Greg - Antibody Structure Flashcards
What are antibodies?
Glycoprotein molecules
Produced by plasma cells
They bind to antigenic targets
They are also called immunoglobubins/gamma globulins
What are antibodies?
Glycoprotein molecules
Produced by plasma cells
They bind to antigenic targets
They are also called immunoglobulins/gamma globulins
What is a glycoprotein?
A protein with a carbohydrate post translation modification
How can antibodies neutralise/block entry of pathogens?
Antibody against viral surface proteins block entry to human cell by binding to the protein - prevents it from binding to host’s cell
= neutralisation
Explain agglutination/precipitation
(3)
Antibodies can clump antigens together into an immune complex for removal
Agglutination = insoluble target
Precipitation = soluble target
What effector functions do antibodies have?
(3)
Activate complement
Aid phagocytosis via opsonisation
Mediate antibody mediated cellular cytotoxicity by NKCs
List the functions of antibodies
(5)
Block entry/neutralisation of pathogen
Agglutination/precipitation
Complement activation
Opsonisation
Aids NKCs
What is a myeloma?
Cancer of plasma cells
i.e. one plasma cell keeps producing it’s antibody continuously
Why were antibodies hard to study?
Due to their heterogenous nature i.e. they are all different
What research allowed us to study antibodies?
Studies of myelomas as these cells produce a monoclonal antibody population which is identical and can therefore be separated and studied
How are the different parts of an antibody examined?
(3)
We cleave the antibody into parts
Papain cleaves the antibody at the middle and creates three fragments - we can then make crystals of Fc because the Fc molecule is the same in every antibody (uniform) -> can examine Fc now
Pepsin cleaves antibody at hinge - gives us a F(ab’)2 with the hinge and a partial Fc fragment (pFc) - research can now be carried out on the Fab
What joins the light and heavy chains of an antibody?
Disulphide bonds between cysteine residues
What does mercaptoethanol do to antibodies?
(3)
This chemical destroys the disulfide bridges between the light and heavy chains
The four chains fall apart
This gives us 2 heavy chains and 2 light chains which can be examined further
What are the two types of disulphide bonds?
Inter-chains
Intra-chains
List the variable and constant regions
VL & CL
VH & CH
List the parts of an antibody
(7)
Fab region
Fc region
Intra-chain
Inter-chain
Variable region
Hinge region
Constant region
What is the hinge region?
(3)
Part of the molecule that can move
It gives the antibody some flexibility
It makes binding to antigens easier
The functions of the antibody molecule are spatially segregated, what does this mean?
Some functions of antibodies relate only to the Fab region while others relate only to the Fc region
What function is the Fab site responsible for?
Antigen binding
What functions is the Fc site responsible for?
(3)
Complement binding site
Binding to Fc receptors
Placental transfer
Write about the antibody-antigen reaction?
(2)
A non-covalent bond
Reversible reaction - i.e. antigen has affinity for antibody but can be pulled apart
What is affinity in terms of antigens?
The strength of the reaction between a single antigenic determinant and a single combining site
What is avidity in terms of antigens?
The overall strength of binding between antigen with many determinants and multivalent antibodies
The total attractive force = the total affinity
What type of antibody is first made by plasma cells?
IgM
What is specificity?
The ability of an antibody binding site to react with only one antigenic deteminant
What is cross-reactivity?
The ability of an individual antibody binding site to react with more than one antigenic determinant
What is an epitope?
Regions of an antigen recognized by B cell antibody and T cell receptor
What is the difference between T cell dependant antibody production and T cell independent antibody production?
T-dependent results in high affinity antibodies, memory B cells, longer lived plasma cells, and isotype switching of antibodies (i.e. switch from IgM)
T-independent results in low affinity antibodies, short lived plasma cells and mainly IgM antibodies
What determines the classes or isotypes of antibodies?
Their heavy chain amino acid sequences
List the five antibody isotypes and their heavy chains
IgG - Gamma heavy chain
IgA - alpha heavy chain
IgM - mu heavy chain
IgD - delta heavy chain
IgE - epsilon heavy chain
Where is IgD found?
The surface of B cell receptors where it acts as a receptor
Where is IgA found?
The IgA monomer is found in blood but the dimer is found on cell surfaces
What determines amino acid subclasses?
Heavy chain amino acid sequences
How many subclasses does IgG have?
4
What are the four main igG subclasses?
IgG1 -> gamma 1 heavy chain
IgG2 -> gamma 2 heavy chain
IgG3 -> gamma 3 heavy chain
IgG4 -> gamma 4 heavy chain
How many subclasses does IgA have?
2
What are the subclasses for IgA?
IgA1 -> alpha 1 heavy chain
IgA2 -> alpha 2 heavy chain
How are the types of light chains determined?
They are determined by sequence
Either kappa (K) or lambda
How many kappa subtypes are there?
None
How many lambda subtypes are there?
1-4 subtypes
How would IgA , subtype 1, lambda 2 be written?
IgA1(lamda2)
List the subtypes of IgG in order of population?
IgG1
IgG2
IgG3 = IgG4
How much of the gamma globulins in serum are IgG?
80%
What is the only class of antibody that can cross the placenta?
IgG2
What subtypes of IgG can fix complement?
IgG3 and IgG4
Which IgG subtypes act as opsonins and how?
