This document describes the development of monoclonal antibodies that target the DSVVYG peptide sequence of human osteopontin. Several antibody clones were selected and tested in ELISA and cell adhesion assays. Six antibody clones emerged as most promising based on their ability to detect recombinant and native osteopontin in ELISA and influence breast cancer cell adhesion. Further analysis is planned using these six clones, including western blotting of native osteopontin and additional cell adhesion assays using native osteopontin to better understand post-translational modifications.
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
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)
Notes de l'éditeur
Additional studies currently underway include a western blot analysis using native purified OPN against all six of the selected clones as well as cell adhesion assays using the native OPN. If the DSVVYG peptide is linear, the western blot data will shed light on the integrity of the purified native OPN by immunostaining full length and /or OPN fragments containing the DSVVYG, indicated by molecular weight of bands. Additionally, the ability of these clones to inhibit cell adhesion by binding to native OPN will be of significant value to cancer researchers and is the motivation to continue these studies. Results of the extended studies will be made available at a later date. Please contact Sales to access samples of this unique panel of Osteopontin Monoclonal antibodies.