Antibody Production

Adapted by:
dr. Bagus Hermansyah, M.Biomed
thAfter completion of is lecture, you
will be able to:
1) define the terms antibody and antigen.
2) describe the structure and preparation of antibodies
3) give a brief description of the production of polyclonal
and monoclonal antibodies, and antiglobulins.
 What is an antigen?
• An antigen is any substance that elicits an
immune response and is then capable of binding
to the subsequently produced antibodies.
• Antigens are generally proteins or
polysaccharides, but other substances such as
nucleic acids can also be antigens.
 What is an Epitope
• An epitope is the small site on the antigen
which is recognized by the antibody.
• Usually between one and six sugars or
amino acids on the surface of the antigen.
 Antibodies, Antigens and Analytes
Defined
• An antibody is a protein that is produced by the body
in response to an “invading” (foreign) substance.

• Antibodies are produced as part of the body’s

immune response to protect itself. For instance, some
immunoassays test for the presence of antibodies to
cancer molecules. Thus, if the antibodies are present,
it means invading cancer cells are, too.
 Antibodies
• Secreted by B lymphocytes
• Great diversity and specificity: >109
different antibodies; can distinguish
between very similar molecules
• Antibodies possess high affinity
• Protect against re-infection (vaccines)
 Structure of Antibodies

• Antibodies (Ab) are a type of protein called

immunoglobulins.
• The most common one is immunoglobulin
G (IgG).
• IgG is a protein composed of two main
structural and functional regions
 Antibody Structure

Ig domain: 110 amino

acids; globular domain
used in many proteins.
Variable domains,
Constant domains,
Hinge.
Fab: fragment antigen
binding
Fc: fragment
crystallizable (effector
functions)

© New Science Press Ltd. 2003

Antibody Classes: Structure

© New Science Press Ltd. 2003

 The B cells can make a unique antibody for each antigen
presented. It is estimated that there is the potential to
produce up to 1 x 1010 structurally different IgG
antibodies. Antigen binding site Disulfide bonds Antigen binding site

Fab region
Fab region

Fc region

Antigen A Antigen B

Antigen does not fit Antigen fits correctly

 Affinity and Avidity
•Affinity: the strength of binding between a single binding
site and a single ligand.
[A][B]
KD = [AB]

•Avidity: the strength of binding between a molecule and a

complex ligand, e.g. if there are multiple binding sites then
the avidity may be increased by increasing the number of
binding sites or by increasing the affinity of those binding
sites.
 Affinity and Avidity, continued

IgM is produced early in an immune response when the

affinity for antigen often is low; as an immune response
continues, antibody affinity is improved, this is combined by
“class switching” to the use of smaller molecules (IgG, IgE and
IgA). The increased affinity compensates for the decrease in
number of binding sites in maintaining the overall avidity for
antigen.
© New Science Press Ltd. 2003
 Cont’ed
• The animals produce the antiserum - just as
a human would - as a defense mechanism
when exposed to an antigen.
• Antiserum contains a mixture of antibodies,
each of which may bind to different antigen
binding sites, or epitopes.
 Producing antiserum
• The process of making an antiserum begins by injecting
a solution that contains the antigen of interest into an
animal. This antigen of interest is sometimes called an
immunogen, because it can stimulate an immune
response.
Over time, and in some cases with multiple injections,
the immune system of the animal produces antibodies to
the antigen that was injected.
Blood is collected from the animal, and serum is isolated
from the blood. This serum is usually rich in antibodies
that recognize the antigen, and is called the antiserum.
 Polyclonal Antibody Production

Polyclonal Antibody Production

23
 Polyclonal Antibodies
• Antiserum usually contains a mixture of
antibodies that recognize and bind to the same
antigen, but they may attach to different
epitopes An antigen that has multiple sites for
antibodies to bind is called a multivalent
antigen. These types of antibodies, present as
a diverse mixture, are called polyclonal
antibodies.
 Polyclonal Antibodies
• Polyclonal antibody reagents are produced as different
classes of immunoglobulins by many B-cells clones and
react with various epitopes on an antigen.

• They are more tolerant of small changes in the nature of

the antigen since they often recognize multiple epitopes.

• They may be generated in a variety of animals like rabbit,

goat, sheep, horse, etc. The rabbit is the most commonly
used animal for generating polyclonal antibodies.
 Polyclonal Antibodies
Figure 2

Polyclonal antibodies reacting with various epitopes

Each antibody is made by a different B-cell

22
 Production of antibodies
1. Polyclonal antibodies – a mix of many different antibodies that recognize
different determinants on an antigen. This mix makes standardization of
assays difficult.
Step 2. Blood containing the antibody
along with blood cells and other
proteins is collected from the
animal.
Step 3. Anticoagulant is added
to the blood to prevent clot
formation.
Step 1. Animal is inoculated with
several doses of antigen
over several weeks to
induce production of antibodies.
Step 4. Blood sample is centrifuged
to sediment the blood Plasmacontaining
cells antibodies and
other proteins
Pelleted blood
cells
Step 5. Plasma is heated to 65oC to
Polyclonal antibodies
destroy contaminating
proteins leaving the antibodies intact
 Polyclonal Antibody Production
antigen

A rabbit is injected (intradermally or

subcutaneously) with a purified dose of
antigen.
24
 Polyclonal Antibody Production

antibodies

Y
Y
Y

The rabbit’s immune system responds by

producing antibodies specific to the injected
antigen.
25
 Polyclonal Antibody Production

Blood is harvested from the ear at the peak of

antibody production.

