D1 The immune system
D2 Antibody structure
D3 Polyclonal and monoclonal antidies
D4 Antibody synthesis
D5 Antibodies as tools
Section D ---Antibodies
? Functions
The immune system has two main function to
recognize invading pathogens and then to trigger
pathways that will destroy them,The humoral immune
system relies on β lymphocytes to produce soluble
antibodies that will bind the foreign antigens,The
cellular immune system uses killer T lymphocytes that
recognize and invading cells directly,
D1 The Immune System
Primary and secondary
immune responses
? The primary immune responses occurs on initial
contract with a foreign and results in production of
immunoglobulin M (lgM) and then immunoglobulin G
(lgG),If the same antigens encountered again,immuno-
logical memory leads to a secondary immune responses
that produces a much more rapid and larger increase in
specific IgG production,
Primary and secondary immune
responses
? The primary immune response occurs on initial
contact with a foreign antigen and results in
production of immunoglobulin M and then IgG,
? If the same antigen is encountered
again,immunological memory leads to a secondary
immune rsponse that produces a much more rapid
and larger increase in specific IgG production,
Primary and secondary immune
responses
Clonal selection theory
? A large number of antibody-producing cells
exist in an animal even before it encounters a foreign
antigen,each cell producing only one specific
antibody and displaying this on its cell surface,An
antigen binds to cells that display antibodies with
appropriate binding sites and causes proliferation of
those cells to form clones of cells secreting the
same antibody in high concentration,
Clonal selection
Self-tolerance
? Cells that produce antibody that reacts with normal
body components are killed early in fetal life so that
the adult animal normally is unable to make antibodies
against self,a condition called self-tolerance,
Strategy to circumvent self-tolerance
Complement
Antibodies bound to an invading microorganism
activate the complement system via the classical
pathway,This consists of a cascade of proteolytic
reactions leading to the formation of membrane attack
complexes on the plasma membrane of the
microorganisms that cause its lysis,Polysaccharides
on the surface of infecting microorganism can also
activate complement directly in the absence of
antibody via the alternative pathway,
Complement system
1.Light and heavy chains
Each IgG antibody molecule consists of four polypeptide
chains (two identical light chains and two identical heavy
chains joined by disulfide bonds) and has two antigen-
binding sites (i.e,is bivalent),
D2 Antibody structure
ANTIBODY STRUCTURE
Structure of an antibody molecule
Variable and constant regions
? Each light chain and each heavy chain
consists of a variable region and a
constant region,Variability in the
variable regions is largely confined to
three hypervariable regions ;the
remaining parts of the variable regions
are far less variable and are called the
framework regions,
IgG
Antibody domains
? Each light chain folds into two domains,one
for the variable region and one for the
constant region, Each IgG heavy chain
folds into four domains,one for the variable
region and three in the constant region,
Antibody domains
Fab and Fc fragments
? Papain IgG into two Fab fragments
(each of which has an antigen for
complement activation and
phagocytosis ).Pepsin digests IgG to
release an F(ab｀ ) 2fragment that has
two antigen-binding sites,
Fab and Fc fragments
Five classes of immunoglobulins
? Human immmunoglobulins exist as IgA、
IgD,IgE,IgG and IgM classes which
contain αδεγandμ heavy chains，
respectively,IgM is a pentamer that
binds to invading microorganisms and
activates complement killing of the cells
and phagocytosis,
Five classes of immunoglobulins
Five classes of immunoglobulins
IgG
? IgG is the main antibody found in the blood after antigen
stimulation and also has the ability to cross the
placenta,IgA mainly functions in body secretions,IgE
provides immunity against some parasites but is also
respomsible for the clinical symptoms of
allergicteactions.The role of IgD is unknown,
? All antibody molecules contain either kappa or lambda
light chains,
1,Polyclonal antibodies
A preparation of antibody
molecules that arises from several
different clones of cells is called a
polyclonal antibody,It is a mixture
of antibody molecules that bind to
different parts of the antigen and
with different binding affinities,
D3 Polyclonal and monoclonal
antibodies
Monoclonal antibodies
? Antibody produced by a single clone of cells is a
monoclonal antibody; all the antibody molecules are
identical and bind to the same antigenic site with identical
binding affinities,
? Monoclonal antibodies can be ge-nerated in large amounts
by creatint a cell fusion (called a hybridoma) between an
antibody-producing cell and a mye-loma cell,
1,Somatic recombination
No complete antibody gene exists in
germ-line cells,The genes for light
chains and heavy chains assemble by
somatic recombination during
blymphocyte maturation,
D4 Antibody synthesis
2,Recombination of light chain genes
? In the germ-line,each light chain gene exists as
multiple V and J gene segments upstream of a
single C gene segments,During B-lymphocyte
differentiation,one V gene segment joins with
