Applied Science, Faculty of
Medicine, Faculty of
Other UBC
Biomedical Engineering, School of
Pathology and Laboratory Medicine, Department of
DSpace
Alzheimer's Research & Therapy. 2021 Mar 07;13(1):58
Djavad Mowafaghian Centre for Brain Health
International Collaboration on Repair Discoveries
The Author(s)
Button, Emily B.
Cheng, Wai H.
Barron, Carlos
Cheung, Honor
Bashir, Asma
Cooper, Jennifer
Gill, Jasmine
Stukas, Sophie
Baron, David C.
Robert, Jerome
Rowe, Elyn M.
Cripton, Peter Alec, 1965-
Wellington, Cheryl L.
Background: Glial fibrillary acidic protein (GFAP) has emerged as a promising fluid biomarker for several neurological indications including traumatic brain injury (TBI), a leading cause of death and disability worldwide. In humans, serum or plasma GFAP levels can predict brain abnormalities including hemorrhage on computed tomography (CT) scans and magnetic resonance imaging (MRI). However, assays to quantify plasma or serum GFAP in preclinical models are not yet available. Methods: We developed and validated a novel sensitive GFAP immunoassay assay for mouse plasma on the Meso Scale Discovery immunoassay platform and validated assay performance for robustness, precision, limits of quantification, dilutional linearity, parallelism, recovery, stability, selectivity, and pre-analytical factors. To provide proof-of-concept data for this assay as a translational research tool for TBI and Alzheimer’s disease (AD), plasma GFAP was measured in mice exposed to TBI using the Closed Head Impact Model of Engineered Rotational Acceleration (CHIMERA) model and in APP/PS1 mice with normal or reduced levels of plasma high-density lipoprotein (HDL). Results: We performed a partial validation of our novel assay and found its performance by the parameters studied was similar to assays used to quantify human GFAP in clinical neurotrauma blood specimens and to assays used to measure murine GFAP in tissues. Specifically, we demonstrated an intra-assay CV of 5.0%, an inter-assay CV of 7.2%, a lower limit of detection (LLOD) of 9.0 pg/mL, a lower limit of quantification (LLOQ) of 24.8 pg/mL, an upper limit of quantification (ULOQ) of at least 16,533.9 pg/mL, dilution linearity of calibrators from 20 to 200,000 pg/mL with 90–123% recovery, dilution linearity of plasma specimens up to 32-fold with 96–112% recovery, spike recovery of 67–100%, and excellent analyte stability in specimens exposed to up to 7 freeze-thaw cycles, 168 h at 4 °C, 24 h at room temperature (RT), or 30 days at − 20 °C. We also observed elevated plasma GFAP in mice 6 h after TBI and in aged APP/PS1 mice with plasma HDL deficiency. This assay also detects GFAP in serum. Conclusions: This novel assay is a valuable translational tool that may help to provide insights into the mechanistic pathophysiology of TBI and AD.
