Bio 151 lec 2 2012 2013

ANTIGENS &
ANTIBODIES
Biology 151 lecture 2
AY 2012-2013_1st

Main Reference for this Topic: Immunology by KUBY et al.
Monday, June 25, 2012

ANTIGENS


What are antigens?
• any molecule that can bind
speciﬁcally to an antibody
• substances that can be recognized by
the immunoglobulin receptor of B cells
or by the T-cell receptor when
complexed with MHC
• name arises from their ability to
generate antibodies

What are antigens?
• includes sugars, lipids, intermediary
metabolites, hormones, complex
carbohydrates, phospholipids, nucleic
acids and proteins
• found in surface or parts of a microbe
or from the environment (e.g. food,
pollen, etc)


Immunologic
Properties of Antigens
• Allergenicity:
• having the capacity to induce allergy
(hypersensitivity)
• Tolerogenicity:
• capable of inducing immunological tolerance
(immune system does not attack the antigen)
• Immunogenicity*
• Antigenicity*

Biology 151
Introduction to Immunology

ARE YOU
ALLERGIC TO
SOME DRUGS?


Antigens and Immunogens
• ANTIGEN
• A molecule which can be speciﬁcally recognized and bound
by an antibody

• “Antigenic”
• IMMUNOGEN
• A molecule which can elicit the production of speciﬁc
antibody upon injection into a suitable host

• “Immunogenic”
• ALL molecules that are immunogenic are also antigenic..BUT...not
all antigenic molecules are immunogenic! (Example: HAPTENS!)

Antigens: Haptens &
Immunogens
• HAPTENS (incomplete antigen)

• antigens that by themselves do not elicit antibody
production

• unable to induce an immune response alone
but able to react with products (Abs)

• have the property of antigenicity but not
immunogenicity (elicit immune response)

• NOTE: could be rendered immunogenic by covalently
linking them to a carrier molecule


Antigens: Haptens &
Immunogens

• IMMUNOGENS (complete antigen)
• antigens that can elicit antibody production
• stimulate B and/or T cell arms of the immune
response and react with products (Abs)
• both induces an immune response and
reacts with the products of it (Abs)


Factors that Inﬂuence
Immunogenicity
• Nature of the Immunogen
• foreignness
• molecular size
• chemical composition and heterogeneity
• lipids as antigens
• susceptibility to antigen processing and presentation


Foreigness
• Recall: “in order to elicit an immune response, a
molecule must be recognized as NON-SELF by the
biological system”
• tolerance for SELF-antigens
• The greater the phylogenetic distance between two
species, the greater the structural disparity between
them

• EXAMPLE: bovine serum albumin not immunogenic
to cow bt is on chicken (cow > goat > chicken)


Molecular Size
• The most ACTIVE immunogens:
• 100,000 Da
• Substances with a molecular mass of
5,000-10,000 Da are poor immunogens
• EXEMPTIONS: few substances with a
molecular mass less than 1,000 Da have
proven to be immunogenic


Chemical Composition
& Heterogeneity

• chemical
complexity
contributes to
immunogenicity


Lipids as Antigens

• appropriately presented lipoidal
antigens can induce both B-cell and
T-cell responses
• Example: lipid-protein conjugates
(lipids are used as haptens)


Lipids as Antigens


Susceptibility to Antigen
Presentation & Processing
• the development of both humoral and cell-mediated
immune responses requires interaction of T-cells with
antigen that has been processed and presented together
with MHC molecules

• LARGE, INSOLUBLE macromolecules are generally more
immunogenic than SMALL, SOLUBLE ones

• larger molecules are more readily phagocytosed and
processed

• degradative enzymes within antigen-presenting cells can
degrade only proteins containing L-amino acids, polymers of
D-amino acids cannot be processed


Factors that Inﬂuence
Immunogenicity

•Biological System
• genotype of the recipient animal
• immunogen dosage and and route of
administration
• adjuvants


Genotype of
Recipient Animal
• MHC gene products = determines the degree
to which an animal responds to an immunogen
(immune responsiveness)

• response inﬂuenced by genes that encode B-cell
and T-cell receptors

• response inﬂuenced by genes that encode
various proteins involved in immune regulatory
mechanisms

