imm 4 Flashcards
Name the 3 mechanisms our body implements to increase antibody diversity
1, Germ line diversity 2. Combinatorial diversity3. Junctional diversity
What is are antibody repertoire a part of?
Part of our adaptive immune system
What does The random generation of coding joints generate?
Lots of diversity
How many progenitor B cells will not produce a functional immunoglobulin?
2/3| ie only 1/3 will be functional
What do we need to make sure the B cells produces?
Only one type of antigen receptor
Why do we need to make sure B cells only produce one type of antigen receptor?
So that we can screen out any self reactive b cells more efficiently
How do we regulate and make sure each B cells only produce one type of antigen receptor?
Rearrangement is regulated by allelic exclusion.
What does the generation of immunoglobulin diversity all generate?
Some self reactive B cells
Where does a non self reaction B cell go?
It migrates to the periphery to form a mature b cell
Where does a multivalent self molecule go?
It undergoes clonal deletion of receptor editing (this is more likely)ORUndergoes receptor editing generating a non auto reactive mature b cell (This option Is rare)
Where does a soluble self molecule go?
It migrates to the periphery to from an anergic B cell
What are the 4 functions of antibodies?
- Neutralization2. Opsonization3. Activation of Complement4. Binding to TRIM21 intracellularly, targeting virus to proteosome
What does the function of different antibodies classes determined by?
Determined by the Fc region
What are the difference between different antibody classes ?
Different Ig classes have different functions and distributionDetermined by C regions (Fc).
When B cells first reach maturity which class of antibodies do they ALL produce?
IgM and IgD
Name the 5 different classes of antibodies?
IgM (μ)
IgD (δ)
IgG (γ)
IgE (ε)
IgA (α)
What makes the different classes of antibodies discrete from one another?
Each class has a different constant regions on their heavy chain
When does class changing of antibodies occur?
During clonal expansion when the B cells are proliferating in response to a specific antigen
Can one B cell clone produce different classes of antibodies?
YES and they will all have the same specificity to the pathogen
name the 2 forms all antibody classes can be produced as
Secretes form| Transmembrane form
How are both the secreted and transmembrane form transcribed?
They are both transcribed in the same primary RNA transcript
What determines whether the antibody will be produced in the secreted form of the transmembrane form?
Alternative splicing of carboxy exons determines use of either the hydrophilic secretory sequence or hydrophobic transmembrane domain (membrane bound).
When is IgM produced?
It is procured first prior to class switch
In what form is IgM secreted?
Secreted form is a pentamer
What does having a pentamer structure increase
It increases avidity BUT decreases affinity
Where is IgM present?
In the blood stream but not in tissues (as it is very big)
Describe the structure of IgM
Has 3 C regions but not hinge
Describe the structure of IgM
Has 3 exons in its Constant region but no hinge
What functions can IgM carry out?
- Neutralisation 2. Opsonisation 3. Can activate the compliment system
When is the IgD produced?
It is co expressed with IgM on B cells
Do we know what IgD do?
We don’t know as it doesn’t perform any of the antibody functions well
What can IgG, IgE, IgA all do?
They can all readily diffuse from blood to tissues
Where is IgG found?
It is the principal antibody found in our blood and extracellular fluids
What functions can IgG perform?
- Neutralisation 2. Opsonisation 3. Sensitisation for killing by natural killer cells 4. Very good at activating the compliment system
What does IgE defend us against?
Multicellular parasites functions in allergy.
What does IgE antibodies bind to?
Mast cells which can induce pathogen expulsion by coughing, sneezing, vomiting
What function can IgE perform?
Sensitisation of mast cells
What is the main role of IgA?
IgA is the principal antibody in secretions| It is found In our epithelial cells
Which antibody is responsible for allergies?
IgE
What functions can IgA do?
Neutralisation| Opsonisation
What does IgG exist in?
Exists as subclasses
Where are IgG found?
In the blood stream and tissues
What does IgG do?
It strongly activates compliment It is an effective neutraliserIts an efficient opsonin and binds to phagocytes
What happens when IgG binds to phagocytes?
Triggers engulfment
How can IgGs cross the placental barrier?
Via binding to neonatal FcR (FcRn)
What can IgG do when it crosses the placental barrier?
and give passive maternal derived protection to newborns.
What do we describe babies as?
they are not fully immunocompetent
What is passive immunity
When you have gained immunity from a different source| You have not produced the antibodies yourself
Give some examples of passive immunity
Mothers to babies| Anti toxins when people have been poisoned
Where is IgA secreted?
Secreted at mucosal surfaces in the gut and respiratory tract Can be secreted into breastmilk
What is the major function of IgA?
Major function as neutralising antibody.