RESEARCH Open AccessDevelopment of a novel, sensitivetranslational immunoassay to detectplasma glial fibrillary acidic protein (GFAP)after murine traumatic brain injuryEmily B. Button1,2, Wai Hang Cheng1,2, Carlos Barron1,2, Honor Cheung1,2, Asma Bashir1,2, Jennifer Cooper1,2,Jasmine Gill1,2, Sophie Stukas1,2, David C. Baron1,2, Jerome Robert1,2, Elyn M. Rowe1,2, Peter A. Cripton3,4 andCheryl L. Wellington1,2,3,4*AbstractBackground: Glial fibrillary acidic protein (GFAP) has emerged as a promising fluid biomarker for severalneurological indications including traumatic brain injury (TBI), a leading cause of death and disability worldwide. Inhumans, serum or plasma GFAP levels can predict brain abnormalities including hemorrhage on computedtomography (CT) scans and magnetic resonance imaging (MRI). However, assays to quantify plasma or serum GFAPin preclinical models are not yet available.Methods: We developed and validated a novel sensitive GFAP immunoassay assay for mouse plasma on the MesoScale Discovery immunoassay platform and validated assay performance for robustness, precision, limits ofquantification, dilutional linearity, parallelism, recovery, stability, selectivity, and pre-analytical factors. To provideproof-of-concept data for this assay as a translational research tool for TBI and Alzheimer’s disease (AD), plasmaGFAP was measured in mice exposed to TBI using the Closed Head Impact Model of Engineered RotationalAcceleration (CHIMERA) model and in APP/PS1 mice with normal or reduced levels of plasma high-densitylipoprotein (HDL).Results: We performed a partial validation of our novel assay and found its performance by the parameters studiedwas similar to assays used to quantify human GFAP in clinical neurotrauma blood specimens and to assays used tomeasure murine GFAP in tissues. Specifically, we demonstrated an intra-assay CV of 5.0%, an inter-assay CV of 7.2%,a lower limit of detection (LLOD) of 9.0 pg/mL, a lower limit of quantification (LLOQ) of 24.8 pg/mL, an upper limitof quantification (ULOQ) of at least 16,533.9 pg/mL, dilution linearity of calibrators from 20 to 200,000 pg/mL with90–123% recovery, dilution linearity of plasma specimens up to 32-fold with 96–112% recovery, spike recovery of67–100%, and excellent analyte stability in specimens exposed to up to 7 freeze-thaw cycles, 168 h at 4 °C, 24 h at(Continued on next page)© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you giveappropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtainpermission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to thedata made available in this article, unless otherwise stated in a credit line to the data.* Correspondence: wcheryl@mail.ubc.ca1Djavad Mowafaghian Centre for Brain Health, University of British Columbia,2215 Wesbrook Mall, Vancouver, British Columbia V6T 1Z3, Canada2Department of Pathology and Laboratory Medicine, University of BritishColumbia, Vancouver, British Columbia V6T 1Z3, CanadaFull list of author information is available at the end of the articleButton et al. Alzheimer's Research & Therapy (2021) 13:58 https://doi.org/10.1186/s13195-021-00793-9(Continued from previous page)room temperature (RT), or 30 days at − 20 °C. We also observed elevated plasma GFAP in mice 6 h after TBI and inaged APP/PS1 mice with plasma HDL deficiency. This assay also detects GFAP in serum.Conclusions: This novel assay is a valuable translational tool that may help to provide insights into the mechanisticpathophysiology of TBI and AD.Keywords: GFAP, Plasma biomarker, TBI, CHIMERA, ImmunoassayIntroductionOver the past decade, impressive advances have beenmade in the discovery of blood biomarkers for multipleneurological indications including traumatic brain injury(TBI), Alzheimer’s disease (AD) and many others [1, 2].Blood biomarkers have several advantages over neuroim-aging and cerebrospinal fluid (CSF) biomarkers as theyare less expensive, more high-throughput, and less inva-sive [1]. Circulating glial fibrillary acidic protein (GFAP),an intermediate filament protein expressed in astrocytes,is a particularly promising biomarker for predictingbrain damage after TBI. GFAP, along with ubiquitin C-terminal