It is a method for producing large numbers of identical antibodies (also called monoclonal antibodies).

1. POLYCLONAL AND MONOCLONAL
ANTIBODY PRODUCTION
Amer Ali Khaleel
M.SC in Medical Immunology
Hawler Medical University

2. HYBRIDOMA TECHNOLOGY
It is a method for producing large numbers
of identical antibodies (also called
monoclonal antibodies).

3. Hybridoma is immortalized cell derived from
the fusion of B lymphoblast's with a
myeloma cell.

4. ANTIBODY
Antibodies (Abs) or Immunoglobulins
(Igs) are glycoprotein having combing
sites for antigen & are produce by plasma
cells in response to stimulation of B cell
by foreign antigen.

5. Figure: Basic structure of a monomeric immunoglobulin molecule.
Domains
CH3

6. BONDS OF ANTIGEN-ANTIBODY INTERACTION
1. Hydrogen bonds,
2. Electrostatic bonds,
3. hydrophobic interactions
4. van der Waals forces,
Antibody and antigen reactions are reversible

7.  Affinity refers to how a specific combining site on the
antibody is attracted to a specific epitope on the antigen
(remember that the antigen has multiple epitopes).
 Avidity refers to the overall interaction between the
antigen-antibody complex. In other words, avidity is the
sum of all affinities.

8. The most important function of antibodies is to confer
protection against microbial pathogens, by different ways.
1- Neutralization.
2- Opsonization.
3- Killing by Oxidation.
4- Agglutination and precipitation.
5- Antibody-dependent cellular cytotoxicity (ADCC).
6- Antibodies activate the complement system.
FUNCTIONS OF IMMUNOGLOBULINS:

9. IMMUNOGLOBULIN CLASSES (ISOTYPE)
The immunoglobulins can be divided into five different
classes, based on differences in the amino acid sequences
in the constant region of the heavy chains. So, there are 5
classes of Igs are IgM, IgA, IgG, IgE and IgD.
Mnemonic rule = (MAGED)

10. EPITOPES (ANTIGENIC DETERMINANTS)
 Epitope is immunologically active regions of an
immunogen that binds to antigen-specific membrane
receptors on B lymphocyte cell.
 The whole antigen dose not trigger immune response and
only a small part of it induces B and T cell response.
 Each antigen carries many & different epitopes.

11. *Antigens (Ag) are molecules (substances) recognised by
the immune system which induce an immune response.
*Although all antigens are recognized by specific
lymphocytes or by antibodies, only some antigens are
capable of activating lymphocytes.
*Molecules that stimulate immune responses are called
Immunogens.
*All immunogens must be antigens, but not all antigen
must be immunogens.

12. CRITERIA'S INFLUENCING IMMUNOGENICITY:
1.Foreignness:
2. High molecular weight (MW):
3. Chemical complexity:
4. Genetic constitution (code) of the host:
5. Route of administration:

13. (Antigenic determinants)
(paratope)

14. PARATOPE
*Each Y-shaped antibody molecule has at least two binding sites
(paratope) that can attach to a specific epitope on an antigen.
*The site in the variable (V) region of an antibody which is
complementary to the epitope and interacts with the epitope.
*The combination is very much like the lock and key analogy
(identity).

15. MONOCLONAL ANTIBODIES
 Mono=single=one
 These singular types of antibody are produced by
harvesting purified cell lines derived from a single
B cell within the immunity, allowing them to bind to
one unique epitope region of an antigen.
(mAB,moAB)

16. PRODUCING MONOCLONAL ANTIBODIES
 mAbs are produced by immunizing (injection an
immunogen) an animal, often a mouse, multiple times with
a specific antigen.
 After the immunogen has caused an immune response, the
B-cells from the spleen are removed.
 Since normal B cells are unable to proliferate forever, they
are fused with immortal, cancerous B cells called myeloma
cells, to yield hybridoma cells.

17. PRODUCING MONOCLONAL ANTIBODIES
 All of the cells are then placed in a selective medium
(Hypoxanthine Aminopterin Thymidine -HAT) that allows
only the hybridomas to grow; unfused myeloma cells
cannot grow, and any unfused B cells die off.
 Mechanism of grow hybridoma cell in this selective media:
B cell is contain a gene hypoxanthine-guanine
phosphoribosyltransferase (HGPRT) but myeloma don’t
have it this gene.

18. PRODUCING MONOCLONAL ANTIBODIES
 This gene is necessary for replication of the cell.
 This HAT media only cell that have HGPRT
positive will survive ,while cells HGPRT negative
will die.
 So, HAT media only kill HGPRT negative cells only.

