Antigens and Antibodies

Prepared by: Nhoelyn E. Burcao, RMT

Objectives:
At the end of discussion, the students should:
1. Describe the characteristics of Antigens
2. Identify the types of biological molecules that are
immunogenic, weakly immunogenic, and/or non-
immunogenic.
3. Discuss the structure of an antibody molecule and its
properties.
4. Classify the various antibody isotypes, and their
subclasses and variants.
5. Describe the properties and role of the various antibody
isotypes
6. Explain how monoclonal antibodies are generated.
Prepared by: Nhoelyn E. Burcao, RMT
 Structure and Biologic Properties of Antigens

ANTIGEN
• Antibody generator/generation
a. Antibody (Ab)
• Substance with the ability to bind w/
b. T cell antigen receptor

IMMUNOGEN
• Substance that can induce an immune response
a. Formation of Ab
b. Sensitized T cells in immunocompetent host

All immunogens are antigens but not all antigens are immunogens.
Prepared by: Nhoelyn E. Burcao, RMT
 Structure and Biologic Properties of Antigens
CHARACTERISTICS:
Epitope –
• Specific reactivity
antigenic
✓Ability of Ag to react specifically w/ Ab or cells it provoked determinant
• Immunogenicity
✓Ability to provoke an immune response by stimulating the
production of Ab, proliferation of specific T cells, or both
HAPTEN
• Simple antigens, no immunogenicity but has reactivity
• 2 kinds Simple/Nonprecipitating Complex/ Precipitating
✓ Can combine w/ Ab ✓ Can combine w/Ab
✓ Cannot produce precipitates ✓ Produces precipitates

Prepared by: Nhoelyn E. Burcao, RMT

 Structure and Biologic Properties of Antigens
CARRIER/SCHLEPPER MOLECULES
• Larger molecules attached to haptens w/c confer new antigenic specificities

Complete Ag Hapten/ Incomplete Ag

✓ Capable of stimulating antibody synthesis ✓ Cannot by themselves stimulate immune
in the host response
✓ Can also react with homologous Ab ✓ Can react specifically with homologous
✓ Bacterial cells and proteins antibodies

Prepared by: Nhoelyn E. Burcao, RMT

 Factors Affecting Immunogenicity
1. Degree of Foreignness
• Autoantigen: Ag derived from same individual
• Alloantigen: Ag derived from different individual but same species
• Heteroantigen: Ag derived from different individual of different species
✓ Heterophile antigen: heteroantigen found in unrelated plants & animals
• Graft - can cross-react w/ Abs of other individuals
1. Autograft
2. Isograft/ Syngraft
3. Allograft/Homograft
4. Heterograft/Xenograft

Normally an individual’s immune system does not respond to self antigens.

Prepared by: Nhoelyn E. Burcao, RMT
 Factors Affecting Immunogenicity
2. Molecular Weight (MW) / Size 3. Chemical composition and
Complexity
• Potential Ag: • Proteins:
• Good immunogen: • Polysaccharides:
✓ Example: Albumin • Lipids and nucleic acid:
• Excellent immunogen:
✓ Example: Hemocyanin

The greater the molecular weight = the greater the immune response
The more complex an Ag = The greater is its effectiveness
Prepared by: Nhoelyn E. Burcao, RMT
 Factors Affecting Immunogenicity
4. Route, dosage and timing
• Intravenous and intraperitoneal routes: effective
• Intradermal route: stronger stimulus as compared to subcutaneous & intramuscular
route
• Dose response may be partially dependent on the nature of immunogen processing
✓ the smaller the dose, less likely a response

5. Degradability
• Sufficient Ag must be present to induce an immune response
• In vaccination: adequate dose of vaccine at appropriate intervals must be
administered

Prepared by: Nhoelyn E. Burcao, RMT

 Factors Affecting Immunogenicity
6. Adjuvants
• Substances added to vaccine and less immunogenic substance to enhance immune
response
✓ Stimulate T cells
➢ Complete Freund’s Adjuvant: used for vaccinations against M. tuberculosis
✓ Stimulate B cells
➢ LPS
✓ Stimulate phagocytic cells
➢ Alum for humans

Prepared by: Nhoelyn E. Burcao, RMT

 Antibodies

• A.K.A. Immunoglobulins
• Glycoproteins produced in response to antigenic stimulation that is
capable of specified interaction with provoking immunogen
• General functions:
✓Neutralize toxic substances
✓Facilitate phagocytosis and kill microbes
✓Combine w/ antigens of cellular surface and cause
destruction of these cells either extravascularly or
intravascularly

