Production of Rabbit Monoclonal Antibodies Againts Mouse Embryonic Stem Cells and Identification of Pluripotency-associated Surface Antigen Dewi Satwika 116090100011006 PROGRAM PASCA SARJANA JURUSAN BIOLOGI FAKULTAS MATEMATIKA DAN PENGETAHUAN ALAM UNIVERSITAS BRAWIJAYA

? proteomics Monoclonal antibody Embryonic stem cells (ES cells) potential for regenerative medicine pluripotent stem cells ES cells surface molecules detection of antigen genomics proteomics Monoclonal antibody Efficient method for surface marker identification hibridoma Mus muscullus Kohler and milstein ? Smaller Ab repertoire Potential to recogniize new surface molecule on mouse ES cells higher affinity and specificity in recognizing conformational and modified epitopes Immunotolerant to cell surface Ag on mouse ES cells Rabbit New Zealend larger antibody repertoire

METHODS 3 months old hibridoma clone mES cells culture in MEF Injected subcutaneously with 108 mES D3 cell line The mES cell lines D3 were cultured on mitomycin C-treated mouse embryonic fibroblasts (MEFs) 3 months old 1 week serum collection Resuspended in 1 ml PBS 2 weeks injected intravenously with 108 mES cells suspended in 1 ml of PBS emulsified with CFA in a 1:1 (v/v) ratio Booster using mES cell suspension emulsified in IFA Sacrifice Added PEG to fuse 240E-1 cell line hibridoma Spleen centrifuge Supernatant hibridoma clone FITC conjugated goat anti-rabbit sec. Ab mES cells culture in MEF

Protein-A conjugated resin propagated Culture supernatant clone Cells lysate Positive clone 5×108 F9 EC cells by ultrasonication in lysis buffer Culture supernatant Protein-A conjugated resin Western blotting Putative antigens eluted with a 2.5 mM citrate solution SDS PAGE c Staining primary and second Ab Blocked with blocking solution Fluoresence microscopy Fixed in 4% PFA cells Flowcytometry

RESULT 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. 1. Staining patterns of different rMabs on mES D3 cells and MEFs.

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 cells Fig. 2. ICC staining using the 20 rMabs.

From 20 positively rMabs 10 antibodies targeted the extracellular epitopes of cell surface proteins for more than 5% of the mES cells Positively stained by three antibodies, ZjuESrMab3, ZjuESrMab29 and ZjuESrMab61, decreased more than 50% upon mES cell differentiation 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

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 ZjuESrMab29 LC-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)

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

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