Lecture 10; Humoral immunity and Antibodies; Flashcards
What is an antibody?
Soluble, globular proteins produced by B lymphocytes and are secreted into the serum, lymph or at mucosal surfaces.
Describe B cell antibody specificity;
Every B-cell is antigen specific, receptor is the default molecule produced by b-lymphocytes (IgM, explains later)
What do antigens do?
Antibodies confer resistance to pathogens by binding to antigenic epitopes on the surface of the pathogen, initiating a cascade of events which results in the death and clearance of that pathogen.
What is the normal concentration of antibodies in the serum?
Antibodies are found in the gamma globulin fraction of serum and a normal individual will have 20-30mg/ml of serum immunoglobulin
What are antibodies formed by?
Repeats of a basic and highly sucessful protein ; Ig domain
12.5kD in size.
Write some notes on the Ig domain;
- Building blocks of ABs and TcR, also many other proteins
- Always bivalent (2 binding sites)
- Lengths of AB gene dependant
- High affinity Fc receptors for each isotype of leukocytes (Fc on AB binds leukocytes)
What are the basic features of Ig sequence?
- 3 Highly conserved regions called framework (FR1,2,3)
- 3 regions of extreme AA variability called hyper variable regions (HV)
- 2 Completely conserved Cysteine residues located in the FR1 and Fr3 regions. These form intrachain disulphide bonds that gives the Ig domain great stability
Describe the classic Ig domain fold;
Beta-barrel Ig structure.
Ig domain consists of two anti-parrallel beta sheets made up of seven constant or nine variable b-strands.
A single disulphide bonds stabilises the b-barrel
The strands are connected by loops, these are not constrained by the b-barrel structure and therefore have extreme AA diversity without compromising overall stability
These loops form the antigen binding site
Describe the AB light chain;
- 25kD
- One variable Ig domain (V) = 9 strands
- One constant Ig domain (C) = 7 strands
Constant region is invariable beween AB
2 Ig subunits
Describe the AB Heavy chain:
- 50-75kD
- One variable Ig domain (paris with LC V region)
- 3-5 constant Ig domains
- Hinge region where heavy chains join one another (there can be 1-7 disulphide bonds in this region depending on class)
Five different heavy chain classes IgGAMED
What does the antibody molecule consist of?
- 2 heavy chains
- 2 light chains
(generic forumla; H2L2 but IgA and IgM exist as a dimer and pentameric form respectively)
H chains joined at hinge by 1-7 disulphide bonds depending on class
L chains joined to H via interchain disulphide bonds
Can be divided into Fab and Fc regions
2 binding sites are identical
Look at diagram to further understand
How were Fab and Fc discovered?
By using proteases to cleave antibodies into functional domains
Pepsin and Papain
What is the most abundant AB?
IgM
Write some notes on IgG;
- Placental immunity (only one to cross)
- Anti-toxin
- Anti-bacterial
- Opsonin
- Initiaties complement
- Blocks receptor binding
- High affinity and specificity
Soluble
Write some notes on IgM;
- Membrane bound and soluble
- IgM is the default B cell membrane receptor
- Default isotype made by niave b cells
- Low affinity, high avidity due to pentamer shape
- Opsonin
- Initiates complement
First line serum defence
Write some notes on IgA;
- Produced in serum and at mucousal surfaces i.e breast milk
- Primary protection at mucosal surfaces
- Block adhesion and invasion
Write some notes on IgD;
- Membrane and soluble
- Unknown role
- B cells with mIgD but not mIgM resist central tolerance
Write some notes on IgE;
- Defence against large pathogens that cant be engulfed
- Potent activator of mast cells via FceR (high affinity)
- Prime activator of type one hypersensitivity
- Causes anaphylactic shock when it triggers systemic mast cell response
- Low Conc.
- Low dose secretion at mucousal surfaces
Describe Fc receptor expression;
Each Ig isotype has its own unique Fc receptor expressed on many different cells. Role is to connect antibody with cellular mechanism to trigger a much larger response
What may Fc receptors trigger?
