PART III
Immunological Methods
Topics covered:
• Antibody Production
• Monoclonal Antibodies
• Immunoassays
114
, CHAPTER 13--IMMUNIZATION
Antibodies are proteins that specifically recognize and bind to other molecules with a
high affinity and specificity. This high degree of specificity makes antibodies powerful reagents
for the characterization of proteins and other macromolecules. Antibodies are generated against
specific proteins or other macromolecules by immunizing animals and collecting the sera. The
immune sera contains a mixture of antibodies that recognize various epitopes on the antigen.
The immune system can also respond to foreign substances via a cellular immune response.
IMMUNOGENICITY
Immunogenicity (i.e., the level of the immune response) depends on the chemical nature
of the immunogen and the ability of the animal to respond to the immunogen. An immunogen is
a substance that induces a specific immune response, whereas an antigen is defined as a
substance that reacts with the products of a specific immune response. An antigen can be an
immunogen if it is correctly presented to the immune system. In addition, there is a genetic
component to immunogenicity in that some substances are immunogenic in one species but not
in another. Similarly, some substances are immunogenic in one individual but not in others (i.e.
responders and non-responders). Immunogenicity can be manipulated, though, by either
modifying the immunogen or stimulating the animal.
An immunogen generally needs to be a foreign sub- • foreigness
stance since the immune system normally discriminates • molecular complexity
between self and non-self. In addition, immunogens need to • molecular size
have significant chemical complexity and be of a large size • B-cell epitope
(eg., > 10 kDa). Proteins and polysaccharides tend to make • T-cell epitope
good immunogens, whereas nucleic acids and lipids are • class II binding site
• degradation/presentation
usually poorly immunogenic. Lipids can function as
• particulate/phagocytosis
haptens, though (see below).
Optimal immunogens should also have B-cell epitopes, T-cell epitopes and class II
binding sites (Box). Epitopes are the structural determinants recognized by either antibodies or
T-cell receptors. B-cell epitopes bind to membrane bound antibodies on the surface of B-cells
and stimulate the production and secretion of antibodies. This can occur independent of T-cells,
but the antibody response (both in amount and affinity) tends to be lower. Polysaccharides are
generally T-independent immunogens in that they are characterized by the repetition of the
same antigenic determinant (i.e., the saccaride subunits) and are somewhat resistance to
degradation. B-cell epitopes in proteins tend to be surface exposed hydrophilic residues and
usually consist of 4-8 amino acid residues
T-dependent immunogens are characterized by a few copies of a complex mixture of
epitopes as exhibited by proteins. T-dependent antigens are also processed into small peptides
and expressed on the surface of antigen-presenting cells via the MHC class II proteins. For
example, macrophages will take up foreign organisms or particulate matter and degrade these
substances with hydrolytic enzymes in the lysosomes. Immunogens in particulate form are
better since they are more efficiently phagocytosed by macrophages. Proteins will be
115
, proteolytically processed into peptides, some of which will bind to the class II MHC molecules
and transported to the cell surface. Thus a good immungen needs to contain peptide sequences
which bind to the MHC protein. This also explains genetic restrictions in the immune response
since individuals have different repertoires of MHC proteins and will react differently to
immunogens.
Potential Problems Associated with
Immunogens and Possible Remedies
Property Defect Result Remedy
Lacks B-cell No B-cell No response None
epitope recognition
Lacks Class II No presentation No response or only Conjugate with class II
site primary response site or switch animals
Lacks T-cell No T-helper No response or only Conjugate with T-epitope
epitope involvement primary response or switch animals
Non-degradable No presentation No response or only None
primary response
Small size No T-helper No response Conjugate with carrier
involvement (hapten)
Non-particulate Poor Weak response Self-polymerize or
phagocytosis couple to beads
A macrophage with MHC associated
peptides on its surface is an antigen presenting
cell. Antigen presentation is not restricted to
macrophages and other types of cells can also
function as antigen-presenting cells. In the
MCH-bound state the peptides (i.e., T-cell
epitope) are recognized by a T-cell receptor
found on the surface of T-lymphocytes. T-cell
receptors exhibit a high degree of specificity in
regards to the peptide sequence recognized and
only those T-cells expressing the correct recep-
tor will be activated. The exact immune re-
sponse will depend in part on the nature of the
activated T-cells. For example, type 2 T-helper
cells (Th2) will secrete cytokines that stimulate
B-cells in the presence of antigen to replicate
and then differentiate into the antibody-producing plasma cells. Some of the activated B-cells
will differentiate into memory cells. Upon a second encounter with the immunogen memory
cells more rapidly differentiate into plasma cells resulting in a faster and more intense antibody
response. Thus, T-dependent immunogens are also characterized by 'immunologic memory' and
a strong secondary response (i.e., boosting). Immunogens lacking class II sites or T-cell
epitopes can be conjugated to good MHC-binding sites or T-cell epitopes.
