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J Neurosci Methods. 2011 Feb 15;195(2):143-50. doi: 10.1016/j.jneumeth.2010.11.026. Epub 2010 Dec 4.

A method to solubilise protein aggregates for immunoassay quantification which overcomes the neurofilament "hook" effect.

Author information

1
Sobell Department of Motor Neuroscience and Movement Disorders, UCL Institute of Neurology, University College London, London WC1N3BG, UK. clu@ion.ucl.ac.uk

Abstract

INTRODUCTION:

Neurofilament (Nf) aggregates are a common pathological feature of neurodegenerative disorders. Although Nf levels have been investigated as a potential disease biomarker, Nf aggregates may mask Nf epitopes, preventing accurate quantification by immunoassay. Using the SOD1(G93A) mouse model of amyotrophic lateral sclerosis, we developed a method to disrupt Nf aggregates, allowing optimal immunoassay performance.

METHODS:

Phosphorylated (NfH(SMI35)) and hyperphosphorylated (NfH(SMI34)) Nf levels in plasma from 120-day SOD1(G93A) mice were quantified using an in-house ELISA modified for use with small volumes. Different pre-analytical methods were tested for their ability to solubilize Nf aggregates and immunoblotting was used for qualitative analysis.

RESULTS:

A 'hook effect' was observed for serially diluted plasma samples quantified using an ELISA originally developed for CSF samples. Immunoblotting confirmed the existence of high molecular-weight NfH aggregates in plasma and the resolving effect of timed urea on these aggregates. Thermostatic (pre-thawing) and chemical (calcium chelators, urea) pre-analytical processing of samples had variable success in disrupting NfH aggregates. Timed urea-calcium chelator incubation yielded the most consistent plasma NfH levels. A one hour sample pre-incubation with 0.5M urea in Barbitone-EDTA buffer at room temperature resolved the "hook effect" without compromising the ELISA. In SOD1(G93A) mice, median levels of NfH(SMI34) were over 10-fold and NfH(SMI35) levels 5-fold greater than controls.

CONCLUSION:

NfH aggregates can be solubilised and the "hook effect" abolished by a one-hour sample pre-incubation in a urea-calcium chelator-enriched buffer. This method is applicable for quantification of NfH phosphoforms in experimental and disease settings where Nf aggregate formation occurs.

PMID:
21134399
DOI:
10.1016/j.jneumeth.2010.11.026
[Indexed for MEDLINE]

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