Fig. 1

Anti-OPNc monoclonal antibody validation. A Solid-phase ELISA for antibody-binding to GST-Osteopontin splice variants. B2, F1, and F2 are individual clones of the rabbit monoclonal antibodies to OPN-c. The antibody ahOPNc is the polyclonal chicken antibody to OPN splice variant-c. The antibody MAB193P recognizes exon 4. The y-axis indicates absorbance. The antibodies were tested for binding to OPN-a (left panel), OPN-b (middle panel), or OPN-c (right panel). B The antibodies utilized in A) were tested in further solid-phase ELISA binding to the distinct splice variants of GST-Osteopontin. C Antibody neutralization of soft agar colony formation. Stably transfected MCF-7 OPN-c cells were plated in soft agar under standard conditions [31]. On day 0, 2 µg of antibody (clone B2) was added per plate and 0.6 µg every other day with medium (the control received only medium). The clone sizes were measured on day 11 as relative units [32]. The y-axis is displayed on logarithmic scale. The error bars are mean ± sem. The difference is significant according to the t test (p = 0.004). The other antibodies gave similar results (not shown). D Antibody validation in immunohistochemistry of breast cancers. The upper panel of slides is stained with anti-OPN-exon-4 antibody MAB193P (5× and 20× magnification, followed by a zoomed-in 20×), while the lower panel slides are probed with anti-OPN-c antibody clone F2 (5× and 20× magnification, followed by a zoomed-in 20×)