Validation of a hypoxia-inducible factor-1 alpha specimen collection procedure and quantitative enzyme-linked immunosorbent assay in solid tumor tissues.

Abstract

Hypoxia-inducible factor-1 alpha (HIF-1α) is an important marker of hypoxia in human tumors and has been implicated in tumor progression. Drugs targeting HIF-1α are being developed, but the ability to measure drug-induced changes in HIF-1α is limited by the lability of the protein in normoxia. Our goal was to devise methods for specimen collection and processing that preserve HIF-1α in solid tumor tissues and to develop and validate a two-site chemiluminescent quantitative enzyme-linked immunosorbent assay (ELISA) for HIF-1α. We tested various strategies for HIF-1α stabilization in solid tumors, including nitrogen gas-purged lysis buffer, the addition of proteasome inhibitors or the prolyl hydroxylase inhibitor 2-hydroxyglutarate, and bead homogenization. Degassing and the addition of 2-hydroxyglutarate to the collection buffer significantly increased HIF-1α recovery, whereas bead homogenization in sealed tubes improved HIF-1α recovery and reduced sample variability. Validation of the ELISA demonstrated intra- and inter-assay variability of less than 15% and accuracy of 99.8±8.3% as assessed by spike recovery. Inter-laboratory reproducibility was also demonstrated (R(2)=0.999). Careful sample handling techniques allow us to quantitatively detect HIF-1α in samples as small as 2.5μg of total protein extract, and this method is currently being applied to analyze tumor biopsy specimens in early-phase clinical trials.

• Authors: Sook Ryun Park, Robert J Kinders, Sonny Khin, Melinda Hollingshead, Smitha Antony, Ralph E Parchment, Joseph E Tomaszewski, Shivaani Kummar, James H Doroshow
• Journal: Analytical biochemistry (Anal Biochem) Vol. 459 Pg. 1-11 (Aug 15 2014) ISSN: 1096-0309 [Electronic] United States
• PMID: 24799347 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Validation Study)
• Copyright: Copyright © 2014 Elsevier Inc. All rights reserved.

Chemical References

• Biomarkers, Tumor
• Hypoxia-Inducible Factor 1, alpha Subunit

Topics

• Animals
• Biomarkers, Tumor (metabolism)
• Cell Line, Tumor
• Cell Transformation, Neoplastic
• Enzyme-Linked Immunosorbent Assay
• Female
• Humans
• Hypoxia-Inducible Factor 1, alpha Subunit (metabolism)
• Mice
• Neoplasms (pathology)
• Specimen Handling (methods)