hydrolase 1 (UCH-L1), was the first biomarkerfor concussion to be approved by the United States Foodand Drug Administration (FDA) in 2018 [3] using anenzyme-linked immunosorbent assay (ELISA)-basedmethod by Banyan Biomarkers Inc. to predict intracra-nial injuries on computed tomography (CT) head scanwith 97.6% sensitivity. Overall, it is estimated that up toa third of TBI patients could avoid unnecessary CTscans and the associated radiation using the GFAP/UCH-L1 blood test [4]. Many other studies have sup-ported these findings using various assays and analyticalplatforms [5–7] and show that post-TBI serum andplasma GFAP levels can predict magnetic resonance im-aging (MRI) abnormalities [6–8], mortality [5, 9–11], de-lays to return to work or return to play [6, 12], and poorscores on the Glasgow Outcome Scale [5, 11, 13]. Inmany studies, blood GFAP was a better predictor of im-aging findings or other outcomes than other biomarkerstested [5, 7, 13].Emerging evidence suggests that circulating GFAPmay also be an effective blood biomarker for otherneurological indications. For example, serum GFAP cor-relates with multiple sclerosis (MS) disease severity [14]and tumor volume in glioblastoma patients [15]. Severalstudies have found an acute increase in serum or plasmaGFAP after intracerebral hemorrhage [16–18] and an as-sociation between serum GFAP and stroke severity [17]or stroke volume [18]. Circulating GFAP has also shownpromise as an Alzheimer’s disease (AD) biomarker inseveral recent studies. In a study of n = 28 AD patientsand n = 34 controls, serum GFAP concentrations werehigher in the AD patients and correlated with cognitiveimpairment [19]. In a study of n = 252 subjects in theAmsterdam Dementia Cohort, plasma GFAP concentra-tions predicted brain amyloid positivity in positron emis-sion tomography scans and were associated withcognitive performance [20].Despite the promise of circulating GFAP as a biomarkerfor multiple neurological conditions, there is currently novalidated method to quantify GFAP in mouse plasma. Ani-mal models are invaluable research tools for many neuro-logical conditions and a robust assay of blood GFAP couldcatalyze many cost-effective translational studies with im-proved rigor by which serial blood samples could be mea-sured. For example, murine models of TBI allow for greatercontrol over injury severity and timing of blood collection,which can be challenging in human studies. Indeed, manyanimal models of TBI have been developed to better under-stand mechanisms of injury and potential therapeutic path-ways. Our laboratory developed a closed-head TBI modelcalled CHIMERA (Closed-Head Impact Model of Engi-neered Rotational Acceleration) that uses impact from apneumatic piston resulting in rotational acceleration in mice,rats, and ferrets [21]. In a recent study using CHIMERA toinduce a moderate-severe injury, mice exhibited acute neuro-logical deficits, long-term memory deficits, neuroinflamma-tion, axonal injury, and blood-brain barrier leakage [22].We aimed to bridge the translational gap between hu-man and rodent TBI research by using the Meso ScaleDiscovery platform to develop and validate a sensitiveELISA assay to quantify GFAP in murine plasma. Wefirst validated assay performance according to standardoperating procedures outlined by Andreasson et al. [23].We then demonstrated that this assay reliably detects anacute and robust elevation of plasma GFAP after murineTBI, providing proof-of-concept for the utility of thisassay to accelerate translational research in animalmodels of brain injury. We also show that plasma GFAPis elevated in aged APP/PS1 mice and in APP/PS1 micethat are deficient in plasma HDL, providing preliminaryevidence supporting the use of this assay to study plasmaGFAP in rodent models of AD.Materials and methodsAnimals and tissue collectionAll procedures were approved by the University of Brit-ish Columbia Committee on Animal Care (protocolA15-0096, A19-0264, A15-0040, A14-0057, A14-0003)Button et al. Alzheimer's Research & Therapy (2021) 13:58 Page 2 of 27and were performed in strict compliance with the Can-adian Council on Animal Care guidelines. Animals wereanesthetized with