Validation of a hypoxia-inducible factor-1 alpha specimen collection procedure and quantitative enzyme-linked immunosorbent assay in solid tumor tissues.
Anal Biochem
; 459: 1-11, 2014 Aug 15.
Article
em En
| MEDLINE
| ID: mdl-24799347
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.
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Texto completo:
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Base de dados:
MEDLINE
Assunto principal:
Manejo de Espécimes
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Ensaio de Imunoadsorção Enzimática
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Subunidade alfa do Fator 1 Induzível por Hipóxia
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Neoplasias
Limite:
Animals
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Female
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Humans
Idioma:
En
Ano de publicação:
2014
Tipo de documento:
Article