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Open Access New method

A quantitative homogeneous assay for fragile X mental retardation 1 protein

Gabi Schutzius12, Dorothee Bleckmann12, Sandra Kapps-Fouthier3, Francesco di Giorgio12, Bernd Gerhartz3 and Andreas Weiss14*

Author Affiliations

1 Neuroscience Discovery, Novartis Pharma AG, Novartis Institutes for Biomedical Research, Postfach, Basel, CH-4002, Switzerland

2 Developmental and Molecular Pathways, Novartis Pharma AG, Novartis Institutes for Biomedical Research, Postfach, Basel, CH-4002, Switzerland

3 Center for Proteomic Chemistry, Novartis Pharma AG, Novartis Institutes for Biomedical Research, Postfach, Basel, CH-4002, Switzerland

4 Present address: IRBM Promidis, Via Pontina Km 30.600, Pomezia, Roma, 00040, Italy

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Journal of Neurodevelopmental Disorders 2013, 5:8  doi:10.1186/1866-1955-5-8

Published: 2 April 2013

Abstract

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.

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