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Immunoglobulin and
Monoclonal antibodies
Reported by
M.Sc.D endo student
What is immunoglobulin ?
Immunoglobulin's are glycoprotein
molecules that are produced by
plasma cells in response to an
immunogen and which function as
antibodies. The immunoglobulins
derive their name from the finding that
they migrate with globular proteins
when antibody-containing serum is
placed in an electrical field
Immunoglobulin Fragments
Structure/Function Relationships
Ag binding
Valence = 1
determined by VH
and VL
Fc ( crystallizable)
Effector functions
Immunoglobulin Fragments
Structure/Function Relationships
Complement Binding Site
Binding to Fc
IgM normally exists as a pentamer (19S
immunoglobulin) but it can also exist as a
monomer. In the pentameric form all
heavy chains are identical and all light
chains are identical. Thus, the valence is
theoretically 10. IgM has an extra domain
on the mu chain (CH4) and it has another
protein covalently bound via a S-S bond
called the J chain. This chain functions in
polymerization of the molecule into a
IgM Properties
a) IgM is the third most common serum
b) IgM is the first Ig to be made by the
fetus and the first Ig to be made by a
virgin B cells when it is stimulated by
c) As a consequence of its pentameric
structure, IgM is a good complement
fixing Ig. Thus, IgM antibodies are very
efficient in leading to the lysis of
IgM Properties
d) As a consequence of its structure,
IgM is also a good agglutinating Ig .
Thus, IgM antibodies are very good in
clumping microorganisms for eventual
elimination from the body.
e) IgM binds to some cells via Fc
f) B cell surface Ig
Surface IgM exists
as a monomer and
lacks J chain but it
has an extra 20
amino acids at the
C-terminus to
anchor it into the
membrane. Cell
surface IgM
functions as a
receptor for
antigen on B cells.
Surface IgM is non
covalently associated
with two additional
proteins in the
membrane of the B
cell called Ig-alpha
and Ig-beta. These
additional proteins act
as signal transducing
molecules since the
cytoplasmic tail of the
Ig molecule itself is
too short to transduce
a signal.
IgD Properties
a) IgD is found in low levels in serum;
its role in serum uncertain.
b) IgD is primarily found on B cell
surfaces where it functions as a
receptor for antigen. IgD on the
surface of B cells has extra amino
acids at C-terminal end for anchoring
to the membrane. It also associates
with the Ig-alpha and Ig-beta chains.
c) IgD does not bind complement.
IgE structure
IgE Properties
a) IgE is the least common serum Ig
since it binds very tightly to Fc receptors
on basophils and mast cells even before
interacting with antigen.
b) Involved in allergic reactions - As a
consequence of its binding to basophils
an mast cells, IgE is involved in allergic
reactions. Binding of the allergen to the
IgE on the cells results in the release of
various pharmacological mediators that
result in allergic symptoms.
IgE Properties
c) IgE also plays a role in parasitic
helminth diseases. Since serum IgE
levels rise in parasitic diseases,
measuring IgE levels is helpful in
diagnosing parasitic infections.
Eosinophils have Fc receptors for IgE
and binding of eosinophils to IgEcoated helminths results in killing of
the parasite.
d) IgE does not fix complement.
clinical implications of human
immunoglobulin classes
IgM Increases (in adults) in:
a) Waldenström's macroglobulinemia
b) Trypanosomiasis
c) Actinomycosis
d) Carrión's disease (bartonellosis)
e) Malaria
f) Infectious mononucleosis
g) Lupus erythematosus
h) Rheumatoid arthritis
I) Dysgammaglobulinemia (certain cases)
Note: In the newborn, a level of IgM above 20
ng./dl is an indication of in utero stimulation of
the immune system and stimulation by the
rubella virus, the cytomegalovirus, syphilis, or
clinical implications of human
immunoglobulin classes
IgM Decreases in
a) Agammaglobulinemia
b) Lymphoproliferative disorders
(certain cases)
c) Lymphoid aplasia
d) IgG and IgA myeloma
e) Dysgammaglobulinemia
f) Chronic lymphoblastic leukemia
clinical implications of human
immunoglobulin classes
IgD Increases in
a) Chronic infections
b) IgD myelomas
clinical implications of human
immunoglobulin classes
IgE Increases in
a) Atopic skin diseases such as
b) Hay fever
c) Asthma
d) Anaphylactic shock
e) IgE-myeloma
clinical implications of human
immunoglobulin classes
IgE Decreases in
a) Congenital agammaglobulinemia
b) Hypogammaglobulinemia due to
faulty metabolism or synthesis of
Monoclonal antibodies are antibodies
that are identical because they were
produced by one type of immune cell, all
clones of a single parent cell
Polyclonal antibodies are antibodies
that are derived from different cell lines
Epitope, also known as antigenic
determinant, is the part of an antigen that
is recognized by the immune system
specifically by antibodies, B cells, or T
In 1975, Georges Kohler and
Cesar Milstein first fused lymphocytes to
produce a cell line which was both
immortal and a producer of specific
antibodies. The two scientists were
awarded the Nobel Prize for Medicine in
1984 for the development of this
"hybridoma." The value of hybridomas to
the field was not truly appreciated until
about 1987, when MAbs were regularly
produced in rodents for diagnostics.