IgG2 and IgG3 act as opsonins
They can be bound by the Fcgamma receptor
What is responsible for the long serum half-life of IgG?
FcRn
What is FcRn?
The neonatal Fc receptor for IgG2
Describe the structure of IgA in secretions
A dimer with a J-chain and secretory component (hence why it’s found in secretions)
Hoe much of the gamma globulins in serum are IgA
Between 10 and 15 percent
List some places IgA can be found
(3)
Saliva
Breast milk
Tears
What is the J-chain of IgA similar to?
Similar to the J-chain of IgM
Write a note on the structure of IgM
(5)
Pentameric in serum but a monomer on B-cell surface
In pentamer - all light and heavy chains are identical
Extra domain (CH4)
J chain
Valency of 10
What is the extra domain in IgM called?
CH4
Write about the properties of IgM
(5)
3rd highest concentration in serum
First Ig made by foetus and B cells
Best complement fixer
Agglutination
IgMFc receptor -> binds Fc receptors on B cells
Anchors in B cell membrane but must partner with other proteins on the surface to act as a receptor and activate the cell
Write about IgE structure
(3)
Monomer in structure
Extra CH4 domain
Only survives for 2 and a half days
Write about the properties of IgE
(6)
Very potent activity
Very low serum concentrations
Hypersensitivity reactions
Hay fever, asthma hives etc
Binds Fce receptors on basophils and mast cells (degranulation)
Anti-parasitic defence
Explain what happens in an allergic reaction
IgE has been produced against a harmless environmental molecule
When you first come across the allergen you won’t have a reaction but IgE will be made and it will bind to Fc epsilon receptors on mast cells -> next time you meet the allergen the IgE will cross bind and send an activation into the mast cell which releases histamine
Write about the structure and properties of IgD
(5)
Monomer
Tail piece to be anchored in B cell membrane along with IgM
2nd lowest concentration in blood
Most ancient antibody class
We though it had no function but now see it is produced in the lower respiratory tract during infection
What do most cells express that act as receptors for the Fc portions of antbiodies
Glycoprotin
What do most cells express that act as receptors for the Fc portions of antbiodies
Glycoprotiens
Why are Fc receptors needed on cells?
Opsonisation
Antibody-dependent cellular cytotoxicity
Cell activation
Cell differentiation
What is the requirement to be part of the immunoglobulin superfamily
Must have at least one domain homologous to other members
e.g. Immunoglobulin-fold domain structure
Why might specificity be measured in the lab?
Chemical methods could not distinguish between similar proteins (e.g. Human vs. pig insulin) but antibodies can do this absolutely
Why might avidity be used in the lab?
Affinity dictates the effectiveness of an antibody. For example, the higher the affinity of an antibody, the less you might need for a procedure
Under what conditions might patient antibodies be measured?
Inflammation/infection
Cancer
Allergy
Parasitic infection
Immunodeficiency
Autoimmunity
Why might antibodies be measured during inflammation?
Due to increase in antibody response due to immune responses
Why might antibodies be measured during cancer?
Cancer of B cells result in monoclonal antibody type
Why might antibodies be measured during allergic reaction
Increase in IgE
Increase in specific IgE
Why might antibodies be measured during parasitic infection
Increased total IgE
Why might antibodies be measured in immunodeficiency
Can the patient make normal levels of all antibody classes?
Why might antibodies be measured during autoimmunity
What antibody is being produced against self
What antibodies are used in the lab?
Anti-sera = antibodies produced in animals - a mixture (cheap)
Monoclonal = antibodies are identical to each other and bind the exact same epitope
How are anti-sera antibodies produced?
(5)
We take antigen A and vaccinate a rabbit using it
The animal produces different types of antibodies against it
We take antibodies from rabbit
Purify antibodies
We now have a polyclonal mixture of antibodies which can be used to detect Antigen A
How do anti-immunoglobulin antibodies worj
Antibodies against antibodies
Human antibody injected into animal -> provokes an immune response as it is seen as an antigen
Antibodies produced can be used to detect antibody
How is anti sera used?
(5)
ELISA test
Antigen bound to well
Human antibody (if present) binds to antigen
Anti human antibody is added and binds to antibody
Anti-human antibody is marked with a horse radish peroxidase which creates a colour
What is a monoclonal antibody?
An artificially created antibody from a single cell clone - binds same epitope
Who immortalised plasma cells?
Georges Kohler
Cesar Milstein
How were plasma cells immortalised?
Plasma cell were fused with myeloma cell which resulted in immortal cell producing monoclonal antibodies
Describe the process of making monoclonal antibodies
Inject animal with antigen (multiple epitopes)
Animal makes antibodies - for each epitope
Kill the animal and take its spleen
Take plasma cell of spleen making the antibody of interest
Immortalise plasma cells by fusing them with myeloma cells
Dilute wells until we have a single cell per well (you have created an eternal source of antibodies for a specific antigen)
Explain the relationship between HGPRT and spleen plasma cells
(3)
Myeloma cells are engineered so that they are deficient in HGPRT
Without HGPRT the myeloma cells will die
The spleen plasma cells gain immortality by providing the myeloma cell with HGPRT
What is HGPRT?
Hypoxanthine-guanine phosphoribosyl transferase
Enzyme involved in purine synthesis
