02 Antibody, Ag-Ab Interaction, Cytokines, Complement
02 Antibody, Ag-Ab Interaction, Cytokines, Complement
02 Antibody, Ag-Ab Interaction, Cytokines, Complement
Antibody
Ag-Ab Interaction
Cytokine
Complement
Emilio Q. Villanueva III, MD, DPSP, RMT, MT(ASCPi)
Anatomic and Clinical Pathologist
Antigens
Immunogens
• Immune response is triggered by immunogens
• macromolecules capable of triggering an adaptive immune response
by inducing the formation of antibodies or sensitized T cells in an
immunocompetent host
• can specifically react with corresponding antibodies or sensitized T
lymphocytes
Antigens
• A substance that stimulates antibody formation and has the ability to
bind to an antibody or a T lymphocyte antigen receptor but may not
be able to evoke an immune response initially
• e.g. lower molecular weight particles, haptens, can bind to an
antibody but must be attached to a macromolecule as a carrier to
stimulate a specific immune response
all immunogens are antigens, but not all antigens are immunogens
Epitope
• Foreign substances can be immunogenic or antigenic if their
membrane or molecular components contain(s) structures recognized
as foreign by the immune system
• Antigenic determinants
• Part of an antigen that reacts specifically with an antibody or T
lymphocyte receptor
Chemical Nature of Ag
• Large organic molecules that are proteins or large polysaccharides
and, rarely, if ever, lipids
• Antigens, especially cell surface or membrane-bound antigens, can be
composed of combinations of biochemical classes (e.g., glycoproteins,
glycolipids)
Chemical Nature of Ag
• Proteins are excellent antigens because of their high molecular weight
and structural complexity.
• Lipids are considered inferior antigens because of their relative
simplicity and lack of structural stability.
• Nucleic acids are poor antigens because of relative simplicity,
molecular flexibility, and rapid degradation.
• Carbohydrates (polysaccharides) by themselves are considered too
small to function as antigens.
Physical Nature of Antigens
• Foreignness
• The degree to which antigenic determinants are recognized as nonself by an
individual’s immune system.
• The immunogenicity of a molecule depends to a great extent on its degree of
foreignness.
• Degradability
• For an antigen to be recognized as foreign by an individual’s immune system,
sufficient antigens to stimulate an immune response must be present.
Physical Nature of Antigens
• Molecular Weight
• The higher the MW, the better the molecule will function as an antigen.
• The number of antigenic determinants on a molecule is directly related to its
size.
• Structural Stability
• If a molecule is an effective antigen, structural stability is mandatory.
• If a structure is unstable (e.g., gelatin), the molecule will be a poor antigen.
• Similarly, totally inert molecules are poor antigens.
Physical Nature of Antigens
• Complexity
• The more complex an antigen, the greater is its effectiveness.
• Complex proteins are better antigens than large repeating polymers such as
lipids, carbohydrates, and nucleic acids, which are relatively poor antigens.
Antibodies
Immunoglobulin
• Antibodies are specific proteins referred to as immunoglobulins.
• Many antibodies can be isolated in the gamma globulin fraction of
protein by electrophoresis separation.
• The term immunoglobulin (Ig) has replaced gamma globulin because
not all antibodies have gamma electrophoretic mobility.
• Antibodies can be found in blood plasma and in many body fluids
Immunoglobulin
• The primary function of an antibody in body defenses is to combine
with antigen, which may be enough to neutralize bacterial toxins or
some viruses
• A secondary interaction of an antibody molecule with another
effector agent (e.g., complement) is usually required to dispose of
larger antigens (e.g., bacteria).
Immunoglobulin Classes
• Five distinct classes of immunoglobulin molecules are recognized in
most higher mammals—IgM, IgG, IgA, IgD, and IgE
IgM
• Accounts for about 10% of the Ig pool and is largely confined to the
intravascular pool because of its large size
• This antibody is produced early in an immune response and is largely
confined to the blood
• IgM is effective in agglutination and cytolytic reactions
IgG
• The major immunoglobulin in normal serum is IgG.
• It diffuses more readily than other immunoglobulins into the
extravascular spaces and neutralizes toxins or binds to
microorganisms in extravascular spaces.
• IgG can cross the placenta.
• In addition, when IgG complexes are formed, complement can be
activated.
IgA
• It is the predominant immunoglobulin in secretions such as tears, saliva,
colostrum, milk, and intestinal fluids.
• IgA is synthesized largely by plasma cells located on body surfaces.
• IgA may pass directly into the intestinal lumen or diffuse into the blood
circulation.
• As IgA is transported through intestinal epithelial cells or hepatocytes, it
binds to a glycoprotein called the secretory component. The secretory
piece protects IgA from digestion by gastrointestinal proteolytic enzymes.
• It forms a complex molecule termed secretory IgA, which is critical in
protecting body surfaces against invading microorganisms because of its
presence in seromucous secretions
IgD
• Immunoglobulin D is found in very low concentrations in plasma,
accounting for less than 1% of the total Ig pool.
