T4.

Polyclonal and monoclonal

antibodies.
1. Polyclonal antibodies.
Polyclonal antibodies come from a heterogeneous cell population, where they are products of
several B-cell clones, resulting in a mixture of several molecules with varying affinity and
binding site specificity. The origin is the immunization of an individual by an immunogen.

2. Monoclonal antibodies.
Monoclonal antibodies come from a homogenous cell population, where they are products of
1 clone of plasma cells derived from B-cells, resulting in identical copies of immunoglobulins
with equal primary structure, binding site specificity and effector functions. The origin can be
spontaneous in case of cancer growth of plasma cells, for example, or artificial in case of
lymphocyte hybridoma.

Some properties of these monoclonal antibodies are that they are chemically identical,
monospecific (against only 1 epitope), effective in lower concentrations, non-precipitating and
they can be prepared in unlimited amounts.

3. Antibody production.
Antibodies can be produced by exposing an
organism to an antigen, but we can also increase
this production by hybridizing plasma cells to
myeloma cells, creating hybridomas that can be
then separated by properties.

Hybridoma technology consist in fusing B-cells,

which contain certain selector gen and
immunoglobulins gens but that are mortal, with
myeloma cells without the selector or
antibodies gens but that are
immortal. By putting them into
polyethylene glycol, we can obtain
heterokaryions, which will be selected
in a concrete culture. Unfused B-cells
or double fused B-cells will die
because they are mortal, myeloma
cells or double fused myeloma cells
will die because they don’t have the
selector gen, and only hybrid cells will
survive.

In case of human antibodies, we can

use mice with SCID (severe combined
immunodeficiency) which not have humoral immunity (but it’s very dangerous for the animal)
or cloning large fragments of DNA in artificial yeast chromosomes (but glycosylation may isn’t
correctly produced).
 3.1. Cell fusion
To promote cell fusion, we can use fusogens substances, which are substances that ease the
cell joining. Most commonly used are inactivated Sendai virus or other viruses, and
polyethylene glycol, so we can obtain unfused cells, hybrid cells or 2 equal cells, but after
selection only hybrid cells will remain. Like this, we will obtain cells that have sustained growth
in vitro and in vivo, which cannot produce myeloma antibodies and which are differentiated to
produce antibodies after fusion.

3.2. Selection.
Method based on enzyme defects are usually used in selection. This consist in inducing enzyme
defects in myeloma cells, so they won’t be able to grow in selection media but hybrid cells do
will be able, because they compensate the loss of enzyme from the other cell. We normally
produce nucleotide synthesis defects, most used are:

 HGPRT (hypoxanthine-guanine phosphoribosyltransferase), which are mutants

obtained from cultivation with 8-azaguanine.
 TK (thymidine kinase), which are mutants obtained from cultivation with 5-
bromodeoxyuridine.

The selection medium use to be HAT (hypoxanthine, aminopterin and thymidine) and it’s
known that addition of insulin into HAT medium increases hybridoma formation. Hypoxanthine
and thymidine are XXXXXXXXXXXXXXXXXXXXX of the enzymes, and aminopterin is a molecule
that blocks nucleotide synthesis de novo, that is, from amino acid and saccharides.

4. Use of monoclonal antibodies

There are a lot of uses for them. Among other applications, they are used for:

 Immunochemical methods, such us specific protein isolation, specific labelling or

determination of specific protein quantity/quality.
 Epitope mapping, that would be the discovery of structure of proteins and sites of
antibody binding.
 Cell surface antigen mapping, obtaining information like quantification of individual
cell populations and subpopulations or determining differentiation stages of cells.
 Infectious disease diagnosis, by determining the pathogen that caused a disease.
 Diagnosis and experimental cancer treatment. This consist in the localization of tumors
in an organism by radioimmune scintigraphy and preparation of immunotoxins for
targeted cancer treatment (immunotoxins are antibodies conjugated with toxins).
 Experimental treatment of autoimmune diseases, using antiidiotypic monoclonal
antibodies against autoantibodies.

5. Antibody genetic engineering.

If we obtained a mouse monoclonal anti-tumor antibody
and we wanted to use it in human, we couldn’t do it
because human body would refuse it. Because of this,
we can do some genetic engineering, by placing variable
domains or CDRs of human antibodies by the equivalent
of the previous ones. We can also obtain with this tool
chimeric immunotoxins or heteroconjugated antibodies.
In case of heteroconjugated antibodies, we are talking of quadroma technology (fusion of 2
hybridomas), trioma technology (fusion of hybridoma and plasma cell) or genetic engineering
(fusion of 2 variable genes).

With genetic engineering we can increase immunocompatibility, make changes in biological

average life or in production of alternative isotypes, also increase in affinity or avidity, change
effector functions and make hybrid and heteroconjugated antibodies.

With this technology we can avoid problems like biocompatibility, such us in use of mouse
antibodies in humans, because they have a short average life in serum, they don’t substitute
effector function and they can induce undesirable immune reactions or formation of human
anti-mouse antibodies.

6. Other types of antibodies.

There is a very big diversity of antibodies:

 Single chain antibodies. They are created by covalent bond between variable
fragments and various effector molecules.
 Catalytic antibodies. They have an enzyme instead of FC domains or their parts.
 Abzymes. They have catalytic site equivalent to the binding site and specifically bind a
haptene. Either this and the previous ones are effective catalysts of organic synthesis
reactions, so we can create antibodies, for example, against the substrate or an
intermediate product of the enzyme reaction.

7. Antibody isolation methods.

Most used are non-specific methods, like:

 Fractionated precipitation. Antibodies are going to precipitate in a differential way

regarding on their properties, by using neutral salts, organic solvents, metal ions,
organic cations or polymers.
 Electrophoresis. Separation according to charge and size.
 Isoelectric focusing. Separation according to isoelectric point.
 Ion exchange chromatography.
 Gel filtration chromatography.
 Hydrophobic chromatography.
 Chromatofocusing.

We can also use specific methods, like immunoafinity chromatography.