Hybridoma 😊

🧬 Hybridoma Monoclonal Antibody Production

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Overview

  • Used to produce monoclonal antibodies (mAbs)

Steps

Step
Description
1. Immunization
Mouse is immunized with antigen to generate antibody response.
2. Booster Dose
Given after measuring antibody titers.
3. Spleen Cell Isolation
Mouse is euthanized
Spleen cells (B-lymphocytes) are harvested.
4. Fusion with Myeloma Cells
Spleen cells + myeloma cells (unlimited growth)
â–º using PEG
5. Hybridoma Formation
Cells with antibody production + immortal growth.
6. Selection in HAT Medium
(Hypoxanthine, Aminopterin and Thymidine)
Only hybridomas survive
(via salvage pathway;
myeloma cells die due to lack of HGPRT).
7. Screening & Cloning
Screen for desired antibody-producing hybridomas

and clone them.
8. Antibody Harvesting
Cultured to harvest monoclonal antibodies.

HAT Medium Components & Function

Component
Function
Hypoxanthine
Substrate for salvage pathway.
Aminopterin
Folate antagonists

â›” de novo nucleotide or purine synthesis.
Thymidine
Allows DNA synthesis via salvage pathway.

Enzymes

  • HGPRT (Hypoxanthine-Guanine Phosphoribosyltransferase)
  • Thymidine Kinase

Applications

Field
Application
Therapeutics
Immunosuppressants,
mAb therapy (e.g., for cancer, autoimmune diseases)
Research
Listing lymphocyte subpopulations,
antigen quantification
Diagnostics
ELISA, blood component assays
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