Antibodies Flashcards
What is the structure of an antibody?
2 heavy chains and 2 light chains.
Heavy chains are joined by disulphide bonds adn each heavy chain is linked to light chain by disulphide bond.
Each chain contains several Ig domain repeats.
Heavy chains have hinge regions.
The two heavy and light chains are identical - allowing antibody to have two identical antigen binding sites and bind simultaneously to 2 identical structures.
Stalk = Fc region = contains Fc receptor/complement binding sites.
Antigen binding sites are in FAb region.
What are the different antibody classes?
The antibody’s characteristics are determined by heavy chain class.
IgM are encoded by mu genes and are the first Ig’s produced - effective complement activators in opsonisation to enhance phagocytosis.
IgD contains a TM sequence which enables insertion into plasma membranes = expressed on cell surface = as antigen receptor on naive B cells.
IgA = used in covid vaccine and involved in epithelial/mucosal immunity.
IgE = in response to parasites and allergic responses = these IgE antibodies cause histamine release and mast cell degranulation.
IgG = produced by memory B cells upon secondary exposure to antigen! - involved in neonatal immunity and antibody dependent cell-mediated toxicity = opsonisation + complement activation.
How are the different classes of antibody arranged as oligomers/monomers?
IgA are joined as dimers/trimers via J chains.
IgE and IgG are monomers.
IgM exists as pentamers, with 5 Ig’s joined by J chain.
= as each ab has 2 Fab binding sites = pentamer has 10 low affinity binding sites.
Why is there such antibody diversity?
Can produce approx 10^7 functional antibodies.
Variable regions of heavy and light chains usually form the antigen-binding sites in Fab region.
Each light chain variable region is encoded by a DNA sequence assembled from 2 gene segments - V and J, encoded separately in the genome.
Each heavy chain variable region is encoded by a DNA sequence assembled from 3 gene segments - V, J and D segments…
Gene duplication means that there are already large numbers of inherited V, D, and J segments in the genome, but combinatorial joining diversity greatly increases the diversity of possible variable regions.
What is the process of combinatorial diversification?
During B cell development, V and J segments (Light chain) and V,D,J segments (Heavy chain) are joined by V(D)J recombinases - which mediate the joining of single gene segments.
V,D,J segments are joined together to form functional DNA assmeblies encoding for variable regions for light and heavy chains.
What group of genes control V(D)J joining?
RAG genes encode for V(D)J recombinases.
Individuals with RAG gene mutations cannot carry out V(D)J joining and as such do not have functional B or T cells.
TCR use the same V(D)J recombinase to assemble gene segments for TCR’s.
What is junctional diversification?
During B cell development, during V(D)J joining of gene segments for DNA assemblies of light and heavy chain variable regions, there is a random loss/gain of nucleotides at the joining sites from the ednds of recombining gene segments.
This causes reading frame shifts which can introduce further diversity, but usually produce non-functional genes…
How do antibodies become higher affinity to antigen during immunisation?
Affinity maturation = following immunisation, there is a progressive increase in the affinity of antibodies produced against the antigen.
Affinity maturation is driven by somatic hypermutation - accumulation of point mutations in both heavy and light chain variable region coding segments…
Believed to involve an error-prone DNA repair mechanism targeted to rearranged V-region coding sequences.
= as it introduces mutations at much higher rates than spontaneous mutation.
Whilst only a minority of antibodies ay have increased affinity, the B cells which express these are preferentially stimulated to survive and proliferate by antigen engagement.
= Antigen-driven proliferation of selected memory B cell clones with higher affinity antibodies.
What are the hypervariable regions?
Complementarity determining regions (CDRs)….
3 regions within variable gene segment with highly variable amino acid sequences - CDR1,2,3.
3 sites of highly variable AA sequences in variable regions of both light and heavy chains..
Combining CDRs from heavy and light chains increases diversity of the antigen-binding site possibilites.
The main antigen binding site in Fab region is between CDR folds of heavy and light chain..
How are antibodies produced?
B cells undergo irreversible genetic recombiation to produce an antibody of restricted specificity as they mature in bone marrow.
First to form is the TM bound IgM, then IgD.
Antibody typically binds antigen with weak affinity/high avidity.
When B cells are activated, Ig class switching to IgG, IgA, IgE occurs followed by affinity maturation driven by somatic hypermutation….
Affinity increases…
Activated B cells become plasma cells, some become memory B cells, Plasma cells produce approx 2000 abs per second.
What are the Fc mediated effects of an antibody?
Fc region = stalk.
Fc region mediates many of effector functions of Abs.
Class affects function.
IgG bind FC receptors on neutrophils, macrophages to promote phagocytosis… opsonisation.
IgE Fc binding to eosinophils leads to eosinophil activation and mast cell degranulation in allergy…
IgG and IgM antibodies can bind C1q via Fc region and therefore trigger complement activation.
BUT… antigen needs to initate cross linking of antibodies.
What part of antigen does antibody recognise?
Antibody only recognises small portion, the epitope, of an antigen which typically are large macromolecules…
The antibody binds the epitope in the grooves between CDR’s of heavy and light chains in the Fab region…
Most macromolecules activate many different B cells, producing a polyclonal antibody response. However, activating only a single B cell causes a monoclonal antibody response.
How can antibodies be used as tools?
HLA testing - antibodies can be used to determine blood groups.
As a diagnostic tool - antibody detection in patient sera can indicate disease.
Treatment of disease - vaccinations, or inject antibodies for short-term protection against disease = holiday vaccinations….
How do vaccinations work?
The immune system is challenged with a non-pathogenic form of antigen which is avirulent/heat killed.
In order to confer immunity to future pathogenic challenge.
Usually requires adjuvants to stimulate innate immune system…
What are monoclonal antibodies?
How are they made?
Identical antibodies produced from a single B cell clone.
Tend to be chimeric - made from mice.
Mice immunised with antigen of interest.
B cells harvested from spleen and B cells fused with myeloma cells (eternal, as a single B cell would die quite quickly)…. The resulting hybridomas are grown on selection mediate.
