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


CONCLUSIONS
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.
Sobell Department of Motor Neuroscience and Movement Disorders, UCL Institute of Neurology, University College London, London WC1N3BG, UK. clu@ion.ucl.ac.uk
Ching-Hua
CH
Lu
Bernadett
B
Kalmar
Andrea
A
Malaspina
Linda
L
Greensmith
Axel
A
Petzold
D000367:Age Factors; D000818:Animals; D002614:Chelating Agents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D051379:Mice; D008822:Mice, Transgenic; D009154:Mutation; D016900:Neurofilament Proteins / Q000097:blood* / Q000187:drug effects; D010949:Plasma / Q000187:drug effects / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000097:blood* / Q000235:genetics; D014508:Urea / Q000494:pharmacology
D002614:Chelating Agents; D016900:Neurofilament Proteins; C051774:neurofilament protein H; D004533:Egtazic Acid; D014508:Urea; D004492:Edetic Acid; C109415:SOD1 G93A protein; D013482:Superoxide Dismutase