(T0930-09-49) Validation of Ultra-High Sensitive SIMOA Assays for the Quantitation of Neurofilament Light Chain (NF-L) and Glial Fibrillary Acidic Protein (GFAP)

Purpose: Neurodegenerative diseases affect reportedly fifty million people in the United States each year. There are over 600 types of neurodegenerative diseases and the underlying causes can vary significantly. Over the past decade, biomarker has played more and more important role in diagnosis, prognosis and clinical research. A series of neurodegenerative biomarkers have been identified and studied using various types of technologies. Blood based biomarker testing provides a non-invasive tool to study the neurodegenerative biomarkers. However, it usually suffers from low concentration, thus, needs ultra-high sensitive assays to support. Biomarker analysis has been progressed in two directions in the past few decades. One is focusing on the increase the multiplexing capacity and the other is to improve the sensitivity. Quanterix SIMOA, for example, provide ultra-high sensitive detection and quantitation of a variety of neurodegenerative biomarkers such as neurofilament light chain (NF-L) and Glial Fibrillary Acidic Protein (GFAP). Herein, we report validation of NF-L and GFAP in human plasma/serum and CSF using Quanterix SIMOA following FDA bioanalytical validation guidance.

Methods: The assays use commercial Simoa Advantage NF-Light and GFAP Discovery assay kits provided by Quanterix. For the methods, as the first step, an anti-biomarker antibody-coated paramagnetic capture beads and an anti-biomarker antibody conjugated with biotin are incubated with samples diluted in sample diluent. Biomarker molecules present in the samples are captured by the antibody-coated capture beads. The detection antibody will then bind to the biomarker molecules. The beads are washed and a streptavidin-ß-galactosidase (SßG) conjugate is mixed with the capture beads, where the SßG binds to the biotinylated detection antibody, resulting in the enzymatic labeling of the captured biomarker. Followed by wash steps, the capture beads are re-suspended in a resorufin ß-D-galactopyranoside (RGP) substrate solution and transferred to the Simoa Disc where image was taken and data analyzed.

Results: All standards and validation samples were tested in duplicate wells. The number of standards/validation samples indicated in the following sections always refers to the total number of sets (replicates) being evaluated (note: n=1 is equivalent to 2 wells). Parameters include standard calibration model, accuracy and precision, interference, detectability and reproducibility, dilution linearity, and stability (bench‑top at room temperature, refrigerator at 4°C, and freeze/thaw) have been successfully validated. The quantitative range of the NF-L method was 0.673–472 pg/mL. Human CSF samples could be diluted with sample diluent to a maximum dilution of 1:2700 (1:27 in addition to an MRD of 1:100); Human serum and plasma samples could be diluted with sample diluent to a maximum dilution of 1:108 (1:27 in addition to an MRD of 1:4). NF-L in human plasma remains stable under the following conditions: up to 5 freeze/thaw cycles, 4 hours and 2 minutes at ambient temperature, and 24 hours and 10 minutes stored at 4°C (nominal). NF-L in human serum remains stable under the following conditions: up to 5 freeze/thaw cycles, 5 hours and 35 minutes at ambient temperature, and 20 hours and 40 minutes stored at 4°C. The quantitative range of the GFAP method was 1.37–1000 pg/mL. MRD of 1:4 and 1:40 were applied for plasma and CSF samples, respectively. GFAP in human plasma remains stable under the following conditions: up to 5 freeze/thaw cycles, 23 hours at ambient temperature and 4°C (nominal). Hemolysis of up to 5% hemolyzed blood and lipemic effect up to 500 mg/dL were also evaluated and no significant impact on the quantitation was observed.

Conclusion: The validation data presented in this poster demonstrates that the following parameters met all acceptance criteria: standard calibration model, accuracy and precision, interference, detectability and reproducibility, dilution linearity, and stability (bench‑top at room temperature, refrigerator at 4°C, and freeze/thaw). Both NF-L and GFAP are highly reliable, reproducible and are used for supporting high sensitivity biomarker profiling and analysis.