1. Monoclonal Antibodies Specific for DSVVYG Peptide of Human Osteopontin

2. Functional Domain Structure of Osteopontin Immunogen to target cryptic site of OPN: DSVVYG 3. Assays: Indirect ELISA Peptide Recombinant N-term OPN Recombinant Full Length OPN Native OPN (purified from human breast milk), evaluated on SDS-PAGE 4. Clones Selected for Further Study Summary of Indirect ELISAof Selected Clones 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 03H05 09C11 10E02 11H05 13F04 14C08 15C02 16A05 16A11 Clone designation A(450) DSSVYG-BSA recN-terminal OPN recflOPN Native OPN 5. Assays: Sandwich ELISA Nine selected peptide clones as tracer with current 5 MABS as capture, rec-flOPN and native OPN as antigen Nine selected peptide clones as capture with current 5 MABS as tracer, rec-flOPN and native OPN as antigen 6. Matched Pair ELISA, Peptide clones Tracer Match Pair Assay Panel with OPN sandwich 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 MAB193P MAB194P MAB195P MAB196P MAB197P nocoat MAB193P MAB194P MAB195P MAB196P MAB197P nocoat Capture MAb's ODvalue 03H06 09C11 10E02 11H05 13F04 14C08 15C02 16A05 16A11 MAB197 no biotin Native OPN flOPN 7. Matched Pair ELISA, Peptide clones Capture Match Pair Assay Panel w/ OPN Sandwich 9-1-10 MH 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 03H06 09C11 10E02 11H05 13F04 14C08 15C02 16A05 16A11 MAB193 nocapture 03H06 09C11 10E02 11H05 13F04 14C08 15C02 16A05 16A11 MAB193 nocapture Capture Ab's Absorbance450nm MAB193P MAB194P MAB195P MAB196P MAB197P no biotin Native OPN flOPN 8. Cell Adhesion Assays Breast Cancer Cell Line: MDA-MB-231 Percent Cellular Adhesion 0 20 40 60 80 100 120 09B05 16A09 16E12 irrelevant Ab No Mabs Antibody Percent N-terminal FL-OPN 9. Conclusions MBS has developed a panel of antibodies specific to the unique DSVVYG sequence of OPN Six clones emerged based on the data generated from ELISA and Cell Adhesion as being of most interest. Data demonstrates that using native OPN in the assays is optimal, as recombinantly expressed OPN in E. coli will not provide the post translational modifications of native OPN Samples of the six clones are available upon request 10. Next Actions Study of Peptide specific OPN clones by Western Blot of Native OPN Study of Peptide Specific OPN clones on Cell Adhesion Assays using Native OPN References: Alicia Plumer, Hongyi Duan, Sripriya Subramaniam, F Lee Lucas, Susan Miesfeldt, Ah- Kau Ng, Lucy Liaw. Development of fragment specific osteopontin antibodies and ELISA for quantification in human metastatic breast cancer BMC Cancer 2008, 8:38 (31 January 2008)