Monoclonal antibodies
Antibodies produced from a single
clone of B cells.
Produced by fusing a B cell secreting
the desired antibody with a myeloma
cell capable of growing indefinitely in
tissue culture.
Monoclonal antibodies all have
identical antigen-binding sites. Thus
they all bind to the same epitope with
the same affinity. They are all of the
same antibody class (isotype).
Hybridomas Technique
B lymphocytes can mutate into tumor
cells that result in a type of cancer
termed myeloma.
- Myeloma cells become “immortal”
and will grow indefinitely in culture.
- Fusion of a single activated B cell
and a myeloma cell will create a
hybridoma that can grow indefinitely in
Hybridoma Selection
The “HAT Trick”
Myeloma cells have been genetically
engineered 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.
Polyclonal antibodies
Produced by:
Many B cell clones
A single B cell clone
Bind to:
Multiple epitopes of all
antigens used in the
A single epitope of a
Antibody class:
A mixture of different Ab
classes (isotypes)
All of a single Ab class
Ag-binding sites:
A mixture of Abs with
different antigen-binding
All Abs have the same
antigen binding site
Potential for
Monoclonal Antibodies
Purified antigen
Inject antigen into mouse
Kill the mouse and remove
the spleen (containing antibody
generating cells)
Each cell may have produced a different antibody
A Hybrid cell that can be cultured
Select one of these "hybrid" cells to subculture….
… and produce a
mono clonal population
Measuring protein and drug levels in
Typing tissue and blood
Identifying infectious agents
Identifying clusters of differentiation for
the classification and follow-up therapy
of leukemias and lymphomas
Identifying tumor metastasis
Identifying and quantifying hormones
Immuno affinity Purification
Used in several diagnostic tests to
detect small amounts of drugs, toxins
or hormones, e.g. monoclonal
antibodies to HCG used in pregnancy
test kits or diagnosis of AIDS by the
ELISA test.
monoclonal antibody can be coupled
to another molecule like a fluorescent
molecule to aid in imaging the target
Or with a strongly-radioactive atom,
such as Iodine-131 to aid in killing the
Used in the radioimmuno detection
and radioimmuno therapy of cancer,
and some new methods can even
target only cancerous cells.
Monoclonal antibodies can be used to
treat viral diseases, traditionally
considered "untreatable". In fact, there
is some evidence to suggest that
antibodies may lead to a cure for
Monoclonal antibodies can be used to
classify strains of a single pathogen,
e.g. Neisseria gonorrhoeae can be
typed using mAB.
To identify and to trace specific cells or
molecules in an organism.
OKT3 (immunosuppressant ) an
antibody to the T3 antigen of T cells, is
used to alleviate the problem of organ
rejection in patients who have had
organ transplants.
Affinity chromatography:
1. Bind antibody to a support matrix (e.g.
sepharose gel)
2. Add protein mixture - antigen binds to antibody
on support
3. Wash to remove unbound material
4. Lower pH - antibody releases the antigen - which
is now free of contaminants
Affinity chromatography antibody purification.
Antigen can be bound to the
support matrix in order to
purify antigen-specific
antibody from a polyclonal
Tumor diagnosis
in vitro
in vivo
Monoclonal antibodies for cancer
Three mechanisms that could be
responsible for the cancer treatment.
1. mAbs act directly when binding to a
cancer specific antigens and induce
immunological response to cancer
cells. Such as inducing cancer cell
apoptosis, inhibiting growth, or
interfering with a key function
Monoclonal antibodies for cancer
2. mAbs can be modified for delivery
of a toxin, radioisotope, cytokine or
other active conjugates.
3. it is also possible to design
bispecific antibodies that can bind with
their Fab regions both to target antigen
and to a conjugate or effector cell
Drawbacks of Monoclonal
Since monoclonal antibodies produce
antibodies to a single determinant,
they do not form the lattice necessary
for precipitation and so cannot be used
in precipitation assays,
radial immunodiffusion,
immunoelectrophoresis or agar gel
Very high titer antibodies also pose
certain difficulties