Lesson 7: Antibody Discovery

6 steps in hybridoma technology

1. cell isolation

2. fusion of B cells with myeloma

3. HAT selection & culturing cells within the media

4. selection of hybridomas that make the antibody to the antigen

5. monoclonal antibodies are purified and produced in bulk

What is the HAT media for

It is a selective growth environment that is used to isolate successfully fused hybridoma cells while eliminating unfused parent cells

Pros & cons of hybridoma technology

Pros:

• highly reproducible

• animal maintenance isnt required after cloning

Cons:

• need to immunize mice

• system is developed only for rat and mouse

Purpose of display technologies

Couples a protein’s physical function directly to its encoding nucleic acid sequence

Types of display technologies

1. phage display

2. ribosome display

3. yeast display

4. mammalian display

What is a phage

A virus that infects and replicates within bacteria

5 steps of phage display

1. construction of a phage library

2. target binding

3. washing

4. elution

5. amplification

repeat the cycle 2-3x to enrich the library for the best binders

Target binding - phage display

Exposing the library to the target allowing the phages with highest affinity to the target to bind

Washing - phage display

Washing away of the unbound phages

Elution

Phages that are bound to the target will be eluted or recovered from the target

Amplification

Eluted phages are amplified by infecting another host cell (typically E. coli) allowing them to replicate

Pros & cons of phage display

Pros:

• allow production of fully human antibodies

• no animals required

• presents a large library

Cons:

• not all antibodies express well in E. coli

• discovered antibodies are not always manufacturable

Achieving broadly neutralising antibodies by

Consensus antigen

Cross panning

Achieving highly specific antibodies by

Deselection & blocking

Achieving environment sensing antibodies by

Changing selection parameters

Antibody formats clearance by

Renal filtration and endocytosis