Antibodies

Question 1

Give an overview of immunoglobulins?

Answer 1

Immunoglobulins (antibodies) are secreted by B lymphocytes
The function of immunoglobulins is to bind to antigens (epitopes)
Immunoglobulins mediate humoral immunity
A dimer of dimers gives us two binding sites

Question 2

What is the structure of immunoglobulin?

Answer 2

2 heavy chains and 2 light chains linked by disulphide bonds
They can be cleaved into 2 parts using protease papain into a Fab (fragment antigen binding) fragment and an Fc region (fragment crystallisation)

A 2-layer sandwich of between 7 and 9 antiparallel β-strands arranged in two β-sheets found in multiple, each contain about 70-110 amino acids and are categorised according to their size and function
Ig-domains possess a characteristic Ig-fold, which has a sandwich-like structure formed by two sheets of antiparallel beta strands
Interactions between hydrophobic amino acids on the inner side of the sandwich and highly conserved disulphide bonds

Question 3

What are the FAB and Fc regions of an immunoglobulin?

Answer 3

The Fab fragment region is responsible for antigen binding (which there are 2 antigen binding domains per molecule)
The Fc region is bound by Fc receptors expressed by phagocytes and other immune cells

Question 4

What are the two types of domain?

Answer 4

Variable domain - made up of 2 domains: Variable light (VL) and Variable Heavy (VH)
They vary which defines antigen specificity

Constant domain - made up of 4 domains: Constant Light (CL) and 3 Contant Heavy (CH1, CH2 and CH3) 
Within the same class they don't differ

Question 5

What is the antigen binding site made up of?

Answer 5

The variable domain - Variable light (VL) and Varibale Heavy (VH)

The binding site are formed via loops that project form theses regions - called complementary determining regions (CDR) and hypervariable loops
The loops help determine specificty/complementarity

Question 6

What binding do antibodies use?

Answer 6

Antibodies use non-covalent bonds to bind to antigenic epitopes:
Electrostatic interactions
Hydrogen bonds
Hydrophobic interactions
Van-der Waals forces

These non-covalent bonds become stronger as the intermolecular distances become smaller
A complementary binding surface on an antibody maximises the non-covalent interactions with the epitope

Question 7

Describe some physiological functions of antibodies?

Answer 7

Blocking interactions with the cells - neutralising immunoglobulins
Many functions are mediated via constant region of antibody

Immunoglobulins opsonise (coat) antigens for uptake by phagocytes
Fc receptors bind to Fc region of immunoglobulin-antigen complexes
Phagocytosis of immunoglobulin-antigen complexes leads to the degradation of the antigen

Immunoglobulins can activate the classical complement pathway
Complement mediates neutralisation, lysis and phagocytosis

Antibody Dependent cell-mediated cytotoxicity (ADCC)
If something is coated in an antibody e.g. Tumour/virus, allowing neutrophils, NK cells and macrophages to engage

Question 8

What are the types of epitopes?

Answer 8

Continuous epitopes - residues that make up the epitope are all directly linked

Discontinuous epitopes - formed by the apposition of distant residues as a result of molecular folding
Discontinuous epitopes are typically lost by unfolding or denaturing protein antigens

Epitopes can be 12 - 16 amino acids or 5 - 6 sugar residues

Question 9

What are the 5 classes of immunoglobulins? What are they distinguished by?

Answer 9

IgM, IgG, IgA, IgE and IgD

They are distinguished by their heavy chains:
IgM - µ (mu) heavy chain 
IgG - γ (gamma) heavy chain
IgA - a (alpha) heavy chain
IgE - ε (epsilon) heavy chain 
IgD - δ (delta) heavy chain

Question 10

Describe the structure of IgM?

Answer 10

Looks like a star

µ heavy chain has 4 CH domains
It is pentameric (970kDa): 10 antigen binding sites
The J chain promotes polymerisation
The monomers are linked by disulphide bonds to each other and to the J chain
Can be imaged by electron microscopy

Question 11

What are the functions of IgM?

Answer 11

It is the first antibody secreted in response to a foreign antigen (primary response)

Neutralises antigens
Agglutinates (clumps) microbes
Activates classical complement pathway
IgM is effective in limiting the spread of microorganisms via the bloodstream

Question 12

Facts about IgM?

Answer 12

Confined to the vascular system due to large size

It has a low affinity for an antigen, but has 10 binding sites so can vind to polyvalent antigens e.g. Bacterial surfaces

Question 13

Describe the structure of IgG?

Answer 13

Looks normal - monomeric

4 distinct isotypes: IgG1, IgG2, IgG3 and IgG4
IgG1, IgG2 and IgG4 - 146 kDa
IgG3 - 165 kDa
Each gamma-heavy chain has 3 CH domains

Question 14

What are the functions of IgG?

Answer 14

Neutralises antigens
Bound by Fc receptors on phagocytes
Activates the classical complement pathway
During pregnancy it is actively transported across placenta into the foetus to protect new born babies for the first 3-6 months

Question 15

Facts about IgG?

Answer 15

Produced in secondary response to an antigen by B-cells in the lymph nodes and spleen
Major antibody in normal human blood, IgG1 is the most abundant isotype in the blood (9 mg/ml in serum)
Can enter the extra-vascular sites due to small size i.e. the tissues of the body

IgG opsonises microbes for uptake by phagocytes

Question 16

Describe the structure of IgA?

