(T1130-02-08) Evaluation of the MSD S-Plex Neuropanel 1 Multiplex Assay for Neurological Biomarkers

Purpose: The purpose of this study is to evaluate the feasibility of the MSD S-Plex Neuropanel 1 assay in detecting three critical neurological biomarkers—Glial Fibrillary Acidic Protein (GFAP), Neurofilament Light (NFL), and Tau—simultaneously. This evaluation aims to determine the assay's bioanalytical robustness and operational value, specifically in terms of increased data efficiency, cost and time savings, and reduced sample handling. By leveraging the multiplex capability of the assay, this study seeks to extract rich information from a single sample, thereby enhancing the overall efficiency and accuracy of neurological biomarker detection.

Methods: The MSD S-PLEX Neurology Panel 1 kit is MSD’s ultrasensitive multiplex assay that uses proprietary TURBO-TAG and TURBO-BOOST reagents for improved sensitivity. The neurology panel 1 captures GFAP, NFL, and Tau simultaneously in a multi-spot microplate, with each capture antibody coated in a different spot. The MSD platform uses electrochemiluminescence (ECL) technology for signal readout. ECL technology works by using electrochemiluminescent labels that emit light when electricity is applied. In the S-PLEX MSD assay, capture antibodies are immobilized on specific spots of a multi-spot microplate. When a sample containing the target analytes is introduced, these analytes bind to the capture antibodies. After the targets are captured, detection antibodies, labeled with TURBO-BOOST tags, are then added, and bind to the captured analytes, forming an immunocomplex. Subsequently enhancer and TURBO-TAG is added. Finally, after addition of Read Buffer, an electrical current is applied and the immunocomplexes with labels emit light, which is measured to quantify the analytes.

Results: The evaluation parameters included calibration curves, parallelism in plasma and CSF, accuracy and precision, dilution linearity and hook effect, lot-to-lot variation, and cross-reactivity between analytes/reagents. All three analyte calibration curves were fitted with 4PL curve fitting and 1/Y² weighting, achieving an R² > 0.9900, and back-calculated calibrator % recovery (%RE) within ± 20%. Four commercial endogenous plasma and CSF samples were titrated and tested multiple times for parallelism. Plasma samples were diluted from neat to 32X, and at the experimentally determined minimum required dilution (MRD) of 4X and subsequent dilutions, the % recovery ranged from 94% to 108%. CSF samples were diluted from neat to 80X, and at the MRD of 10X and subsequent dilutions, the % RE ranged from 86% to 112%. Internal quality control (QC) samples were tested for accuracy and precision. Buffer-spiked samples at the upper limit of quantitation (ULOQ), the high end (HQC), middle of the calibration range (MQC), low end (LQC), and lower limit of quantitation (LLOQ) were tested over 16 separate runs. For all accepted runs, the %CV was ≤ 20% for HQC, MQC, and LQC, and ≤ 25% for ULOQ and LLOQ. The %RE to nominal concentrations was ≤ 25% for HQC, MQC, and LQC, and ≤ 30% for ULOQ and LLOQ. During the evaluation of accuracy and precision, some failed runs showed high recovery for GFAP and NFL and low recovery for Tau. Further investigation and troubleshooting led to a modification in the protocol for the last manual wash step, requiring the plate to be rotated 180 degrees between wash steps, which improved accuracy for all analytes. Additionally, minimizing sample transfers between vials helped better recover Tau levels in the QC samples. The assay did not present a prozone effect, and the titration of the spiked buffer samples demonstrated good linearity. Three different lots of the S-PLEX Neuropanel kit were tested using 17 plasma and 21 CSF samples. Lot-to-lot variability was < 20% for plasma and < 25% for CSF for all analytes. Lastly, cross-reactivity was evaluated by adding a single analyte or a single detector antibody in the well. Clinically relevant concentrations of these 3 analytes were tested to demonstrate potential analyte cross-reactivity in real patient samples. When only GFAP was added, there was 6% cross-reactivity with NFL and 0.2% with Tau. Similarly, NFL showed cross-reactivity of 0.6% with GFAP and 0.07% with Tau. Tau protein showed almost zero cross-reactivity with both GFAP and NFL. No significant cross-reactivity was observed when only a single detector antibody was added to the well.

Conclusion: The MSD S-Plex Neuropanel for GFAP, NFL, and Tau was evaluated in-house, demonstrating promising results. The assay quantitatively measures three biomarkers in a single well, exhibiting good parallelism, accuracy and precision, and lot-to-lot variability. Technical considerations, such as rotating the plate 180 degrees during last manual wash step and minimizing sample transfers between vials, contributes to better accuracy. However, at high concentrations of GFAP and NFL, analyte cross-reactivity between GFAP and NFL was observed, ranging from 0.6% to 6%.

References: 1. Park Y, Kc N, Paneque A, Cole PD. Tau, Glial Fibrillary Acidic Protein, and Neurofilament Light Chain as Brain Protein Biomarkers in Cerebrospinal Fluid and Blood for Diagnosis of Neurobiological Diseases. Int J Mol Sci. 2024 Jun 7;25(12):6295. doi: 10.3390/ijms25126295. PMID: 38928000; PMCID: PMC11204270.


Figure 1. Parallelism in endogenous plasma and CSF


Figure 2. Improved accuracy with a modified protocol for the final manual wash step


Table 1. Analyte Cross-reactivity