ketamine (150 mg/kg) and xylazine(20 mg/kg). A surgical plane of anesthesia was confirmedwith a toe-pinch test then whole blood was collected bycardiac puncture into a tube containing ethylenedi-aminetetraacetic acid (EDTA). Collected blood wasstored on ice up to 4 h then centrifuged to plasma at3000g for 10 min at room temperature (RT) to isolateplasma. Isolated plasma was then stored at − 80 °C untilanalysis. To assess assay selectivity, blood collected fromcardiac puncture was divided into tubes with and with-out EDTA to prepare plasma and serum, respectively,from the same animals. Serum was allowed to clot for30 min at RT prior to centrifugation at 3000g for 10 min.Following cardiac puncture, animals were perfused withheparinized ice-cold phosphate-buffered saline (PBS).Half-brains were collected for biochemistry and snapfrozen on dry ice then stored at − 80 °C. Where indi-cated, blood was collected from the saphenous vein im-mediately before anesthesia into micro capillary tubeswith EDTA (14-915-85, Thermo Fisher Scientific, Wal-tham, MA).Plasma specimens for assay development and validationMurine EDTA plasma specimens, collected as describedin the “Animals and tissue collection” section, were usedto create plasma pools with low, intermediate, or highexpected GFAP concentrations. Expected GFAP concen-trations were estimated based on observed bloodGFAP concentrations in human studies. Specifically,GFAP levels have been shown to be moderately elevatedin the blood of people with AD [20] and robustly ele-vated in blood collected within 24 h of TBI [10, 11, 13,24–26]. Plasma specimens with low expected GFAP con-centrations were collected from female wildtype C57Bl/6mice aged 3 to 6 months (Jackson labs, line 85, mixedC57Bl/6 and C3H/HeJ backgrounds) and pooled whereindicated. Plasma specimens with intermediate expectedGFAP concentrations were collected from APP/PS1mice aged 9 to 24 months and wildtype littermates aged18 to 24 months (Jackson labs, line 85, mixed C57Bl/6and C3H/HeJ background) and pooled where indicated.Plasma specimens with high expected GFAP concentra-tions were collected from mice 6 h post-TBI as describedin the “Proof-of-concept studies in TBI and AD mice”section and pooled where indicated. Aliquots werestored at − 80 °C until analysis and each assay used afreshly thawed aliquot. To assess assay selectivity, humanAPOE3 targeted replacement mice [27] were used inaddition to C57Bl/6 mice. Targeted replacement micewere obtained from the Cure Alzheimer Fund and havehumanized APOE introns between exons 2–3 and exons3–4 but retain the murine ApoE 5′ and 4′ untranslatedregions.Assay procedureA novel highly sensitive ELISA to detect murine GFAPin plasma was developed using the Meso Scale Discovery(MSD) platform and materials detailed in Table 1. Acapture and detection antibody pair reactive againstmice, rats, and humans was obtained (ab244094, Abcam,Cambridge UK). This antibody pair was chosen for sev-eral reasons. First, this antibody pair was previously re-ported by the supplier to have cross-reactivity with mice,rats, and humans. Second, this antibody pair was avail-able in a buffer free of sodium azide and carrier proteins,which was necessary for optimal antibody conjugationwith capture and detection tags. Finally, this antibodypair was available for purchase in relatively large quan-tities. Biotinylation of the capture antibody was per-formed at a 1:10M ratio of IgG:biotin by incubating theantibody with EZ-LinkTm NHS-biotin at 4 °C overnight(20217, Sigma, Burlington MA). Labeling of the detec-tion antibody with MSD gold sulfo-tag NHS-ester (150nM, R91A0, Meso Scale Discovery, Rockville MD) wasperformed according to the manufacturer’s recommen-dations. Optimal capture and detection antibody con-centrations were determined through a preliminary testof the signal to background ratio when measuring 100pg/mL of recombinant GFAP protein. RecombinantGFAP stock (ab68428, Abcam, Cambridge, UK) was di-luted to 10 μg/mL working stocks in 1% BSA in PBS.Details of the assay standard operating procedure(SOP) are as follows:1. Add blocking buffer: add 150 μL/well Incubate for 1 h at room temperature withshaking (150 