Synthetic Immunology/Ab Discovery

Question 1

3 main methods of Ab discovery and their differences.

Answer 1

1) Hybridomas:
- Immunize mouse with antigen of interest and isolate spleen B cells
- Mix isolated B cells with myeloma cells (immortal cancerous B cell line)
- Fusion is a hybridoma, can produce specific Ab on surface and proliferate indefinitely in vitro
- Screen hybridomas to identify those that produce desired Ab via ELISA assay
- Hybridomas that produce desired ab are clones -> monoclonality, can be cultured and expanded
- Cheap, easy, can use flow cytometry for screening

2) Phage Display:
- Immunize mouse and amplify Ab V gene fragments (scFv domain, VL or VH domain or Fab domain) from mouse B cells
- Clone Ab V gene fragments into phage display vector (encodes fusion protein of scFv-coat protein of bacteriophage)
- Introduce vectors into host bacterila strain to build phage display library
- Selection and screening by incubation library with immobilized target antigen
- Binding scFvs are selected and amplified
- No cytometry, not grown in culture

3) Yeast Display:
- Immunize mouse and amplify Ab scFv from B cells
- Ab scFvs are cloned into yeast display vector (encodes fusion protein of scFv-yeast surface protein)
- Introduce vesctors into yeast cells by transformation to build library expressing scFv on surface
- Selection and screening by incubation with immobilized target antigen
- Binding scFvs are selected and amplified
- Can make coupled display method for FACS
- Not in culture, bound to surface