one J gene segment (VJ joining) to assemble
the complete light chain gene,usually by
deletion of inter-vening DNA,
3,Recombination of heavy chain genes
? Heavy chains are encoded by multiple V,J and D gene
segments which lie upstream of a single copy of C gene
segments for each of the constant regions of μ δ γ ε and
α chains,During B-lymphocyte differentiation,a D gene
segment joins a J segment (DJ joining)and then the
recombined DJ joins a V gene segment(VDJ joining),
4,Class switching
? A B lymphocyte can change the class of
antibody being expressed by moving a
new C gene segment into position after
the recombined VDJ segment,deleting
the intervening DNA,The new heavy
chain has a different constant region but
retains the same antigen-binding
specificity of the previous heavy chain,
1,Immunolocalization methods
Because of the high specificity of an antibody
for its epitope,an antibody raised against a
particular protein antigen can be used to
determine the location of that antigen in a cell
using immu-nofluorescence light microscopy
or immuno-electron microscopy,
D5 Antibodies as tool
2,ELISA
? ELISA can be used to quantify the amount
of a specific protein antigen in a sample,The
antibody is bound to an inert polymer
support,then exposed to reacts with the
antigen at a different epitope is added,
The second antibody
? The second antibody used is one that has an
enzyme attached to it that coverts a colorless
or nonfluorescent substrate into a colored or
fluorescent product,The amount of second
antibody bound,and hence the amount of
protein antigen present in the original sample,
is determined by quan-tification of the
intensity of color or fluorescence produced,
3,Western blotting
? Protein samples are separated by one-
dimensional SDS-PAGE of two-dime-
nsional gel electrophoresis in polya-
crylamide gels,The separated proteins
are then transferred to a nitrocellulose
or nylon sheet,
Autoradiography
? This is incubated with specific antibody to
the protein and then unbound antibody is
washed away,Those proteins in the gel that
bind the antibody are detected either by
autoradiography (if the specific antibody
was radiolabeled) or by using a second
labeled antibody that binds to the primary
antibody,
4,Immunoaffinity chromato-graphy
? Immunoaffinity chromatography can be used to
purify protein antigens by immo-bilizing the
relevant antibodies on an inert matrix such as
polysaccharide beads,When exposed to a
protein mixture,only the protein recognized by
that antibody will bind to the beads and can be
eluted later in pure or almost pure form,Cells
bearing the antigen on their surface can also be
purified using a similar procedure,
D2 Antibody structure
D3 Polyclonal and monoclonal antidies
D4 Antibody synthesis
D5 Antibodies as tools
Section D ---Antibodies
? Functions
The immune system has two main function to
recognize invading pathogens and then to trigger
pathways that will destroy them,The humoral immune
system relies on β lymphocytes to produce soluble
antibodies that will bind the foreign antigens,The
cellular immune system uses killer T lymphocytes that
recognize and invading cells directly,
D1 The Immune System
Primary and secondary
immune responses
? The primary immune responses occurs on initial
contract with a foreign and results in production of
immunoglobulin M (lgM) and then immunoglobulin G
(lgG),If the same antigens encountered again,immuno-
logical memory leads to a secondary immune responses
that produces a much more rapid and larger increase in
specific IgG production,
Primary and secondary immune
responses
? The primary immune response occurs on initial
contact with a foreign antigen and results in
production of immunoglobulin M and then IgG,
? If the same antigen is encountered
again,immunological memory leads to a secondary
immune rsponse that produces a much more rapid
and larger increase in specific IgG production,
Primary and secondary immune
responses
Clonal selection theory
? A large number of antibody-producing cells
exist in an animal even before it encounters a foreign
antigen,each cell producing only one specific
antibody and displaying this on its cell surface,An
antigen binds to cells that display antibodies with
appropriate binding sites and causes proliferation of
those cells to form clones of cells secreting the
same antibody in high concentration,
Clonal selection
Self-tolerance
? Cells that produce antibody that reacts with normal
body components are killed early in fetal life so that
the adult animal normally is unable to make antibodies
against self,a condition called self-tolerance,
Strategy to circumvent self-tolerance
Complement
Antibodies bound to an invading microorganism
activate the complement system via the classical
pathway,This consists of a cascade of proteolytic
reactions leading to the formation of membrane attack
complexes on the plasma membrane of the
microorganisms that cause its lysis,Polysaccharides
on the surface of infecting microorganism can also
activate complement directly in the absence of
antibody via the alternative pathway,
Complement system
1.Light and heavy chains
Each IgG antibody molecule consists of four polypeptide
chains (two identical light chains and two identical heavy
chains joined by disulfide bonds) and has two antigen-
binding sites (i.e,is bivalent),
D2 Antibody structure