rpm)2. Wash: add 300 μL/well wash buffer Repeat 3 times, decant the wash buffer inbetween additions, remove excess wash bufferfrom the wells following the final wash bytapping on a paper towel3. Add capture antibody: add 30 μL/well captureantibody diluted to 0.5 μg/mL in PBS Incubate for 1 h at room temperature withshaking (150 rpm)4. Prepare standard curve Dilute recombinant protein stock first to100,000 pg/mL in assay diluent Dilute from 100,000 to 40,000 pg/mL in assaydiluent for the highest standard point (point 1) Perform 1:4 serial dilutions in assay diluent forpoints 2–7 Point 8 will be blank assay diluentButton et al. Alzheimer's Research & Therapy (2021) 13:58 Page 3 of 275. Add samples and standards: final volume of 50 μL/well Standards: add 25 μL/well assay diluent plus25 μL/well prepared standard points 1–8 Samples: add 37.5 μL/well assay diluent plus12.5 μL/well plasma samples Incubate 2 h at room temperature with shaking(150 rpm)6. Wash: as in step 27. Add detection antibody: add 25 μL/well detectionantibody diluted to 0.5 μg/mL in assay diluent Incubate for 1 h at room temperature withshaking (150 rpm)8. Wash: as in step 29. Read plate: add 150 μL/well Read buffer, read plateusing an MSD instrumentAssay validationSummary of assay validation parametersA partial validation of assay performance was performedfollowing the SOP outlined by Andreasson et al. for theBIOMARKAPD project, supported by the EuropeanUnion initiative joint program for neurodegenerative dis-ease research [23]. Validation parameters are summa-rized in Table 2 and detailed in the "Assay validation"sub-sections below.RobustnessAssay robustness was evaluated over 6 assays run in par-allel with planned variations in the duration of captureantibody, sample/standard, or detection antibody incu-bation. Assay 1 followed the SOP described in the “Assayprocedure” section, assay 2 reduced the capture antibodyincubation to 30min, assay 3 extended the capture anti-body incubation to overnight at 4 °C, assay 4 reducedthe sample/standard incubation to 1 h, assay 5 extendedthe sample/standard incubation to overnight at 4 °C, andassay 6 reduced the detection antibody incubation to 30min. Each assay included duplicate specimens from thelow, intermediate, and high concentration plasma pools.PrecisionAssay precision was evaluated over 5 assays run on sep-arate days each measuring 5 replicates from low, inter-mediate, and high concentration plasma pools.Limits of quantificationAssay limits of quantification were determined using 2methods. First, 8 low-concentration and 8 high-concentration specimens were assayed in duplicate todetermine the lowest (lower limit of quantification(LLOQ)) and highest (upper limit of quantification(ULOQ)) concentration that could be quantified with <20% coefficient of variation (CV). Second, 16 replicatesof blank assay diluent were assayed and a calculationwas made based on the standard deviation (SD) of theelectrochemiluminescent (ECL) signal of the blank repli-cates. Lower limit of detection (LLOD) was calculated as2.5 SD above the blank signal and LLOQ as 10 SD abovethe blank signal.Dilution linearityDilution linearity of the recombinant GFAP calibratorand endogenous GFAP in plasma specimens was deter-mined. Calibrator linearity was determined by gathering3 plasma specimens with very low endogenous GFAPconcentrations then spiking recombinant GFAP intothem at a concentration 50-fold higher than the higheststandard curve point. Serial dilutions of the spiked speci-mens were performed such that the final dilution wasexpected to be below the LLOD of the assay, as deter-mined by methods described in the “Limits of quantifi-cation” section. Specimens were assayed in duplicate.Dilution linearity of endogenous GFAP in plasma wasdetermined by gathering 3 specimens with high GFAPconcentrations. Serial dilutions of each specimen weremade and specimens were assayed in duplicate.Table 1 Assay materialsMaterial Manufacturer Catalog numberMSD GOLD Small Spot Streptavidin SECTOR pate Meso Scale Discovery (Rockville MD) L45SAGFAP antibody pair Abcam (Cambridge UK) Ab244094Capture label (EZ-link NHS-biotin) Sigma (Burlington MA) 20217Detection label (MSD gold sulfo-tag NHS-ester) Meso Scale Discovery (Rockville MD) R91A0 (150 nM)Recombinant GFAP Abcam (Cambridge UK) Ab68428Blocking buffer (5% BSA in PBS) Sigma (BSA) (Burlington MA) A7906Assay diluent (1% BSA in PBS) Sigma (BSA) (Burlington MA) A7906Wash (PBS-T) VWR (Tween) (Radnor PA) CAJTX251-7Read buffer (MSD GOLD Read buffer A) Meso Scale Discovery (Rockville MD) R92TGMESO QuickPlex SQ 120 Meso Scale Disocvery (Rockville MD) AI0AA-0Button et al. Alzheimer's Research & Therapy (2021) 13:58 Page 4 of 27Table 2 Parameters for partial assay validationParameter Definition Experimental details Sample type TechnicalreplicatesDeviation fromAndreasson et alRobustness The ability of the assay toaccurately and preciselymeasure the analyte undersmall protocol variations [23].Modify theincubation duration ofcapture antibody (1 h vs. 2 hvs. overnight), sample (2 h vs.1 h vs. overnight), anddetection antibody (1 h vs.30 min) parallel assays.Analyze %CV and %recovery.Plasma pools (low GFAP: 3–6-month-old wildtype mice,intermediate GFAP: 9–24-month-old APP/PS1 mice and18–24-month-old wildtypelittermates, high GFAP: 6 hpost-TBI).2 No deviations.Precision The variation in analytemeasurement within a singleassay (repeatability, intra-assay variation) and betweenindependent assays per-formed on different days(intermediate precision, inter-assay variation) [23].Repeat the assay with thesame specimens on 5independent days. Analyzeintra-assay and inter-assay%CV.Plasma pools (low GFAP: 3–6-month-old wildtype mice,intermediate GFAP: 9–24-month-old APP/PS1 mice and18–24-month-old wildtypelittermates, high GFAP: 6 hpost-TBI).5 No deviations.Limits ofquantificationThe lowest and highestanalyte concentrations inplasma that can be measuredby the assay with a givenlevel of precision (< 20% CV)[23].LLOD: Assay replicates ofdiluent, calculate LLOD as 2.5SD above the blank.Diluent (1% BSA in PBS). 16 No recommendationavailable in Andreassonet al [23], followed MesoScale Discoveryrecommendations.LLOQ: Assay replicates ofdiluent, calculate LLOQ as 10SD above the blank.Diluent (1% BSA in PBS) 16 No deviations.LLOQ: Assay specimens withvery low GFAPconcentrations. LLOQ =lowest measuredconcentration with duplicates< 20%CV.Plasma specimens with lowexpected concentrations (n =8, 3–6-month-old wildtypemice).2 No deviations.ULOQ: Assay specimens withhigh GFAP concentrations.ULOQ = highest measuredconcentration with duplicates< 20%CV.Plasma specimens with highexpected concentrations (n =8, 6 h post-TBI).2 No deviations.DilutionlinearityThe ability of the assay toaccurately and reliably detectthe analyte in plasma spikedwith the calibrator at a veryhigh concentration afterdilution [23].The ability of endogenousanalyte in plasma to bedetected at various dilutionsaccurately and reliably [28].Spike plasma specimens withrecombinant protein 120-foldabove the estimated ULOQ(2,000,000 pg/mL), performserial dilutions until theoret-ical concentration is belowLLOQ (1-fold to 1,000,000-fold). Analyze %CV and%recovery.Plasma specimens with lowexpected concentrations (n =3, 3–6-month-old wildtypemice).2 No deviations.Parallelism Comparison of the signal vs.dilution factor response ofthe calibrator andendogenous analyte inplasma [23].Perform serial dilutions (1-foldto 64-fold) of specimens withhigh expected concentration.Analyze %CV and %recovery.Plasma specimens with highexpected concentrations (n =3, 6 h post-TBI).2 3 specimens were usedrather than therecommended 4specimens, no otherdeviations.Perform serial dilutions (1-foldto 64-fold) of specimens withhigh expected concentration,analyze response of signal todilution factor compared toresponse of recombinantprotein.Definition from Sweeneyet al [28]. 