This study reports novel biochemical characterization of the same Capture and Detection antibodies used in the anti-GFAP swELISA (Banyan Biomarkers).17
These antibodies were examined here by western blot against human post-mortem brain lysate and the ELISA calibrator, recombinant human GFAP (rhGFAP; ). When control (non head-injured) human post-mortem brain lysate was blotted and probed with the anti-GFAP antibodies, similar banding patterns were observed (). Both antibodies detected full length GFAP protein at 50 kDa, as well as additional bands from 48–38 kDa (). By contrast, when rhGFAP was blotted and probed with the two anti-GFAP antibodies, each antibody detected GFAP primarily in a single band at 50 kDa, with a few minor smaller bands (). Since human brain tissue was obtained from normal individuals after their death, the banding pattern shown in suggested that GFAP might have been subjected to death-associated proteolysis. To test this, rhGFAP was digested with the cell death enzyme calpain-2. When calpain-cut rhGFAP was blotted and probed with the Detection antibody, a range of rhGFAP bands was detected between 50 and 38 kDa (), which resembled those in human post-mortem brain lysate (). This result, not previously shown, indicated that calpain-2 was capable of digesting GFAP into multiple GFAP-BDPs, with the limit of digestion at 38 kDa. To examine the tissue specificity of the two anti-GFAP antibodies, proteins from a panel of human organs were examined by western blot (). Both antibodies detected GFAP from 50–38 kDa exclusively in the brain, and did not cross-react with proteins extracted from 9 other human organs.
The anti-GFAP ELISA antibodies recognized full length human GFAP (50 kDa) and GFAP-BDP (48–38 kDa)
The anti-GFAP ELISA antibodies detected GFAP from 50–38 kDa specifically in human brain
The anti-GFAP swELISA was developed to measure GFAP in biofluids from human TBI patients, such as CSF or serum.17
Although our previous findings suggested that GFAP-BDPs were likely to be present in TBI-associated biofluids,17
this has not yet been characterized in the literature. In order to examine GFAP protein in TBI-associated samples, aliquots of CSF, drawn at one day after injury from 12 human severe TBI patients, were immunoblotted and compared to CSF from 5 healthy control individuals. Representative data from 8 TBI patients and one normal control are shown (). When blots were probed with the anti-GFAP Detection antibody, GFAP bands from 50–38 kDa were detected in most of the TBI patients examined (n = 12; 10/12 were GFAP positive; 8/12 positives are shown), but not in healthy control CSF (5/5 were GFAP negative; one representative control CSF sample is shown, ). The Capture antibody showed similar results (not shown). The GFAP-positive CSF samples varied by patient in terms of overall GFAP levels and extent of GFAP breakdown ( and not shown). The GFAP bands in human TBI CSF were from 50–38 kDa and appeared similar to those in post-mortem human brain lysate, or calpain-cut rhGFAP ( and ).
In human severe TBI CSF, GFAP was present in a range of bands from 50–38 kDa
Next, experiments were performed to investigate which GFAP bands in the 50–38 kDa range were recognized by the anti-GFAP swELISA antibodies on immunoblots, and whether the antibodies showed a preference for GFAP-BDP over intact GFAP. To produce human GFAP-BDPs in a controlled manner, human HEK293 cells were transiently transfected with a construct encoding full length human GFAP, isoform 1. Lysates from these cells were then in vitro
digested with low or high calpain-2. Digested and undigested lysates were then examined by immunoblotting (). αII-spectrin (spectrin) was also examined in the same lysates, as a control for enzyme activity.30,31
In lysates from GFAP-transfected HEK293 cells, both GFAP and spectrin were cleaved by calpain. Spectrin was first cleaved to SBDP150, and then to a mixture of SBDP150 and SBDP145 with increasing enzyme concentration (), as expected.30,31
When blots were probed with the two anti-GFAP antibodies, both antibodies detected full length GFAP at 50 kDa, calpain-cleaved GFAP at 38 kDa, and intermediate GFAP-BDPs (). Both antibodies showed that GFAP was cleaved to bands migrating between 50 and 38 kDa with a low amount of calpain-2, and then mostly to 38 kDa with more enzyme ().
In order to examine GFAP from healthy brain, fresh rat and mouse brain lysates were analyzed similarly by calpain-2 digestion. Rat brain was digested with two levels of calpain-2, low and high, and mouse brain was digested with a high level of calpain-2 only. Calpain-2 cleaved spectrin as expected in both rat and mouse brain lysates (). The anti-GFAP Detection antibody recognized rat GFAP from 50–38 kDa, and mouse GFAP at 50 and 38 kDa (). This analysis revealed that calpain was able to cleave rodent GFAP, and that the Detection antibody recognized intact GFAP and GFAP-BDP from healthy brain. The Capture antibody did not detect GFAP from rat or mouse brain (). Under some conditions such as increased loading and/or increased anti-GFAP Capture antibody concentration, the Capture antibody did detect some rat GFAP bands, but the bands detected varied from blot to blot (not shown). The anti-GFAP Capture antibody did not detect mouse brain GFAP even when blots were probed with high concentration of antibody (up to 11 μg/mL; not shown).
Antigen binding by antibodies in western blot and ELISA formats is different. On SDS-PAGE immunoblots, the antigen is denatured and reduced, while in biofluids, the native antigen is folded. To provide insight into the ability of the anti-GFAP Capture antibody to bind native GFAP, immunoprecipitations (IPs) were performed. CSF samples were selected and pooled from 3 human TBI patients that exhibited robust GFAP breakdown, displaying together a greater quantity of 38 kDa GFAP-BDP compared to full length 50 kDa GFAP by immunoblot (, lane 1). In the IP experiments, conditions were designed to mimic those used in the swELISA assay. IPs were performed with biotinylated anti-GFAP Capture antibody and streptavidin-coupled beads. Post-mortem normal human brain lysate served as the positive control. As a negative control, streptavidin beads only were used (no antibody). Following incubation, washing, and elution, immunoprecipitates (eluates) were blotted and probed with the anti-GFAP Detection antibody. Samples of diluted brain lysate or CSF before (pre) and after (post) IP were also run on the same blots, for comparison (). From human brain lysate, the biotinylated Capture antibody pulled down GFAP bands from 50–38 kDa (, lane 3). The pattern of GFAP bands that were pulled down (, lane 3) appeared identical to those in the original lysate (, lane 1). The wash lane (lane 4) was clean indicating good elution. By contrast, the streptavidin beads alone (without Capture antibody) did not recover GFAP from brain lysate (, lane 6). From human TBI CSF, the biotinylated Capture antibody pulled down full length human GFAP at 50 kDa, GFAP-BDP at 38 kDa, and minor intermediate bands (, lane 3). Again, the pattern of GFAP bands that were eluted was very similar to that in the starting pooled CSF (, lane 1). In the CSF after IP (, lane 2), there was a slight reduction in GFAP signal compared to CSF before IP (, lane 1). Again the wash lane was relatively clean (, lane 4), and the streptavidin beads alone did not precipitate GFAP (, lane 6). These data reveal that the anti-GFAP Capture antibody was able to bind to full length GFAP and GFAP-BDP from 50–38 kDa, without appearing to enrich for any particular band.
The Capture antibody pulled down full length human GFAP and GFAP-BDP