In what form is IgA usually found?
Monomeric or dimeric form
Where can the dimeric form be transported?
Can cross the epithelia to our outside mucosal surfaces
How can the dimeric form be transported across the epithelia?
We have a receptor called polymeric Ig receptor that binds to the dimeric form of IgA IgA is then transported across the epithelia cells to the outside apical surfaces of the mucosaThe receptor is then cleaved and this process releases the IgA
Where does clonal expansion of B cells to fight off infection occur?
Occurs in the secondary lymphoid organs
What steps have B cells taken before they can be activated to produce antibodies?
- B cells at immature stage 2. B cells produce their specific antigen receptor 3. Made sure B cell isn’t reactive 4. Made sure each B cell is only producing one the of antigen receptor (allelic exclusion)5. Cell exit bone marrow and go our to secondary lymphoid organs
What are the 2 different types of antigens?
- T independent antigens| 2. T dependent antiegns
How do T independent antigens activate B cells
Can activate B cells alone, without T cell help.| They engage a lot of different B cell receptors and other receptors at the same type (Like toll receptors)
What type of antigens are produced by T independent antigens?
Low affinity just IgM antibodies made
Most antigens are os what type?
They are T dependent antiegns
How do T dependent antigens activate B cells
to T dependant antigens can only activate B cells with the help of T cells To initiate a response they must be a sufficient number of T cells helping to provide 2 signals
What are the 2 signals that the B cell needs to receive to be able to be activated?
- When the antigen binds to the B cell a signal is sent| 2. When a helper T cell binds to the epitome presented by an MHC molecule it sends a signal to the B cell
What signal does the helper T cell send to the B cell when it recognises an epitope on an MHC molecule?
It sends:CD40/ CD40LCytokines IL-4, IL-5, IL-6To the B cell
What do T cells help to do?
triggers B cells to proliferate and differentiate.
What type do epitopes do B cells tend to recognise?
Discontinuous Epitopes| Epitopes produced by 3d structures
What type do epitopes do T cells tend to recognise?
Continuous EpitopesLittle bits of peptides that have been chopped up Can only recognise Epitopes on a MHC molecule
What must the T cell respond to to activate the B cell?
T cell must respond to the same antigen as the B cell.Though not necessarily the same epitope,They must be physically associated = linked recognition.
What is the T cell that is specific and primed for the same antigen called?
Cognate T cells
What happens once a B cell is activated?
It undergoes rounds of proliferation (clonal expansion)| It also undergoes the final stages of differentiation
What can B cells differentiate to in their final stage?
Either:1. Effector cells2. Memory cells
What do effector cells do?
They are plasma cells that secrete antibodies
What do memory cells leave us with?
Long lasting memory incase of future re infection
Where is the final differentiation of B cells occurring?
In the B cell follicle in secondary lymphoid organs.
What do B cells produce very earl yon when they discovery a pathogen?
They produce primary focus
What is a primary focus
It is when a B cell produces a very small amount of antibodies very early on in the infection
Where doe the mass germination of antibodies occur?
In the germinal centre
What is produced by the germinal centre of a B cell?
A more effective humoral response occurs This is where good quality antibodies are produces This response is still present for 3-4 weeks post infection
Where are stromal cells found?
In the lymph nodes
What do stromal cells secrete
They express chemokines which signal for the B cells to remain in the follicle
What happens to plasma cells following clonal expansion and final differentiation?
They move into the medullary cords| Some plasma cells migrate to the bone marrow
Over time what happens to then cells ability to recognise the antigen overtime in the adaptive response?
It improves over time in the germinal centre
What is happening in our general centre reaction?
While the B cells are proliferating they need to replicate their DNA So when their DNA is being replicated mutations in Ig Variable regions occur Point mutations occur one every 1x 10^3 base pairs in our B cells
What is the mutation rate in our normal cells compared our B cells?
Normal cells= 1x 10^10| B cells= 1x 10^3
What Is the increased never of point mutations in B cells called?
Somatic hypermutation
What is the mutating of the immunoglobulin variable region do and what is this process called?
Can lead to possible changes in affinity| This is called affinity maturation
What triggers the increased number of mutations in B cells?
Triggered by the enzyme AID (activation induced cytidine deaminase).
What does AID do?
This enzyme is able to attack the DNA of our immunoglobulin genes as they are being transcribed The AID will swap some of our cytosine bases for uracil bases
What happens when the body resales the AID enzyme has swapped a cytosine for a uracil in our immunoglobulin gene?
It triggers our DNA repair machine to correct the alterationsBUT the machinery sometimes doesn’t correct the gene accurately leading to a mutation
Why does somatic hypermutation only occur in B cells?
