Validation of a hypoxia-inducible factor-1 alpha specimen collection procedure and quantitative enzyme-linked immunosorbent assay in solid tumor tissues |
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Authors: | Sook Ryun Park Robert J Kinders Sonny Khin Melinda Hollingshead Smitha Antony Ralph E Parchment Joseph E Tomaszewski Shivaani Kummar James H Doroshow |
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Institution: | 1. Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MD 20892, USA;2. Laboratory of Human Toxicology and Pharmacology, Applied/Developmental Research Directorate, Leidos Biomedical Research, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA;3. Biological Testing Branch, Developmental Therapeutics Program, Frederick National Laboratory for Cancer Research, Frederick, MD 20892, USA;4. Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA |
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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. |
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Keywords: | Hypoxia-inducible factor HIF-1α Quantitative ELISA Pharmacodynamics Solid tumors |
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