A novel, ultrasensitive assay for tau: potential for assessing traumatic brain injury in tissues and biofluids

Abstract

Traumatic brain injury (TBI) is a cause of death and disability and can lead to tauopathy-related dementia at an early age. Pathologically, TBI results in axonal injury that is coupled to tau hyperphosphorylation, leading to microtubule instability and tau-mediated neurodegeneration. This suggests that the forms of this protein might serve as neuroinjury-related biomarkers for diagnosis of injury severity and prognosis of the neurological damage prior to clinical expression. We initially determined whether we could detect tau in body fluids using a highly sensitive assay. We used a novel immunoassay, enhanced immunoassay using multi-arrayed fiberoptics (EIMAF) either alone or in combination with rolling circle amplification (a-EIMAF) for the detection of total (T) and phosphorylated (P) tau proteins from brains and biofluids (blood, CSF) of rodents following controlled cortical impact (CCI) and human patients post severe TBI (sTBI). This assay technology for tau is the most sensitive to date with a detection limit of approximately 100 ag/mL for either T-tau and P-tau. In the rodent models, T-tau and P-tau levels in brain and blood increased following CCI during the acute phase and remained high during the chronic phase (30 d). In human CSF samples, T-tau and P-tau increased during the sampling period (5-6 d). T-tau and P-tau in human serum rose during the acute phase and decreased during the chronic stage but was still detectable beyond six months post sTBI. Thus, EIMAF has the potential for assessing both the severity of the proximal injury and the prognosis using easily accessible samples.

Keywords: EIMAF; brain and biofluids; rolling circle amplification; severe traumatic brain injury; total and phosphorylated tau.

FIG. 1.

Sensitivity limits of enhanced immunoassay using multi-arrayed fiberoptics alone (EIMAF) and EIMAF coupled to rolling circle amplification (a-EIMAF) for rTau.

FIG. 2.

Sensitivity limits of enhanced immunoassay using multi-arrayed fiberoptics alone (EIMAF) and EIMAF coupled to rolling circle amplification (a-EIMAF) for P-tau using rTau-TTBK-1 with Mab CP13 (A) and rTau-GSK3β with Mab RZ3 (B).

FIG. 3.

Enhanced immunoassay using multi-arrayed fiberoptics (EIMAF; ––) vs. sandwich enzyme-linked immunosorbent assay (ELISA; – –) for T-tau (●, ■) and P-tau (○, □) detection sensitivity. Detection of T-tau and P-tau was performed using serial dilutions of JNPL3 (P301L; ●, ○) and T-tau knockout (TauKO; ■, □) mouse brain homogenates. EIMAF has a fivefold to sixfold more sensitive detection limit than sandwich ELISA.

FIG. 4.

Detection of tau by enhanced immunoassay using multi-arrayed fiberoptics coupled to rolling circle amplification (a-EIMAF) in rat serum following severe traumatic brain injury. Adult male Sprague-Dawley rats were subjected to controlled cortical impact and blood was collected at various time points as indicated. The levels of T-tau and P-tau in serum was determined by a-EIMAF. Statistical analysis (t-test) was based on comparison to naïve rats. *p<0.001; **p<0.0001; ***p<0.00001.

FIG. 5.

Detection of tau by enhanced immunoassay using multi-arrayed fiberoptics coupled to rolling circle amplification (a-EIMAF) in mouse serum following severe traumatic brain injury. At Days 1 (D1), 3 (D3), and 7 (D7) post–controlled cortical impact (CCI) or sham treatment, blood was collected and the serum was assayed for T-tau and P-tau by a-EIMAF. The levels of T-tau and P-tau in the sham-treated mice at D1 (shown), D3, and D7 did not change significantly. Statistical analysis (t-test) was based on comparison to sham-treated mice. *p<0.01; **p<0.001; ***p<0.00001.

FIG. 6.

Western blotting of mouse brain lysates for tau. At Days 1, 3, and 7 post– controlled cortical impact (CCI) or sham treatment, soluble fractions (25 μg) of ipsilateral cortex from CD-1 mouse brain lysates were electrophoresed and Western blotted for T-tau with Mab DA9 (A) and P-tau with Mab CP13 (B). Western blotting of naïve (N) mouse brain lysate also was performed. Shown are representative Western blots. Densitometric quantitation (C) of the most intensely immunostained T-tau (white bars) and P-tau (black bars) bands was performed using Image J software. Statistical analysis (t-test) compares densitometry of CCI vs. sham-treatment. *p<0.01; **p<0.001; ***p<0.00001.

FIG. 7.

Detection of T-tau and P-tau in human cerebrospinal fluid (1:100 dilution) during the acute and chronic phases post severe brain injury using enhanced immunoassay using multi-arrayed fiberoptics coupled to rolling circle amplification (A) and enzyme-linked immunosorbent assay (B).

FIG. 8.

Detection of T-tau and P-tau in human serum post severe traumatic brain injury (sTBI) by enhanced immunoassay using multi-arrayed fiberoptics coupled to rolling circle amplification (a-EIMAF). Human serum samples were diluted 1:20 in phosphate-buffered saline and assayed by a-EIMAF for T-tau (Mab DA31) and P-tau (Mab RZ3) in combination with biotinylated Mab DA9 for detection. Serum levels of both T-tau and P-tau are elevated in the acute phase (range, Days 1–3), compared with control serum. In the chronic serum samples (range, Days 27–190), the majority of T-tau returned to control levels. In contrast, P-tau levels in most sTBI serum samples remained higher than control levels.