• IgD is extremely susceptible to proteolysis and is primarily a cell
membrane Ig found on the surface of B lymphocytes in association
with IgM.
IgE
• IgE is crucial because it mediates some types of hypersensitivity
(allergic) reactions, allergies, and anaphylaxis and is generally
responsible for an individual’s immunity to invading parasites.
• The IgE molecule is unique in that it binds strongly to a receptor on
mast cells and basophils and, together with antigen, mediates the
release of histamines and heparin from these cells.
Antibody Structure
• Each Ig molecule is bifunctional;
• one region of the molecule involves binding to antigen,
• and a different region mediates binding of the immunoglobulin to host tissues,
including cells of the immune system and the first component (C1q) of the classic
complement system.
• The primary core of an antibody consists of the sequence of amino acid
residues linked by the peptide bond.
• All antibodies have a common, basic polypeptide structure, with a three-
dimensional configuration.
• The polypeptide chains are linked by covalent and noncovalent bonds,
which produce a unit composed of a four-chain structure based on pairs of
identical heavy and light chains.
Antibody Structure
• IgG, IgD, and IgE occur only as monomers of the four-chain unit
• IgA occurs in both monomeric and polymeric forms
• IgM occurs as a pentamer with five four-chain subunits linked
together
Antibody Structure
• The basic unit of an antibody structure is the homology unit, or
domain
• A typical molecule has 12 domains, arranged in two heavy (H) and
two light (L) chains, linked through cysteine residues by disulfide
bonds so that the domains lie in pairs
• The antigen-binding portion of the molecule (N-terminal end) shows
such heterogeneity that it is known as the variable (V) region; the
remainder is composed of relatively constant amino acid sequences,
the constant (C) region.
Antibody Structure
• Short segments of about 10 amino acid residues within the variable
regions of antibodies (or T cell receptor [TCR] proteins) form loop
structures called complementary-determining regions (CDRs).
• Three hypervariable loops, also called CDRs, are present in each
antibody H chain and L chain. Most of the variability among different
antibodies or TCRs is located within these loops.
Antibody Structure
Antibody Structure
• The IgG molecule provides a classic model of antibody structure,
appearing Y-shaped under electron microscopy
• If the molecule is studies by chemical treatment and the interchain
disulfide bonds are broken, the molecule separates into four
polypeptide chains.
Antibody Structure
• A typical monomeric IgG molecule consists of three globular regions
(two Fab regions and an Fc portion) linked by a flexible hinge region.
• If the molecule is digested with a proteolytic enzyme such as papain,
it splits into three approximately equal-sized fragments.
• Two of these fragments retain the ability to bind antigen and are
called the antigen-binding fragments (Fab fragments).
• The third fragment, which is relatively homogeneous and is
sometimes crystallizable, is called the Fc portion.
Antibody Structure
• If IgG is treated with another proteolytic enzyme, pepsin, the molecule
separates somewhat differently.
• The Fc fragment is split into tiny peptides and thus is completely destroyed.
• The two Fab fragments remain joined to produce a fragment called F(ab)′
2. This fragment possesses two antigen-binding sites.
• If F(ab)′ 2 is treated to reduce its disulfide bonds, it breaks into two Fab
fragments, each of which has only one antigen-binding site.
• Further disruption of the interchain disulfide bonds in the Fab fragments
shows that each contains a light chain and half of a heavy chain, which is
called the Fd fragment.
Primary Antibody Response
• IgM antibody response proceeds in the following four phases after a
foreign antigen challenge:
1. Lag phase—no antibody is detectable.
2. Log phase—the antibody titer increases logarithmically.
3. Plateau phase—the antibody titer stabilizes.
4. Decline phase—the antibody is catabolized.
Secondary (Anamnestic) Response
• Subsequent exposure to the same antigenic stimulus produces an
antibody response that exhibits the same four phases as the primary
response
• Repeated exposure to an antigen can occur many years after the
initial exposure, but clones of memory cells will be stimulated to
proliferate, with subsequent production of antibody by the individual.
Secondary (Anamnestic) Response
• An anamnestic response differs from a primary response as follows:
1. Time. A secondary response has a shorter lag phase, longer plateau, and
more gradual decline.
2. Type of antibody. IgM-type antibodies are the principal class formed in the
primary response. Although some IgM antibody is formed in a secondary
response, the IgG class is the predominant type formed.
3. Antibody titer. In a secondary response, antibody levels attain a higher titer.
The plateau levels in a secondary response are typically 10-fold or greater than
the plateau levels in the primary response.
Monoclonal Antibodies
• Monoclonal antibodies are purified antibodies cloned from a single
cell.
• These antibodies exhibit exceptional purity and specificity and are
able to recognize and bind to a specific antigen.
• In 1975, Kšhler, Milstein, and Jerne discovered how to fuse
lymphocytes to produce a cell line that was both immortal and a
producer of specific antibodies.
• Hybridoma (cell hybrid) from different lines of cultured myeloma
cells (plasma cells derived from malignant tumor strains).
Monoclonal Antibodies
• Hybrid cells secrete the antibody that is characteristic of the parent
cell (e.g., anti–sheep erythrocyte antibodies).
