Fig 1.
Types of OPN antigens and their characterization.
A) Map of human OPN protein showing the various fragments (OPN-O, OPN-P) produced as GST fusion proteins in bacteria and as V5-tagged fusion proteins in insect cells. Numericals denote amino-acid numbering of protein. Shown are the cleavage sites of caspase-8 and various metalloproteinases (MMP-3, -7, -9), the integrin binding sites (αvβ1, αvβ3, αvβ5, α9β1 and α4β1) and the CD44-binding site. Binding sites of the pair of antibodies used in the OPN detection kit marketed by IBL, Gunma, Japan, are indicated by dashed lines. The sequence surrounding the thrombin-sensitive site is also shown. B) Demonstration of the purity of the bacterially-derived recombinant fusion proteins (OPN-O-GST and OPN-P-GST) isolated by affinity chromatography, separated on 12.5% SDS-PAGE gel and stained by Coomassie Blue. The starting material (crude lysate), the OPN-O-GST antigen (*), the OPN-P-GST antigen (#), and mol. wt. markers, are shown. C) WB results identifying the fusion proteins (OPN-O-V5 [*], OPN-P-V5 [#]) derived from insect cells. Cell lysates were separated on 12.5% SDS-PAGE gel and probed with anti-V5 antibody. Lysate of un-infected insect cells (Sf9) included as negative control.
Fig 2.
Characterization of the OPN-specific mouse mAbs.
A) ELISA results showing binding specificity of the various mAbs (mAb 659, 446, and 492) to various OPN antigens (OPN-O-GST, OPN-P-GST, OPN-O-V5, OPN-P-V5, and full-length OPN), including TCA-GST (control negative containing human telomerase segment). Anti-V5 antibody (V5) and BSA are included as positive and negative control, respectively. B) ELISA results showing titration curves of individual mAbs (mAb 659, 446, and 492) against OPN-O-GST, OPN-P-GST or full-length OPN. C) Inhibition ELISA results showing mAb 659 and 446 do not cross-inhibit each other. Left panel shows the inability of mAb 659 to inhibit binding of biotin-labeled mAb 446 and the converse for right panel. D) Western blot results showing specific detection of the various insect cells-derived OPN antigens (OPN-O-V5, OPN-P-V5) by the various mAbs; 15% gel used; Sf9 is control insect-cell lysate. E) IFA results showing specific binding by various mAbs to insect cells expressing the appropriate OPN-O-V5 or OPN-P-V5 protein (magnification: 400 x). Anti-V5 antibody (V5) and normal mouse serum (N) used as positive and negative control, respectively.
Fig 3.
mAb 659 binds to the thrombin-cleavage site of OPN.
A) ELISA results showing digestion of OPN by thrombin (0–5 U/ml), as evidenced by loss of antigenic activity determined using mAb 659 in an inhibition assay; such detection could not be made using mAb 446. B) WB results showing digestion of OPN by thrombin (0–5 U/ml) using mAb 659 (top panel) or mAb 446 (bottom panel) as probe. Digested fragment (*) revealed by mAb 446 but not mAb 659. Duplicate results shown (left and right panels). C) WB results of protection experiment showing the resistance of OPN to thrombin digestion when bound by mAb 659, using mAb 659 as probe.
Fig 4.
Detection of OPN in buffer solutions by various inhibition ELISAs.
A) Dose-dependent inhibition of binding of various mAbs to target OPN (immobilized) by full-length OPN. Data represent mean +/- SD of three samples. B) Results showing specificity of the mAb 659 and mAb 446 inhibition ELISAs. OPN proteins (OPN, OPN-O-GST, OPN-O-V5, OPN-P-GST, and OPN-P-V5) and non-OPN proteins [TCA-GST and Sb-V5 (SARS-CoV spike protein)] were used as inhibitors. C) Comparison between the mAb 659 and mAb 446 inhibition ELISA on the detection of full-length OPN produced by HL60 cells [following stimulation or absence of stimulation with phorbol myristate acetate (PMA)]. NS0-derived recombinant human full-length OPN was used as positive control. Data represent mean +/- SD of three samples.
Fig 5.
Comparison of various inhibition ELISAs and a commercial capture-ELISA in the detection of circulatory OPN from cervical cancer patients.
A) Plasma OPN levels (ng/mL) detected by the mAb 659 ELISA from cervical cancer patients (n = 33) or normal subjects (n = 31). Shaded area denotes cut-off based on mean + 2 SD of OPN levels in normal subject. B) Same as in (A) except serum used instead of plasma. C) Regression analysis between plasma and serum OPN levels found in the cervical cancer patients. D) Same as in (A) except using the mAb 446 inhibition ELISA. The top eight responders are encircled with the OPN levels shown; these measurements were used in regression analysis against the OPN levels obtained from the mAb 659 inhibition ELISA–the results are indicated. E) Same as in (D) except using the commercial capture ELISA. F) Plasma OPN levels (ng/mL) detected by the mAb 659 inhibition ELISA from a new cohort of 147 cervical cancer patients and 29 healthy subjects; also shown is a breakdown of the patient group into the four stages of disease. In all cases, each point represents mean of duplicates. G) Regression analysis between plasma OPN levels and OPN gene expression levels determined from the tumor tissues of 20 cervical cancer patients.
Fig 6.
Gel fractionation of plasma OPN from cervical cancer patients and healthy subjects.
Gel chromatography of pooled plasma from cervical cancer patients or healthy subjects using Bio-Gel of different retention limit: (A) P10 [20 kDa], (B) P30 [40 kDa], (C) P60 [60 kDa], and (D) P100 [100 kDa]. OPN antigenic content determined by mAb 659 inhibition ELISA; concentration of BSA (included as control) determined by BCA. (E) Western blot results showing the antigen detected in individual P60 gel fractions from patients or healthy people, probed by mAb 659.