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. 2014 Jan 5;5(2):115-24.
doi: 10.7150/jca.8084. eCollection 2014.

An ultra-sensitive immunoassay for quantifying biomarkers in breast tumor tissue

Carol B Fowler  1 , Yan-Gao Man  2 , Jeffrey T Mason  1
Affiliations

An ultra-sensitive immunoassay for quantifying biomarkers in breast tumor tissue

Carol B Fowler et al. J Cancer. .

Abstract

Urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) have been validated at the highest level of evidence as clinical biomarkers of prognosis in breast cancer. The American Society of Clinical Oncology recommends using uPA and PAI-1 levels in breast tumors for deciding whether patients with newly diagnosed node-negative breast cancer can forgo adjuvant chemotherapy. The sole validated method for quantifying uPA and PAI-1 levels in breast tumor tissue is a colorimetric ELISA assay that takes 3 days to complete and requires 100-300 mg of fresh or frozen tissue. In this study we describe a new assay method for quantifying PAI-1 levels in human breast tumor tissue. This assay combines pressure-cycling technology to extract PAI-1 from breast tumor tissue with a highly sensitive liposome polymerase chain reaction immunoassay for quantification of PAI-1 in the tissue extract. The new PAI-1 assay method reduced the total assay time to one day and improved assay sensitivity and dynamic range by >100, compared to ELISA.

Keywords: breast cancer; high-pressure tissue extraction; immunoassay; immunoliposome-PCR; plasminogen activator inhibitor type-1; tissue biomarkers.

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Conflict of interest statement

Competing Interests: Carol B. Fowler and Jeffrey T. Mason are named as lead contributors on two patents (USPTO numbers: 20130011854 and 20100136613) entitled “Pressure-Assisted Antigen Retrieval (PAAR), Pressure-Assisted Molecule Recovery (PAMR), and Pressure-Assisted Tissue Histology (PATH)”. Jeffrey T. Mason is named as a lead contributor on two patents (USPTO numbers: 20090176250 and 20050158372) entitled “Immunoliposome-Nucleic Acid Amplification (ILNAA) Assay”. The assignee on all four patents is the United States of America as represented by the Secretary of the Army and/or the Secretary of Veterans Affairs.

Figures

Figure 1
Figure 1
Diagram showing a cross-section of the liposome detection reagent. The reporter DNA (green and red) is encapsulated inside the liposome (yellow). The polyethylene glycol polymers (green) with biotin receptors (red) are conjugated to phospholipids located within the outer surface of the liposome.
Figure 2
Figure 2
Assay of PAI-1 concentration standards by colorimetric ELISA and ILPCR. A) Colorimetric ELISA using the Sekisui Diagnostics ELISA assay kit performed by following the manufacturer's instructions. B) ILPCR assay performed suing PAI-1 standards from the ELISA assay, but with additional serial dilutions of 100, 10, and 1 pg/mL. The Streptavidin-HRP conjugate with colorimetric development was replaced with NeutrAvidin plus detection liposomes with qPCR quantification. Data points (black dots), standard deviations (blue bars), regression analysis (solid orange plot lines), ILPCR assay blank (solid red line), IPCR assay threshold (magenta dotted line).
Figure 3
Figure 3
Assay of PAI-1 in human breast tumor tissue extracts. A) Conventional ELISA and ILPCR assays of PAI-1 concentration in total protein extracts derived from 1 to 150 mg of tumor tissue. Total protein extracts were prepared by conventional overnight stirring at 4°C for the ELISA assay and by pressure-assisted protein extraction for the ILPCR assay. B) The PAI-1 values plotted after being normalized to the total protein concentration of the corresponding tissue extracts. Results from the ELISA assay as shown as black squares and results from the ILPCR assay are shown as black dots. Standard deviations are shown as horizontal blue bars. The orange line in A shows the linear regression fit of the data and the magenta line in B is the average normalized PAI-1 value for all of the tumor tissue samples analyzed.
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References

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