Development of a novel, sensitive translational immunoassay to detect plasma glial fibrillary acidic protein (GFAP) after murine traumatic brain injury

Abstract

Background Glial fibrillary acidic protein (GFAP) has emerged as a promising fluid biomarker for several neurological indications including traumatic brain injury (TBI), a leading cause of death and disability worldwide. In humans, serum or plasma GFAP levels can predict brain abnormalities including hemorrhage on computed tomography (CT) scans and magnetic resonance imaging (MRI). However, assays to quantify plasma or serum GFAP in preclinical models are not yet available. Methods We developed and validated a novel sensitive GFAP immunoassay assay for mouse plasma on the Meso Scale Discovery immunoassay platform and validated assay performance for robustness, precision, limits of quantification, dilutional linearity, parallelism, recovery, stability, selectivity, and pre-analytical factors. To provide proof-of-concept data for this assay as a translational research tool for TBI and Alzheimer’s disease (AD), plasma GFAP was measured in mice exposed to TBI using the Closed Head Impact Model of Engineered Rotational Acceleration (CHIMERA) model and in APP/PS1 mice with normal or reduced levels of plasma high-density lipoprotein (HDL). Results We performed a partial validation of our novel assay and found its performance by the parameters studied was similar to assays used to quantify human GFAP in clinical neurotrauma blood specimens and to assays used to measure murine GFAP in tissues. Specifically, we demonstrated an intra-assay CV of 5.0%, an inter-assay CV of 7.2%, a lower limit of detection (LLOD) of 9.0 pg/mL, a lower limit of quantification (LLOQ) of 24.8 pg/mL, an upper limit of quantification (ULOQ) of at least 16,533.9 pg/mL, dilution linearity of calibrators from 20 to 200,000 pg/mL with 90–123% recovery, dilution linearity of plasma specimens up to 32-fold with 96–112% recovery, spike recovery of 67–100%, and excellent analyte stability in specimens exposed to up to 7 freeze-thaw cycles, 168 h at 4 °C, 24 h at room temperature (RT), or 30 days at − 20 °C. We also observed elevated plasma GFAP in mice 6 h after TBI and in aged APP/PS1 mice with plasma HDL deficiency. This assay also detects GFAP in serum. Conclusions This novel assay is a valuable translational tool that may help to provide insights into the mechanistic pathophysiology of TBI and AD.

Age dictates brain functional connectivity and axonal integrity following repetitive mild traumatic brain injuries

“…Inflammation is a prevailing pathological response found in aged or injured brains 17,33,36 . Like young mice 17 , injury increased levels of Iba-1 and GFAP inflammatory markers in the optic tract of aged animals ( Figure 7A-7D ).…”

Translating from mice to humans: using preclinical blood-based biomarkers for the prognosis and treatment of traumatic brain injury

“…Injury severity was moderate-to-severe TBI in 26 studies, smTBI in 15, and rmTBI in 7 studies. Twenty-six studies performed a longitudinal assessment of GFAP levels after TBI (41,42,55–80).…”

“…In moderate-to-severe TBI models, circulating GFAP levels were assessed at ’hours’ in 18 studies (57–59,62,64,65,68–72,74,75,78,80–83), at ’days’ in 11 studies (41,57,67,68,72,75,78,80,84–86), ’weeks’ in 3 studies (67,75,87), and ’months’ in 1 study (88) ( Fig. 4A ).…”

“…One study did not report the timepoint of GFAP sampling (89). Following moderate-to-severe TBI, GFAP sharply increased within 2h post-injury (57,80), peaked at 4h-24h post-injury (57–59,64,69–71,74), and returned to sham levels at one week post- injury (41,57,75,80). Only one study reported GFAP levels increased at 2 months post-injury (88).…”

“…GFAP studies Asterisks indicate that age had to be inferred from the animal weight reported. ,57,75,80). Only one study reported GFAP levels increased at 2 months post-injury(88).…”