1. Creating and Characterizing Monoclonal Antibodies Against Native Human Milk OPN Josephine Cassella, Tanya Gordonov, Christian C. Kazanecki, Bhumika Desai, David T. Denhardt What is Osteopontin? Future Work ProceduresResults 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. OPN’s modifications, receptors, and structure allow it to take on many various roles in the body. 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) 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.. 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. Various experiments performed to characterize the antibodies 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 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. 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. Our Goals How antibody-producing hybridoma cells are created (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.htmwww.cmb.lu.se/ctb/html/OPN.htm (6)http://www.uccs.edu/~rmelamed/MicroFall2002/Chapter%2017/ch17.htmhttp://www.uccs.edu/~rmelamed/MicroFall2002/Chapter%2017/ch17.htm 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. *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 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. 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 OPN’s structure (5) An example of a developed Western. The bands show where an antibody bound to OPN. (6) Magnetic beads coupled to protien G