Monoclonal antibody-based screening assay for factor inhibiting hypoxia-inducible factor inhibitors

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Title
Monoclonal antibody-based screening assay for factor inhibiting hypoxia-inducible factor inhibitors
Author(s)
S H Lee; Jeong Hee Moon; Eun-Ah Cho; Seong Eon Ryu; Myung Kyu Lee
Bibliographic Citation
Journal of Biomolecular Screening, vol. 13, no. 6, pp. 494-503
Publication Year
2008
Abstract
The factor-inhibiting hypoxia-inducible factor (FIH) hydroxylates the asparagine 803 (Asn803) residue of the hypoxia-inducible factor 1α (HIF-1α), and the modification abrogates the transcriptional activity of HIF-1α. Because FIH is more active on HIF-1α than prolyl hydroxylase domain proteins under hypoxic conditions, its inhibitors have potential to be developed as anti-ischemic drugs targeting normal cells stressed by hypoxia. In this study, the authors developed the first monoclonal antibody, SHN-HIF1α, specifically to Asn803 hydroxylated HIF-1α and a sensitive assay system for FIH inhibitors using the monoclonal antibody (Mab). SHN-HIF1α showed 740 times higher affinity to the Asn803 hydroxylated HIF-1α peptide than the unmodified one. An enzyme-linked immunosorbent assay-based system using SHN-HIF1α displayed at least 30 times more sensitivity than previous methods for screening FIH inhibitors and was easily applicable to develop a high-throughput screening system. SHN-HIF1α also showed an Asn803 hydroxylation-dependent specificity to HIF-1α in cells. Taken together, the results suggest that it may be used to analyze the in vivo and in vitro activities of FIH inhibitors.
Keyword
ELISAFactor-inhibiting hypoxia-inducible factorHypoxia-inducible factor-1αInhibitor screeningMonoclonal antibody
ISSN
1087-0571
Publisher
Sage
DOI
http://dx.doi.org/10.1177/1087057108318800
Type
Article
Appears in Collections:
Division of Bio Technology Innovation > Core Research Facility & Analysis Center > 1. Journal Articles
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