IMMU lecture note 5

IMMU3201 – L5 – Lymphocyte Receptors and Antibodies

Recognition of Antigens -

Molecules that recognise antigen include Antibodies, T-cell receptor (TCR) and MHC molecules

Antibodies and antigens -

Antibodies recognise macromolecules

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Macromolecules include proteins, lipids, polysaccharides

How are specific antibodies produced? -

Immature B cell clones mature in lymphoid organs

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Each immature lymphocyte clone is specific to one type of antigen

The immature clones enter peripheral lymphoid tissue to search for their respective antigens

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Activated clones proliferate to generate antigen-specific clones

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The antigen-specific clones then create antigen-specific antibodies specific to that antigen

What are Antigens? -

Antigens are substances that bind to antibodies, TCR or MHC molecules (i.e. antigenrecognising molecules)

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They are initiators  they initiate the adaptive immune response

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Examples of antigens include:

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Microbes, foreign cells, foreign serum, pollens, food, drugs, chemicals

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Antigens are large complex molecules (or whole cells e.g. virus)

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Whole antigen molecules are not recognised by the immune system

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Small regions on the antigen are recognised  antigenic determinant or epitope

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Epitopes are immunologically active regions that bind to antigen-specific receptors

Are all antigens good antigens? -

Not all antigens are good antigens

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Antigens that stimulate an immune response are called immunogens

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Not all antigens are good immunogens

What makes a good antigen? What makes it more immunogenic? -

Foreignness

o o -

whole cell antigens have more epitopes purified antigens

molecular size

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How different it is to “Self”

Complexity

o o -

How foreign is the antigen to the host

the larger the size the better

Stability

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Hold itself long enough to be recognised by receptors and not degrade

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Proteins are good immunogens

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Polysaccharides are good immunogens (e.g. LPS)

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DNAs are poor immunogens

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Lipids are poor immunogens

Antigens and lymphocytes -

Not all antigens can activate lymphocytes

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Immunogens are molecules that stimulate immune response

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B cells are activated by macromolecules that cross-link B cell receptors

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Proteins and polysaccharides can activate B cells

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Haptens are small chemicals that will only bind to B cell receptors when conjugated to a protein carrier

Hapten-carrier complex -

Haptens are small and are chemically active

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They are antigenic but NOT immunogenic

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Antigenic in that they are foreign to a host

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Haptens alone cannot induce an immune response

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Haptens are chemically coupled to different carriers to produce an immunogenic hapten-carrier complex

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The complex induces an immune response

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Therefore haptens can only induce immunity when bound to a carrier

Size of Antigens matters! -

Antigen needs to be reasonably large to be immunogenic

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Hapten is antigenic but not immunogenic alone as its too small

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Hapten-carrier complex is both antigenic AND immunogenic

Antigen stability and immunogenicity -

Antigens need to be stable to bind to an antibody

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They need a stable tertiary structure

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Change in antigen shape prevents antigen from binding to antibody

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Therefore denaturation of protein prevents antigen binding and thus prevent antigen recognition

3 ways an antibody can bind to antigens -

Antibodies can bind to 3 different types of determinants (epitopes): 1. conformational determinants 2. linear determinants 3. neoantigenic determinants

Antibodies and Conformational Determinants -

protein antigens fold together in a tertiary shape to form conformational determinants

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parts of the epitope along the protein come together to form one epitope for the antibody to recognise

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denaturation unfolds conformation  epitope is discontinuous  protein not in a stable tertiary structure  antibody cannot recognise antigen

Antibodies and Linear Determinants -

Antibody binds to visible epitope of the linear determinant

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There are hidden epitopes within the tertiary structure

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denaturation does not prevent binding of antigen to antibody (in contrast to conformational determinants)

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The denatured protein antigen unveils the previously inaccessible determinant or “hidden” epitope that the antibody can now bind to

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Therefore antibody binds to antigen regardless of denaturation

Antibodies and Neoantigenic Determinants -

With neoantigenic determinants or epitopes, the epitope needs to be “created” by proteolysis in order for the antibody to bind to the antigen

Difference between Surface and Secreted Immunoglobulin (Antibody) -

Antibody=Immunoglobulin (Ig)

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Membrane bound antibodies are called B cell receptors (BCR)

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Secreted antibodies mediated the effector functions

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Both membrane-bound and secreted forms bind antigen

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Naïve B cells express IgM and IgD

Antibody Features -

Y-shape molecule

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2 heavy + 2 light chains

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Variable region + constant region

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ALL antibodies are bi-functional  both the variable region and constant region have a function

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the constant region determines the effector function of the antibody

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the variable region determines what type of antigen the antibody binds to

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constant part is conserved between clones

variable part is vary between clones

hinge allows flexibility

Antibody Structure -

4 polypeptide chains assembled into Y-shape molecule

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2 identical light (L) chains

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2 identical heavy (H) chains

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Light chains = 1 variable domain, 1 constant domain

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Heavy chain = 1 variable domain, 3-4 constant domains (depend on isotype)

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Hinge region  allow movement of variable domain  increase flexibility

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Fab = variable region

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Fc = constant region

Binding Site of antibodies -

The variable region of the heavy chain (VH) and light chain (VL) each contain 3 hypervariable (HV) regions

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Therefore VH and VL each have 3 HV regions

Hypervariable regions are also called Complementarity-Determining Regions (CDRs)

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CDR1, CDR2, CDR3

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CDRs contribute to antibody specificity

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The 6 CDRs come together to form the

antigen binding site -

Sequence differences within the CDRs

contribute to distinct interaction surfaces  determine the specificities of individual antibodies