19. PRODUCING MONOCLONAL ANTIBODIES
 The hybridomas, which are capable of growing
continuously in culture while producing antibodies, are then
screened for the desired mAb. Those producing the desired
mAbs are grown in tissue culture; the culture medium is
harvested periodically and mAbs are purified from the
medium.

20. PRODUCING MONOCLONAL ANTIBODIES
 Once a monoclonal antibody hybridoma clone is created, it
is a stable renewable source of antibodies. This ensures
that each batch is identical to the previous one.
 Hybridomas can also be injected in the peritoneal cavity of
a mouse in order to create a tumor that secrets a fluid rich
in antibodies called ascites fluid.

21. Figure: Production of Monoclonal antibodies
(mAbs).
specific
immortal
fused

22. MONOCLONAL ANTIBODIES: ADVANTAGES
1. Can produce large quantities of identical
antibodies.
2. Batch to batch homogeneity.
3. Have high specificity to a single epitope which
reduces the risks for cross reactivity.
4. Better results in assays requiring quantification of
the protein levels
5. Very efficient in affinity purification.

23. MONOCLONAL ANTIBODIES: DISADVANTAGES:
1. more expensive.
2. Requires significantly more time 6 months.
3. More demanding storage conditions for the clone.
4. Less strong for detecting the protein in a denatured state
or altered conformation. Why?
5. Less ideal for application requiring quick capture of the
target protein
6. More sensitive to pH and buffer conditions.
7. More susceptible to binding changes when labelled.

24.  Poly=many
 Describe a collection of antibodies which are
grown from different B cells. This makes them
capable of recognizing multiple epitopes on the
same antigen.
POLYCLONAL ANTIBODIES

25.  Antibodies used for research and diagnostic purposes are
often obtained by injecting a lab animal such as a rabbit
with a specific antigen.
 Within a few weeks, the animal’s immune system will
produce high levels of antibodies specific for the antigen.
 These antibodies can be harvested in an antiserum, which
is whole serum collected from an animal following
exposure to an antigen.
PRODUCING POLYCLONAL ANTIBODIES

26.  Because most antigens are complex structures with
multiple epitopes, they result in the production of multiple
antibodies in the lab animal.
 This so-called polyclonal antibody response is also
typical of the response to infection by the human immune
system.
 Antiserum drawn from an animal will thus contain
antibodies from multiple clones of B cells, with each B cell
responding to a specific epitope on the antigen.
PRODUCING POLYCLONAL ANTIBODIES

27.  Antiserum obtained from animals will not only contain
antibodies against the antigen artificially introduced in the
laboratory, but it will also contain antibodies to any other
antigens to which the animal has been exposed during its
lifetime.
 For this reason, antisera must first be “purified” to remove
other antibodies before using the antibodies for research or
diagnostic assays.
PRODUCING POLYCLONAL ANTIBODIES

28. Figure: This diagram illustrates the process for harvesting polyclonal antibodies produced in
response to an antigen.
specific
specific epitope
multiple
Varying paratope

29. POLYCLONAL ANTIBODIES: ADVANTAGES
1. Inexpensive.
2. Easy to store.
3. Quick to produce.
4. Ready to use in under four months.
5. Highly stable and tolerant of pH or buffer changes.
6. High affinity as the antibodies bind to more than one
epitope, they can help amplify the signal from target
protein even with low expression level.

30. POLYCLONAL ANTIBODIES: DISADVANTAGES
1. Higher potential for cross reactivity.
2. High variability between different batches produced in
different animals at different times.
3. The affinity purification of the serum will typically be
required to minimize cross reactivity.

31. POLYCLONAL VS. MONOCLONAL ANTIBODIES.
Monoclonal antibodies Polyclonal antibodies
Expensive production Inexpensive production
Long production time Rapid production
More Specificity less Specificity
Recognize a single epitope on an
antigen
Recognize multiple epitopes on an
antigen
Production is continuous and uniform
once the hybridoma is made
Different batches vary in composition
Few host species options Many host species options
A homogenous antibody population. A heterogeneous antibody population.

32. ANTIBODY PURIFICATION
 Antibody purification is a multistep process by which
contaminants of source is removed and antibody with
high purity is obtained.
 Antibodies are widely used as injectables and parenteral
products for human use.

33. POSSIBLE CONTAMINANTS
Antibodies are produced in mice, rabbits, etc. Nowadays
antibodies are expressed in cell culture with good yield.
When antibodies are taken from animal source possible
contaminants could be serum proteins such as albumin,
transferrins and cell degradation products like DNA and
cellular proteins.
Currently serum free media for cell culture are developed
which can ease the antibody purification process. With a
combination of chromatographic steps and precipitation
methods one can purify the antibody with good yield and
resolution.