Because they are the only cell that have the AID enzyme
How is improved affinity an advantage for B cells?
It means the B cell can more strongly pull the antigen towards itThis is an advantage as their aren’t that many antigens so the B cells need to compete for antigens so that they can proliferate
What do mutated B cells compete for?
They compete to bind to antigens provided by follicular dendritic cells.
What happens if a B cell doesn’t receive a signal from a B cell?
B cells apoptose.
What do signals from T cells allow theB cells to do?
allows for survival, proliferation and differentiation of B cells
What are the 2 paths an antigen activated B cell can take?
- Somatic hypermutation| 2. Class switching
What is class switching?
Within the same clone, different antibody classes can be produced.
What type of event is class switching?
A recombination event
What do different classes of antibodies all produced by the same clone have in common?
They’ll have the same antigen specificity| They’ll have the same variable region
What happens when a mutated gene is transcribed?
It will go immediately from the variable region to the IgE constant region
What does a natural immune response produce?
a heterogeneous mix of antibodies.
Whta is hybridoma?
An immortal “cell line” which produces lots of a single antibody.
How are antibodies used outside are bodies?
ResearchDiagnostics of diseasesTreatment tools
How do scientists generate monoclonal antibodies
- Inoculate a mouse with antigen2. Fuse splenic cells with immortal “B cell line” with polyethylene glycol3. Eliminate unfused cells4. Separate individual cells 5. Grow in culture to produce a clone.6. This produces cells combining the characteristics of producing antigen specific antibody with ability to grow in culture with unlimited lifespan.
What are monoclonal antibodies used for?
Immunotherapy
What are monoclonal antibodies?
They are cells combining the characteristics of producing antigen specific antibody with ability to grow in culture with unlimited lifespan.
What are myeloma cells?
Plasma cells that are immortal
What are some immunological techniques based on monoclonal antibodies?
- ELISA2. SDS PAGE / western blotting/ immunoblotting3. Immunochemistry/ immunocytochemistry4. Flow cytometry and FACS
What does ELISA stand for?
Enzyme-linked Immunosorbent Assay
What does ELISA help us identify?
Identifies specific protein and permits determination of| their concentration in mixtures / physiological fluids.
What is ELISA used in?
diagnostic tests.
What does indirect ELISA detect?
Detects the presence of specific antibodies in body fluids
How do you carry out indirect ELISA?
- Have an antigen coated wall in a beaker2. Wash3. Add sample which includes 1st antibody4. Specific antibodies will bind to the antigen 5. Wash6. Add enzyme that is complementary to the 1st antibody with a 2nd antibody attached to it 7. Enzyme linked 2nd antibody will bind to the 1st antibody8. Wash9. Add a substrate complimentary to the enzyme10. Substrate is converted by the enzyme into a coloured product if the correct antibody was in the sample
What is the rate of colour formation in an indirect ELISA proportional to?
Proportional to the amount of specific antibody present
What does direct ELISA detect?
Detects the the presence of a particular protein in body fluid.
How do you carry out direct ELISA?
- Have a monoclonal antibody coated wall in a beaker2. Wash3. Add sample which includes 1st antigen4. Specific antigen will bind to the antibody5. Wash6. Add a second monoclonal antibody linked ti an enzyme7. The monoclonal antibody enzyme complex will bind to immobilised antigen 8. Wash9. Add a substrate complimentary to the enzyme10. Substrate is converted by the enzyme into a coloured product if the correct antibody was in the sample
What is the rate of colour formation in an direct ELISA proportional to?
Proportional to the amount of specific antigen resent
What does a SDS PAGE/ Western blotting / Immunoblotting identify?
Identifies specific proteins and provides information on molecular weight and quantity.
What does SDS PAGE stand for?
SDS polyacrylamide gel electrophoresis (PAGE)
What is Western blotting / Immunoblotting routinely used as?
a research method
What can the Western blotting / Immunoblotting technique be used to analyse?
samples that contain a mixture of proteins
Describe the steps involved in Western blotting / Immunoblotting
- Samples must first be separated by SDS gel electrophoresis
How are samples separated by SDS gel electrophoresis?
- The samples are mixed with SDS 2. Samples and SDS are loaded in lanes on the polyacrylamide gel3. Gel is surrounded by buffer containinG electrolytes 5. Voltage applies across the equipment 6. Causes SDS coated proteins to move towards the positive electrodethey migrate through the gel matrix at varying speed, their mobility through the gel determined by their size (molecular weight) and overall charge7. proteins are then transferred sideways 8. membrane is then “probed” with a specific antibody which will only bind to the band composed of the antigen which the specific antibody binds to. antibodies are labelled so we can see which antibody has bound
What is SDS?
