Immunochemical techniques Flashcards
Does antibodies bind to antigen with their variable- or constant regions?
Variable region (FAB)
avg Total weight Ab : heavy chain weight : light chain weight
150kDa : 50kDa : 25kDa
Which of IgG and IgM is most relevant in the lab?
IgG - These have undergone affinity maturation.
What’s an epitope?
The domain to which Ab binds Ag.
How large to antigens have to be in order to elicit an immune response? Why?
2kDa. The reason is that macromolecules such as glucose or TAGs shouldn’t be recognized as foreign.
What’s the difference between mono- and polyclonal antibodies?
Monoclonal antibodies bind to the same epitope. These derive from the same B-cell.
Polyclonal antibodies bind to different epitopes. These derive from several different B-cells.
What is an adjuvant? Why use them?
Adjuvants are chemicals that elicit a strong immune response. One application is that adjuvants facilitate the immunological resistance from vaccines.
Describe how monoclonal antibodies are generated.
- Inject animal with antigen.
- Screen the animal for B-cells that bind the antigen the most efficiently. (ex w/ phage display)
- Hybridize the B-cell with the genome of a myeloma cancer cell (to incorporate the proliferative properties of cancer).
- Screen for hybridoma cells that produce antibodies and bind antigens.
- Cultivate the best hybridoma cells.
What are the pros/cons of monoclonal antibodies compared to polyclonal antibodies?
PROS
1. Identifies if a certain part of the antigen is present.
2. Available in unlimited supply, although initially expensive.
CONS
1. Monoclonal antibodies may cross-react with other proteins that share the same epitope.
2. Usually less sensitive to the presence of Ag due to the ab only recognizing a small part of it.
What are the pros/cons to polyclonal antibodies (2 each)?
PROS
1. Identifies epitopes all over the antigen, thus, it’s more sensitive for antigen prevalence.
2. The detection signal is strong due to many antibodies binding the same antigen.
CONS
1. Variation between batches.
2. Supply is limited to immunized animals.
What are nanobodies? Give an example of therapeutic use.
Nanobodies only contain the variable domain (FAB) of an antibody.
Nanobodies are used to intercept IL17 in psoriasis patients, this calms down the inflammation.
What three ways are used to emulate affinity maturation when developing antibodies w/o animals?
- Phage display
- Cell surface display
- Ribosomal display
What are the steps to creating a phage display?
- Gene library is cloned into phagemids (phage plasmids).
- Active viruses are produced which are all ontaining a phagemid. The phagemid –> exterior antibody.
- The active phages are added to immobilized antigens.
- Phages that are poorly bound to the antigen are washed away.
- Only high-affinity phages remain.
- Incubate the most high-affinity phage in bacteria.
EXTRA PRO: You can sequence the bacterial genome to find what antibody-antigen interaction was the strongest.
What are the most common antibody labels for detection?
- Enzymatic labeling: HRP
- Fluorescent labeling
The aforementioned are both indirect labels. They are more versatile as they detect the presence of the first antibody.
Name as many antibody-dependent methods as possible (6 total).
- Western blot
- ELISA
- Co-IP
- ChIP
- Immuohistochemistry (microscopy)
- Flow cytometry
Describe the western blot method in a nutshell
- Protein electrophoresis.
- Transfer to membrane.
- Detect protein of interest with primary and secondary antibody.
What does SDS do in a western blot?
SDS = Sodium dodecyl sulphate. It binds to amino acids and gives them a negative charge. SDS also linearize the protein.
What are isoelectric focusing gels?
Gels in which the pH differs. The proteins will migrate in different directions depending on their isoelectric points.
The proteins are initially in the same well. When a current is applied, the current pulls the proteins to their respective isoelectric points.
Explain ELISA in a nutshell.
- There are two methods: DAS and TAS.
DAS (double sandwich)
1. Antibody is fixed to 96-well plate (recognises Ag-A).
2. Antigen is added to the well.
3. Second antibody is added (recognises Ag-B).
4. Third antiboy is added (recognises second ab)
TAS (no sandwich)
2. Ag is trapped.
3. ab is added (w/ different epitope recog.)
4. Second Ab is added, binds to secondary.
Explain what immunohistochemistry is.
It is when you embed tissue in paraffin, cut it thinly, then stain it with fluorescent antibodies.
Explain differences between Co-IP and ChIP.
Co-IP: Protein-protein interactions.
Ch-IP: DNA-protein interactions.
What are catalytic antibodies?
They can act as a substrate which would otherwise stabilise an intermediary product in a reaction, thus shifting the equilibrium.
