Bio151lec22012 2013 120625102153 Phpapp01

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
specically to an antibody the immunoglobulin receptor of B cells or by the T-cell receptor when complexed with MHC name arises from their ability to generate antibodies
substances that can be recognized by
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 Immunogenicity* Antigenicity*

(immune system does not attack the antigen)
Biology 151 Introduction to Immunology
ARE YOU ALLERGIC TO SOME DRUGS?
Antigens and Immunogens

ANTIGEN A molecule which can be specically recognized and bound
by an antibody
IMMUNOGEN A molecule which can elicit the production of specic

antibody upon injection into a suitable host
Antigenic
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) reacts with the products of it (Abs)
both induces an immune response and

Factors that Inuence 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 molecular mass less than 1,000 Da have proven to be immunogenic
EXEMPTIONS: few substances with a

Chemical Composition & Heterogeneity
chemical complexity contributes to immunogenicity
Lipids as Antigens
appropriately presented lipoidal

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

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 Inuence 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 inuenced by genes that encode B-cell and T-cell receptors response inuenced by genes that encode various proteins involved in immune regulatory mechanisms immunogenicity in different animals
THUS: genetic variability affects

Dosage and Route

experimental immunogen exhibits unique doseresponse curve
DOSE insufcient 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 antigenspecic 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 inuence which immune organs and cell populations will be involved in the response intravenous = carried rst to the spleen subcutaneous = moves rst to local lymph nodes
Adjuvants
substances that, when mixed with an
antigen and injected with it, ENHANCE the immunogenicity of that antigen an antigen has LOW IMMUNOGENICITY or when only SMALL AMOUNTS of an antigen is available
used to boost the immune response when
Adjuvants
IN SUMMARY...
Parameter
Size
Increased Immunogenicity Large >10,000; best >100,000 Intermediate Subcutaneous/IM > Intraperitoneal > Complex Particulate Denatured Multiple differences Slow release Bacteria Effective
Decreased Immunogenicity Small MW<2500 High or Low Intravenous > Oral or intragastric Simple Soluble Native Few differences Rapid release No bacteria Ineffective
Dose Route Composition Form
Similarity to self proteins Adjuvants
Interaction with host MHC
EPITOPES: Antigenic Determinants

Antigenic Determinants or Epitopes
DEFINITION:
immunologically active regions of an immunogen that interacts with the specic 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 (Blymphocytes/ 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
Antigenic Determinants 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 epitope will be the most highly immunogenic/ antigenic? on Tuesday
QUESTION: How will you know which fraction/ Handwritten: Yellow Paper to be submiited
Antigen Classication
T cell dependent Ags or TD much more complex than TI
Ags
usually proteins stimulate a full complement of immunoglobulins with all ve classes represented elicit an anamnestic or memory response & are present in most pathogenic organisms
Viral proteins = HA and NA
bacterial protein = agellin
Fungal proteins = keratinases

Antigen Classication
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 Molds Yeasts
Yeasts
Fungi Aspergillus
Candida albicans Cryptococcus neoformans
Polysaccharide Ag (TI) galactomannan Alpha mannan

galactoxylomannan
Protein Ag (TD) glycoproteins proteinase
Dermatophytes
Trichophyton
Galactomannan peptides
Keratinases I, II. III
Zygomycetes Dimorphic systemic fungi
Rhizopus Histoplasma capsulatum
peptidofucomannan galactomannan
Proteinase 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 Classication

Thymus dependent Structural properties Chemistry Complex Proteins; proteinnucleoprotein conjugates; glycoproteins; lipoproteins IgG, IgM, IgA, (+ IgD and IgE) YES Thymus independent Simple Polysaccharide of pneumococcus; dextran polyvinyl pyrolidone; bacterial Lipolysaccharide IgM
Antibody class induced Immunological memory response Present in most pathogenic microbes
NO
YES
NO
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 Structure
Tetramer
Tetramer
Polymeric
Ligand
A-D-mannose & aD-glucose
NDi-Nacetylgalactosamine 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 specicity on B-cells Common to all antibodies: structural features binds to antigen participate in effector function
Antibody: Name Game

Antitoxin Agglutinins Precipitins Lysins Opsonins
Neutralizing antibodies
Neutralize toxins Clumps cells Ppt soluble antigens Lyses cells ! phagocytosis Neutralize viruses Activate complement
Complement fixing antibodies

