Monoclonal Antibodies to Osteopontin: Generation and Characterization Tanya Gordonov, Christian C. K

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Monoclonal Antibodies to Osteopontin Generation
and CharacterizationTanya Gordonov, Christian C.
Kazanecki, Josephine Cassella, Lotte Schack,
Bhumika Desai, David T. Denhardt

• What is Osteopontin?

Procedures
Results
Although this project is far from over,
during the 10 weeks 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 I have 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. The antibodies in blue
and green have been developed and tested during
my stay over the summer.

• 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
  post-translationally phosphorylated and
  glycosylated, and is conserved among vertebrates.
• OPNs modifications, receptors, and
  structure allow it to take on many various roles
  in the body.
• Some of OPNs 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)

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.
(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
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 if they bind to a functional
part of the protein and if so what are the
consequences of an antibody impeding that
function.
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
OPNs 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.

• 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

• PEPTIDE ASSAY
• This is the assay that I have been concentrating
  on.
• Used to test which specific peptide of OPN, if
  any, an antibody binds to
• Similar to an ELISA, only wells are coated with
  non-phosphorylated peptides instead of
  full-length OPN.
• Peptides cover the entire OPN molecule in smaller
  overlapping pieces.
• If the reading is high that means the antibody is
  binding to the peptide to some degree (we test 19
  peptides)
• This gives us an idea where on the protein the
  antibody binds

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. The
support of an Aresty Summer Fellowship to Tanya
Gordonov is gratefully acknowledged. We thank Dr.
Larry Steinman for his generous contribution of
the peptides used in this research. TG
would also personally like to thank Dean Levine
for all of her efforts to make the program
successful, the Aresty Research Center for
Undergraduates, and Dr. Denhardt for his guidance
and support.
References

• Weidner M. Developing and Characterizing Novel
  Anti-Osteopontin Monoclonal Antibodies Against
  Post-Translational Modifications. G.H. Cook
  Honors Thesis. April 11, 2006
• 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.
• 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 May107(9)1055-61.
• Nemoto H et al., Osteopontin deficiency reduces
  experimental tumor cell metastasis to bone and
  soft tissues. J Bone Miner Res. 2001
  Apr16(4)652-9.
• www.cmb.lu.se/ctb/html/OPN.htm
• http//www.uccs.edu/rmelamed/MicroFall2002/Chapte
  r2017/ch17.htm

An example of a developed Western. The bands
show where an antibody bound to OPN.
An example of an ELISA plate.