Lecture 7 - Antigens and Antibodies Flashcards
What do B cells use to detect antigens?
Membrane-bound antibodies
What is a plasma cell?
An activated B cell.
Secretes antibodies
True of False: B cells have multiple antigen receptor types on their cell membrane.
FALSE
B cells are specific to only one antigen.
What happens if a B cell encounters its antigen?
Clonal expansion
Some divide into plasma cells
Some divide into long-lived memory cells
What is a BCR?
B cell receptor
Membrane-bound antibodies that are specific to a single epitope
What happens if a B cell never encounters its antigen?
Circulates for 3-4 months, then dies
True or False: The antigen secreted by a B cell recognizes the same epitope as the BCR
TRUE
What differentiates a BCR from a secreted antibody?
BCRs have a transmembrane portion to keep them bound in the membrane.
Antibodies do not.
Identify the following regions on this IgG molecule
- Variable region
- Antigen binding sites
- Heavy chain
- Hinge region
- Light chain
In total, how many peptide chains are in an IgG molecule?
4
2 heavy chains, 2 light chains
What is meant by the term “domain” or “homology region” on an antibody?
A 110 amino acid segment that folds into a loop and is held in place with a disulfide bond.
Essentially the building blocks of the antibody molecule.
How many amino acids are in a single antibody domain?
110
Which 2 domains make up the antigen binding site?
VH and VL domains
What composes the Fab portion of the antibody?
Composed of VH and CH1, VL and CL domains.
What does Fab stand for?
“Fragment antigen binding”
This is the portion of the antibody that binds antigen.
What does Fc stand for?
“Fragment crystallizable”
The tail of the antibody that binds with complement and leukocytes.
What is the Fc portion of the antibody composed of?
CH2 domain and onward
In terms of antibody domains, what does C stand for?
“Constant”
This domain does not vary within the class (isotype) of antibody. I.e. All IgM will have the same C domain.
Gives antibody its biological functions like fixing complement and binding to mast cell receptors.
In terms of antibody domains, what does V stand for?
“Variable”
Varies between antibodies. Gives the antibody the ability to bind to a specific antigen.
What is the function of the antibody’s hinge region?
Gives antibody flexibility, like a joint
What is an epitope?
The portion of a molecule to which an antibody binds.
Note that an epitope does not have to be a continuous chain of atoms on a molecule.
Also note that one molecule can have many epitopes.
True or False: Antibodies bind to antigens covalently, meaning that they will never be able to separate.
FALSE
Ag/Ab binding is always non-covalent. How long they stay bound is based on how attracted the molecules are to each other.
What 4 forces promote binding of antigen to antibody?
Electrostatic
Hydrogen bonding
Van der Waal’s forces
Hydrophobic bonds
An epitope is usually composed of how many amino acids?
4-5
Take some time to draw an IgG molecule. Try to keep in mind chains, domains, and binding sites.
Things that you should have included:
Heavy chain, light chain, VH and VL domain, CL, CH1, CH2 and CH3 domain, hinge region, Fab and Fc, and antigen-binding site.
True or False: IgM in the membrane of a B cell is a monomer.
TRUE
The secreted form is a pentamer
True or False: Antigen/Antibody bonding is reversible.
TRUE
Remember, the bonding is not covalent.
What are the 2 factors that promote the strength of Ag/Ab binding?
pH
Ionic strength
Note that in vivo, pH is relatively constant, so this is usually not a factor.
True or False: Lower temperature will slow the rate of Ab/Ag binding.
TRUE
What is immuno-affinity chromatography?
A purification method that uses antigen and antibody binding.
Used to purify antibody that is specific to an antigen and vice versa.
Describe the four steps of immuno-affinity chromatography for purifying an antigen.
- Antibodies specific to the desired antigen are covalently bound to beads and placed in solution.
- Add a solution with a mixture of antigens. The antibody on the beads will bind to the desired antigen.
- Wash away the unbound antigens. Because the beads are too large to cross the filter, they will not be washed away.
- Elute the antigen - change the pH to release the antigen from the antibody.
Suppose you want to purify equine IgG that will bind to tetanus toxin. You have purified tetanus toxin and rabbit anti-equine antibody. WHAT DO YOU DO!?!?
- Inject tetanus toxin into a horse so that it will produce antibodies against tetanus.
- In the first column, mix beads covalently bound to tetanus toxin with serum from the horse. Antibodies will bind to the beads. Note that this will be a mixture of antibodies, not just IgG. Wash, and acidify to separate the antibodies.
- In the second column, mix your antibody mixture with beads covalently bound to rabbit anti-equine IgG molecules. These will bind to only equine IgG. Wash and acidify to separate the antibodies. You now have a mixture of pure IgG.
What size range is necessary to make a good antigen (i.e. one that the body will actually respond to)?
Greater than 10,000 molecular weight
What is a hapten?
A small molecule bound to a carrier protein.
Small molecules by themselves are not very immunogenic. However, when bound to a carrier protein, they can produce an immune response. This has implications for vaccines, etc.
Which molecular class (protein, lipid, etc.) makes the best antigens (i.e. most likely to elicit an immune response?
Proteins
The more complex the molecule is, the more likely it is to elicit an immune response. Proteins have positive and negative charges, hydrophobic areas, etc., and are thus good antigens. Lipids are simple and have no charges, and thus are poor antigens.
True or False: A less rigid molecule will make a better antigen.
FALSE
Rigidity is important for antibody binding. A molecule that is constantly changing shape makes a poor antibody binding surface.
Name the four major factors that contribute to the strength of an antigen (i.e. the likelihood that it will elicit an immune response).
Foreignness
Size
Molecular complexity
Rigidity
Is a steroid hormone a strong antigen?
Steroid characteristics: small, not a protein, conserved, not foreign
Steroids would make weak antigens
Is insulin a strong antigen?
Insulin characteristics: peptide hormone, small, some variance between species.
Insulin would likely elicit a small immune response.
Is antibody from a different species a strong antigen?
Foreign antibody characteristics: large, foreign, protein
Antibody from a different species would elicit a strong immune response.
What is an adjuvant?
Any substance that, when given with an antigen, enhances the immune response to that antigen.
Many vaccines contain adjuvants to create a stronger immune response.
What is tolerance in terms of immunity?
Tolerance suggests that an antigen is less likely to elicit an immune response.
If an animal has a prolonged exposure to an antigen (as opposed to short days to weeks exposure), what will the immune response be?
Will favor tolerance
Which portal of entry will elicit a stronger immune response?
Intradermal
Intravenous
Intradermal
Antigens that enter IV tend to promote tolerance. Think of nutrients being absorbed through the gut.
If an antigen-presenting cell presents an antigen but there are no associated danger signals, what will the response of the immune system be?
Tolerance
Immune response occurs when an antigen is presented with associated danger signals.
Name the four factors that will influence whether or not an antigen will be tolerated by the immune system.
Persistence
Portal of entry
Presence of adjuvants
Properties of the antigen presenting cell (level of costimulation, release of cytokines, etc.)
Why doesn’t the immune system respond to the food we eat (most of the time)? Explain in terms of the 4 factors that promote tolerance.
High level of persistence
Absorbed intravenously
No associated adjuvants
No danger signals from antigen-presenting cells