• THUS: genetic variability affects
immunogenicity in different animals

Dosage and Route
• experimental immunogen exhibits unique dose-
response curve

• DOSE
• insufﬁcient dose will not stimulate an immune
response (fails to activate lymphocytes or
tolerance)

• excessively high dose = tolerance

• THUS....repeated adminsitrations or BOOSTERS
are done if a single dose will not induce a strong
response = increase clonal proliferation of antigen-
speciﬁc T cells or B-cells = increase the lymphocyte
populations SPECIFIC for the immunogen

Dosage and Route
• experimental immunogen exhibits unique dose-response curve

• ROUTE

• generally administered parenterally = other than the GIT

• Subcutaneous route > Intramuscular > Intraperitoneal >
Intraveous > Oral route

• Can you recall the route of your vaccine shots?

• strongly influence which immune organs and cell populations will be
involved in the response

• intravenous = carried first to the spleen

• subcutaneous = moves first to local lymph nodes


Adjuvants

• substances that, when mixed with an
antigen and injected with it, ENHANCE
the immunogenicity of that antigen
• used to boost the immune response when
an antigen has LOW IMMUNOGENICITY
or when only SMALL AMOUNTS of an
antigen is available


Adjuvants


IN SUMMARY...
Parameter Increased Decreased
Immunogenicity Immunogenicity
Size Large >10,000; Small MW<2500
best >100,000
Dose Intermediate High or Low
Route Subcutaneous/IM > Intravenous > Oral or
Intraperitoneal > intragastric
Composition Complex Simple
Form Particulate Soluble
Denatured Native
Similarity to self proteins Multiple differences Few differences
Adjuvants Slow release Rapid release
Bacteria No bacteria
Interaction with host MHC Effective Ineffective


EPITOPES:
Antigenic Determinants

or Epitopes
• DEFINITION:

• immunologically active regions of an immunogen that interacts with the
speciﬁc antigen binding site in the variable region of the antibody molecule
(PARATOPE) or to secreted antibodies

• EXCELLENT FIT between epitope and paratope: based upon their 3-D
interaction and non covalent union

• are discrete site on the macromolecule recognized by the lymphocytes (B-
lymphocytes/ T-lymphocytes)

• NOTE: B and T cells recognize DIFFERENT epitopes on the SAME antigenic
molecule

• THUS: the ability to function as a B-cell epitope is determined by the
nature of the ANTIGEN-BINDING site of the antibody molecules
DISPLAYED by B-cells


or Epitopes
• An antigen molecule has 2 or more epitopes or antigenic determinants per
molecule

• Epitopes consist of approximately 6 amino acids or 6 monosaccharides

• Immunodominant Epitope: stimulate a greater antibody response


APPLICATIONS:
Epitope Mapping
• Assignment: Diagram an experiment on how to
carry out epitope mapping and determination of
epitopes
• QUESTION: How will you know which fraction/
epitope will be the most highly immunogenic/
antigenic?
• Handwritten: Yellow Paper to be submiited
on Tuesday


Antigen Classiﬁcation
• T cell dependent Ags or TD
• much more complex than TI
Ags

• usually proteins
• stimulate a full complement of
immunoglobulins with all ﬁve
classes represented
• elicit an anamnestic or
memory response & are
present in most pathogenic
organisms

Viral proteins = HA and NA

bacterial protein = ﬂagellin

Fungal proteins = keratinases

Antigen Classiﬁcation
•T cell independent
Ags or TI
• often polysaccharides
or
lipopolysaccharides
• elicit an IgM response
only and fail to
stimulate an amnestic
response

Bacterial membrane/OM components

capsular polysaccharide

Fungal Mannans & Glucans

Example: Fungi
Group of fungi Fungi Polysaccharide Ag (TI) Protein Ag (TD)

Molds Aspergillus galactomannan glycoproteins

Yeasts Candida albicans Alpha mannan proteinase

Yeasts Cryptococcus neoformans galactoxylomannan

Dermatophytes Trichophyton Galactomannan peptides Keratinases I, II. III

Zygomycetes Rhizopus peptidofucomannan Proteinase

Dimorphic systemic fungi Histoplasma capsulatum galactomannan “h” & ‘m” factors

“ Paracoccodioides galactomannan E2 factor

“ Coccidioides imitis Methyl-mannose polymer Coccidiodin factor

Subcutaneous Sporothrix schenckii Peptido-L-rhamno-D-mannan


Summary of Antigen Classiﬁcation
Thymus dependent Thymus independent

Structural Complex Simple
properties
Chemistry Proteins; protein- Polysaccharide of
nucleoprotein conjugates; pneumococcus; dextran
glycoproteins; polyvinyl pyrolidone;
lipoproteins bacterial Lipolysaccharide

