Presentation on theme: "antibodies produced by differentiated B-cells"— Presentation transcript:
1 antibodies produced by differentiated B-cells recombination results in B-cell lineages producing distinct antibodiesserum contains antibodies representing many lineagesdifficult to isolate B-cells of defined specificitycannot grow B-cells in cultureKohler and Milstien (1975)fused B-cells with myeloma cells stable hybridomahybridoma secretes monoclonal antibody
2 Monoclonal Antibodies PreparationImmunization*FusionSelectionScreening & CloningProductionAdvantagesunlimited supplydefined reagentDisadvantagessingle epitopetime consumingrodents, especially mice, are animal of choicebest characterized myeloma lines are from micematch strains for ascites productionpossible to use crude antigens
3 mouse making desired antibodies boost 3-4 days before fusion remove spleen (lymph nodes etc) and harvest cellsmix with myeloma cells in presence of fusogenic agentBalb/c MouseMOPC 21 (tumor)P3K (cell line)P3-X63Ag8 (HGPRT-)X63-Ag8.653 (IgG-)
4 HAT Medium:Hypoxanthine(purine salvage)Aminopterin(DHFR inhibitor)Thymidine(pyrimidine salvage)Nucleotide Metabolism:salvage and de novo pathwaysHGPRT essential for purine salvageDHFR essential for de novo synthesisHGPRT = hypoxanthine-guanine phosphoribosyl transferaseDHFR = dihydrofolate reductase
5 Selection in HAT Medium Human mAbs produced by trans-forming lymphocytes with EBV
7 Production produce mAbs in vitro or with ascites harvest culture media (supernatant)in vitro material is less concentrated and contains bovine serumascites are tumors grown in peritoneal cavityascites fluid and sera contain high [mAb]minor contamination with mouse IgG
8 Affinity Purification use Ag or epitope to make affinity columnrecover Abs from blots, etc.