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

Sook Ryun Park, Robert J. Kinders, Sonny Khin, Melinda Hollingshead, Smitha Antony, Ralph E. Parchment, Joseph E. Tomaszewski, Shivaani Kummar, James H. Doroshow

Research output: Contribution to journalArticlepeer-review

10 Scopus citations

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 (R2 = 0.999). Careful sample handling techniques allow us to quantitatively detect HIF-1α in samples as small as 2.5 μg of total protein extract, and this method is currently being applied to analyze tumor biopsy specimens in early-phase clinical trials.

Original languageEnglish (US)
Pages (from-to)1-11
Number of pages11
JournalAnalytical Biochemistry
Volume459
DOIs
StatePublished - Aug 15 2014
Externally publishedYes

Keywords

  • HIF-1α
  • Hypoxia-inducible factor
  • Pharmacodynamics
  • Quantitative ELISA
  • Solid tumors

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Validation of a hypoxia-inducible factor-1 alpha specimen collection procedure and quantitative enzyme-linked immunosorbent assay in solid tumor tissues'. Together they form a unique fingerprint.

Cite this