Ab-mediated Effector Functions
Opsonization: promotion of phagocytosis

of antigens by macrophages and nuetrophils nuetrophils) bind to Ig molecules which induces a signal transduction pathway resulting in phagocytosis of the Ag-Ab complex
Fc receptors (in surfaces of macrophages and Actions: enzymatic digestion, oxidative

damage, membrane-disrupting effects of bacterial peptides
Complement Activation (IgM and C3b: important by-product; binds nonmacrophages leading to phagocytosis)
IgG): induces a collection of proteins that can perforate cell membranes specically to cell and Ag-Ab complexes
many cell types have receptors for C3b (e.g. signicance: 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 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
Pre-requisite: linking of Ab bound to target
Basic Structure of Antibodies
Antibodies found in
serum protein fractions revealed four peaks: albumin, alpha, beta and gamma
electrophoretic mobility Gamma globulin factors
= immunoglobulins (IgG) which contains serum antibodies
Antibodies are Heterodimers

consist of two identical heavy chain: has an
side light chains & two identical heavy chains linked by disulde bonds 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 ve 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)
xes complement (IgG4) binds to Fc receptors (iGg2 and IgG4)
phagocytes - opsonization Killer cells - ADCC
PROPERTIES pentamer 3rd highest serum

immunoglobulin
Antibody Classes : IgM
rst immunoglobulin made by fetus and B cells xes complement
C1r C1 s
C1q
C1r C1 s
C1q
Figure: xation of C1 by IgG and IgM

No activation
agglutinating immunoglobulin binds to Fc receptors B-cell surface immunoglobulins
Activation
Antibody Classes : IgM & IgG
Antibody Classes : IgA

PROPERTIES OF IgA

serum monomer secretions (sIgA) 2nd highest serum immunoglobulins major secretory Ig (tears, saliva, gastric and pulmonary secretions) = mucous and local immunity DO NOT x complement (unless aggregated binds to Fc receptors on some cells
Secretory Piece J Chain

FORMATION OF SECRETORY IgA
Antibody Classes : IgD

PROPERTIES OF IgD
monomer tail piece 4th highest serum Ig B-cell surface Ig DOES NOT BIND
complement
Tail Piece
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)
C! 4
allergic reaction parasitic infections (helminths)
binds to Fc receptors on eosinophils
DOES NOT x complement
Allergies
IMMUNOGLOBULINS & ALLERGIES

Georges Kohler and Cesar Milstein in 1975
MONOCLONAL ANTIBODIES
Polyclonal antibodies : arise from MANY Bcell 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
CLINICAL UTILITY OF MONOCLONAL ANTIBODIES

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
NEXT MEETING
Innate and Adaptive Response

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ANTIGENS & ANTIBODIES

Biology 151 lecture 2 AY 2012-2013_1st

Main Reference for this Topic: Immunology by KUBY et al.

Monday, June 25, 2012

What are antigens?

any molecule that can bind

substances that can be recognized by

Monday, June 25, 2012

What are antigens?

Monday, June 25, 2012

Immunologic Properties of Antigens

Tolerogenicity: capable of inducing immunological tolerance Immunogenicity* Antigenicity*

(immune system does not attack the antigen)

Biology 151 Introduction to Immunology

ARE YOU ALLERGIC TO SOME DRUGS?

Monday, June 25, 2012

Monday, June 25, 2012

Monday, June 25, 2012

Antigens and Immunogens

IMMUNOGEN A molecule which can elicit the production of specic

Monday, June 25, 2012

Antigens: Haptens & Immunogens

HAPTENS (incomplete antigen)

Monday, June 25, 2012

Monday, June 25, 2012

Monday, June 25, 2012

Antigens: Haptens & Immunogens

both induces an immune response and

Factors that Inuence Immunogenicity

Monday, June 25, 2012

EXEMPTIONS: few substances with a

Chemical Composition & Heterogeneity

chemical complexity contributes to immunogenicity

Monday, June 25, 2012

appropriately presented lipoidal

antigens can induce both B-cell and T-cell responses

Example: lipid-protein conjugates

Monday, June 25, 2012

Susceptibility to Antigen Presentation & Processing

larger molecules are more readily phagocytosed and processed

Factors that Inuence Immunogenicity

Genotype of Recipient Animal

THUS: genetic variability affects

Dosage and Route

DOSE insufcient dose will not stimulate an immune

Monday, June 25, 2012

Dosage and Route

Can you recall the route of your vaccine shots?

Monday, June 25, 2012

used to boost the immune response when

Monday, June 25, 2012

Monday, June 25, 2012

Dose Route Composition Form

Similarity to self proteins Adjuvants

Interaction with host MHC

Monday, June 25, 2012

EPITOPES: Antigenic Determinants

Antigenic Determinants or Epitopes

Monday, June 25, 2012

Antigenic Determinants or Epitopes

Immunodominant Epitope: stimulate a greater antibody response

Monday, June 25, 2012

Monday, June 25, 2012