A quantitative homogeneous assay for fragile X mental retardation 1 protein

Journal of Neurodevelopmental Disorders

Andreas Weiss²  Bernd Gerhartz¹  Francesco di Giorgio³  Sandra Kapps-Fouthier¹  Dorothee Bleckmann³  Gabi Schutzius³ 

[1] Center for Proteomic Chemistry, Novartis Pharma AG, Novartis Institutes for Biomedical Research, Postfach, Basel, CH-4002, Switzerland;Present address: IRBM Promidis, Via Pontina Km 30.600, Pomezia, Roma, 00040, Italy;Developmental and Molecular Pathways, Novartis Pharma AG, Novartis Institutes for Biomedical Research, Postfach, Basel, CH-4002, Switzerland

关键词: Immunoassay;    Time-resolved Förster’s resonance energy transfer;    FMRP;    Fragile X syndrome;   

Others  :  811349
DOI  :  10.1186/1866-1955-5-8

 received in 2012-12-05, accepted in 2013-03-15,  发布年份 2013

【 摘 要 】

Background

Hypermethylation of the fragile X mental retardation 1 gene FMR1 results in decreased expression of FMR1 protein FMRP, which is the underlying cause of Fragile X syndrome – an incurable neurological disorder characterized by mental retardation, anxiety, epileptic episodes and autism. Disease-modifying therapies for Fragile X syndrome are thus aimed at treatments that increase the FMRP expression levels in the brain. We describe the development and characterization of two assays for simple and quantitative detection of FMRP protein.

Method

Antibodies coupled to fluorophores that can be employed for time-resolved Förster’s resonance energy transfer were used for the development of homogeneous, one-step immunodetection. Purified recombinant human FMRP and patient cells were used as control samples for assay development.

Results

The assays require small sample amounts, display high stability and reproducibility and can be used to quantify endogenous FMRP in human fibroblasts and peripheral blood mononuclear cells. Application of the assays to FXS patient cells showed that the methods can be used both for the characterization of clinical FXS patient samples as well as primary readouts in drug-discovery screens aimed at increasing endogenous FMRP levels in human cells.

Conclusion

This study provides novel quantitative detection methods for FMRP in FXS patient cells. Importantly, due to the simplicity of the assay protocol, the method is suited to be used in screening applications to identify compounds or genetic interventions that result in increased FMRP levels in human cells.

【 授权许可】

   
2013 Schutzius et al.; licensee BioMed Central Ltd.

【 图 表 】

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