Answer 16

Either normal - monomeric (160 kDa) or two joined together at the bottom - dimer (320 kDa)
In the dimer disulphide bonds and the J chain hold them together

Question 17

What are the functions of IgA?

Answer 17

The principal function of IgA is to neutralise antigens:
IgA prevents bacterial toxins binding their cellular targets
IgA inhibits microbial adhesion to epithelia
IgA inhibits viral infectivity

Question 18

Facts about IgA?

Answer 18

IgA is produced by B-cells in the mucosal associated lymphoid tissues during secondary immune response
Majority is secreted onto mucosal epithelial surfaces as Ig receptor transports it across the epithelium

Question 19

Describe the structure of IgE?

Answer 19

Monomeric - 188 kDa

The ε heavy chain has 4CH domains

Question 20

What are the functions of IgE?

Answer 20

Important for immune responses against helminths (worms) and other parasites
Also involved in type I hypersensitivity responses (allergic reactions) eg hay fever, food allergies
Binding of IgE molecules to polyvalent antigens cross-links Fc(epsilon) receptors and triggers release of inflammatory mediators

Question 21

Facts about IgE?

Answer 21

Produced in response to some antigens, usually by B-cells in the mucosal associated lymphoid tissues• Low concentration in the blood
Vast majority of IgE is bound to Fc(epsilon) receptors on mast cells, basophils and eosinophils

Question 22

Describe IgD?

Answer 22

Monomeric - 160 kDa
The δ heavy chain has 3CH domains

Acts as antigen receptor on immature B-cells and is present in the blood at very low concentrations
No other biological function has yet been described

Question 23

What is antibody diversity? How does it arise?

Answer 23

Antibody diversity refers to the array of different antigenic specificities produced in the humoral immune response
Antibody diversity is generated by recombination of the antibody genes during the antigen-independent differentiation of B-cells in the bone marrow (antibody gene rearrangement)

Question 24

Facts about antibody diversity?

Answer 24

5 X 10^13 different specificities of antibodies can be produced
This allows enough antibodies for every antigen it encounters

Diversity is achieved by recombination of less than 1,000 gene segments in the DNA of developing B-cells

Question 25

How does specificity arise?

Answer 25

Antigenic specificity of an antibody arises from the sequence of the VL and VH domains

In order to encode an antibody molecule, immunoglobulin genes must combine

Question 26

What are all the gene segments that are used within the chains?

Answer 26

V coded for by variable (V)-gene segment [98aa]
D coded for by diversity (D)-gene segment [16-21aa]
J coded for by joining (J)-gene segment [1-15aa]
C coded for by constant gene segment

Typical H-chain - 1000 V, 10 D, 4 J and 8 C
Typical L-chain - 90-300 V, 4 J, 1 C

Question 27

What is an overview of the gene recombination?

Answer 27

Immunoglobulin genes are non-functional until they are rearranged
They are rearranged during antigen independent differentiation of B cells in the bone marrow

Each light and heavy chain uses only one of each gene segment
This is called V(D)J recombination
It allows a large number of possible combinations from only a few gene segments

Question 28

How is the variable light chain developed?

Answer 28

Somatic recombination VJ joining
Any combination is technically possible during recombination
After this recombination we get transcription to form a primary RNA transcript
Splicing will then remove J segments that aren’t needed to form mRNA in a virgin B-cell
The light chain mRNA is then translated into a protein in the RER

Question 29

How is the variable heavy chain developed?

Answer 29

First there is somatic recombination DJ joining
Then there is somatic recombination VDJ joining to form a rearranged DNA pre-B-cell
Transcription will then form a primary RNA transcript
RNA splicing will remove unwanted J segments to form mRNA in virgin B-cells
This mRNA is translated into the heavy chain proteins in the RER

Question 30

What happens when the VL and the VH chains are produced?

Answer 30

The heavy chains can now associate with light chains in the ER to form antibody molecules
They are sent to the Golgi which can then be secreted

Question 31

What is combinatorial diversity?

Answer 31

This refers to the different combinations of heavy and light chains that can be used to form an antibody
Theoretically any heavy and light chain can interact to form an antibody molecule
Combinatorial diversity further increases antigen specificity

Question 32

What is junctional diversity

Answer 32

Junctional diversity describes the DNA sequence variations introduced by the imprecise joining of gene segments during the process of V(D)J recombination
The random generation of sequences and the addition of nucleotides being added and removed at random (at the sites of V, D and J gene segments)

The junction corresponds to the 3rd hypervariable loop of the variable domain

Question 33

How can immunoglobulins switch class?

Answer 33

B-cells switch class as the immune response matures
Naïve B-cells exit the bone marrow expressing IgM and IgD on their cell surface
IgM is the first immunoglobulin secreted in response to antigens
As the humoral response matures B-cells undergo class switching and one IgG, IgA and IgE are expressed instead of IgM/IgD by B-cells
Class switching tailors the humoral response to the antigen

Question 34

What is significant with immunoglobulin switching?

Answer 34

Only the constant regions of the heavy chains are switched
Therefore class switching doesn't alter the antigenic specificity because the light chain and VH domains are maintained