Presentation on theme: "Production of Rabbit Monoclonal Antibodies Againts Mouse Embryonic Stem Cells and Identification of Pluripotency-associated Surface Antigen Dewi Satwika."— Presentation transcript:
Production of Rabbit Monoclonal Antibodies Againts Mouse Embryonic Stem Cells and Identification of Pluripotency-associated Surface Antigen Dewi Satwika PROGRAM PASCA SARJANA JURUSAN BIOLOGI FAKULTAS MATEMATIKA DAN PENGETAHUAN ALAM UNIVERSITAS BRAWIJAYA
detection of antigen Embryonic stem cells (ES cells) potential for regenerative medicine pluripotent stem cells ES cells surface molecules genomics proteomics Monoclonal antibody Efficient method for surface marker identification Kohler and milstein Mus muscullus hibridoma Smaller Ab repertoire Immunotolerant to cell surface Ag on mouse ES cells Potential to recogniize new surface molecule on mouse ES cells Rabbit New Zealend larger antibody repertoire higher affinity and specificity in recognizing conformational and modified epitopes ?
METHODS Injected subcutaneously with 10 8 mES D3 cell line The mES cell lines D3 were cultured on mitomycin C- treated mouse embryonic fibroblasts (MEFs) 3 months old Resuspended in 1 ml PBS emulsified with CFA in a 1:1 (v/v) ratio Booster using mES cell suspension emulsified in IFA injected intravenously with 10 8 mES cells suspended in 1 ml of PBS serum collection 1 week Sacrifice Spleen 240E-1 cell line Added PEG to fuse hibridoma mES cells culture in MEF Supernatant hibridoma centrifuge FITC conjugated goat anti-rabbit sec. Ab clone 2 weeks
clone Positive clone propagated Culture supernatant Cells lysate 5×10 8 F9 EC cells by ultrasonication in lysis buffer Culture supernatant Protein-A conjugated resin Putative antigens eluted with a 2.5 mM citrate solution c SDS PAGE Fluoresence microscopy cells Fixed in 4% PFA Blocked with blocking solution Staining primary and second Ab Western blotting Flowcytometry
RESULT Fig. 1. Staining patterns of different rMabs on mES D3 cells and MEFs. After subcloning, 240 remained positive. Six monoclones of each subclone plate were expanded and cryopreserved. These antibodies can be divided into four major types based on the cellular locations of their targeted antigens: membrane (A), extracellular matrix (B), nucleus (C) and cytoplasm (or cytoskeleton) (D).
Fig. 2. ICC staining using the 20 rMabs. 7 antibodies showed nuclear or diffused staining while 13 were cytosolic/membraneous. Twenty Mab that were able to bind to mES cells. These Ab showed different staining patterns. All but two antibodies stained positively on mES cells
Positively stained by three antibodies, ZjuESrMab3, ZjuESrMab29 and ZjuESrMab61, decreased more than 50% upon mES cell differentiation 10 antibodies targeted the extracellular epitopes of cell surface proteins for more than 5% of the mES cells Fig. 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 From 20 positively rMabs
ZjuESrMab29 and ZjuESrMab61 did not detect any protein in the western blot analysis, suggesting that they might target conformational epitopes on live mES cells. one specifically stained band of approximately 42 KDa was observed for ZjuESrMab29 LC-MS/MS analysis identified the protein as GM-CSFR α. 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%. 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)
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 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. 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.