Transcript Review Journal-Produksi Antibodi Monoklonal

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
Embryonic
stem cells
(ES cells)
pluripotent stem cells
ES cells surface
molecules
genomics
potential for regenerative
medicine
detection of antigen
proteomics
Monoclonal antibody
Efficient method for surface marker identification
hibridoma
Mus muscullus
Kohler and milstein
?
Potential to
recogniize new
surface
molecule on
mouse ES cells
Smaller Ab repertoire
higher affinity and
specificity in recognizing
conformational and
modified epitopes
Rabbit New Zealend
larger antibody repertoire
Immunotolerant to
cell surface Ag on
mouse ES cells
METHODS
Injected
subcutaneously with
108 mES D3 cell line
The mES cell lines D3 were cultured on mitomycin Ctreated 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
Spleen
240E-1 cell
line
hibridoma
centrifuge
Supernatant
hibridoma
mES cells culture in MEF
FITC conjugated
goat anti-rabbit
sec. Ab
clone
propagated
clone
Positive clone
Culture supernatant
Cells lysate
5×108 F9 EC cells
by ultrasonication in
lysis buffer
Culture supernatant
Protein-A conjugated resin
Fluoresence
microscopy
Staining
primary and
second Ab
SDS
PAGE
Blocked with
blocking
solution
c
Western
blotting
Putative antigens eluted with a
2.5 mM citrate solution
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.
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 GMCSFR 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
Thank You