/she is mounting an immune
response to his/her insulin-producing
cells. Remember, once activated by
exposure to an antigen, B-lymphocytes
continue to produce antibodies,
although at decreasing levels. The
detection of circulating antibodies is a
very sensitive test for the presence of
the antigen somewhere in the body. 11
 • This antibody attaches to the antibodies
that are attached to the antigen simply
because they are human antibodies.
The excess is washed away. Where the
In this case, you would use a second antibody has attached, the
modification of ELISA, enzyme digests the substrate so that
whereby the antigen is you can see the immunocomplex. It’s
attached to the substrate, and
the patient’s blood containing another sandwich!
the antibody is allowed to
react with the antigen. to
detect the formation of this
immunocomplex, you will
need another antibody with
an enzyme link. This antibody
is engineered to react to any
human anti-body. It is an anti-
human antibody. 12
One note of caution: most assays
need to determine the trade-off
between sensitivity and accuracy
based on the use of the results.
In the case of HIV, you may want to
make sure that you don’t tell patients
that they are HIV negative when in fact
they are HIV positive.

• As a result, you would rather

err on the conservative side
even though sometimes the
assay will determine that a
patient is HIV positive when in 13

fact he or she is not.

In other words, the
assay for HIV is very
sensitive but is prone
to false positives. Final
determination of the
presence of HIV
therefore requires a
repeat of the test or the
performance of
additional, more
accurate tests. 14