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