Prepared by: Nhoelyn E. Burcao, RMT

 Typical Antibody Structure
• Each immunoglobulin(Ig) is bifunctional
• Fab : binding antigen
• Fc : mediates binding of Ig to host
tissues, cells of the immune system,
C1q component of classical complement
system

Prepared by: Nhoelyn E. Burcao, RMT

 Typical Antibody Structure
Functions Mediated by Interactions with Antibody Fc Region
FUNCTON Fc REGION INTERACTS WITH:

Opsonization Fc receptors on macrophages and neutrophils

Killing by means of ADCC Fc receptors on neutrophils, macrophages, NK

cells, eosinophils
Degranulation leading to allergic and Fc receptors for IgE on mast cells
antiparasitic responses
Activation of cells Fc receptors on lymphocytes
Transmucosal movement Fc receptors for dimeric IgA on epithelial cells
Activation of classical complement pathway Initial component of pathway (C1)
leading to cell lysis (especially of bacteria),
opsonization, and inflammatory response
Prepared by: Nhoelyn E. Burcao, RMT
 Typical Antibody Structure
• 12 domains
• 2 Heavy chains
• 2 Light chain
• 2 Kappa (65%) only or 2 Lambda
(35%) only
• Binding
• Heavy chain – Heavy chain
• Light chain – Heavy chain

Normally no light chain- light chain binding (L-L): Bence Jones Proteins
Prepared by: Nhoelyn E. Burcao, RMT
 Typical Antibody Structure
• Variable region
• Hypervariable loops/
complementary-determining
regions (CDR)
Heavy
• First 110-120 amino acids chain

• Constant region
• Determines the class and
subclass of the Ig
• Remaining amino acids

Prepared by: Nhoelyn E. Burcao, RMT

 Typical Antibody Structure
Hinge region: flexible portion
✓ ~15amino acids (between
CH1 and CH2 regions)
✓Proline residues

microbiologyinfo.com Prepared by: Nhoelyn E. Burcao, RMT

Immunoglobulin(Ig) Classes

Antibody for ADCC cell function

Antibody with the longest half life

Prepared by: Nhoelyn E. Burcao, RMT Clinical Immunology & Serology by Stevens
 Immunoglobulin(Ig) Classes
Immunoglobulin (Ig) G
• Present at the highest concentration in plasma
• Produced during secondary immune response
• It mediates antibody-dependent cellular cytotoxicity (ADCC)
• Functions:
✓Providing immunity for the newborn
✓Fixing complement
✓Opsonization
✓Neutralizing toxins and viruses
✓Agglutination
✓Precipitation (more efficient)

Prepared by: Nhoelyn E. Burcao, RMT

 Immunoglobulin(Ig) Classes

Immunoglobulin (Ig) M
• Pentameric: with 5 basic subunits
• Held together by J (joining) chain
• First to appear in the B cells
• Surface receptor in B cells

• Functions:
✓Complement fixation (most efficient)
✓Agglutination
✓Opsonization
✓Toxin neutralization
Prepared by: Nhoelyn E. Burcao, RMT
 Immunoglobulin(Ig) Classes
Immunoglobulin (Ig) A
• Found in the breastmilk
• IgA1 (Serum IgA) and IgA2 (Secretory IgA)
• Secretory component
✓Protects the IgA from degradation of enzymes
✓Prevents attachment of pathogens to mucosal surfaces
✓Produced by epithelial cells near IgA-producing plasma cells
• Functions:
✓Aggregates of this antibody can activate the antibody-independent complement
pathway
✓Respiratory burst and degranulation of neutrophils, macrophages and
monocytes
Secretory IgA
✓Patrol mucosal surfaces and act as a first line of defense.
✓It plays an important role in neutralizing toxins produced by microorganisms
✓Helps to prevent bacterial adherence to mucosal surfaces.

Prepared by: Nhoelyn E. Burcao, RMT

 Immunoglobulin(Ig) Classes
Immunoglobulin (Ig) D
• Most are present on the surface of immunocompetent
but unstimulated B lymphocytes
• Second to appear in B cells
• With tail piece
• Has a longer hinge region

• Function:
✓Antibody for immunoregulation

Prepared by: Nhoelyn E. Burcao, RMT

 Immunoglobulin(Ig) Classes
Immunoglobulin (Ig) E
• Least abundant in the serum
• Heat-labile antibody
• Originally known as reaginic antibody
• Functions:
✓Mediates some hypersensitivity (allergic reactions)
allergies, anaphylaxis
✓Immunity to invading parasites
✓Binds strongly to a receptor on mast cells and basophils
with antigen → release of histamine & heparin
• TESTS:
✓RIST (Radioimmunosorbent test)
✓RAST (Radioallergosorbent test)
✓FAST (Fluorescentallergosorbent test)
Prepared by: Nhoelyn E. Burcao, RMT
 Antibody Fragmentation
• Used for detecting specific parts of Ab
• PROTEOLYTIC ENZYMES
• peptide bond-splitting enzymes
• Degrade immunoglobulins molecules into definable
fragment
• Papain
• Cleaves the molecule into 2 antigen-binding
fragments (Fab) and 1 crystallizable fragment
(Fc)
• Directly on the hinge region/above
• Fab has a univalent binding capacity

