Lecture 14 - Hybridoma and Phage Display

Question 1

What are polyclonal antibodies and how is a polyclonal antisera germinated?

Answer 1

A collection of Aby that recognized many epitopes on the same antigen. The antigen is multivalent. Inoculation of this antigen into a suitable host species generated polyclonal antibodies which can be separated and purified.

Question 2

What are some phylogenetic or other considerations when choosing a host species for inoculation of an antigen for the creation of polyclonal antibodies?

Answer 2

How evolutionarily related the species are:
more distant - less genetic conservation - more immune response in generating Aby.
Picking a specific pathogen free animal to minimize infectious disease cross over. (more important for vaccine)
Size of animal influences amount of antigen produced.

Question 3

What are some examples of diseases that are treated using polyclonal aby?

Answer 3

Tetanus, botulism, diphtheria.

Neutralize toxins created by these pathogens.

Question 4

How are monoclonal antibodies produces?

Answer 4

Hybridoma technology (cell fusion of B cell of interest with an immortal cancer cell or high replication cell)
Phage display

Question 5

Describe the process that selects for hybridoma cells in monoclonal aby production.

Answer 5

Moral spleen antibody producing cells with a selectable marker are fused with immortal myeloma cells without the marker to produce fusion cells. Spleen cells not merged will eventually die. Myeloma cells without the selectable marker will die due to not having it. Merged cells will be immortal and will have selectable marker so will be the only cells that will proliferate. These immortal cells will produce monoclonal antibodies with a single antigen specificity.

Question 6

Describe the experimental process in the hybridoma production of mAb.

Answer 6

Step 1.
3 or so mice are immunized with an adjuvant within their spleen. Booster immunizations are given. Mice who have the desired amount of Aby in their blood are selected for.
Step 2: Fusion (day 21)
spleen cells are harvested and fused with myeloma cells in a selectable media (HAT, HGPRT).
Step 3: Screening
Screening is done to ensure Aby has required specificity by ELISA, Flow cytometry, or western blotting.
Step 4: Subcloning:
Positive parental hybridomas are separated / sub cloned and screened by elisa for specific mAb.
Isotype determined.
Aby of interest mass produced in culture, harvested by centrifugation, buffer, freeze dry.

Question 7

What are adjuvants?

Answer 7

Insoluble immune-stimulating agents enhance the immune response to an Ag.
Result in delayed release of antigen and enhanced uptake and presentation by macrophages.

Question 8

What is Freund’s Complete Adjuvant and incomplete adjuvant?

Answer 8

Oil in water emulsion of dead mycobacteria and oil-in-water emulsion of muramyldipeptide.

Question 9

What is HAT selection?

Answer 9

Hypoxanthine aminopterin thymine (HAT) requires thymidine kinase (TK) for DNA synthesis. TK- cells cannot survive without the enzyme TK. Mortal splenic cells are TK+ but will eventually die naturally. A fusion cell of TK- myeloma and TK+ splenic cell created an immortal cell that can undergo DNA synthesis.

Question 10

What is phage display?

Answer 10

Generation of Aby like fragments called scFv (28 kDa) which recognize antigens by in vitro screening in phage. this by-passes the need for animal immunization / hybridoma.
scFv = Vl+Vh

Question 11

What is the method for Phage Display?

Answer 11

Stage 1: Screening
Phage library has 10^10 scFv-displaying phage.
Immobilize receptor RANK to surface of tube.
Add library, wash,elute, amplify eluted phage, bind, elute, amplify and repeat until only those phage that are bound best remain.
Stage 2: Expression
Infect E coli with Phage to amplify the amount.
Use Elisa to identify RANK-binding clones.
Induce further large scale expression with enhancer protein like IPTG

Question 12

What are the 6 therapeutic immune-targeting strategies?

Answer 12

1. Naked antibody targeting
2. Ab-drug conjugates.
3. Ab-isotyope conjugates for imagine and radiotherapy.
4. Ab-tosin/cytokine/conjugate/fusions
5. Immunoliposomes
6. Bispecific/bifunctional Aby (quadroma) for drug delivery targeting

Question 13

What is naked antibody targeting?

Answer 13

Biologic response modifiers.
cytokine scavenging.
Receptor agonist/antagonist.
Aby targeted against a ligand or receptor to block its action

Question 14

What are the differences between chimeric and humanised mAb?

Answer 14

Humanized mAb is achieved by drafting the HV/CDR from a mouse Aby onto a human variable region framework. Chimeric mAb involve grafting of full mouse variable region onto human H and L framework.

Question 15

Differentiate the naming system for Aby.

Answer 15

umab: fully human
zumab: humanized
ximab: chimeric
xizumab: both chimeric and humanized
omab: fully mouse

Question 16

What are antibody-drug conjugates?

Answer 16

Aby delivers cargo of drug.

Cancer therapy

Question 17

What are are Ab-isotope conjugates for imagine and radiotherapy?

Answer 17

These direct isotopes to specific areas such as in cancer therapy for imaging or therapeutic purposes.

Question 18

What are Ab-fusion protein constructs?

Answer 18

These act like a receptor or contain a receptor to clear substances (ligands) from the system.

Question 19

What are immunoliposomes?

Answer 19

These are used to target cargo like toxic drugs to specific areas of the body.
Preferentially localize to sites of cancer via tumour EPR effect where there is enhanced permeability and retention of fluid

Question 20

What are bispecific / bifunctional Aby (quadroma)?

Answer 20

These are Aby with two different FAB; useful for cross-linking two different targets like vaccine and immune cell or is immuno-diagnostic kits.