Serum neurogranin measurement as a biomarker of acute traumatic brain injury

Abstract

Objectives: Neurogranin (NRGN) is a small neuronal protein that plays an important role in synaptic signaling by regulating calmodulin (CaM) availability. In this study, we developed an ELISA to measure NRGN quantitatively in serum samples from a cohort of acute traumatic brain injury (TBI) patients and a non-TBI control cohort, and explored the potential value of NRGN as a circulating biomarker for TBI.

Design and methods: Recombinant His-NRGN protein was used to develop mouse monoclonal capture and rabbit polyclonal detection antibodies, and they were used to develop a sandwich ELISA. After validation, we used this ELISA to measure serum samples from a cohort of typical adult acute TBI patients (N=76 TBI cases) and non-TBI control patients (N=150 controls).

Results: The NRGN ELISA lower limit of detection was 0.055ng/mL, lower limit of quantification was 0.2ng/mL, and interassay CVs were ≤10.7%. The average recovery was 99.9% (range from 97.2-102%). Serum NRGN concentrations in TBI cases were significantly higher than in controls (median values were 0.18ng/mL vs. 0.02ng/mL, p<0.0001), but did not discriminate TBI cases with intracranial hemorrhage (p=0.09).

Conclusions: We have developed a highly sensitive and reproducible ELISA for measuring circulating NRGN in blood samples. Serum NRGN concentrations in acute TBI patients were significantly higher than in controls, indicating that NRGN could have utility as a circulating biomarker for acute TBI. This report provides evidence to support larger and controlled TBI clinical studies for NRGN validation and prediction of outcomes.

Keywords: Biomarker; ELISA; Neurogranin; Traumatic brain injury.

Fig. 1

SDS-PAGE of His-NRGN in reduced condition. Recombinant His-NRGN (1 μg) was run on 4–12% gradient SDS-PAGE in MES buffer, and the gel was then stained with Coomassie blue. Two samples of His-NRGN were loaded; molecular weight standard is indicated in kDa.

Fig. 2

Typical NRGN ELISA standard curve. A typical calibration curve is shown. His-NRGN was used as calibrator, and 7 points of duplicated calibrators were assayed by NRGN ELISA (40.0–0.055 ng/mL).

Fig. 3

NRGN values among acute TBI cases and controls. This figure is a graphical representation of the distribution of neurogranin among TBI cases and non-TBI control subjects. Neurogranin values are higher in TBI cases than in non-TBI controls. The rectangle of the box plot represents the interquartile range (25th–75th percentiles) and the thick line within the box represents the median value of neurogranin. LOQ = limit of quantitation for neurogranin assay. LOD = limit of detection for neurogranin assay. Statistically significant difference between the control cohort and TBI cohort was observed, p < 0.0001.