26
 Polyclonal Antibody Production

Y
Y Y
Y Y
Y

Red blood cells and clotting proteins are

removed and the antiserum is purified.

27
 Polyclonal Antibody Production
• Polyclonal antibodies are purified either by Protein
Purification or Antigen Affinity Chromatography.

• Protein Purification eliminates the bulk of serum proteins

but does not eliminate nonspecific immunoglobulin
fraction.

• Antigen Affinity Purification eliminates the bulk of the

nonspecific immunoglobulin fraction using antigen to
capture the antibody leaving only the immunoglobulin of
desired specificity.

28
Cross-reactivity

if two antigens share an epitope

an antibody recognizes an unrelated,
but chemically similar, epitope
Cross reactivity
 Monoclonal Antibody Production

23
 Monoclonal Antibodies

• Single antibody (all same H and L chains)

• Made by fusion of B cells to a transformed
cell line of the plasma cell type and selection
for “hybridomas” that produce antibody with
the desired properties
• Standardized, unlimited reagent for diagnosis
or therapy (human antibodies or “humanized”
antibodies can be made)
Generation of Monoclonal Antibodies

© New Science Press Ltd. 2003

 Monoclonal antibodies
• Monoclonal antibodies differ from polyclonal
antibodies in that they are highly specific for a
single epitope on a multivalent antigen.
• They are produced from a single cell line using
hybridoma technology and mouse myeloma cell
lines.
• Hybridomas are antibody-producing tumor cells
that produce many copies of the same antibody
and grow easily in laboratory cell culture.
 An advantage
• An advantage of monoclonal antibodies is that the
hybridoma cell line that produces them is
potentially “immortal” and can produce the same
antibodies consistently and indefinitely.
• A polyclonal antisera produced by immunization
of animals can vary from animal to animal, and a
useful antiserum may no longer be available if the
single animal that produces it dies.
 Monoclonal Antibodies
Figure 3

Monoclonal antibodies reacting with similar epitopes

32
 Procedure
Hybridomas are produced in a multi-step procedure :
• injecting a specific antigen into a host animal (typically a
mouse);
• isolating antibody-producing cells (B cells) from the
spleen of the mouse;
• fusing these B cells with a specific type of tumor cell that
grows easily in culture and produces antibodies;
• isolating successful hybridomas (fused cells) that produce
antibodies specific for the antigen of interest.
 Preparations of mAb
• Step 1: Immunization of mice
• Mice are immunized with an antigen (attached to adjuvant). The
antigen can be whole cells, membrane fragment, or complex
molecules.
• Mice serum’s are screened using various techniques such as ELISA.
• When sufficient titer is reached the mice are euthanize and spleen is
removed as a source of cells for cell fusion.

• Step 2: Preparation of Myeloma Cells

• Myeloma cells are immortalized cells that are capable of dividing
indefinitely.
• These cells are treated with 8-azaguanine to ensure sensitivity to HAT
 Preparations of mAb
• Step 3: Fusion of myeloma cells with Spleen cells
• Spleen cells harvested from mice are fused with myeloma
cells.
• Fission is done through co-centrifusing in polyethylene glycol.
• Cells are plated in selection medium
• hypoxanthine-aminopterin-thymidine (HAT 1) selection
medium – inhibitor of aminoterin which blocks nucleotide
synthesis.
• Only fused cells with grow on HAT!
• Cells are distributed on feeder cells (murine bone-marrow 2) to
promote growth of the hybridomal cells.
 Preparations of mAb
• Step 4: Cloning of Hybridoma cells.
• A mouse is inoculated with the cell and thereby becomes a
factory for producing the mAB.
• Ascites are collected from the mouse.

• Step 5: Ab are screened and Purified

• Ab are screened using specific Ag binding.
• Advantage of in vivo process
• Relatively inexpensive and easy
• Disadvantage:
• Ethical concerns with using animals.

• Step 6: Desired Ab are cloned

• This is done in vitro on culture bottles
 Monoclonal Antibody Production
antigen

A mouse is injected (intradermally or

subcutaneously) with a purified dose of
antigen.
34
 Monoclonal Antibody Production
antibodies

YY
Y

The mouse’s immune system responds by

producing antibodies specific to the injected
antigen.
35
 Monoclonal Antibody Production

spleen

B-lymphocytes

The antibody-producing B-cells are harvested

from the spleen or lymph nodes.

