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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.

SUBMITTER: Park SR

PROVIDER: S-EPMC4810780 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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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.

Park Sook Ryun SR Kinders Robert J RJ Khin Sonny S Hollingshead Melinda M Antony Smitha S Parchment Ralph E RE Tomaszewski Joseph E JE Kummar Shivaani S Doroshow James H JH

Analytical biochemistry 20140504

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 immunoso ...[more]

PMID: 24799347

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Project description:Background:The enzyme-linked immunosorbent assay (ELISA) is an indispensable tool for clinical diagnostics to identify or differentiate diseases such as autoimmune illnesses, but also to monitor their progression or control the efficacy of drugs. One use case of ELISA is to differentiate between different states (e.g. healthy vs. diseased). Another goal is to quantitatively assess the biomarker in question, like autoantibodies. Thus, the ELISA technology is used for the discovery and verification of new autoantibodies, too. Of key interest, however, is the development of immunoassays for the sensitive and specific detection of such biomarkers at early disease stages. Therefore, users have to deal with many parameters, such as buffer systems or antigen-autoantibody interactions, to successfully establish an ELISA. Often, fine-tuning like testing of several blocking substances is performed to yield high signal-to-noise ratios. Methods:We developed an ELISA to detect IgA and IgG autoantibodies against chitinase-3-like protein 1 (CHI3L1), a newly identified autoantigen in inflammatory bowel disease (IBD), in the serum of control and disease groups (n = 23, respectively). Microwell plates with different surface modifications (PolySorp and MaxiSorp coating) were tested to detect reproducibility problems. Results:We found a significant impact of the surface properties of the microwell plates. IgA antibody reactivity was significantly lower, since it was in the range of background noise, when measured on MaxiSorp coated plates (p < 0.0001). The IgG antibody reactivity did not differ on the diverse plates, but the plate surface had a significant influence on the test result (p = 0.0005). Conclusion:With this report, we want to draw readers' attention to the properties of solid phases and their effects on the detection of autoantibodies by ELISA. We want to sensitize the reader to the fact that the choice of the wrong plate can lead to a false negative test result, which in turn has serious consequences for the discovery of autoantibodies.

Dataset Information

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

Publications

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

Similar Datasets

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