1. 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

2. ? 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

3. 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

4. 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

5. 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.

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

7. 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

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 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)

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

10. Thank You