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A Magnetic Bead-Based Sensor for the Quantification of Multiple Prostate Cancer Biomarkers.

Jokerst JV, Chen Z, Xu L, Nolley R, Chang E, Mitchell B, Brooks JD, Gambhir SS - PLoS ONE (2015)

Bottom Line: The results were analyzed with receiver operator characteristic curve analysis.The highest AUC discrimination was achieved with a spondin-2 OR free/total PSA operation--the area under the curve was 0.84 with a p value below 10(-6).This bead-based system offers important advantages in assay building including low cost, high throughput, and rapid identification of an optimal matched antibody pair.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiology, Molecular Imaging Program at Stanford (MIPS), Stanford University, Stanford, California, United States of America.

ABSTRACT
Novel biomarker assays and upgraded analytical tools are urgently needed to accurately discriminate benign prostatic hypertrophy (BPH) from prostate cancer (CaP). To address this unmet clinical need, we report a piezeoelectric/magnetic bead-based assay to quantitate prostate specific antigen (PSA; free and total), prostatic acid phosphatase, carbonic anhydrase 1 (CA1), osteonectin, IL-6 soluble receptor (IL-6sr), and spondin-2. We used the sensor to measure these seven proteins in serum samples from 120 benign prostate hypertrophy patients and 100 Gleason score 6 and 7 CaP using serum samples previously collected and banked. The results were analyzed with receiver operator characteristic curve analysis. There were significant differences between BPH and CaP patients in the PSA, CA1, and spondin-2 assays. The highest AUC discrimination was achieved with a spondin-2 OR free/total PSA operation--the area under the curve was 0.84 with a p value below 10(-6). Some of these data seem to contradict previous reports and highlight the importance of sample selection and proper assay building in the development of biomarker measurement schemes. This bead-based system offers important advantages in assay building including low cost, high throughput, and rapid identification of an optimal matched antibody pair.

No MeSH data available.


Related in: MedlinePlus

Assay Construction.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).
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pone.0139484.g002: Assay Construction.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).

Mentions: We first had to identify an optimal antibody pair, and the following section describes this approach using PAP as an example—results for other assays are presented in Table 1. Fig 2 shows representative data of how this was achieved for the PAP biomarker with a so-called “checkerboard assay.” Here, four different PAP antibodies as well as an isotype control were used as both the c.Ab and d.Ab. All four antibodies and an isotype control was used at antigen concentrations of 0 and 1 ng/mL. Fig 2A plots the signal difference between these two concentrations. For PAP the highest signal was with the Cos1 d.Ab and Cos2 c.Ab with a signal of 0.65 a.u. (Fig 2A). Inverting the order, i.e. Cos2 d.Ab and Cos1 c.Ab had a value of 0.43 a.u. The pair from R&D Systems had lower signal, and the isotope control antibody had signal <0.01 a.u. Thus, Cos1 and Cos2 were used for the remainder of the experiments.


A Magnetic Bead-Based Sensor for the Quantification of Multiple Prostate Cancer Biomarkers.

Jokerst JV, Chen Z, Xu L, Nolley R, Chang E, Mitchell B, Brooks JD, Gambhir SS - PLoS ONE (2015)

Assay Construction.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).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4589536&req=5

pone.0139484.g002: Assay Construction.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).
Mentions: We first had to identify an optimal antibody pair, and the following section describes this approach using PAP as an example—results for other assays are presented in Table 1. Fig 2 shows representative data of how this was achieved for the PAP biomarker with a so-called “checkerboard assay.” Here, four different PAP antibodies as well as an isotype control were used as both the c.Ab and d.Ab. All four antibodies and an isotype control was used at antigen concentrations of 0 and 1 ng/mL. Fig 2A plots the signal difference between these two concentrations. For PAP the highest signal was with the Cos1 d.Ab and Cos2 c.Ab with a signal of 0.65 a.u. (Fig 2A). Inverting the order, i.e. Cos2 d.Ab and Cos1 c.Ab had a value of 0.43 a.u. The pair from R&D Systems had lower signal, and the isotope control antibody had signal <0.01 a.u. Thus, Cos1 and Cos2 were used for the remainder of the experiments.

Bottom Line: The results were analyzed with receiver operator characteristic curve analysis.The highest AUC discrimination was achieved with a spondin-2 OR free/total PSA operation--the area under the curve was 0.84 with a p value below 10(-6).This bead-based system offers important advantages in assay building including low cost, high throughput, and rapid identification of an optimal matched antibody pair.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiology, Molecular Imaging Program at Stanford (MIPS), Stanford University, Stanford, California, United States of America.

ABSTRACT
Novel biomarker assays and upgraded analytical tools are urgently needed to accurately discriminate benign prostatic hypertrophy (BPH) from prostate cancer (CaP). To address this unmet clinical need, we report a piezeoelectric/magnetic bead-based assay to quantitate prostate specific antigen (PSA; free and total), prostatic acid phosphatase, carbonic anhydrase 1 (CA1), osteonectin, IL-6 soluble receptor (IL-6sr), and spondin-2. We used the sensor to measure these seven proteins in serum samples from 120 benign prostate hypertrophy patients and 100 Gleason score 6 and 7 CaP using serum samples previously collected and banked. The results were analyzed with receiver operator characteristic curve analysis. There were significant differences between BPH and CaP patients in the PSA, CA1, and spondin-2 assays. The highest AUC discrimination was achieved with a spondin-2 OR free/total PSA operation--the area under the curve was 0.84 with a p value below 10(-6). Some of these data seem to contradict previous reports and highlight the importance of sample selection and proper assay building in the development of biomarker measurement schemes. This bead-based system offers important advantages in assay building including low cost, high throughput, and rapid identification of an optimal matched antibody pair.

No MeSH data available.


Related in: MedlinePlus