Phage display for identification of serum biomarkers of traumatic brain injury

Abstract

Background: The extent and severity of traumatic brain injuries (TBIs) can be difficult to determine with current diagnostic methods. To address this, there has been increased interest in developing biomarkers to assist in the diagnosis, determination of injury severity, evaluation of recovery and therapeutic efficacy, and prediction of outcomes. Several promising serum TBI biomarkers have been identified using hypothesis-driven approaches, largely examining proteins that are abundant in neurons and non-neural cells in the CNS.

New method: An unbiased approach, phage display, was used to identify serum TBI biomarkers. In this proof-of-concept study, mice received a TBI using the controlled cortical impact model of TBI (1mm injury depth, 3.5m/s velocity) and phage display was utilized to identify putative serum biomarkers at 6h postinjury.

Results: An engineered phage which preferentially bound to injured serum was sequenced to identify the 12-mer 'recognizer' peptide expressed on the coat protein. Following synthesis of the recognizer peptide, pull down, and mass spectrometry analysis, the target protein was identified as glial fibrillary acidic protein (GFAP).

Comparison with existing methods and conclusions: GFAP has previously been identified as a promising TBI biomarker. The results provide proof of concept regarding the ability of phage display to identify TBI serum biomarkers. This methodology is currently being applied to serum biomarkers of mild TBI.

Keywords: Animal models; Biomarkers; Blood; Concussion; Rodents; Traumatic brain injury.

Figure 1. ELISA with individual phage clones

Each phage clone was evaluated using ELISA against serum samples from uninjured (white bars) and injured (black bars) mice. Anti M13-HRP antibody (GE Healthcare, Cat#27-9421-01) was used to detect the phage, the signal was detected using 3,3′,5,5′-tetramethyl benzidine (TMB) substrate and the absorbance was read at 450nm. Individual phages 1, 2, 3,and 4 showed increased binding to serum from injured (black) as compared to uninjured (blue)mice. Data are the mean ± SEM, n=3.

Figure 2. Peptide sequence of individual phage clones

The DNA insert encoding the 12-mer peptide expressed on the P8 coat protein for the six individual phages was sequenced using primer sequences provided by the manufacturer (New England Biolabs) and translated using a standard amino acid codon table. Note that the sequence for clones 2, 3 and 4 are identical (highlighted), indicating that they were amplified from the same original sequence.

Figure 3. ELISA for Phages 1 and 2 vs. novel serum samples

Phages 1 and 2 were evaluated using novel serum samples collected from injured (1 mm CCI, 6h post-injury, n=8) and uninjured (n=3) mice. Phage 1, but not Phage 2, exhibited preferential binding to the injured serum. (* denotes P<0.05)