36
 Monoclonal Antibody Production

myeloma cells B-lymphocytes

The B-cells are fused with mouse myeloma

cells forming immortal hybrid cells or
hybridomas.
37
 Monoclonal Antibody Production

The generated hybridomas will produce many

copies of the exact same antibody.

38
 Monoclonal Antibody Production

The hybridomas are either transplanted into

the peritoneal cavity of a syngeneic mouse
and the antibodies produced are harvested as
ascitic fluid.
39
 Monoclonal Antibody Production

Or are propagated in a tissue culture medium

and the antibodies produced are harvested as a
hybridoma supernatant
40
PRODUCTION OF MONOCLONAL ANTIBODY

HYBRIDOMA TECHNOLOGY
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 HYBRIDOMA TECHNOLOGY
• 1) Immunize animal (mouse or rabbit)

• 2) Isolate spleen cells (containing antibody-

producing B cells)

• 3) Fuse spleen cells with myeloma cells (e.g.

using PEG - polyethylene glycol)

• 4) Allow unfused B cells to die

• 5) Add HAT culture to kill unfused myeloma

cells

• 6) Clone remaining cells (place 1 cell per well

and allow each cell to grow into a clone of
cells)

• 7) Screen supernatant of each clone for

presence of the desired antibody (ELISA)

• 8) Grow the chosen clone of cells in tissue

culture indefinitely.

• 9) Harvest antibody from the culture

supernatant.
 Fusion of Myeloma Cells with Immune Spleen Cells &
Selection of Hybridoma Cells
PEG
(polyethylene
glycol)

FUSION (by making the

SPLEEN CELLS cell membranes more MYELOMA CELLS
permeable) HGPRT-
HGPRT+

1. Plating of Cells in
HAT selective
HYBRIDOMA CELLS Medium
2. Scanning of Viable
HAT Medium Hybridomas

Hypoxanthine-guanine phosphoribosyltransferase - is an enzyme in purine

metabolism.
 The hybridomas now are ready to be ELISA PLATE
diluted and grown, thus obtaining a number of
different colonies, each producing only one type
of antibody. The desired antibodies from the
different colonies are then should be tested for
their ability to bind to the antigen ( ELISA), and
the most effective one is picked out.

Reclone and cultivate positive clones

•Monoclonal antibodies can be
produced in cell culture or in
animals.
•Unethical to inject
hybridoma cells in mice!!!
 Hybridoma Selection
The “HAT Trick”

• Myeloma cells have been genetically engineered

(HGPRT-) such that they can not use Hypoxanthine,
Aminopterin, and Thymidine (HAT medium) as a
source for nucleic acid biosynthesis and will die in
culture

• Only B cells that have fused with the engineered

myeloma cells will survive in culture when grown in
HAT medium
Formation and Selection of
Hybrid Cells
• Hybridoma: the B cell X myeloma cell
– To be produce by using polyethylene glycol (PEG) to
fuse cells
– The myeloma cells: immortal growth properties
– The B cells: to contribute the genetic information for
synthesis of specific antibody
– Selected by using HAT medium (hypoxanthine,
aminoprotein, and thymidine)
• Myeloma cells are unable to grow
• B cells are able to survive, but can not live for extended
periods
 Monoclonal antibodies used in
medicine
Standardized, unlimited amounts of reagents for diagnosis or therapy
(human antibodies or “humanized” antibodies can be made).
1975, by Georges Köhler and Cesar Milstein
- Be awarded a Nobel Prize in1984
Commercial production of antibodies:
polyclonal vs monoclonal
• Host animals ca be used to raise antibodies
against a given antigen
• Slected clones from a polyclonal each
recognizing a single epitope can be fused to
a tumor cell (hybridoma) to proliferate
indefinitely
 Clinical use of antibodies
• Diagnostic
– Detection of peptides and other molecules in various diseases
• Endocrine diseases: hyperinsulinemia, diabetes, hyperparatyroidism
• Tumor antigens (p53 tumor suppressor, PSA, a-foetoprotein)
• Antibodies against viral proteins (AIDS, hepatitis)

• Therapeutic
– Neutralizing antibodies
• Anti-ErbB2 for breast and ovarian cancer
• Anti-CD20 for B-cell non-Hodgkin's lymphoma
• Antisera and antidotes (viruses and venoms)

• Drug discovery
– Identification of therapeutic targets (phage display)
 Therapeutic applications

• Neutralizing antibodies
– Antidotes and antivenin (snake & spider bites)
– Tumor antigens ErbB-2, melanoma and T-cell leukemia,
antibodies coupled to toxins
– Autoimmune antibodies, cytokines TNF-a
– Antisera aigainst virus, bateria and toxins (vaccine)
– Anti IgE and IgM for allegies (experimental)
– Quantitation of blood peptides (hormones metabolites)

• Activating antibodies
– Complement activating for uncontrolled bleeding (hemophilia)