• The multiplying hybrid cell culture is a hybridoma.
• Hybridoma cells can be cloned.
• The immunoglobulins derived from a single clone of cells are termed
monoclonal antibodies (MAbs).
Monoclonal Antibodies
• The greatest impact of MAbs in immunology has been on the analysis of
cell membrane antigens.
• Because they have a single specificity rather than the range of antibody
molecules present in the serum, MAbs have multiple clinical applications,
including the following:
• Identifying and quantifying hormones
• Typing tissue and blood
• Identifying infectious agents
• Identifying clusters of differentiation for the classification of leukemias and
lymphomas and follow-up therapy
• Identifying tumor antigens and autoantibodies
• Delivering immunotherapy
Antigen-Antibody Interaction
Specificity
• The ability of a particular antibody to combine with a particular
antigen is referred to as its specificity.
• This property resides in the portion of the Fab molecule called the
combining site, a cleft formed largely by the hypervariable regions of
heavy and light chains.
• Specificity exists when the binding sites of antibodies directed against
determinants of one antigen are not complementary to determinants
of another dissimilar antigen.
Specificity
• The closer the fit between this site and the antigen determinant, the
stronger are the noncovalent forces between them, and the higher is
the affinity between the antigen and antibody.
• Binding depends on a close three-dimensional fit, allowing weak
intermolecular forces to overcome the normal repulsion between
molecules.
• When more than one combining site interacts with the same antigen,
the bond has greatly increased strength.
Cross-reactivity
• When some of the determinants of an antigen are shared by similar
antigenic determinants on the surface of apparently unrelated
molecules, a proportion of the antibodies directed against one type of
antigen will also react with the other type of antigen.
• Antibodies directed against a protein in one species may also react in
a detectable manner with the homologous protein in another species.
Affinity
• The initial force of attraction that exists between a single Fab site on
an antibody molecule and a single epitope or determinant site on the
corresponding antigen
• The antigen is univalent and is usually a hapten
• Several types of noncovalent bonds hold an epitope and binding site
close together
Avidity
• Each four-polypeptide–chain antibody unit has two antigen-binding
sites, which allows them to be potentially multivalent in their reaction
with an antigen
• The functional combining strength of an antibody with its antigen is
called avidity
• When a multivalent antigen combines with more than one of an
antibody’s combining sites, the strength of the bonding is significantly
increased.
Immune Complexes
• The noncovalent combination of antigen with its respective specific
antibody is called an immune complex.
• An immune complex may be of the small (soluble) or large
(precipitating) type, depending on the nature and proportion of
antigen and antibody.
• Under conditions of antigen or antibody excess, soluble complexes
tend to predominate.
• If equivalent amounts of antigen and antibody are present, a
precipitate may form.
Molecular Basis of Antigen-Antibody
Reactions
• Bonding
• Bonding of an antigen to an antibody results from the formation of multiple,
reversible, intermolecular attractions between an antigen and amino acids of
the binding site.
• These forces require proximity of the interacting groups.
Molecular Basis of Antigen-Antibody
Reactions
• The optimum distance separating the interacting groups varies for different
types of bond; however, all these bonds act only across a very short distance
and weaken rapidly as that distance increases.
• The bonding of antigen to antibody is exclusively noncovalent. The attractive
force of noncovalent bonds is weak compared with that of covalent bonds,
but the formation of multiple noncovalent bonds produces considerable total
binding energy.
• The strength of a single antigen-antibody bond (antibody affinity) is produced
by the summation of the attractive and repulsive forces.
Molecular Basis of Antigen-Antibody
Reactions
• The four types of noncovalent bonds involved in antigen-antibody reactions
are:
• hydrophobic bonds
• The major bonds formed between antigens and antibodies are hydrophobic.
• Many of the nonpolar side chains of proteins are hydrophobic.
• When antigen and antibody molecules come together, these side chains interact and exclude
water molecules from the area of the interaction.
• The exclusion of water frees some of the constraints imposed by the proteins, which results in
a gain in energy and forms an energetically stable complex.
Molecular Basis of Antigen-Antibody
Reactions
• hydrogen bonds
• Hydrogen bonding results from the formation of hydrogen bridges between appropriate
atoms.
• electrostatic forces.
• Electrostatic forces result from the attraction of oppositely charged amino acids located on
the side chains of two amino acid residues.
Molecular Basis of Antigen-Antibody
Reactions
• Goodness of Fit
• The strongest bonding develops when antigens and antibodies are close to
each other and when the shapes of the antigenic determinants and the
antigen-binding site conform to each other
• This complementary matching of determinants and binding sites is referred to
as goodness of fit
Molecular Basis of Antigen-Antibody
Reactions
• Goodness of Fit
• If a poor fit exists, repulsive forces can overpower any small forces of
attraction.
• Variations from the ideal complementary shape will produce a decrease in the
total binding energy because of increased repulsive forces and decreased
attractive forces.
Cytokines
Cytokines
• Cytokines are synthesized and secreted by the cells associated with
innate and adaptive immunity in response to microbial and other
antigen exposures