Fig 1.
A. The c.Ab. is covalently bound to a magnetic bead that is incubated with sample and fluorescein-tagged d.Ab. This sample is introduced into a flow cell capped with a piezo-electric membrane coated with an anti-fluorescein antibody. An electromagnet above the membrane is controlled with embedded software. B. Upon magnetic perturbation, all beads (black circles) move towards the membrane (Bi), and beads with a completed immunocomplex (black circles coated with red dots) bind to the anti-fluorescein antibody (Bii). After the field is removed and flow restored, only beads with a completed immunocomplex remain bound (Biii). These beads alter the oscillation of the membrane (represented as orange sinusoidal curve), which is interpreted as signal in arbitrary units [36]. See S1 Video. In B, the solid blue line labeled Pos refers to beads in the presence of antigen, and the dotted red line labeled Neg refers to beads in the absence of antigen.
Fig 2.
A) Optimal antibody pairs were identified with a “checkerboard” assay that evaluated different antibody pairs as well as isotype controls. Metric plotted here is signal difference between the negative control and 1 ng/mL recombinant PAP. The different antibodies are defined in the Materials and Methods section. B) A full calibration curve illustrates the different sensitivities of different antibody pairs for PAP. Pair 1: Cos2 c.Ab.; Cos1 d.Ab. Pair 2: RDPo c.Ab.; RDMo. d.Ab. The red error bars represent the standard deviation of at least three replicate measurements. C) Reproducibility from day to day is <8%. D) The piezo-based approach shows 3 log orders improvement in analytical sensitivity versus direct ELISA when identical antibody pairs are used (Cos1/Cos2).
Table 1.
The best-matched pair with resulting dynamic range, reference range, and dilution factors are shown for the 7 biomarker assays as well as representative intra-assay variation values.
Fig 3.
A) Percent recovery for three representative biomarkers. Matrix effects can either dampen signal (CA1, PAP) or inflate signal (SPARC). Ideal dilution factors were dependent on sensitivity of the assay and reference range of the biomarker (Table 1). Black dashed line indicates 100% spike recovery. B) Bland-Altman plot [38] validating specimen integrity of a subset (N = 35) of the clinical samples with high sample volumes. Red dashed line indicates 95% confidence interval.
Fig 4.
Raw values with means for BPH, CaP, and post-surgery (PS) samples. Biomarkers include tPSA (A), fPSA(B), CA1 (C), PAP (D), IL6-sr (E), SPARC (F), and SPON2 (G). Horizontal lines indicate mean values.
Fig 5.
A) Mean values with standard deviation for the seven biomarkers with BPH, CaP, and post-surgery [39] samples. Units are ng/mL. The p value reported here is the significance between the BPH and CaP samples. AUC values are also given and report discrimination between CaP and BPH. Lower panel presents ROC curves for PSA, SPON2, and PSA OR SPON2. The OR operator increases the AUC to 0.84 from 0.80 for PSA alone.