Characterization of Antibodies that Detect Human GFAP after Traumatic Brain Injury

Abstract

After traumatic brain injury (TBI), glial fibrillary acidic protein (GFAP) and other brain-derived proteins and their breakdown products are released into biofluids such as CSF and blood. Recently, a sandwich ELISA was constructed that measured GFAP concentrations in CSF or serum from human mild-moderate TBI patients. The goals of the present study were to characterize the same two antibodies used in this ELISA, and to determine which GFAP bands are detected by this antibody combination. Here, both antibodies recognized GFAP specifically in human brain and post-TBI CSF in a cluster of bands ranging from 50–38 kDa, that resembled bands from calpain-cleaved GFAP. By immunoprecipitation, the anti-GFAP Capture antibody recovered full length GFAP and its breakdown products from human brain lysate and post-TBI CSF. These findings demonstrate that the anti-GFAP ELISA antibodies non-preferentially detect intact GFAP and GFAP breakdown products, underscoring their utility for detecting brain injury in human patients.

Acute Biomarkers of Traumatic Brain Injury: Relationship between Plasma Levels of Ubiquitin C-Terminal Hydrolase-L1 and Glial Fibrillary Acidic Protein

“…Outcome was assessed at 3 and 6 months after injury. An initial analysis of TRACK-TBI data 20 confirms the findings of Papa et al, 17 and further indicates that elevated GFAP selects for patients who have persistent disabilities 3 months after injury.…”

“…The antibodies detect both whole GFAP molecules as well as GFAPBDPs, potentially resulting in a more complete measure of GFAP levels in circulation. 20 The estimated limit of detection (LOD) for GFAP is 0.1 ng/mL. The test sample was allowed to react sequentially with the capture and detection antibodies.…”

Glial fibrillary acidic protein is a body fluid biomarker for glial pathology in human disease

“…Responsible is calpain-mediated digestion, a broadly expressed calciumdependent protease Zoltewicz et al, 2012). There is also evidence for caspase-3 and caspase-9 GFAP cleavage products (Acarin et al, 2007;Rohn et al, 2002Rohn et al, , 2013bMouser et al, 2006).…”

GFAP-BDP as an Acute Diagnostic Marker in Traumatic Brain Injury: Results from the Prospective Transforming Research and Clinical Knowledge in Traumatic Brain Injury Study

“…The GFAP ELISA utilized a proprietary mouse monoclonal antibody for solid phase immobilization, and a proprietary polyclonal rabbit antibody for detection. 4,8 The test sample was allowed to react sequentially with the capture and detection antibodies, resulting in GFAP molecules being sandwiched between the two antibodies. The antibodies detected both whole GFAP molecules and GFAPBDPs, potentially resulting in a more complete measure of GFAP levels in circulation.…”

“…The antibodies detected both whole GFAP molecules and GFAPBDPs, potentially resulting in a more complete measure of GFAP levels in circulation. 8 Detection was mediated by addition of a tertiary anti-rabbit-horseradish peroxidase (HRP) conjugated antibody and a colorimetric (tetramethylbenzidine [TMB]) substrate. Quantitative determination of the biomarker concentration was achieved by comparing the unknown sample results to a standard curve obtained from the same assay.…”