(M1430-06-36) Qualification of a Neurology 2-Plex B (Nf-L, GFAP) Quanterix Simoa® Assay Using a Novel Approach

Purpose: Biomarkers associated with neurodegenerative diseases and traumatic brain injuries, including Nf-L (Neurofilament light) and GFAP (Glial fibrillary acidic protein) have been identified as potential prognostic tools. Nf-L provides structural support to the axon, and elevated levels are seen following axonal damage or neuronal degeneration and are also increased following traumatic brain injury and with neurodegenerative diseases (Alzheimer’s, dementia, Parkinson’s, etc) (3,4). GFAP plays an important role in repair after CNS injury and concentrations are increased after acute brain/spinal cord injury, and in neuroinflammatory diseases (MS) (5,6). Decreases in GFAP have also been reported in Down’s Syndrome, schizophrenia, bipolar disorder, and depression (7). Quanterix Neurology 2-Plex B (N2PB) Assay Kits measure both Nf-L and GFAP simultaneously in human serum, plasma, and CSF matrices in the fully automated Simoa® HD-X instrument. To qualify the method quantitative ranges in each matrix, high concentrations of proteins are required to achieve the necessary ranges, while maintaining the matrix integrity. However, obtaining reference material for both analytes with a high concentration to prepare controls in each matrix was not an option. Therefore, a novel approach was utilized when, during screening of individuals, a high endogenous concentration for both Nf-L and GFAP was observed in one human CSF individual. The CSF individual was used to prepare controls in human serum, plasma, and CSF to qualify the assay successfully on the Simoa® instrument.

Methods: Neurology 2-Plex B (N2PB) Assay kits were procured from Quanterix (Billerica, MA). Normal human serum and plasma were procured from BioIVT (Westbury, NY). Normal human cerebrospinal fluid (CSF) was procured from BioChemed (Winchester, VA). The method was conducted according to the manufacturer’s recommendations and qualified according to current industry standards and regulatory guidance (FDA, 2018), (EMEA, 2011). A 3-step assay was performed autonomously in the HD-X analyzer, where a capture antibody coated bead was incubated with the sample in a cuvette. Target molecules (Nf-L, GFAP) that are present bind to the coated beads. After washing, biotinylated detector antibodies are added, mixed, and incubated with the beads. The detector antibodies will bind to the Nf-L and GFAP captured on the beads. After another wash, streptavidin-β-galactosidase (SBG) is mixed with the beads and binds to the biotinylated detector antibodies, resulting in enzyme labeling of captured Nf-L and GFAP. Following a final wash, the beads are resuspended in a resorufin β-D-galactopyranoside (RGP) substrate solution and transferred to the Simoa® disk. Individual beads are sealed within microwells in the array. If the target has been labeled and captured on the bead, SBG hydrolyzes the RGP substrate into a fluorescent product that will provide the signal (AEB, average enzymes per bead) for measurement. Back-calculated concentrations (BCC) of analyte are generated using an appropriate fit model (e.g., 4PL fit model), The minimum required volume of sample for one run in serum/plasma is 65 µL and for CSF is 7 µL and one plate can run 34 samples.

Results: A high endogenous concentration CSF individual was run at multiple dilutions on the HD-X instrument to measure the concentration of both Nf-L and GFAP in order to utilize it as the reference material for the assay’s qualification. The qualified calibration curve range is from 0.318 to 336.00 pg/mL for Nf-L and from 8.47 to 8,733.00 pg/mL for GFAP. For human plasma, the qualified ranges are from 2.56 to 834.60 pg/mL for Nf-L and from 52.90 to 20,732.50 pg/mL for GFAP. For human serum, the qualified ranges are from 3.48 to 945.40 pg/mL for Nf-L and from 62.60 to 25,053.40 pg/mL for GFAP. For human CSF, the qualified ranges are from 19.60 to 10,793.30 pg/mL for Nf-L and from 425.70 to 258,877.70 pg/mL for GFAP. Selectivity was tested in 10 normal human serum and plasma individuals, and all passed acceptance criteria. Dilutional linearity was prepared by spiking the ULOQ concentration into 4 normal human serum and plasma individuals and diluting from 1/1-1/64, all samples measuring within the curve range passed acceptance criteria. Both analytes in human serum, plasma, and CSF were stable up to 24 hours at both room temperature and refrigerated. Both analytes in human serum, plasma, and CSF were stable up to 5 freeze/thaw cycles. Both analytes in human serum, plasma, and CSF were stable for up to 6 months for long term stability at -80°C.

Conclusion: A multianalyte method for the quantitation of Nf-L and GFAP in human serum, plasma, and CSF was qualified using a novel approach and meets current industry standards for all parameters evaluated. This method will provide data that may help discern subtle changes in responses to therapeutic treatments and improve diagnostic and prognostic efforts in various neurodegenerative diseases and following traumatic brain or spinal cord injuries. This method can help streamline clinical trials by providing decision makers with high-quality biomarker data which can lead to a more targeted and effective way of treating patients and improving their lives.

References: 1. US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research. (2018) “Guidance for Industry: Bioanalytical Method Validation”. US FDA, Rockville, MD.
2. European Medicines Agency, Committee for Medicinal Products for Human Use, 192217 (2011) “Guideline on Bioanalytical Method Validation”, London, UK
3. Shilpa Narayanan, Akshay Shanker, Tanvi Khera, and Balachundhar Subramaniam. Neurofilament light: a narrative review on biomarker utility. 2021 May 7. doi: 10.12703/r/10-46. PMID: 34131656; PMCID: PMC8170685.
4. Gaetani L, Blennow K, Calabresi P, et al. Neurofilament light chain as a biomarker in neurological disorders Journal of Neurology, Neurosurgery & Psychiatry 2019;90:870-881.
5. Zhihui Yang and Kevin K.W. Wang. Glial Fibrillary acidic protein: From intermediate filament assembly and gliosis to neurobiomarker. 2015 May 11. Doi: 10.1016/j.tins.2015.04.003. PMID: 25975510; PMCID: PMC4559283.
6. Abdelhak, A., Foschi, M., Abu-Rumeileh, S. et al. Blood GFAP as an emerging biomarker in brain and spinal cord disorders. Nat Rev Neurol 18, 158–172 (2022). https://doi.org/10.1038/s41582-021-00616-3
7. Johnston-Wilson, N., Sims, C., Hofmann, JP. et al. Disease-specific alterations in frontal cortex brain proteins in schizophrenia, bipolar disorder, and major depressive disorder. Mol Psychiatry 5, 142–149 (2000). https://doi.org/10.1038/sj.mp.4000696

Characterizing high endogenous CSF individuals to measure Nf-L and GFAP concentrations.

Qualification Executive Summary of Results

Simoa Multiplex Assay Format