L3 4 Antibodies

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Immunology

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Introduction

Antibodies (Immunoglobulin / Ig)The major agents of the humoral immune responseGlycoproteinsMembrane-bound (acting as receptors) or soluble (secreted)Bi-functional molecules: Bind antigen (Ag) & bind to receptors on cells & bind complementEvery antibody has a highly specific binding site

Structure

Four polypeptide chains

2 identical large chains (heavy) ~450AA, ( / K / E / H / I)2 identical small chains (light) ~250AA, (O /P)

The different types ofheavy / light chain are encoded by different genes


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The heavy chains define the class of antibody and the properties they have:

locus = IgMK locus = IgGE locus = IgAH locus = IgDI locus = IgE

The ability to bind to antigens lies in the light chain

The ability to bind to cells and activate the complement lies in the heavy chain (biological activity)

Amino terminus is at the top of the Y

Carboxyl terminus is at the bottom of the Y

Heavy Chain:

Each is broken up into 4 domains

VHVariable heavyCH1-3 Constant heavy (3 domains)

It is the constant regions that define the properties of the heavy chain, and therefore the properties of the antibody

Separated by a hinge region which gives the antibody flexibility in the arms of the Y

Connected by disulphide bonds

Constant heavy domains are numbered from amino (1)p carboxyl (3)

Every domain is stabilised by a single disulphide bond

This stabilises the internal folding

IgM and IgEhave no hinge region, and also have an additional CH domain (CH 4)

Less flexibility for lateral movement

Light Chain:

Broken up into 2 domains:

CLConstant lightVLVariable light

The light chain is connected to the heavy chain by a single disulphide bond

Each light chain is ~25 kDa (50 kDa total)


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Eachheavy chain is ~55 kDa (110 kDa total)

An average antibody has a total MW of 160 kDa

A reducing agent (F-mercaptoethanol) can be used to break disulphide bonds leaving 4 chains

Antibodies are glycoproteins contain a carbohydrate part

No known function other than to promote folding


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Proteolytic digestion was used to classify the antibodies

They were digested into smaller parts, then analysed

Papain cleaves the heavy chains to produce 3 molecules

Fab = fragment antigen binding

Determined that binding was monovalent, each Fab fragment

bound a single antigen

Fc = fragment crystallisable

Fc is constant domain which is why it can be made into a

crystal

It is this region that activates the complement

It is also this region that binds to receptors on cells

Most cells of the immune system have FC receptors (FCR)

The soluble antibody can bind the FCR

There are different FCRs for the different classes of antibody

All classes of IgG can cross the placenta in man

There are receptors that bind the FC region of IgG to mediate placental transfer

This allows the antibody to be passed from the mother to the offspring

This confers mothers immunity p offspring

In other species the antibodies are acquired from the mother through milk

Antibodies selectively cross the gastrointestinal tract

He also used a different enzyme (pepsin) which cut in a different place

An Fc region containing 2 polypeptide chains F(ab)2

This was bivalent and could bind 2 antigens

The variable domain is not uniformly variable

Thousands of different molecules were studied and the AA sequence was determined


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Variability was plotted against position

There are 3 regions ofhypervariability (HV)

CDR = Complementarity Determining Region

25-30 CDR1

50-60 CDR1

90-95 CDR1

Complementarity refers to the region that

determines how an antibody fits to an antigen

The HV region makes up the binding site,

antigens obviously have to be very variable in

this region

The regions in between are know as the

framework regions

Below is a light chain

Immunoglobulin fold is a defined secondary

structure element

Foundin proteins that are part of the immunoglobulin gene superfamily

Comprised of2F-sheets stabilized by a disulphide bond

Found in a lot of molecules that are involved in receptor-ligand interactions

Although in a linear arrangement the CDR are far apart, the folding brings them together at the tip

The 6 CDRs come

together to form a


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binding site for the antigen

The binding site can accommodate for 6-12 AAs at most

The antigen makes close contact with the binding site

The binding is non-covalent, but by a combination of multiple non-covalent bonds

H-bondsElectrostatic interactionsHydrophobic interactions

Individually, the attractive forces are weak, but the bonds multiple provided that the antigen fits the site very closely

(lock + key hypothesis)

Antigen binds very tightly to the binding site

Antibodies do no bind all of a molecule, just small regions of it

e.g. haemaglutinin has 4 / 5 sites for antibody binding

The site of antibody binding is known as an epitope, usually just a few AAs (~6)

Continuous epitopes consecutive residues

Discontinuous epitopes residues that are separate that have been brought together by fold

A single mutation in the epitope will completely disrupt antibody binding

This is why new strains of influenza arrive from a single point mutation

This allows the new strain to escape the immune surveillance

1st exposure to an antigen = primary immune response2nd exposure to same antigen = secondary immune response

IgG:

3 week half-life

Monomeric one four-chain unit (MW ~160 kDa)

~75% of antibody in serum (major serum antibody)

3 constant domains

Distributed evenly in the bloodstream as well as throughout the body in tissue fluids