ANTIBODY STRUCTURE
Structure of an antibody molecule
Variable and constant regions
? Each light chain and each heavy chain
consists of a variable region and a
constant region,Variability in the
variable regions is largely confined to
three hypervariable regions ;the
remaining parts of the variable regions
are far less variable and are called the
framework regions,
IgG
Antibody domains
? Each light chain folds into two domains,one
for the variable region and one for the
constant region, Each IgG heavy chain
folds into four domains,one for the variable
region and three in the constant region,
Antibody domains
Fab and Fc fragments
? Papain IgG into two Fab fragments
(each of which has an antigen for
complement activation and
phagocytosis ).Pepsin digests IgG to
release an F(ab｀ ) 2fragment that has
two antigen-binding sites,
Fab and Fc fragments
Five classes of immunoglobulins
? Human immmunoglobulins exist as IgA、
IgD,IgE,IgG and IgM classes which
contain αδεγandμ heavy chains，
respectively,IgM is a pentamer that
binds to invading microorganisms and
activates complement killing of the cells
and phagocytosis,
Five classes of immunoglobulins
Five classes of immunoglobulins
IgG
? IgG is the main antibody found in the blood after antigen
stimulation and also has the ability to cross the
placenta,IgA mainly functions in body secretions,IgE
provides immunity against some parasites but is also
respomsible for the clinical symptoms of
allergicteactions.The role of IgD is unknown,
? All antibody molecules contain either kappa or lambda
light chains,
1,Polyclonal antibodies
A preparation of antibody
molecules that arises from several
different clones of cells is called a
polyclonal antibody,It is a mixture
of antibody molecules that bind to
different parts of the antigen and
with different binding affinities,
D3 Polyclonal and monoclonal
antibodies
Monoclonal antibodies
? Antibody produced by a single clone of cells is a
monoclonal antibody; all the antibody molecules are
identical and bind to the same antigenic site with identical
binding affinities,
? Monoclonal antibodies can be ge-nerated in large amounts
by creatint a cell fusion (called a hybridoma) between an
antibody-producing cell and a mye-loma cell,
1,Somatic recombination
No complete antibody gene exists in
germ-line cells,The genes for light
chains and heavy chains assemble by
somatic recombination during
blymphocyte maturation,
D4 Antibody synthesis
2,Recombination of light chain genes
? In the germ-line,each light chain gene exists as
multiple V and J gene segments upstream of a
single C gene segments,During B-lymphocyte
differentiation,one V gene segment joins with
one J gene segment (VJ joining) to assemble
the complete light chain gene,usually by
deletion of inter-vening DNA,
3,Recombination of heavy chain genes
? Heavy chains are encoded by multiple V,J and D gene
segments which lie upstream of a single copy of C gene
segments for each of the constant regions of μ δ γ ε and
α chains,During B-lymphocyte differentiation,a D gene
segment joins a J segment (DJ joining)and then the
recombined DJ joins a V gene segment(VDJ joining),
4,Class switching
? A B lymphocyte can change the class of
antibody being expressed by moving a
new C gene segment into position after
the recombined VDJ segment,deleting
the intervening DNA,The new heavy
chain has a different constant region but
retains the same antigen-binding
specificity of the previous heavy chain,
1,Immunolocalization methods
Because of the high specificity of an antibody
for its epitope,an antibody raised against a
particular protein antigen can be used to
determine the location of that antigen in a cell
using immu-nofluorescence light microscopy
or immuno-electron microscopy,
D5 Antibodies as tool
2,ELISA
? ELISA can be used to quantify the amount
of a specific protein antigen in a sample,The
antibody is bound to an inert polymer
support,then exposed to reacts with the
antigen at a different epitope is added,
The second antibody
? The second antibody used is one that has an
enzyme attached to it that coverts a colorless
or nonfluorescent substrate into a colored or
fluorescent product,The amount of second
antibody bound,and hence the amount of
protein antigen present in the original sample,
is determined by quan-tification of the
intensity of color or fluorescence produced,
3,Western blotting
? Protein samples are separated by one-
dimensional SDS-PAGE of two-dime-
nsional gel electrophoresis in polya-
crylamide gels,The separated proteins
are then transferred to a nitrocellulose
or nylon sheet,
Autoradiography
? This is incubated with specific antibody to
the protein and then unbound antibody is
washed away,Those proteins in the gel that
bind the antibody are detected either by
autoradiography (if the specific antibody
was radiolabeled) or by using a second
labeled antibody that binds to the primary
antibody,
4,Immunoaffinity chromato-graphy
? Immunoaffinity chromatography can be used to
purify protein antigens by immo-bilizing the
relevant antibodies on an inert matrix such as
polysaccharide beads,When exposed to a
protein mixture,only the protein recognized by
that antibody will bind to the beads and can be
eluted later in pure or almost pure form,Cells
bearing the antigen on their surface can also be
purified using a similar procedure,
