Establishment of a novel high throughput screening assay for identifying small molecular antagonists of human interleukin-6 receptor
10.7644/j.issn.1674-9960.2014.12.001
- VernacularTitle:人白介素－6受体小分子拮抗剂高通量筛选模型的建立
- Author:
Yu YAN
;
Yangyang HE
;
Chang ZHANG
;
Xiaobin PANG
;
Peng DU
;
Zhiwei SUN
;
Shuang WANG
;
Guanhua DU
- Publication Type:Journal Article
- Keywords:
receptors,interleukin-6;
antagonist;
enzyme-linked immunosorbent assay;
screening model
- From:
Military Medical Sciences
2014;(12):921-926
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish a high throughput screening assay for identifying human small molecular antagonists targeted IL-6R.Methods The full length gene of the human IL-6R extracellular region was amplified by PCR and cloned into a eukaryotic expression vector to construct recombination expression plasmid pABHis -IL6R that was then transfected transiently into HEK293T cells to prepare recombination protein IL-6R.Western blotting assay and receptor-ligand binding experiment were used to analyze the bioactivity of IL-6R.A new screening method based on ELISA was established using the function of IL-6R binding to its ligand and the characteristics of Fc fragment binding to IgG-HRP.Then Z′-factor was calculated and a known antagonist ab 47215 was used to assess the stability and reliability of the new assay .Results Recombination plasmid pABHis-IL6R was constructed and soluble IL-6R was prepared.IL-6R reported herein could be recognized by an anti-IL-6R antibody and specifically bind to its ligand in a dose response manner .A Z′-factor of 0.53 was obtained that could serve high throughput screening assay .Ab47215 , as a known specific antagonist , was able to block rhIL-6 from binding to the receptor in a dose-dependent manner in the new screening assay , the IC50 of which was (0.55 ± 0.11)μg/ml.Conclusion An innovative and easy screening assay for identifying human IL-6R antagonists is established , which might help discover potent and specific antagonists .