3 specimenswere used rather thanthe recommended 5specimens, no otherdeviations.Recovery Ability to accurately andreliably measure theconcentration of plasmaspiked with calibrator [23].Divide specimens into 4aliquots, spike 3 of thealiquots with recombinantprotein at concentrationsacross the range of thePlasma specimens with low(n = 2, 3–6-month-oldwildtype mice) and high (n =3, 6 h post-TBI)concentrations.1 No deviations.Button et al. Alzheimer's Research & Therapy (2021) 13:58 Page 5 of 27ParallelismThe parallelism of the response of the recombinantGFAP calibrator to that of endogenous GFAP was deter-mined using data collected from the serial dilution ofspecimens with high GFAP concentrations performed asdescribed in the “Dilution linearity” section. The ECLsignal was plotted against the dilution factor for eachspecimen and the slope of the response curve was com-pared to that of diluted calibrator.Spike recoveryAssay spike recovery was determined by spiking low andhigh concentration plasma specimens with recombinantGFAP (calibrator) at 3 concentrations. First, 2 plasmaspecimens with low GFAP concentrations and 3 plasmaspecimens with high GFAP concentrations, collected asdescribed in the “Animals and tissue collection” and“Plasma specimens for assay development and valid-ation” sections, were thawed and divided into 4 aliquotsof 13.5 μL each. Recombinant GFAP was diluted in 1%BSA in PBS to stocks of 20,000, 2000, and 200 pg/mL.The following was then added into each aliquotedplasma specimen: 1.5 μL 1% BSA in PBS into aliquot 1,1.5 μL of 20,000 pg/mL spike into aliquot 2, 1.5 μL of2000 pg/mL spike into aliquot 3, and 1.5 μL of 200 pg/mL spike into aliquot 4. Each aliquot was then assayedat a 2-fold dilution. The final theoretical concentrationsof recombinant GFAP in each aliquot after adjusting fordilution factor are therefore as follows: 0 pg/mL in ali-quot 1 (neat aliquot), 10,000 pg/mL in aliquot 2, 1000pg/mL in aliquot 3, and 100 pg/mL in aliquot 4. Thepercent recovery of each spiked specimen was then cal-culated as recommended by Andreasson et al [23] by theformula below:Table 2 Parameters for partial assay validation (Continued)Parameter Definition Experimental details Sample type TechnicalreplicatesDeviation fromAndreasson et alstandard curve (100, 1000,and 10,000 pg/mL), add anequivalent volume of diluentto the fourth aliquot. Analyze%CV and %recovery.Stability The stability of the analyte inplasma after a given numberof freeze-thaw cycles or agiven amount of time at agiven temperature [23].Divide specimens into 19aliquots. Aliquot 1 store at −80 °C. Aliquots 2–6 exposedto 1, 2, 3, 5, or 7 freeze-thawcycles (2 h at RT then store at− 80 °C), respectively. Aliquots7–12 store at RT for 1, 2, 4,24, 72, and 168 h, respect-ively. Aliquots 13–18 store at4 °C as above. Aliquot 19store at −20 °C for 1 month.Assay together and analyze%CV and %recovery.Plasma pools (low: 3–6month- old wildtype mice,intermediate: 9–24-month-old APP/PS1 mice and 18–24-month-old wildtypelittermates, high: 6 h post-TBI).2 No deviations.Selectivity The ability to measure theanalyte in the presence ofother substances expected tobe present [23].A) Compare assayperformance in plasma andserum.B) Measure the analyte inspecimens with hemolysis.A) Plasma and serum fromsham (n = 6) or TBI (n = 7)mice collected 6 h post-TBI.B) 5%, 25%, or 50% red bloodcells spiked into plasma fromsham (n = 3) or TBI (n = 3)mice collected 6 h post-TBI.Spiked specimens were fro-zen at − 80 °C then thawedand assayed.2 Andreasson et alrecommends spiking insubstances with similarphysiochemicalstructure.Pre-analyticalfactorsThe stability of the analytewith respect to bloodcollection and processingmethods.Blood was collected bysaphenous vein and cardiacpuncture. Blood from cardiacpuncture was divided into 2aliquots. One aliquot wascentrifuged to plasma < 1 hafter collection and onealiquot was incubated on ice4 h before centrifugation.Plasma from sham (n = 3) orTBI (n = 3) mice collected 6 hpost-TBI.2 No recommendationavailable in Andreassonet al [23].LLOD lower limit of detection, LLOQ lower limit of quantification, ULOQ upper limit of quantification, TBI traumatic brain injury, CV coefficient of variation, SDstandard deviation, RT room temperatureButton et al. Alzheimer's Research & Therapy (2021) 13:58 Page 6 of 27percent recovery ¼ measured concentrationspiked specimen −measured concentrationneat specimentheoretical concentrationspike 100%StabilityGFAP stability to plasma temperature modulationThe stability of GFAP in plasma exposed to various tem-peratures and freeze-thaw cycles was determined. Sets of19 aliquots were prepared from low, intermediate, andhigh