Sensitive quantitation of glial fibrillary acidic protein in human plasma and cerebrospinal fluid: Validation of single-molecule assay detection method

Objectives Neurodegenerative disease biomarkers are often present at low concentrations in their native matrices; thus, accurate measurements of these biomarkers rely on ultra-high-sensitivity assays. This study validated an ultrasensitive single-molecule array assay for detecting glial fibrillary acidic protein (GFAP) in human plasma and cerebrospinal fluid. Methods We report validation of an ultra-high-sensitivity Quanterix single-molecule array (SIMOA) GFAP discovery assay for GFAP detection in human plasma and cerebrospinal fluid. The assay range, accuracy and precision, detectability and reproducibility, parallelism, specificity (interference), and short-term stability were evaluated. All method validation procedures were designed and performed in accordance with the 2018 U.S. Food and Drug Administration Bioanalytical Method Validation. Results First, we determined the quantitative range of the assay to be 1.37–1000 pg/mL, with minimum required dilutions of 1:4 and 1:40 for plasma and cerebrospinal fluid samples, respectively. For stability, we found that GFAP in human plasma remained stable up to five freeze/thaw cycles and up to 23 hours at ambient temperature and 4°C. Hemolysis of up to 5% and lipemic effects up to 500 mg/dL were also evaluated, with no major impact on assay quantitation. This fit-for-purpose validation demonstrates that the following parameters met acceptance criteria, including standard calibration model, accuracy and precision, interference, detectability, and reproducibility, dilution linearity, and stability. Conclusion The results suggest that GFAP is a reliable and reproducible biomarker in human plasma and cerebrospinal fluid, and that the single-molecule array GFAP discovery assay supports high-sensitivity profiling.