Phage display part 2

Question 1

Murine monoclonal antibody side effects?

Answer 1

Side effects and no cure due to
HAMA = Human Anti Mouse Antibody

Question 2

Phage Display – How does it work?

Answer 2

1.Creation of a Phage Library:

Gene Library Construction: A library of DNA sequences encoding various peptides or protein variants is synthesized. These sequences are inserted into a phage vector, typically a filamentous phage such as M13.
Phage Vector: The DNA sequences are inserted into a gene encoding a coat protein (e.g., gene III or gene VIII) of the bacteriophage. This results in the fusion of the peptide or protein to the coat protein, displaying it on the surface of the phage.
Infection of Bacteria:

The recombinant phage vectors are used to infect a host bacterium, usually Escherichia coli (E. coli). The bacteria produce phage particles displaying the encoded peptides or proteins on their surfaces.
Phage Display Library:

After infection and replication, a large library of phage particles is generated, each displaying a different peptide or protein variant.
Selection (Biopanning):

Binding to Target: The phage library is exposed to a target molecule (e.g., protein, peptide, DNA, or small molecule) immobilized on a solid surface (e.g., a microtiter plate, magnetic beads, or a chromatography column).
Washing: Non-binding phages are washed away, leaving only phages that have bound to the target.
Elution: Bound phages are eluted, often using a competitive binder, a change in pH, or another method to disrupt the interaction between the displayed peptide or protein and the target.
Amplification:

The eluted phages, which are enriched for high-affinity binders, are used to infect fresh bacteria. This allows amplification of the selected phages.
Multiple rounds of selection and amplification (typically 3-5 rounds) are performed to enrich for phages displaying peptides or proteins with the highest affinity and specificity for the target.
Analysis of Selected Phages:

After several rounds of selection and amplification, individual phage clones are isolated.
The DNA encoding the displayed peptides or proteins is sequenced to identify the binding sequences

Question 3

what can you use for Antibody library screening?

Answer 3

ELISA plates, but it is Costly!
Time consuming !

Phage display is cheaper and faster!!

Question 4

Phage display vectors

Answer 4

Phage display vector inside the phage vector. DNA encoding
the antibody fragment
“Genotype”

Question 5

Displayed antibody fragment

Answer 5

Antibody fragment – protein
exhibiting binding properties, on the outside attached to the phage.
“Phenotype”

Question 6

Humira (Adalimumab)

Answer 6

Adalimumab was the first fully human monoclonal antibody approved by the (FDA) on December 31, 2002, for the
treatment of rheumatoid arthritis by inactivating tumor necrosis factoralpha (TNFα).
It is derived from phage display