A negatively charged molecule
Why then carrying out SDS gel electrophoresis do the proteins have to be moved after they ave been separated out?
The gel is flimsy and not stable and can dry out| They are transferred sideways so their positions respective to each other remain intact onto an impermeable membrane
What is the intact membrane the proteins are transferred onto after separating made of?
nitrocellulose
What is a common label we use to identify which antibody has bonded when carrying out a western blotting test?
Horseradish peroxidase enzyme (hrp) which cleaves a chemiluminescent substrate to give off lightThe position of both antibody and protein of interest can then be detected on photographic film which is laid on top of the membrane to be exposed before then being developed.
Which out of all the immunological techniques the most commonly used diagnostic tool in pathology?
Immunohistochemistry / Immunocytochemistry
What do the Immunohistochemistry / Immunocytochemistry techniques identify?
identifies specific proteins in tissue slices (which can be biopsies) and permits their localisation by microscopy.
What is the main difference between Immunohistochemistry and Immunocytochemistry ?
Immunocytochemistry applies the same principles but at the ultrastructural level, revealing the location of specific proteins (antigens) within cells using a transmission electron microscope (TEM).
What molecules are used in Immunohistochemistry and Immunocytochemistry
antibodies are used to determine the presence and distribution of antigens in tissues and cells.
Describe the process of Immunohistochemistry
- Thin slices of tissues cut from the sample after embedding it in wax or resin 2. Antibodies that are specific for the protein of interest are applied to the surface of the tissue3. Wash4. only the antibodies bound to their specific antigen (the molecule of interest) will remain. 5. A second antibody is then added (engineered to recognise the first type of antibody used) also carries a fluorescent marker on its Fc portion6. location of the second immunoglobulin can then be seen in the fluorescent light microscope7. Wherever fluorescence is observed, this means that second antibody has recognised the first, which in turn has recognised and bound to the specific antigen.
How does Immunocytochemistry work?
- works on the same principles as Immunohistochemistry a tiny gold ball is attached to the second immunoglobulin instead of the fluorescent marker2. position of the second immunoglobulin can be seen in the transmission electron microscope and the ultrastructural localisation of the antigen is revealed.
How big is the gold ball used in the Immunocytochemistry technique?
only 15 nm in diameter
Why is a gold ball used in the Immunocytochemistry technique?
Because gold is electron-dense, the position of the second immunoglobulin can be seen in the transmission electron microscope and the ultrastructural localisation of the antigen is revealed.
What does flow cytometry analyse?
- It allows for the analysis of different populations of cells within a mixture2. It also allows for the separation of different populations following this analysis3. can also show changes in the level of expression of proteins in populations of cells.4. allows for the quantitation of the proportions of different cells in samples of tissue and blood.
How is flow cytometry carried out?
- The cells are diluted2. Cells fired past a laser one droplet at a time 3. Each droplet contains just one cell. 4. The laser excites the fluorescent tag on the monoclonal antibody and the emitted light at this particular wavelength is detected.
How can flow cytometry allow for the separation of different populations following analysis?
- Cells identified as having certain characteristics due to their protein expression detected by antibodies are given a charge. 2. The charged droplets are deflected by charged metal plates and separated into different collection tubes.
What is the brightness of fluorescence proportional to in flow cytometry?
to amount of antibody and therefore amount of specific protein expressed by the cell.
Other than diagnostic tools what can monoclonal antibodies also be used for?
therapeutic uses
How can monoclonal antibodies have therapeutic uses?
- Can give Autoimmunity2. Help treat cancer3. Used in transplantation
How can monoclonal antibodies offer autoimmunity?
They can bind to target molecules and block their interactions with other molecules
Give an example of how monoclonal antibodies can block a reaction
monoclonal antibodies can block inflammatory cytokines binding to their cellular receptors of block cell adhesion molecules on leukocytes interacting with vascular endothelial cellsThis inhibits the migration of leukocytes out of the blood stream and into tissues thus reducing inflammatory / autoimmune damage against our own tissues
How are monoclonal antibodies used in transplantation?
Block immune response to prevent graft rejection following transplantation.
Name the fragment used to help treat cancer
F(ab)’2
What is the F(ab)’2 fragment useful in?
might be particularly useful in cancer treatment as they can bind and block interactions without eliciting responses via the Fc region.Antibodies can be engineered with F(ab)2 fragments that will recognise specific targets such as tumour antigens. These fragments can be fused to a toxin or radioactive isotope, the specific binding of the antibody will deliver the toxic drug to the tumour cells allowing it to be targeted where needed.
Examples of passive cancer immunotherapy?
Herceptin/ Avastin
Cancer vaccines: HPV vaccine Gardasil
