What is Osteopontin?. Future Work. OPN’s structure (5). Monoclonal Antibodies to Osteopontin: Generation and Characterization Tanya Gordonov, Christian C. Kazanecki, Josephine Cassella, Lotte Schack, Bhumika Desai, David T. Denhardt. Procedures. Results.
OPN’s structure (5)
Monoclonal Antibodies to Osteopontin: Generation and CharacterizationTanya Gordonov, Christian C. Kazanecki, Josephine Cassella, Lotte Schack, Bhumika Desai, David T. Denhardt
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.
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
*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.
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
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.
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.
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.
An example of a developed Western.
The bands show where an antibody bound
An example of an ELISA plate.