Antibodies

Question 1

What is the structure of immunoglobulin?

Answer 1

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)

Question 2

What are the FAB and Fc regions of an immunoglobulin?

Answer 2

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 3

What are the two types of light chain?

Answer 3

λ (lambda)

k (kappa)

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)

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

Question 6

How were epitopes discovered?

Answer 6

Hapten - a small molcule that can act as an epitope but wouldn’t induce antibodies on their own

1. Hapten is combined with a protein (ovalbumin)
2. Give the compound to a mouse
3. This will produce antibodies to ovalbumin and the hapten
4. Take the hapten antibodies and bind to hapten in vitro

Question 7

What can act as an epitope?

Answer 7

Any chemical can act as an epitope (hapten) in this way; inorganic chemical, peptide, sugar, lipid, nucleic acid

Antibodies recognise antigenic epitopes by molecular complementation i.e. can’t have steric interferences/hindrances

Question 8

What is important about the epitope shape?

Answer 8

Variation in hapten structure (chemical positions of functional groups) can lead to different affinities of binding to an antibody

Question 9

What binding do antibodies use?

Answer 9

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

A complementary binding surface on an antibody maximises the non-covalent interactions with the epitope

Question 10

What are the types of epitopes?

Answer 10

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 11

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

Answer 11

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 12

Describe the structure of IgM?

Answer 12

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 13

What are the functions of IgM?

Answer 13

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 14

Facts about IgM?

Answer 14

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 15

Describe the structure of IgG?

Answer 15

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 16

What are the functions of IgG?

Answer 16

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 17

Facts about IgG?

Answer 17

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 18

Describe the structure of IgA?

Answer 18

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 19

What are the functions of IgA?

Answer 19

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 20

Facts about IgA?

Answer 20

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 21

Describe the structure of IgE?

Answer 21

Monomeric - 188 kDa

The ε heavy chain has 4CH domains

Question 22

What are the functions of IgE?

Answer 22

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 23

Facts about IgE?

Answer 23

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 24

Describe IgD?

Answer 24

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 25

What is antibody diversity? How does it arise?

Answer 25

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 26

Facts about antibody diversity?

Answer 26

5 X 10^13 different specificities of antibodies can be produced This allows enough antibodies for every antigen it encounters The total number of genes in the human genome is insufficient to accommodate one complete gene for each antibody molecule (there are only around 21,000 protein coding genes in the human genome) Three of these genes are for immunoglobulin Solution: Diversity is achieved by recombination of less than 1,000 gene segments in the DNA of developing B-cells

Question 27

How does specificity arise?

Answer 27

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 28

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

Answer 28

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

Question 29

What is the Variable light chain coded for by? Example?

Answer 29

Variable (V) and Joining (J) K-light chain of chromosome 2 39 V gene segments and 5 J gene segments It also has a constant gene segment (C)

Question 30

What is the Variable heavy chain coded for by? Example?

Answer 30

Variable (V), Diversity (D) and Joining (J) Chromosome 14 Heavy chain 46 V gene segments, 23 D gene segments and 6 J gene segments It has 5 constant gene segments

Question 31

What is an overview of the gene recombination?

Answer 31

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 32

How is the variable light chain developed?

Answer 32

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 33

How is the variable heavy chain developed?

Answer 33

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 34

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

Answer 34

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 35

What is combinatorial diversity?

Answer 35

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 36

What is junctional diversity

Answer 36

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 37

How can immunoglobulins switch class?

Answer 37

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 38

What is significant with immunoglobulin switching?

Answer 38

``` 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 ```