Antibody class IgG, IgM, IgA, IgM
induced (+ IgD and IgE)

Immunological YES NO
memory response

Present in most YES NO
pathogenic
microbes


Superantigens
• a substance such as a bacterial toxin capable of stimulating MANY
CD4+ T lymphocytes leading to the release of relatively large
quantities of cytokines that provoke pathophysiologic manifestations

• NOTE: Superantigens are TD antigens = THUS, Do not
require phagocyte processing

• stimulate multiple T cells that augment a protective T & B cell
response


Superantigens


Superantigens
• Examples:

• Staphylococcal enterotoxins (food poisoning)

• Staphylococcal toxic shock toxin (toxic shock
syndrome)

• Staphylococcal exfoliating toxins (scalded skin
syndrome)

• Streptococcal pyrogenic exotoxins (shock)

• The diseases associated with exposure to
superantigens are, in part, due to hyper activation
of the immune system and subsequent release of
biologically active cytokines by activated T cells

Mitogens
• substance, often derived from plants, that
causes DNA synthesis and induces blast
transformation and division by mitosis

• Lectins, representing plant-derived
mitogens or phytomitogens are used in
experimental and clinical immunology to
evaluate T and B lymphocyte function in vitro


Mitogens
Characteristic Concanavalin A Phytohemagglu Pokeweed
(Con A) tinin (PHA) mitogen
(PWM)

Source Jack beans Kidney beans Pokeweed

Molecular Tetramer Tetramer Polymeric
Structure

Ligand A-D-mannose & a- N- Di-N-
D-glucose acetylgalactosamine acetylchitobiose

Target cell/s T cells T cells T cells and B cells


SPECIAL ISSUE:
Antigenic Variation


How are antigens recognized?

• Pattern Recognition
Receptors


DIFFER IN INNATE AND IN ADAPTIVE
RESPONSES


ANTIBODIES
(Immunoglobulins or Igs)


What are antibodies?
• Antibodies : antigen-binding proteins present on
the B-cell membrane and secreted by plasma
cells
• when bound confers antigenic speciﬁcity on B-cells
• Common to all antibodies:
• structural features
• binds to antigen
• participate in effector function

Antibody: Name Game
Antitoxin Neutralize toxins

Agglutinins Clumps cells

Precipitins Ppt soluble antigens

Lysins Lyses cells

Opsonins ! phagocytosis

Neutralizing antibodies Neutralize viruses

Complement fixing Activate complement
antibodies

Ab-mediated Effector Functions
• Opsonization: promotion of phagocytosis
of antigens by macrophages and nuetrophils
• Fc receptors (in surfaces of macrophages and
nuetrophils) bind to Ig molecules which
induces a signal transduction pathway
resulting in phagocytosis of the Ag-Ab
complex
• Actions: enzymatic digestion, oxidative
damage, membrane-disrupting effects of
bacterial peptides


• Complement Activation (IgM and
IgG): induces a collection of proteins that
can perforate cell membranes
• C3b: important by-product; binds non-
speciﬁcally to cell and Ag-Ab complexes
• many cell types have receptors for C3b (e.g.
macrophages leading to phagocytosis)
• signiﬁcance: removal and killing of pathogens

• Antibody-dependent cell-mediated
Cytotoxicity (ADCC): kills cells
• Ab acts as a newly acquired receptor
enabling the attacking cell to recognize and
kill the target cell
• Pre-requisite: linking of Ab bound to target
cells (e.g. virus in host cells) with the Fc
receptor of many cell types can direct the
cytotoxic activities of the effector cell against
the target cell

Basic Structure of Antibodies

• Antibodies found in
serum protein fractions
• electrophoretic mobility
revealed four peaks:
albumin, alpha, beta and
gamma
• Gamma globulin factors
= immunoglobulins (IgG)
which contains serum
antibodies

