Human Ab Production

Question 1

What is phage display?

Answer 1

Using bacteriophage as a vector to produce human mAbs

Question 2

What is a phage library?

Answer 2

Heterogenous mix of phage clones, with different scFv on PIII protein

Question 3

What is phage panning?

Answer 3

Using phage library, mix with antigen bound to surface (ELISA). Wash away those that dont bind, and elute (lower pH and sonification) those that do. Reinfect into bacteria to multiply and repeat. Results in a very specific antibody for the desired antigen.

Question 4

What is ribosomal display?

Answer 4

In vitro, cell free selection of peptides from a library

Question 5

What is a PRM? How do we produce these?

Answer 5

Protein Ribosome mRNA complex - produced via ribosome stalling - antibiotics (halt translation e.g. rifampicin) or deletion of stop codon

Question 6

Example of a transgenic mouse Ab?

Answer 6

Adalimumab - anti TNF a - RA

Question 7

What do we need to ensure we do during ribosomal display?

Answer 7

Keep protein attached
Keep mRNA attached
Ensure protein produced is folded correctly while still attached

Question 8

What do we do with the ARM complexes that have a high affinity for the ligand?

Answer 8

Perform reverse transcription on the mRNA to produce DNA and then amplify using PCR. Results in a nucleotide sequence that has a high affinity for a target.

Question 9

How can we stabilise the PRM complex before panning?

Answer 9

Lower Temp
Add cations (e.g. Mg2+)

Question 10

What is the advantage of ribosomal display over phage display?

Answer 10

1. Diversity of gene library not limited by the transformation efficiency of the bacteria
2. Can easily introduce random mutations after each round of panning
3. Can produce larger proteins