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.

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)