3 Hybrid Cell Lines Normal Cells Are Fused With A Cancerous Cell Line E.x myeloma, lymphomaFusion Is Accomplished With PEG (polyethylene glycol)The New Hybrid Cell Exhibits Properties Of Both Cell TypesUnlimited growthSecretes monoclonal antibodyOr Secretes cytokines
4 Normal Cell For Monoclonal Antibody Production Animal Is Immunized With AntigenSpleen Cells Are IsolatedIntraperitoneal ImmunizationBalb/c MouseDay 0: 50 g of Ag In Complete Freund’s AdjuvantDay 14: 25 g of Ag In Incomplete Freund’s AdjuvantDay 28: 25 g of Ag In D-PBSABleed Animal Test Reactivity To AntigenSerum Is Diluted 1:30Final Boost of 10 g Antigen i.v or i.p 3 days before fusionAseptically Isolate Spleen Cells
5 P3.653 MyelomaThis Cell Line Is Deficient In HGPRT (hypoxantine guanine phosphoribosyl transferase)Alternatively TK (thymidine kinase deficient)Cell Line Cannot Survive In Selection MediumAminopterin Inhibits “De novo Pathway”, “Salvage Pathway” Is Not Possibe Due To HGPRT or TK DeficiencyIt Is Also Ig DeficientIt can not secret any immunoglobulinsAminopterin (folic acid antagonist) Blocks De novo PathwayP3.653 cells die in the presence of aminopterinThey cannot utilize the “salvage pathway” because they are HGPRT deficient
12 Fusing Spleen Cells With Myeloma Purify Spleen cellsT-cell Depletion (anti-Thy 1.2 followed by complement lysis)MACS purification (CD138)FusionMix spleen cells and myeloma in 50 mL tube (4:1)Spin and resuspend pellet with 1 mL PEG over 15 sMix by swirling for 75 sAdd 1 mL of SFM (serum free medium) over 15s, swirl for 45sAdd 2 mL SFM over 30s, swirl 90sAdd 4 mL HAT over 30s, swirl 90sAdd 8 mL HAT over 30s, swirl 90sAdd Above volume in a new container, add HAT till cell concentration is 1x106 cells/mLAdd 200 L per well 96 well/plate (200,000 cells/well)
13 Selection Of Hybridomas Selecting ClonesFeed Wells With New HAT Medium (remove old, add 150 L new)Feed Cells Twice A WeekAfter about 2 weeks Test Supernatants Of Potential Clones Using ElisaRetest +ve Clones in 48 hrsExpand Clones In New 96-well plate with HT NOT HATRetest Sups, Expand In 24-well Plate, Switch To Normal Medium, RetestExpand in 4-well PlateCryopreserve
14 Ensuring Monoclonality To Ensure Monoclonality Sub-clone HybridomaSerially 1 cell per 3 wellsUse a spleen cells as feeder cellsFeeder cells 1x106 cells/mL96 well plate, 2x105 cells/wellColonies are observed at 5 daysReplenish with fresh day 7Screening Is done at day 10 and 14Clones are cryopreserved same way as parental clones
15 Antibody ProductionLarge Amounts Of Antibody Can Be Produced Using AnimalsPrime Balb/c animal with IFAInject clone i.pCollect peritoneal ascitesAlternatively Bioreactors Are UsedCells are cultured in hollow fibersFresh media and waste are recircuilatedHigh concentrations of Ab produced in cell compartmentCollected at different time points
Your consent to our cookies if you continue to use this website.