Transcript OPN also has been found to have a role in: Cancer cell metastasis (4)

Creating and Characterizing Monoclonal Antibodies Against Native Human Milk OPN
Josephine Cassella, Tanya Gordonov, Christian C. Kazanecki, Bhumika Desai, David T. Denhardt
Procedures
What is Osteopontin?
Osteopontin, or OPN, is a protein that is secreted
by cells and is found in all body fluids and in the ECM
of bone and other mineralized tissue (1). It is secreted
in high levels by kidney, bone, mammary gland, and
epithelial tissue, as well as immune cells. It is posttranslationally phosphorylated and glycosylated, and is
conserved among vertebrates.
OPN’s modifications, receptors, and structure allow
it to take on many various roles in the body.
How antibody-producing hybridoma cells are created
The HAT medium that the fused cells
are placed in selects against any
myeloma-myeloma fusions, and since
spleen-spleen fusions have a limited
lifespan, only myeloma-spleen
fusions survive.
Some of OPN’s roles include:
•Bone remodeling
•Inflammatory and immune responses
•Cell signaling (2)
•Anti-apoptotic (pro-survival) signal (3)
OPN also has been found to have a role in:
•Cancer cell metastasis (4)
•Various bone diseases (e.g. osteoporosis)
•Various vascular diseases (e.g. stroke and
atherosclerosis)
•Autoimmune disease (1)
Results
During the past year that I have worked at the lab we have been
able to identify several new monoclonal antibodies that bind to OPN.
We have also been able to locate them to a specific peptide or several
peptides where their epitope may be located.
The OPN diagram below (adapted from Sodek et al. Crit Rev Oral
Biol Med. Vol 11: 279-303 (2000)) shows some of our results so far –
the antibodies have been mapped to specific peptides by peptide
assays. We continue to test all the existing monoclonal antibodies and
plan to test additional antibodies using the methods mentioned earlier..
(6)
*During the expansion process
a lot of the cells die at different
stages. This is because of the
genetic instability of the fused
cells (too much DNA). Only
genetically stable cells survived*
The hybridomas are placed into a 96-well plate and
expanded to 24 and 6 well plates if growth is shown.
The cells are finally grown up in 10cm plates and some
are frozen for future use.
The antibody-containing medium is then concentrated
using filters from about 50 mL to 0.5 – 1.0 mL. These
concentrated samples are the ones we use in our
experiments.
Future Work
Our Goals
Various experiments performed to characterize the antibodies
WHAT?
We are generating monoclonal antibodies
to OPN, produced by hybridoma cells, and
characterizing where on the OPN molecule
they bind. We especially want antibodies that
bind to a phosphorylated section of the protein.
WHY?
Since OPN seems to have a variety of
functions in the body under normal and
diseased conditions, and many depend on its
post-translational modifications, having an
antibody that binds to a phosphorylated
epitope and possibly inhibits OPN’s actions will
be a very interesting find and could have a
wide range of applications.
ELISA (Enzyme Linked ImmunoSorbent Assay):
Performed to see how well an antibody binds to
modified (Native) and non-modified (Recombinant)
forms of OPN.
Fluorescence Assay:
This is the assay that I have
been concentrating on.
1. Protein G is coupled to
magnetic beads
2. mAbs are added to the
protein G/magnetic beads
(protein G binds IgG at the
Fc portion leaving the
antigen binding site
available).
3. Fluorescently labeled OPN
is added to the beads.
4. The antibody/antigen
complex is cleaved from the
beads by using low pH
solution.
5. The supernatant is added to
a fluorescent reading plate
and fluorescence is measured.
WESTERN BLOTTING:
Used to test whether an antibody binds
to Native or Recombinant OPN
•Run Native and Recombinant OPN in
SDS PAGE gel
•Transfer OPN in gel onto a PVDF
membrane
•Add antibodies to the strips of the
membrane
•Add a secondary antibody (same as in
ELISA)
•Add a chemiluminescence reagent
•Develop on film
•A band will show up only if the antibody
bound to the protein
Magnetic beads coupled to protien G
Acknowledgements
This research was supported by funds from a Busch Biomedical Research Award,
the Rutgers Technology Commercialization fund, and by a grant from the National
Multiple Sclerosis Society. I would like to thank Dr. David T. Denhardt for all of his help
and guidance on this project, and Dr. Laura Lorentzen for assistance in editing this
project paper write up. I would like to thank Tany Gordonov for her poster work,
Christian Kazanecki, Melissa Weidner, Bhumika Desai, Tanya Gordonov, Sara
Ghobraiel and Nina Ren for their help with assays and procedures
References
An example of a developed Western.
The bands show where an antibody
bound
to OPN.
OPN’s structure (5)
In the near future we intend to continue characterizing our existing
monoclonal antibodies. We are also going to immunize mice with
phosphorylated peptides instead of with the whole protein in order to induce
the mice to produce antibodies to the specific phosphorylated regions of
OPN. After these mouse spleens are used in fusions, we will test the new
monoclonal antibodies against newly generated phosphorylated peptides.
Once we have a collection of antibodies and know the region where they
bind we will start testing to see if they bind to a functional part of the protein
and if so what are the consequences of an antibody impeding that function.
(1) Weidner M. Developing and Characterizing Novel Anti-Osteopontin Monoclonal Antibodies Against
Post-Translational Modifications. G.H. Cook Honors Thesis. April 11, 2006
(2) Khan et al., Enhanced cell surface CD44 variant (v6, v9) expression by osteopontin in breast
cancer epithelial cells facilitates tumor cell migration: Novel post-transcriptional, post-translational
regulation.Clin Exp Metastasis. 2006 May 12.
(3) Denhardt D.T. et al, Osteopontin as a means to cope with environmental insults: regulation
of inflammation, tissue remodeling, and cell survival. J Clin Invest. 2001 May;107(9):1055-61.
(4) Nemoto H et al., Osteopontin deficiency reduces experimental tumor cell metastasis to bone and
soft tissues. J Bone Miner Res. 2001 Apr;16(4):652-9.
(5) www.cmb.lu.se/ctb/html/OPN.htm
(6) http://www.uccs.edu/~rmelamed/MicroFall2002/Chapter%2017/ch17.htm