1. Hybridomas

3. Hybrid Cell Lines Normal Cells Are Fused With A Cancerous Cell Line
E.x myeloma, lymphoma
Fusion Is Accomplished With PEG (polyethylene glycol)
The New Hybrid Cell Exhibits Properties Of Both Cell Types
Unlimited growth
Secretes monoclonal antibody
Or Secretes cytokines

4. Normal Cell For Monoclonal Antibody Production
Animal Is Immunized With Antigen
Spleen Cells Are Isolated
Intraperitoneal Immunization
Balb/c Mouse
Day 0: 50 g of Ag In Complete Freund’s Adjuvant
Day 14: 25 g of Ag In Incomplete Freund’s Adjuvant
Day 28: 25 g of Ag In D-PBSA
Bleed Animal Test Reactivity To Antigen
Serum Is Diluted 1:30
Final Boost of 10 g Antigen i.v or i.p 3 days before fusion
Aseptically Isolate Spleen Cells

5. P3.653 Myeloma
This Cell Line Is Deficient In HGPRT (hypoxantine guanine phosphoribosyl transferase)
Alternatively TK (thymidine kinase deficient)
Cell Line Cannot Survive In Selection Medium
Aminopterin Inhibits “De novo Pathway”, “Salvage Pathway” Is Not Possibe Due To HGPRT or TK Deficiency
It Is Also Ig Deficient
It can not secret any immunoglobulins
Aminopterin (folic acid antagonist) Blocks De novo Pathway
P3.653 cells die in the presence of aminopterin
They cannot utilize the “salvage pathway” because they are HGPRT deficient

7. Possible Fusion Products
Myeloma HGPRT Deficient And Ig Deficient +
Plasma Cells From Immunized Animal 
Senescence 
Can Use Salvage Pathway
No Senescence 
Senescence 
HAT Medium 
HAT Medium

12. Fusing Spleen Cells With Myeloma
Purify Spleen cells
T-cell Depletion (anti-Thy 1.2 followed by complement lysis)
MACS purification (CD138)
Fusion
Mix spleen cells and myeloma in 50 mL tube (4:1)
Spin and resuspend pellet with 1 mL PEG over 15 s
Mix by swirling for 75 s
Add 1 mL of SFM (serum free medium) over 15s, swirl for 45s
Add 2 mL SFM over 30s, swirl 90s
Add 4 mL HAT over 30s, swirl 90s
Add 8 mL HAT over 30s, swirl 90s
Add Above volume in a new container, add HAT till cell concentration is 1x106 cells/mL
Add 200 L per well 96 well/plate (200,000 cells/well)

13. Selection Of Hybridomas
Selecting Clones
Feed Wells With New HAT Medium (remove old, add 150 L new)
Feed Cells Twice A Week
After about 2 weeks Test Supernatants Of Potential Clones Using Elisa
Retest +ve Clones in 48 hrs
Expand Clones In New 96-well plate with HT NOT HAT
Retest Sups, Expand In 24-well Plate, Switch To Normal Medium, Retest
Expand in 4-well Plate
Cryopreserve

14. Ensuring Monoclonality
To Ensure Monoclonality Sub-clone Hybridoma
Serially 1 cell per 3 wells
Use a spleen cells as feeder cells
Feeder cells 1x106 cells/mL
96 well plate, 2x105 cells/well
Colonies are observed at 5 days
Replenish with fresh day 7
Screening Is done at day 10 and 14
Clones are cryopreserved same way as parental clones

15. Antibody Production
Large Amounts Of Antibody Can Be Produced Using Animals
Prime Balb/c animal with IFA
Inject clone i.p
Collect peritoneal ascites
Alternatively Bioreactors Are Used
Cells are cultured in hollow fibers
Fresh media and waste are recircuilated
High concentrations of Ab produced in cell compartment
Collected at different time points