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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
- Macromolecules include proteins, lipids, polysaccharides


How are specific antibodies produced?
- Immature B cell clones mature in lymphoid organs
o Each immature lymphocyte clone is specific to one type of antigen
- The immature clones enter peripheral lymphoid tissue to search for their respective
antigens
- Activated clones proliferate to generate antigen-specific clones
- 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. antigen-
recognising molecules)
- They are initiators they initiate the adaptive immune response
- Examples of antigens include:
o Microbes, foreign cells, foreign serum, pollens, food, drugs, chemicals
- Antigens are large complex molecules (or whole cells e.g. virus)
- Whole antigen molecules are not recognised by the immune system
- Small regions on the antigen are recognised antigenic determinant or epitope
- Epitopes are immunologically active regions that bind to antigen-specific receptors


Are all antigens good antigens?
- Not all antigens are good antigens
- Antigens that stimulate an immune response are called immunogens
- Not all antigens are good immunogens


What makes a good antigen? What makes it more immunogenic?
- Foreignness
o How foreign is the antigen to the host
o How different it is to Self
- Complexity
o whole cell antigens have more epitopes
o purified antigens
- molecular size
o the larger the size the better
- Stability
o Hold itself long enough to be recognised by receptors and not degrade
- Proteins are good immunogens
- Polysaccharides are good immunogens (e.g. LPS)
- DNAs are poor immunogens
- Lipids are poor immunogens






Antigens and lymphocytes
- Not all antigens can activate lymphocytes
- Immunogens are molecules that stimulate immune response
- B cells are activated by macromolecules that cross-link B cell receptors
- Proteins and polysaccharides can activate B cells
- 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
- They are antigenic but NOT immunogenic
o Antigenic in that they are foreign to a host
- Haptens alone cannot induce an immune response
- Haptens are chemically coupled to different carriers to produce an immunogenic
hapten-carrier complex
- The complex induces an immune response
- Therefore haptens can only induce immunity when bound to a carrier


Size of Antigens matters!
- Antigen needs to be reasonably large to be immunogenic
- Hapten is antigenic but not immunogenic alone as its too small
- Hapten-carrier complex is both antigenic AND immunogenic


Antigen stability and immunogenicity
- Antigens need to be stable to bind to an antibody
- They need a stable tertiary structure
- Change in antigen shape prevents antigen from binding to antibody
- 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
- parts of the epitope along the protein come together to form one epitope for the
antibody to recognise
- 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
- There are hidden epitopes within the tertiary structure
- denaturation does not prevent binding of antigen to antibody (in contrast to
conformational determinants)
- The denatured protein antigen unveils the previously inaccessible determinant or
hidden epitope that the antibody can now bind to
- 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)
- Membrane bound antibodies are called B cell receptors (BCR)
- Secreted antibodies mediated the effector functions
- Both membrane-bound and secreted forms bind antigen
- Nave B cells express IgM and IgD


Antibody Features
- Y-shape molecule
- 2 heavy + 2 light chains
- Variable region + constant region
- ALL antibodies are bi-functional both the variable region and constant region
have a function
- the constant region determines the effector function of the antibody
o constant part is conserved between clones
- the variable region determines what type of antigen the antibody binds to
o variable part is vary between clones
- hinge allows flexibility



Antibody Structure
- 4 polypeptide chains assembled into Y-shape molecule
- 2 identical light (L) chains
- 2 identical heavy (H) chains
- Light chains = 1 variable domain, 1 constant domain
- Heavy chain = 1 variable domain, 3-4 constant domains (depend on isotype)
- Hinge region allow movement of variable domain increase flexibility
- Fab = variable region
- Fc = constant region










Binding Site of antibodies
- The variable region of the heavy chain (V
H
) and light chain (V
L
) each contain 3
hypervariable (HV) regions
o Therefore V
H
and V
L
each have 3 HV regions
- Hypervariable regions are also called Complementarity-Determining Regions (CDRs)
o CDR1, CDR2, CDR3
- CDRs contribute to antibody specificity
- 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

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


Antibody Affinity
- Affinity is the SUM of the attractive and repulsive forces at the binding site between
antibody and ONE epitope
- High affinity High attractionLow repulsion
- Low affinity Low attractionHigh repulsion
Valence and Avidity of antibody-antigen interaction
- Valence how many interactions can the antibody have

