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.

Keywords: GFAP; GFAP-BDP; human; traumatic brain injury.

Figure 1. The anti-GFAP ELISA antibodies recognized full length human GFAP (50 kDa) and GFAP-BDP (48–38 kDa)

(A) Control (normal, non head-injured) human post-mortem brain lysate (20 μg/lane) was separated by SDS-PAGE, blotted, and probed in parallel with the anti-GFAP Capture and Detection antibodies, which yielded similar banding patterns from 50–38 kDa. (B) Recombinant full length human GFAP (rhGFAP) was purified from E. coli, and then 4 μg was loaded and visualized with Coomassie stain. Also, 50 ng/lane was loaded, and blots were probed with the anti-GFAP Capture or Detection antibodies, which showed predominantly 50 kDa protein. (C) rhGFAP was cut or not with rat calpain-2 (capn2), then blotted at 50 ng/lane and visualized with the anti-GFAP Detection antibody. Capn2 digestion of rhGFAP yielded bands from 50–38 kDa.

Figure 2. The anti-GFAP ELISA antibodies detected GFAP from 50–38 kDa specifically in human brain

Protein lysates from the indicated human organs (20 μg/lane) were blotted and probed with the indicated antibodies. Both the Capture (A) and Detection (B) antibodies recognized GFAP from 50–38 kDa exclusively in the brain.

Figure 3. In human severe TBI CSF, GFAP was present in a range of bands from 50–38 kDa

Equal aliquots of CSF drawn at one day after injury from 8 severe human TBI patients were blotted and probed with the anti-GFAP Detection antibody. CSF from one representative control patient is also shown. Full length GFAP and GFAP-BDP were detected from 50–38 kDa. All samples were run together on the same gel/blot; intervening lanes were removed.

Figure 4. The anti-GFAP Capture antibody detected human GFAP and GFAP-BDP, but had poor affinity for rodent GFAP

Lysates from the indicated sources were digested with increasing amounts of rat capn2 (triangles) and probed with the antibodies labeled on the right. Spectrin served as a positive control for digestion efficiency, and was cleaved to SBDP150 and SBDP145 in a capn2 concentration-dependent manner. (A) In lysates from human HEK293 cells over-expressing human GFAP, both antibodies detected GFAP from 50–38 kDa, with capn2 cleaving GFAP to a 38 kDa limit fragment. (B) The anti-GFAP Detection antibody recognized full length endogenous GFAP and capn2-generated GFAP-BDPs from rat and mouse brain, while the Capture antibody did not.

Figure 5. The Capture antibody pulled down full length human GFAP and GFAP-BDP

Immunoprecipitations (IP) were performed with biotinylated anti-GFAP Capture antibody and streptavidin (SA)-coupled beads (Capture SA IP) or with SA beads only as a negative control (SA IP only). GFAP was immunoprecipitated separately from two sources, human post-mortem brain lysate (A) or CSF pooled from 3 severe TBI patients (B). Samples of brain lysate or CSF were resolved prior to IP (pre lane 1), and after IP (post lanes 2, 5). Proteins were eluted from the SA beads (eluate lanes 3, 6), and then the beads were washed with SDS-PAGE buffer (wash lane 4). Blots were probed with anti-GFAP Detection antibody. (A and B). Biotinylated anti-GFAP Capture antibody recovered full length GFAP and GFAP-BDP from human brain lysate and TBI CSF, without appearing to enrich for any particular GFAP band.