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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-1?) is an important marker of hypoxia in human tumors and has been implicated in tumor progression. Drugs targeting HIF-1? are being developed, but the ability to measure drug-induced changes in HIF-1? is limited by the lability of the protein in normoxia. Our goal was to devise methods for specimen collection and processing that preserve HIF-1? in solid tumor tissues and to develop and validate a two-site chemiluminescent quantitative enzyme-linked immunosorbent assay (ELISA) for HIF-1?. We tested various strategies for HIF-1? 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-1? recovery, whereas bead homogenization in sealed tubes improved HIF-1? 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-1? in samples as small as 2.5?g of total protein extract, and this method is currently being applied to analyze tumor biopsy specimens in early-phase clinical trials.


PROVIDER: S-EPMC4810780 | BioStudies | 2014-01-01

REPOSITORIES: biostudies

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