Antibodies are Heterodimers

• consist of two identical
side light chains & two
identical heavy chains
linked by disulﬁde bonds

• heavy chain: has an
amino-terminal variable
region followed by a
constant region


Antibodies are Heterodimers


Immunoglobulin Classes

• in any given antibody
molecule, the constant
region contains one of
ﬁve basic heavy chain
sequences called
isotypes

• the heavy chain
isotype determines
the class of an
antibody


Immunoglobulin Classes

• IgG
• IgD
• IgE
• IgA
• IgM


Antibody Classes


Antibody Classes : IgG


Antibody Classes : IgG
• PROPERTIES:
• major serum immunoglobulins (systemic immunity)
• major immunoglobulin in extravascular spaces
• does not require antigen binding during placental
transfer (IgG2)
• ﬁxes complement (IgG4)
• binds to Fc receptors (iGg2 and IgG4)
• phagocytes - opsonization
• Killer cells - ADCC

Antibody Classes : IgM
• PROPERTIES
• pentamer
• 3rd highest serum
immunoglobulin

• first immunoglobulin made by
fetus and B cells C1r C1
s

• fixes complement C1q
C1r C1
s

• Figure: fixation of C1 by IgG C1q

and IgM

•
No activation Activation
agglutinating immunoglobulin

• binds to Fc receptors

• B-cell surface immunoglobulins

Antibody Classes : IgM & IgG


Antibody Classes : IgA
PROPERTIES OF IgA
• serum monomer

• secretions (sIgA)

• 2nd highest serum Secretory Piece J Chain
immunoglobulins

• major secretory Ig (tears, saliva,
gastric and pulmonary secretions)
= mucous and local immunity

• DO NOT ﬁx complement (unless
aggregated

• binds to Fc receptors on some cells


Antibody
Classes : IgA

FORMATION OF
SECRETORY IgA


Antibody Classes : IgA


Antibody Classes : IgD
PROPERTIES OF IgD

• monomer
• tail piece
• 4th highest serum Ig
• B-cell surface Ig Tail Piece

• DOES NOT BIND
complement


Antibody Classes : IgE
PROPERTIES OF IgE
• monomer

• with extra domain

• least common serum Ig

• binds to basophils and mast cells (DO NOT
require antigen binding)

• allergic reaction
C!4
• parasitic infections (helminths)

• binds to Fc receptors on eosinophils

• DOES NOT ﬁx complement


Allergies

IMMUNOGLOBULINS &
ALLERGIES


Biology 151
Georges Kohler and MONOCLONAL
Cesar Milstein in 1975 ANTIBODIES
Polyclonal antibodies :
arise from MANY B-
cell clones and have a
HETEROGENOUS
collection of binding
sites

Monoclonal
antibodies : derived
from a SINGLE B-cell
clone and is a
HOMOGENOUS
collection of binding
sites


Biology 151

CLINICAL UTILITY OF
MONOCLONAL
ANTIBODIES


Biology 151
CLINICAL UTILITY OF MONOCLONAL ANTIBODIES

•

DIAGNOSTICS:
detect small amounts of drugs, toxins or hormones, e.g. monoclonal antibodies to human chorionic
gonadotropin (HCG) are used in pregnancy test kits (Biotech, 1989); diagnosis of AIDS by the ELISA test.

•

THERAPEUTICS:
radioimmunodetection and radioimmunotherapy of cancer, and some new methods can even target only the cell
membranes of cancerous cells (Chaudhari et al, 1994);

cancer drug based on monoclonal antibody technology = Ritoxin, approved by the FDA in November 1997
(Orrs, 1997)

radioimmunodetection and radioimmunotherapy of cancer, and some new methods can even target only the cell
membranes of cancerous cells (Chaudhari et al, 1994); cancer drug based on monoclonal antibody
technology = Ritoxin, approved by the FDA in November 1997 (Orrs, 1997)

viral diseases, traditionally considered "untreatable" = AIDS (P/S/L, 1997).

•

TRANSPLANTATION
OKT3, 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 (Transweb, 1996).

Parungao-Balolong 2010

Biology 151

NEXT MEETING
Innate and Adaptive
Response


Bio 151 lec 2 2012 2013

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Bio 151 lec 2 2012 2013