To compare different neurofilament light (NfL) chain protein preparations in various assay platforms.

NfL is considered a biomarker of neuro-axonal injury in neurological diseases and a possible surrogate marker in MS. However, most studies are based on a single assays and thorough comparison between measurement techniques are missing.

We used the following NfL preparations: a commercially available bovine NfL from Uman Diagnostics (Uman-NfL), a commercially available recombinant human NfL from Origene (Origene-NfL) and a human NfL expressed in E. coli (hurec-NfL). NfL was detected by ELISA (Uman Diagnostics), Western Blot, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and single molecule array (SIMOA) platform. NfL was diluted in appropriate buffers and Origene-PC was also spiked into 2 sera at different concentrations (200, 20, 2, and 0.2 ng/mL) and tested in SIMOA. Similarly, spiked serum samples with concentrations of 30, 3.3, 0.37, and 0.04 ng/mL were tested in ELISA.

In LC-MS/MS Origene NfL could be detected but not Uman-NfL (hurec-NfL not done). In ELISA Uman-NfL and hurec-NfL returned expected concentrations, but Origene-NfL gave no signal. In western blotting there were NfL-specific bands for Origene-NfL and hurec-NfL but not for Uman-NfL. Average results for spiked samples in SIMOA were 10, 1.5, 0.15, and 0.03 ng/mL, i.e. 8-20 fold less than expected. ELISA results for spiked samples were 3.02, 0.4, 0.1, and 0.08 ng/mL, i.e. 0.5-10 fold less than expected.

There is a striking difference for detection of NfL between various assay platforms. Posttranslational modifications, such as phosphorylation, glycosylation, nitration, oxidation and ubiquitylation might explain some of the observations but not the fact that ELISA was unable to detect pure Origine-NfL whereas it was detected if spiked into serum. This might hint to strong matrix effects or a specificity issue with the assay reagents. Further studies for cross-platform NfL assay validation are needed.