34.  The choice of a purification method is based on these
factors:
 Nature of antibody,
 Nature of feedstock,
 Scale of production,
 Economics – cost and other factors,
 Process Timings, and
 Desired purity.
PURIFICATION METHODS

35. ANTIBODY PURIFICATION PROCESS
 Antibody purification is multistep process, which mainly
includes:
1. Sample Preparation
2. Capture
3. Initial Purification
4. Secondary Purification
5. Polishing / Formulation

36. ANTIBODY PURIFICATION: STEP 1: SAMPLE
PREPARATION
 sample preparation is the initial step in which crude protein
sample is conditioned or making it ready for the initial
capture step.
 Generally this step involves changing pH or Ionic strength,
dilution of the crude sample or addition of salts for the
ionic strength.
 use buffer exchange by size exclusion chromatography or
to use ultrafiltration or diafiltration.

37.  These are the various chromatographic techniques which
are widely used for antibody purification.
 Immunoaffinity (most used).
 Immobilized Metal Affinity Chromatograhy (IMAC)
 Ion – Exchange Chromatograhy (IEC)
 Hydrophobic Interaction Chromatography (HIC)
 Hydroxyapatite
 Size – Exclusion chromatography (SEC)
ANTIBODY PURIFICATION: STEP 2: CAPTURE

38. ANTIBODY PURIFICATION: STEP 3:
SECONDARY PURIFICATION
 The secondary purification step is selected based on the
nature and the optimization requirement of the crude
antibody source.
 In addition to protein contaminants, other impurities such
as DNA, endotoxins, viruses, and aggregates need to be
removed. In such cases, a multistep procedure is almost
inevitable.

39. ANTIBODY PURIFICATION: STEP 4:
POLISHING / FORMULATION
 Final polishing / formulation step can be considered as a
part of purification in which it removes conditions that would
impair the stability or utility of the antibody in its intended
use.
 Ultarfilteration
 Diafiltration
 Lyophilization

40. POLYCLONAL ANTIBODIES VS. MONOCLONAL
ANTIBODIES: DIAGNOSTIC STUDIES
 Which is better, a monoclonal or a polyclonal
antibody?
 It depends on the different characteristics of
monoclonal and polyclonal antibodies.

41. POLYCLONAL ANTIBODIES VS. MONOCLONAL
ANTIBODIES: DIAGNOSTIC STUDIES
 The best use of polyclonal antibodies is to detect unknown antigens.
 Polyclonal antibodies are used as a secondary antibody in
immunoassays (e.g. ELISA, western blotting, microarray
assays, immunohistochemistry, flow cytometry).
 Their role is to bind to different epitopes and amplify the signal, leading
to better detection. (sensitization and stronger)
 Polyclonal Antibodies: Polyclonal antibodies are used in general
research applications.

42. POLYCLONAL ANTIBODIES VS. MONOCLONAL
ANTIBODIES: DIAGNOSTIC STUDIES
 Monoclonal antibodies are often used as primary
antibodies in immunoassays due to their ability of
specifically binding to a single epitope of an antigen.
 Monoclonal antibodies, provide an unlimited source of
antibody that is homogeneous.
 Monoclonal Antibodies: Monoclonal antibodies are used as
therapeutic drugs.

43. WHAT ARE THE USES FOR MONOCLONAL ANTIBODIES?
 The use of monoclonal antibodies to treat diseases is called
immunotherapy because each type of monoclonal antibody
will target a specific targeted antigen in the body.
 Uses for monoclonal antibodies includes: cancer,
MS ,Crohn's disease, ulcerative colitis, rheumatoid
arthritis, systemic lupus erythematosus …

44.  In these conditions the monoclonal antibody targets and
interferes with the action of a chemical or receptor that
is involved in the development of the condition that is being
treated.
 For example, a monoclonal antibody used for treating
cancer may block a receptor that cancer cells use for
preventing the immune system from the destroying the
cancer cell. Blocking this receptor allows the immune
system to recognize cancer cells and destroy them.

45. SUMMARY
 Monoclonal antibodies provide higher specificity than
polyclonal antisera because they bind to a single epitope
and usually have high affinity.
 Polyclonal antisera are useful for some types of
laboratory assays, but other assays require more
specificity. Diagnostic tests that use polyclonal antisera are
typically only used for screening because of the possibility
of false-positive and false-negative results.

46. THANK YOU