Immunochemical techniques

Question 1

Does antibodies bind to antigen with their variable- or constant regions?

Answer 1

Variable region (FAB)

Question 2

avg Total weight Ab : heavy chain weight : light chain weight

Answer 2

150kDa : 50kDa : 25kDa

Question 3

Which of IgG and IgM is most relevant in the lab?

Answer 3

IgG - These have undergone affinity maturation.

Question 4

What’s an epitope?

Answer 4

The domain to which Ab binds Ag.

Question 5

How large to antigens have to be in order to elicit an immune response? Why?

Answer 5

2kDa. The reason is that macromolecules such as glucose or TAGs shouldn’t be recognized as foreign.

Question 6

What’s the difference between mono- and polyclonal antibodies?

Answer 6

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.

Question 7

What is an adjuvant? Why use them?

Answer 7

Adjuvants are chemicals that elicit a strong immune response. One application is that adjuvants facilitate the immunological resistance from vaccines.

Question 8

Describe how monoclonal antibodies are generated.

Answer 8

1. Inject animal with antigen.
2. Screen the animal for B-cells that bind the antigen the most efficiently. (ex w/ phage display)
3. Hybridize the B-cell with the genome of a myeloma cancer cell (to incorporate the proliferative properties of cancer).
4. Screen for hybridoma cells that produce antibodies and bind antigens.
5. Cultivate the best hybridoma cells.

Question 9

What are the pros/cons of monoclonal antibodies compared to polyclonal antibodies?

Answer 9

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.

Question 10

What are the pros/cons to polyclonal antibodies (2 each)?

Answer 10

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.

Question 11

What are nanobodies? Give an example of therapeutic use.

Answer 11

Nanobodies only contain the variable domain (FAB) of an antibody.

Nanobodies are used to intercept IL17 in psoriasis patients, this calms down the inflammation.

Question 12

What three ways are used to emulate affinity maturation when developing antibodies w/o animals?

Answer 12

1. Phage display
2. Cell surface display
3. Ribosomal display

Question 13

What are the steps to creating a phage display?

Answer 13

1. Gene library is cloned into phagemids (phage plasmids).
2. Active viruses are produced which are all ontaining a phagemid. The phagemid –> exterior antibody.
3. The active phages are added to immobilized antigens.
4. Phages that are poorly bound to the antigen are washed away.
5. Only high-affinity phages remain.
6. 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.

Question 14

What are the most common antibody labels for detection?

Answer 14

1. Enzymatic labeling: HRP
2. Fluorescent labeling

The aforementioned are both indirect labels. They are more versatile as they detect the presence of the first antibody.

Question 15

Name as many antibody-dependent methods as possible (6 total).

Answer 15

1. Western blot
2. ELISA
3. Co-IP
4. ChIP
5. Immuohistochemistry (microscopy)
6. Flow cytometry

Question 16

Describe the western blot method in a nutshell

Answer 16

1. Protein electrophoresis.
2. Transfer to membrane.
3. Detect protein of interest with primary and secondary antibody.

Question 17

What does SDS do in a western blot?

Answer 17

SDS = Sodium dodecyl sulphate. It binds to amino acids and gives them a negative charge. SDS also linearize the protein.

Question 18

What are isoelectric focusing gels?

Answer 18

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.

Question 19

Explain ELISA in a nutshell.

Answer 19

1. 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.

Question 20

Explain what immunohistochemistry is.

Answer 20

It is when you embed tissue in paraffin, cut it thinly, then stain it with fluorescent antibodies.

Question 21

Explain differences between Co-IP and ChIP.

Answer 21

Co-IP: Protein-protein interactions.
Ch-IP: DNA-protein interactions.

Question 22

What are catalytic antibodies?

Answer 22

They can act as a substrate which would otherwise stabilise an intermediary product in a reaction, thus shifting the equilibrium.