IMMU lecture note 5

8/10/2019 IMMU lecture note 5

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IMMU3201L5Lymphocyte 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 clonesmature 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 antibodiesspecific to that

antigen

What are Antigens?

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

recognising molecules)

- They are initiatorsthey 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


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- Small regions on the antigen are recognisedantigenic determinantor epitope

- Epitopes are immunologically active regionsthat 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

- Proteinsare good immunogens

- Polysaccharidesare good immunogens (e.g. LPS)

-

DNAs are poor immunogens

- Lipids are poor immunogens


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Antigens and lymphocytes

- Not all antigens can activate lymphocytes

- Immunogensare molecules that stimulate immune response

-

B cells are activated by macromoleculesthat cross-link B cell receptors

- Proteinsand 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 antigenicbut 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 tertiarystructure

- Change in antigen shape prevents antigen from binding to antibody

-

Therefore denaturation of protein prevents antigen binding and thus prevent

antigen recognition


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3 ways an antibody can bind to antigens

- Antibodies can bind to 3 different types ofdeterminants (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 conformationepitope is discontinuousprotein not in a

stable tertiary structureantibody cannot recognise antigen

Antibodies and Linear Determinants

-

Antibody binds to visible epitope of the linear determinant

- There are hidden epitopes within the tertiary structure

- denaturationdoes 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

proteolysisin order for the antibody to bind to the antigen


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Difference between Surface and Secreted Immunoglobulin (Antibody)

- Antibody=Immunoglobulin (Ig)

- Membrane bound antibodiesare called B cell receptors(BCR)

-

Secreted antibodiesmediated the effector functions

- Both membrane-bound and secreted forms bind antigen

- Nave B cellsexpress IgM and IgD

Antibody Features

- Y-shape molecule

-

2 heavy + 2 light chains

- Variable region + constant region

- ALL antibodies are bi-functionalboth 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 regiondetermines what type of antigen the antibody binds to

o

variable part is vary between clones

- hinge allows flexibility


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Antibody Structure

- 4 polypeptide chainsassembled 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 regionallow movement of variable domainincrease flexibility

- Fab = variable region

- Fc = constant region


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- Affinity is the SUM of the attractiveand repulsive forces at the binding site between

antibody and ONE epitope

- High affinityHigh attractionLow repulsion

- Low affinityLow attractionHigh repulsion

Valence and Avidity of antibody-antigen interaction

- Valencehow many interactions can the antibody have

- IgAmonovalentLow avidity

- IgGbivalentHigh avidity

- IgMpentamericformvalence of 10Very High avidity


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How specific is the antibody?

- Antibody can bind specifically

- Bind non-specificallylead to cross-reactivity

- Non-reactivity

Antibody Classes


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- There are 5classes/isotypesof antibodies

- Therefore 5 types of HEAVY chain

o ,, , ,

o

The 5 types of heavy chains differ in their constant (C)domainstherefore

differ in function

- There are 2types 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 cellsare the only cells that synthesis antibodies

-

Antibodies are located in biological fluids throughout the body- Antibodies are present in the plasma, mucosal secretionsand interstitial fluidof the

tissues

- They can attach to Fcreceptors 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 monomericform

- 3 constant regions

- Half-life of 23 days

-

IgG is involved in


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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


- Secreted in a pentamericform5IgM 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 dimericform2IgA antibodies joined by a J chain


- Secreted form of IgA is functionally important in mucosal immunity

- Dimeric form protects IgA from proteolytic attackas it crosses membrane barriers in

the mucosa

o

Prevent degradation of IgA

- Two subclasses of IgAIgA1, IgA2

IgD

- Half-life of 3days

- Secreted in a monomericform

-

Consists of the HEAVY chain


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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 hybridomacells can be frozenin liquid nitrogenfor 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


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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 CD20on B-cellsB-cell depletiontreat rheumatoid arthritis,

multiple sclerosis etc.

- mAbs that are specific to VEGFblock tumour angiogenesistreat breast cancer,

colon cancer

- mAbs that target TNFinhibits T cell mediated inflammationtreat 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


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

- Disadvantages of polyclonal antibodies

- Advantages of monoclonal antibodies

Documents

IMMU lecture note 5