Prepared by: Nhoelyn E. Burcao, RMT

 Antibody Fragmentation
• PROTEOLYTIC ENZYMES
• Pepsin
• Yields a large fragment [ F(ab’)2] that can
precipitate antigen
• Below the hinge region
• F(ab’)2 has a bivalent binding capacity

Prepared by: Nhoelyn E. Burcao, RMT

Antibody Fragmentation

Prepared by: Nhoelyn E. Burcao, RMT

 Antibody Synthesis
Elicit an immune response
Initial encounter with Ag → recognition as nonself (cells of IS)
Tolerance

Cell-mediated immunity (dependent on T cells & macrophages)

Immune reaction
Involve the production of antibodies (B lymphocytes & plasma cells)

PRODUCTION OF ANTIBODIES
• Induced when lymphocyte comes into contact with foreign
antigenic substance that binds to its receptor

Prepared by: Nhoelyn E. Burcao, RMT

 Antibody Diversity
• Ability of the immune system to recognize antigens depends on
• antibodies generated by B cells
• Antigen receptors expressed by T cells
• Antibodies provide enough different combining sites to recognize the millions of antigenic shapes in
the environment
THEORIES
• Side Chain Theory
✓ By Paul Ehrlich, 1900s
✓ Body has preformed B cell receptors (antibodies)
(surface receptors)
✓ Key premises:
(1) the lock- and- key concept of the fit of antibody for antigen
and
(2) the idea that an antigen selected cells with the built-in
capacity to respond to it. Prepared by: Nhoelyn E. Burcao, RMT
 Antibody Synthesis

THEORIES
• Template theory
✓ Instructional hypothesis
➢ By Linus Pauling (1940s)
✓ A flexible antibody molecule is acted on by the antigen to form a
COMPLEMENTARY binding site
✓ Antigens act like templates that direct the folding of a nascent antibody chain

Prepared by: Nhoelyn E. Burcao, RMT

 Antibody Diversity

THEORIES
• Clonal Selection Theory
✓ Fundamental basis of
lymphocyte activation in which
Ag selectively stimulates only
those cells which express
receptors for it to divide and
differentiate
✓ Each lymphocyte produces
one type of immunoglobulin
only and the Ag selects and
stimulates cells carrying that
immunoglobulin type

Prepared by: Nhoelyn E. Burcao, RMT

 Antibody Diversity
THEORIES
• Clonal Selection Theory
✓ Postulates
a. Ab of all specificities are produced prior to contact with the Ag
b. B lymphocytes participating in the immune response have receptors on their surface
membranes that are immunoglobulin molecules of the same specificity as that Ab that will
be produces by their activated and differentiated ontogeny
c. Each lymphocyte carries immunoglobulin molecules of only a single specificity on its
surface
d. Circulating self-antigens that reach the developing lymphoid system prior to its maturation
are recognized as “self” and no subsequent immune response will be induced against them
e. Immunocompetent lymphocytes which are not shut off or deleted in this process can be
stimulated under appropriate conditions by antigen to proliferate and differentiate into
clones of Ab plasma cells and memory cells

Prepared by: Nhoelyn E. Burcao, RMT

Clonal Selection Theory
Each B cell has different Ag receptors

The Ag receptors of any one B cell will combine w/ Ag.

Activation of B cell (by T cells)

In the presence of cytokines, this B cell is stimulated to divide

Clonal Expansion

Plasma Cell Chosen B cell gives rise

to memory cells and Ab-
producing plasma cells
After infection passes,
plasma cells undergo
apoptosis
Prepared by: Nhoelyn E. Burcao, RMT
 Monoclonal Antibody Production

1. Mouse is immunized with a certain antigen → spleen cells are harvested

2. Spleen cells are combined with MYELOMA CELLS in the presence of PEG
3. Plasma cell + Myeloma cell → Hybridoma Cells
4. Cells are placed in culture using HAT medium
5. Myeloma cells and normal B cells die
6. Hybridoma cells are diluted out and placed in microtiter wells where they are allowed
to grow
7. Desired Ab is identified
8. Ab maintained in cell culture to produce a ready supply of monoclonal antibody that
reacts with a single epitopePrepared by: Nhoelyn E. Burcao, RMT