- Phagocytosis
2. Release of mediators and cyototoxins from mast cells, basophils, esinophils and graunlocytes
Do Fc receptors bind with high affinity?
Fc receptors are generally low affinity and requires receptor cross linking by complex Ag/AB
Except FceR (IgE)
What happens when IgE binds FceR?
Causes mast cell degranulation and release of histamine causing atopic allergy, systemic = anaphylatic shock
What is an antigen?
Any molecular surface, the region to which the antibody binds is the epitope.
Some antigens have many epitopes and bacteria have millions of different epitopes
What is an epitope?
The complete region of an antigen encompassed by the antibody binding site
Linear or conformational
Do AB display cross reactivity?
Every antibody is cross reactive to some degree
Every antibody can bind more than one epitope
Affinity in which it does this is more important (describes fit)
Describe the antigen binding site on Ig molecules;
AA variability is confined to a discrete HV region called complementarity Determining Regions (CDR)
Describe the CDRs;
CDRs are 3 hypervariable loops in the VH and VL domains joining the strands.
There are 6 loops all together.
What do two binding sites mean?
- Antigens are bivalent as they have two identical binding sites
What is complementarity?
An antibody can interact with anything so long as there is complementarity between the AB and Ag surface
What is a Hapten?
A region of an epitope that contributes specifically to the AgAb interaction
Define Affinity;
The measure of the thermodynamic desire to bind and stay bound to its antigen.
Sum of attractive forces - repulsive forces
Four main non-covalent forces
What are the non-covalent forces regulating antibody interactions?
Electrostatic forces
H bonds
Van der waals
Hydrophobic interactions
How do we define how well an antibody binds to its antigen?
Simple first order reactions
Binding is directly proportional to the concentration of both reactants.
What is the unique binding constant?
Ratio of bound to free antibody after antibodies had infinite time to react
= Kd (mol/L) (affinity)
Kass = (Kon/Koff) Kdiss= (koff/Kon)
Ab + Ag -> AbAg (Kon)
AbAg -> Ab + Ag (koff)
What is avidity?
Measure of stickiness of an antibody
more relevant measure of antibody function
Why id avidity a better measure?
It accounts for the multiple binding sites.
Therefore an Ab with low affinity for one epitope can bind to an antigen that has many epitopes (mutliple bindings) therefore can have high avidity
Describe the relation of antigen class and affinity / avidity;
Fab - prefers affinity
IgG Soluble - affinity
IgG fixed - avidity
IgM fixed - avidity
Describe affinity and time relationship;
As time to an antigen exposure increases, the affinity of antibodies produced increases to that antigen.
Memory b cells produce very high affinity antibodies
The lifespan of antibodies also increases
Describe affinity maturation;
B cells start by producing IgM (high avidity, low affinity) and over time this switches to IgG with high affinity (as dividing b cells continue being exposed and selected)
THis occurs because of somatic hypermutation (point mutations in the hypervariable regions), this stronger affinity b cells are selected by lower antigens until eventually only very strong affinity IgG are being produced (memory cells that produce high affinity IgG for that epitope)
What are antibody functions?
- Antibody Dependant Cellular Cytotoxicity
- Opsonisation
- Blocking attatchment
- Neutralisation
- Agglutination
- Immobilisation
Describe ADCC;
- Results from Ig Fc receptor binding antibodies bound to antigens
- NK cells, Macrophages, Neutrophils, eosinophils, t cells
Write some notes on opsonisation;
Ab combines with complement to coat targets for phagocytosis
Write some notes on blocking attachment;
Stops antigen binding to mucousal surface for epithelial entry
Write some notes on neutralisation;
Blocks that action of toxins by binding to the active site of receptor binding site.
Mainly IgG b/c high affinity
Write some notes on agglutination;
Clumps bacteria together, stopping their movement.
I.e IgM
Write notes on immobilisation;
Impeding movement i.e Ab binding to bacterial flagellum