Forces involved in Antibody-Antigen interactions 1. Electrostatic forces 2. Hydrogen bonds 3. Van der Waals forces 4. Hydrophobic forces

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Ag-Ab binding is a reversible non-covalent interaction

Antibody-Antigen Binding: Affinity and Avidity -

Affinity is the strength of binding between ONE antigen binding site on antibody molecule and its corresponding epitope

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Avidity is the TOTAL strength of binding of antibody molecule and antigen

Antibody Affinity

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Affinity is the SUM of the attractive and repulsive forces at the binding site between antibody and ONE epitope

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High affinity  High attraction–Low repulsion

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Low affinity  Low attraction–High repulsion

Valence and Avidity of antibody-antigen interaction -

Valence  how many interactions can the antibody have

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IgA  monovalent  Low avidity

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IgG  bivalent High avidity

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IgM  pentameric form  valence of 10  Very High avidity

How specific is the antibody? -

Antibody can bind specifically

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Bind non-specifically  lead to cross-reactivity

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Non-reactivity

Antibody Classes

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There are 5 classes/isotypes of antibodies

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Therefore 5 types of HEAVY chain

o o

µ, δ, γ, ε, α The 5 types of heavy chains differ in their constant (C) domains  therefore differ in function

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There are 2 types of LIGHT chain

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κ, γ the 2 types of light chains differ in their C domains but NO functional difference

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The 5 difference classes of antibodies:

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IgG, IgM, IgA, IgD, IgE

Antibody classes (isotypes) have different functions

Distribution and production of antibodies -

B cells are the only cells that synthesis antibodies

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Antibodies are located in biological fluids throughout the body

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Antibodies are present in the plasma, mucosal secretions and interstitial fluid of the tissues

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They can attach to Fc receptors on the surface of effector cells (mononuclear phagocytes, NK cells, mast cells)

IgG -

Consists of the γ HEAVY chain

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Most abundant antibody class in the Ig pool

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Secreted in a monomeric form

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3 constant regions

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Half-life of 23 days

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IgG is involved in

o o o o o

Opsonization Complement activation ADCC (antibody dependent cell mediated cytotoxicity) Neonatal immunity Feedback inhibition of B cells

IgM -

Half-life of 3 days

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Consists of the µ HEAVY chain

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Secreted in a pentameric form – 5 IgM antibodies joined by a J chain

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Have 4 constant regions

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Functions as Naïve B cell receptor (BCR)

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Involved in Complement activation

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Half-life of 6 days

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Secreted in a dimeric form – 2 IgA antibodies joined by a J chain

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Consists of the α HEAVY chain

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Secreted form of IgA is functionally important in mucosal immunity

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Dimeric form protects IgA from proteolytic attack as it crosses membrane barriers in

IgA

the mucosa

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Prevent degradation of IgA

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Two subclasses of IgA  IgA1, IgA2

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Half-life of 3 days

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Secreted in a monomeric form

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Consists of the δ HEAVY chain

IgD

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Functions as Naïve B cell receptor

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Half-life of 2 days

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Secreted in a monomeric form

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Functions as a defence against helminthic parasite

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Involved in immediate hypersensitivity (by activation mast cells and basophils)

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Consists of the ε HEAVY chain

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4 constant regions

IgE

What is the importance of the variable regions of antibodies? -

The Fab or variable regions binds to antigens

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They can block active sites of pathogen associated molecules

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They can block interactions between host and pathogen associated molecules

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HOWEVER, the variable region CANNOT activate inflammation and effector functions associated with cells

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They also cannot activate inflammation and effector functions of complement

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Therefore antibodies require Fc regions

Polyclonal Antibodies -

Using animals to generate antibodies

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The antibodies produced are polyclonal antibodies

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Major disadvantages of polyclonal antibodies

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The antibodies produced are heterogeneous – i.e. they are not identical (since each animal is different from one another and will produce different antibodies)

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The supply is limited (the animal used to make the antibody has a life-span)

The antibodies produced by one animal is unique and cannot be reproduced exactly produced in a another animal

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We need an endless supply of antibodies with high affinity for their antigen/epitope with DEFINED specificity

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An so we invented monoclonal antibodies

Monoclonal antibodies (mAbs) -

the antibodies produced are homogenous

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the antibodies are derived from a SINGLE B-cell

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We isolate and cultivate this single clone to produce an endless supply of antibodies with high affinity for their antigen/epitope with DEFINED specificity

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The hybridoma cells can be frozen in liquid nitrogen for a long period of time

What are cultured Antibodies used for? -

Antibodies help eradicate infections

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They help us understand the antibody structure and functions

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They are used in

o o o

Research labs Diagnostic labs Tumour detection

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Therapy

Examples of Monoclonal Antibodies used in Diagnostic Labs -

Western blotting

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Immunoprecipitation

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Particle agglutination

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Enzyme linked immunoassay

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Radioimmunoassay

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Flow cytometry

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Immunostaining microscopy

Examples of Monoclonal Antibodies used in Therapies -

mAbs that target CD20 on B-cells  B-cell depletion  treat rheumatoid arthritis, multiple sclerosis etc.

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mAbs that are specific to VEGF  block tumour angiogenesis  treat breast cancer, colon cancer

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mAbs that target TNF  inhibits T cell mediated inflammation  treat Rheumatoid arthritis, Crohn’s disease

lecture Summary -

lymphocyte antigen receptors are specific

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Lymphocytes proliferate and differentiated upon activation

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Features of antigens

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Epitopes

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Haptens

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Antibody (Ig) structure

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Molecular basis of antigen-receptor binding

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There are 5 antibody classes/isotypes: IgM, IgD, IgG, IgA, IgE

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Disadvantages of polyclonal antibodies

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Advantages of monoclonal antibodies