IMMUNOGEN PREPARATION
116
Immunological Methods
Topics covered:
• Antibody Production
• Monoclonal Antibodies
• Immunoassays
114
, CHAPTER 13--IMMUNIZATION
Antibodies are proteins that specifically recognize and bind to other molecules with a
high affinity and specificity. This high degree of specificity makes antibodies powerful reagents
for the characterization of proteins and other macromolecules. Antibodies are generated against
specific proteins or other macromolecules by immunizing animals and collecting the sera. The
immune sera contains a mixture of antibodies that recognize various epitopes on the antigen.
The immune system can also respond to foreign substances via a cellular immune response.
IMMUNOGENICITY
Immunogenicity (i.e., the level of the immune response) depends on the chemical nature
of the immunogen and the ability of the animal to respond to the immunogen. An immunogen is
a substance that induces a specific immune response, whereas an antigen is defined as a
substance that reacts with the products of a specific immune response. An antigen can be an
immunogen if it is correctly presented to the immune system. In addition, there is a genetic
component to immunogenicity in that some substances are immunogenic in one species but not
in another. Similarly, some substances are immunogenic in one individual but not in others (i.e.
responders and non-responders). Immunogenicity can be manipulated, though, by either
modifying the immunogen or stimulating the animal.
An immunogen generally needs to be a foreign sub- • foreigness
stance since the immune system normally discriminates • molecular complexity
between self and non-self. In addition, immunogens need to • molecular size
have significant chemical complexity and be of a large size • B-cell epitope
(eg., > 10 kDa). Proteins and polysaccharides tend to make • T-cell epitope
good immunogens, whereas nucleic acids and lipids are • class II binding site
• degradation/presentation
usually poorly immunogenic. Lipids can function as
• particulate/phagocytosis
haptens, though (see below).
Optimal immunogens should also have B-cell epitopes, T-cell epitopes and class II
binding sites (Box). Epitopes are the structural determinants recognized by either antibodies or
T-cell receptors. B-cell epitopes bind to membrane bound antibodies on the surface of B-cells
and stimulate the production and secretion of antibodies. This can occur independent of T-cells,
but the antibody response (both in amount and affinity) tends to be lower. Polysaccharides are
generally T-independent immunogens in that they are characterized by the repetition of the
same antigenic determinant (i.e., the saccaride subunits) and are somewhat resistance to
degradation. B-cell epitopes in proteins tend to be surface exposed hydrophilic residues and
usually consist of 4-8 amino acid residues
T-dependent immunogens are characterized by a few copies of a complex mixture of
epitopes as exhibited by proteins. T-dependent antigens are also processed into small peptides
and expressed on the surface of antigen-presenting cells via the MHC class II proteins. For
example, macrophages will take up foreign organisms or particulate matter and degrade these
substances with hydrolytic enzymes in the lysosomes. Immunogens in particulate form are
better since they are more efficiently phagocytosed by macrophages. Proteins will be
115
, proteolytically processed into peptides, some of which will bind to the class II MHC molecules
and transported to the cell surface. Thus a good immungen needs to contain peptide sequences
which bind to the MHC protein. This also explains genetic restrictions in the immune response
since individuals have different repertoires of MHC proteins and will react differently to
immunogens.
Potential Problems Associated with
Immunogens and Possible Remedies
Property Defect Result Remedy
Lacks B-cell No B-cell No response None
epitope recognition
Lacks Class II No presentation No response or only Conjugate with class II
site primary response site or switch animals
Lacks T-cell No T-helper No response or only Conjugate with T-epitope
epitope involvement primary response or switch animals
Non-degradable No presentation No response or only None
primary response
Small size No T-helper No response Conjugate with carrier
involvement (hapten)
Non-particulate Poor Weak response Self-polymerize or
phagocytosis couple to beads
A macrophage with MHC associated
peptides on its surface is an antigen presenting
cell. Antigen presentation is not restricted to
macrophages and other types of cells can also
function as antigen-presenting cells. In the
MCH-bound state the peptides (i.e., T-cell
epitope) are recognized by a T-cell receptor
found on the surface of T-lymphocytes. T-cell
receptors exhibit a high degree of specificity in
regards to the peptide sequence recognized and
only those T-cells expressing the correct recep-
tor will be activated. The exact immune re-
sponse will depend in part on the nature of the
activated T-cells. For example, type 2 T-helper
cells (Th2) will secrete cytokines that stimulate
B-cells in the presence of antigen to replicate
and then differentiate into the antibody-producing plasma cells. Some of the activated B-cells
will differentiate into memory cells. Upon a second encounter with the immunogen memory
cells more rapidly differentiate into plasma cells resulting in a faster and more intense antibody
response. Thus, T-dependent immunogens are also characterized by 'immunologic memory' and
a strong secondary response (i.e., boosting). Immunogens lacking class II sites or T-cell
epitopes can be conjugated to good MHC-binding sites or T-cell epitopes.
IMMUNOGEN PREPARATION
116