- IgA monovalent Low avidity
- IgG bivalent High avidity
- IgM pentameric form valence of 10 Very High avidity









How specific is the antibody?
- Antibody can bind specifically
- Bind non-specifically lead to cross-reactivity
- Non-reactivity


Antibody Classes
- There are 5 classes/isotypes of antibodies
- Therefore 5 types of HEAVY chain
o , , , ,
o The 5 types of heavy chains differ in their constant (C) domains therefore
differ in function
- There are 2 types of LIGHT chain
o ,
o the 2 types of light chains differ in their C domains but NO functional
difference
- The 5 difference classes of antibodies:
o 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
- Antibodies are located in biological fluids throughout the body
- Antibodies are present in the plasma, mucosal secretions and interstitial fluid of the
tissues
- They can attach to Fc receptors on the surface of effector cells (mononuclear
phagocytes, NK cells, mast cells)



IgG
- Consists of the HEAVY chain
- Most abundant antibody class in the Ig pool
- Secreted in a monomeric form
- 3 constant regions
- Half-life of 23 days
- IgG is involved in
o Opsonization
o Complement activation
o ADCC (antibody dependent cell mediated cytotoxicity)
o Neonatal immunity
o Feedback inhibition of B cells


IgM
- Half-life of 3 days
- Consists of the HEAVY chain
- Secreted in a pentameric form 5 IgM antibodies joined by a J chain
- Have 4 constant regions
- Functions as Nave B cell receptor (BCR)
- Involved in Complement activation


IgA
- Half-life of 6 days
- Secreted in a dimeric form 2 IgA antibodies joined by a J chain
- Consists of the HEAVY chain
- Secreted form of IgA is functionally important in mucosal immunity
- Dimeric form protects IgA from proteolytic attack as it crosses membrane barriers in
the mucosa
o Prevent degradation of IgA
- Two subclasses of IgA IgA1, IgA2


IgD
- Half-life of 3 days
- Secreted in a monomeric form
- Consists of the HEAVY chain
- Functions as Nave B cell receptor


IgE
- Half-life of 2 days
- Secreted in a monomeric form
- Functions as a defence against helminthic parasite
- Involved in immediate hypersensitivity (by activation mast cells and basophils)
- Consists of the HEAVY chain
- 4 constant regions


What is the importance of the variable regions of antibodies?
- The Fab or variable regions binds to antigens
- They can block active sites of pathogen associated molecules
- They can block interactions between host and pathogen associated molecules
- HOWEVER, the variable region CANNOT activate inflammation and effector functions
associated with cells
- They also cannot activate inflammation and effector functions of complement
- Therefore antibodies require Fc regions





Polyclonal Antibodies
- Using animals to generate antibodies
- The antibodies produced are polyclonal antibodies
- Major disadvantages of polyclonal antibodies
o The antibodies produced are heterogeneous i.e. they are not identical
(since each animal is different from one another and will produce different
antibodies)
o 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

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

- An so we invented monoclonal antibodies


Monoclonal antibodies (mAbs)
- the antibodies produced are homogenous
- the antibodies are derived from a SINGLE B-cell
- We isolate and cultivate this single clone to produce an endless supply of antibodies
with high affinity for their antigen/epitope with DEFINED specificity
- 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
- They help us understand the antibody structure and functions
- They are used in
o Research labs
o Diagnostic labs
o Tumour detection
o Therapy


Examples of Monoclonal Antibodies used in Diagnostic Labs
- Western blotting
- Immunoprecipitation
- Particle agglutination
- Enzyme linked immunoassay
- Radioimmunoassay
- Flow cytometry
- 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.

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

- mAbs that target TNF inhibits T cell mediated inflammation treat Rheumatoid
arthritis, Crohns disease


lecture Summary
- lymphocyte antigen receptors are specific
- Lymphocytes proliferate and differentiated upon activation
- Features of antigens
- Epitopes
- Haptens
- Antibody (Ig) structure
- Molecular basis of antigen-receptor binding
- There are 5 antibody classes/isotypes: IgM, IgD, IgG, IgA, IgE
- Disadvantages of polyclonal antibodies
- Advantages of monoclonal antibodies