The major antibody in the secondary immune response

4 subclasses in humans: IgG 1 4 (different genes for each subclass)

Subclasses in mice are 1, 2A, 2B & 3

Numbered in order of concentration (1 is highest, 4 is lowest)

There are specific FC receptors for each subclass


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IgM:

No subclasses

5 four-chain units (pentameric)

4 constant domains

10% serum antibody

Major antibody involved in the primary immune response

Primarily restricted to the bloodstream due to its size ~1000

kDa

The units are linked by disulphide bonds between the heavy 3

and 4 domains

There is an additional polypeptide (137 resides) known as the J

(joining) chain

It helps to link all of the subunits together

It is added during the assembly of the protein in the ER

Acts as a primary defence against infectious organisms

Very effective at agglutinating and cross-linking

The bacteria are clumped together

IgMhas 10 binding sites for antigens

2 configurations: star (like in diagram, large distance between binding sites) or staple configuration (in which the

molecule has some flexibility) There is no hinge region to provide this flexibility, however there is flexion between the

constant heavy 2 /3 domains. This allows the molecule to sit on a surface with the binding sites oriented in the same

direction, allowing for strong, multivalent binding.

IgA:

2 subclasses: IgA 1 / 2


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~15% serum antibody

May be monomeric or dimeric in serum

Is the predominant antibody at mucosal surfaces

Found at high concentrations in the secretions at mucosal surfaces

e.g. nasopharangeal surfaces, urinogenital tracts etc.

These surfaces are susceptible to infection (cf. skin)

Normally dimeric at mucosal surfaces (can be tetramer or trimer also)

Has a J chain like IgM which is added in the same way

Also has an additional component: secretory component (a separate polypeptide chain)

Pathogen enters at lumen

A plasma cell is a terminally differentiated B-cell

The plasma cell contains a lot ofER and Golgi as its primary role is to produce a lot of antibodies

The plasma cells in the submucosa are primarily programmed to make IgA

1. Plasma cell secretes dimeric IgA

2. IgA binds to the poly-Ig receptor

3. Transcytosis occurs; the protein brings the Ig into the cell

4. During the transport process the receptor is cleaved proteolytically and part of the receptor remains bound to IgA

this is the secretory component

The gut is a very hydrolytic environment. The secretory component is thought to protect the antibody from proteolytic

degradation.

Some bacteria have evolved to produce proteolytic enzymes that specifically degrade Igs


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The gut has specialised lymph nodes known as Peyers patches

The primary follicles are germinal centres where lymphocytes are dividing

In the gut, M-cells take up antigens from pathogens

More in H. Brady lecture

The plasma cells will migrate within the submucosa and produce IgA which will be translocated across the epithelial

later, into the lumen to combat the pathogen.

IgE:

Very low serum concentration


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IgE and mast cells have a particular role against helminth (parasitic worm infections)

IgD:

Single 4-chain unit

Not found in serum

Found on the surface ofB-cells

An immature B-cell has membrane bound IgM and IgD (both are monomeric)

Not bound to receptors, the Igs have a short cytoplasmic tail with a hydrophobic region that interacts well with the lipid

bilayer

When the B-cell is triggered by an antigen, it will differentiate into a plasma cell that will go on to secrete IgM (IgD is

not made, thought to only be there as a receptor)

The IgM that is secreted is modified, it lacks the C-terminal region of the heavy chain by a process of differential

transcription. Inside the plasma cell, the IgM is polymerised into the pentameric structure and is secreted. This is why

IgM is the first antibody produced, its production is the first reaction to an antigen.

Primary Antibody Response:

It takes around 7 days for antibody levels to reach a peak (IgM)

IgM is the major antibody in the primary response

IgG peaks around day 14

Levels of both will slowly fall

Decline in antibody conc. is due to the half-life of the antibody,and immune complexes are removed (antibody +

antigen)

Secondary Antibody Response:

When an antigen is encountered for the 2nd + time

More antigen is produced

The antigen is produced faster

IgM response will be similar in terms of amount produced and time to produce it

IgG response will increase rapidly (3 days) and to a muchhigher level

IgG is therefore the major antibody in a secondary response

This is one of the reasons why we have greater protection from a secondary infection

The antibody affinity (IgG) of binding to antigen will be higher


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Class switching occurs during the primary immune response. IgMp IgG

IgM can alsopIgA /E

Regulation of this is dependent on cytokines

The variable region stays the same; it is just the heavy chain that is altered

The binding specificity is maintained, but given to antibodies with different properties

The higher affinity of binding during the secondary response occurs because during the 2nd infection, mutations occur

in the hypervariable regions of the antibody (certain regions of the genes are hypermutable) this results in a higher

binding strength of the antibody for selected mutants. This process is known as affinity maturation. CDR3 is

particularly hypermutable.

The selection process occurs as when the antigen is at a low concentration, it will natural select a cell that has an

antibody with a better fit, therefore these clones will be expanded.

There are now many more cells that can react to the antigen, but they also have more specific antigens

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L3 4 Antibodies