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

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(SM135)) and hyperphosphorylated (NfH(SM134)) 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 0.5 M urea in Barbitone-EDTA buffer at room temperature resolved the hook effect without compromising the ELISA. In SOD1(G93A) mice, median levels of NfH(SM134) were over 10-fold and NfH(SM135) 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. (C) 2010 Elsevier B.V. All rights reserved.