Presentation on theme: "Production of Rabbit Monoclonal Antibodies Againts Mouse Embryonic Stem Cells and Identification of Pluripotency-associated Surface Antigen Dewi Satwika."— Presentation transcript:
1 Production of Rabbit Monoclonal Antibodies Againts Mouse Embryonic Stem Cells and Identification of Pluripotency-associated Surface AntigenDewi SatwikaPROGRAM PASCA SARJANA JURUSAN BIOLOGIFAKULTAS MATEMATIKA DAN PENGETAHUAN ALAMUNIVERSITAS BRAWIJAYA
2 ? proteomics Monoclonal antibody Embryonic stem cells (ES cells)potential for regenerative medicinepluripotent stem cellsES cells surface moleculesdetection of antigengenomicsproteomicsMonoclonal antibodyEfficient method for surface marker identificationhibridomaMus muscullusKohler and milstein?Smaller Ab repertoirePotential to recogniize new surface molecule on mouse ES cellshigher affinity andspecificity in recognizing conformational and modified epitopesImmunotolerant to cell surface Ag on mouse ES cellsRabbit New Zealendlarger antibody repertoire
3 METHODS 3 months old hibridoma clone mES cells culture in MEF Injected subcutaneously with 108 mES D3 cell lineThe mES cell lines D3 were cultured on mitomycin C-treated mouse embryonic fibroblasts (MEFs)3 months old1 weekserum collectionResuspended in 1 ml PBS2 weeksinjected intravenously with 108 mES cells suspended in 1 ml of PBSemulsified with CFA in a 1:1 (v/v) ratioBooster using mES cell suspension emulsified in IFASacrificeAdded PEG to fuse240E-1 cell linehibridomaSpleencentrifugeSupernatant hibridomacloneFITC conjugatedgoat anti-rabbit sec. AbmES cells culture in MEF
4 Protein-A conjugated resin propagatedCulture supernatantcloneCells lysatePositive clone5×108 F9 EC cells by ultrasonication in lysis bufferCulture supernatantProtein-A conjugated resinWestern blottingPutative antigens eluted with a 2.5 mM citrate solutionSDS PAGEcStaining primary and second AbBlocked with blocking solutionFluoresence microscopyFixed in 4% PFAcellsFlowcytometry
5 RESULTAfter subcloning, 240 remained positive. Six monoclones of each subclone plate were expanded and cryopreserved.These antibodies can be divided intofour major types based on the cellularlocations of their targeted antigens:membrane (A), extracellular matrix (B),nucleus (C) and cytoplasm(or cytoskeleton) (D).Fig. 1. Staining patterns of different rMabs on mES D3 cells and MEFs.
6 All but two antibodies stained positively on mES cells Twenty Mab that were able to bind to mES cells. These Ab showed different staining patterns.7 antibodies showed nuclear or diffused staining while 13 were cytosolic/membraneous.All but two antibodies stained positively on mES cellsFig. 2. ICC staining using the 20 rMabs.
7 From 20 positively rMabs10 antibodies targeted the extracellular epitopes of cell surface proteins for more than 5% of the mES cellsPositively stained by three antibodies, ZjuESrMab3, ZjuESrMab29 and ZjuESrMab61, decreased more than 50% upon mES cell differentiationFig. 3. Flow cytometry analysis using the 20 rabbit monoclonal antibodies. A: Staining of monoclonal antibodies on mES D3 cells. B: Staining of monoclonal antibodies on mES D3 cells that were spontaneously differentiated for 3 days
8 ZjuESrMab29 and ZjuESrMab61 did not detect any protein in the western blot analysis, suggesting that they might target conformational epitopes on live mES cells.ICC staining with ZjuESrMab29 faded after three days of spontaneous differentiation of mES cells cultured in a monolayer.The percentage of positive cells decreased to about 1%.one specifically stained band of approximately 42 KDa was observed for ZjuESrMab29LC-MS/MS analysis identified the protein as GM-CSFR α.A small increase in staining was detected after nine days of monolayer differentiation The number of positively stained cells decreased to about 2% after four days of differentiation into embryoid bodies (Ebs)
9 The expression of GM-CSFR α in the testis may serve as a marker of ES cells and GS cells. expression of GM-CSFR α decreases upon mES cell differentiation and is restricted to adult tissues, suggesting that GM-CSFR α could serve as a new pluripotency- associated surface marker for mES cells.Immunohistochemical staining of adult mouse tissues with ZjuESrMab29. ZjuESrMab29 stained strongly in the testis, stained weakly in the restricted regions of the kidney and spleen and did not stain in the liver, heart, brain or lung.Immunohistochemical analysis showed that ZjuESrMab29 stained strongly in the testis, stained weakly in the restricted, perivascular regions of the kidney and spleen and did not stain in the liver, heart, brain or lung