Development of fragment-specific osteopontin antibodies and ELISA for quantification in human metastatic breast cancer.
BACKGROUND
Osteopontin (OPN) is associated with human cancers, and circulating blood OPN may have diagnostic or prognostic value in clinical oncology.


METHODS
To evaluate OPN as a cancer biomarker, we generated and characterized five novel mouse monoclonal antibodies against the human full-length OPN (fl-OPN). Epitopes recognized by four antibodies (2C5, 2F10, 2H9, and 2E11) map to N-terminal OPN (aa1-166); one (1F11) maps to C-terminal OPN (aa167-314). These antibodies recognize recombinant and native OPN by ELISA and immunoblot, cross reacting with human and mouse OPN. Two of these novel antibodies (2F10 and 1F11) were used to develop a quantitative enzyme linked immunosorbent assay (ELISA) for fl-OPN.


RESULTS
In comparison with commercially available ELISAs, our assay had high accuracy in measuring fl-OPN standards, and high sensitivity. Specifically, our ELISA has a linear dose response between 0.078 ng/ml-10 ng/ml, with a sensitivity of 13.9 pg/ml. We utilized this assay to quantify fl-OPN in the plasma of healthy volunteers in comparison with patients with metastatic breast cancer. The average circulating plasma fl-OPN in healthy volunteers was 1.2 ng/ml, compared to 4.76 ng/ml in patients with metastatic breast cancer (p = 0.0042). Although the increase in fl-OPN in cancer patients is consistent with previous studies, the measured quantity varied greatly between all existing fl-OPN ELISAs.


CONCLUSIONS
Because OPN is a complex molecule with diversity from alternative splicing, post-translational modification, extracellular proteolytic modification, and participation in protein complexes, we suggest that further understanding of specific isoform recognition of multiple OPN species is essential for future studies of OPN biomarker utility.
Plumer
Alicia
A
Dept. Applied Medical Sciences, Univ, Southern Maine, P,O, Box 9300 Portland, ME 04104, USA. alicia.plumer@maine.edu
Duan
Hongyi
H
Subramaniam
Sripriya
S
Lucas
F Lee
FL
Miesfeldt
Susan
S
Ng
Ah-Kau
AK
Liaw
Lucy
L
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000302:isolation & purification; D000915:Antibody Affinity; D014408:Biomarkers, Tumor / Q000097:blood / Q000276:immunology; D001943:Breast Neoplasms / Q000097:blood / Q000175:diagnosis* / Q000473:pathology; D044584:Carcinoma, Ductal / Q000097:blood / Q000175:diagnosis* / Q000473:pathology; D018275:Carcinoma, Lobular / Q000097:blood / Q000175:diagnosis* / Q000473:pathology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D009362:Neoplasm Metastasis; D053495:Osteopontin / Q000032:analysis* / Q000097:blood / Q000276:immunology*; D010446:Peptide Fragments / Q000276:immunology; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; D010446:Peptide Fragments; D011933:Reagent Kits, Diagnostic; D053495:Osteopontin