GFAP concentration plasma pools and treated asoutlined by Andreasson et al. [23]. Briefly, aliquot 1 wasimmediately frozen and stored at − 80 °C. Aliquots 2 to6 were exposed to 1, 2, 3, 5, or 7 freeze-thaw cycleswhere specimens were thawed and stored at roomtemperature for 2 h then stored again at − 80 °C. Ali-quots 7 to 12 were stored at room temperature for 1, 2,4, 24, 72, or 168 h before freezing at − 80 °C. Aliquots 13to 18 were stored at 4 °C for 1, 2, 4, 24, 72, or 168 h be-fore freezing at − 80 °C. Aliquot 19 was stored at − 20 °Cfor 30 days before freezing at − 80 °C. On the day of theassay, all aliquots were thawed together and assayed induplicate.Selectivity Assay selectivity was evaluated by comparingassay measurements between serum and plasma fromthe same animal and investigating assay performance inthe presence of hemolysis. Male and female C57Bl/6mice, aged 2.5 to 4 months old, and APOE3 mice, aged5 months old, were exposed to a 2.5 J head impact withthe CHIMERA device or sham procedures as describedin the “Proof-of-concept studies in TBI and AD mice”section. Blood from each animal was collected 6 h afterTBI by cardiac puncture, as described in the “Animalsand tissue collection” section, into tubes with or withoutEDTA, and then processed into plasma or serum, re-spectively, as described in the “Animals and tissue col-lection” section. Plasma and serum were frozen andstored at − 80 °C then assayed together in duplicate.Thawed plasma specimens were also used to test the ef-fect of hemolysis on assay performance. Red blood cellswere pooled from the blood collected into EDTA-containing tubes from 3 sham animals. Plasma speci-mens were spiked with pooled red blood cells at 0%, 5%,25%, or 50% volume. Assay diluent was added to thespecimens spiked at 0%, 5%, and 25% such that an equaltotal volume of spike and diluent was added to each spe-cimen. Spiked and neat specimens were frozen at −80 °C for 1 h then thawed and assayed in duplicate.Pre-analytical factors The stability of GFAP in bloodcollected and processed by various methods was also de-termined. Male C57Bl/6 mice were exposed to a 2.5 Jhead impact with the CHIMERA device or sham proce-dures as described in the “Proof-of-concept studies inTBI and AD mice” section. Blood was collected 6 h afterTBI, first by the saphenous vein and then by cardiacpuncture as described in the “Animals and tissue collec-tion” section. Blood collected by saphenous vein wasstored on ice then centrifuged at 3000g for 10 min atroom temperature within 1 h of collection. Blood col-lected by cardiac puncture was divided into 2 equal ali-quots. Aliquot 1 was centrifuged as above within 1 h ofcollection. Aliquot 2 was incubated on ice for 4 h thencentrifuged as above. Plasma specimens were assayed induplicate except in the case of most saphenous bloodsamples where volume limitations only allowed a singlemeasurement.Proof-of-concept studies in TBI and AD miceCHIMERA TBI procedureAll CHIMERA procedures were approved by the Univer-sity of British Columbia Committee on Animal Care(protocol A15-0096, A19-0264, and A15-0040) and wereperformed in strict compliance with the Canadian Coun-cil on Animal Care guidelines. Male and female C57Bl/6mice were housed with a reverse 12 h light to dark cycleand exposed to a closed-head TBI in the sagittal planewith a CHIMERA device at 3.5 to 5 months of age aspreviously described [22]. Briefly, animals were anesthe-tized with isoflurane (induction 5%, maintenance 2–4%in oxygen 0.8 L/min), administered meloxicam (1 mg/kg)and saline (10 mL/kg) via subcutaneous injection, thenplaced in the supine position on the CHIMERA im-pactor. A toe-pinch test was used to confirm a surgicalplane of anesthesia, and once confirmed, the flow of iso-flurane was stopped and a single 2.5 J closed-head im-pact was administered using a 50-g steel piston thatimpacted a 3D-printed polylactic acid (PLA) interfacepositioned below the head to prevent skull fracture asdescribed previously [22]. Sham animals received equiva-lent isoflurane, meloxicam, saline, and toe-pinch expo-sures without an impact. Neurological impairment wasassessed using the neurological severity score (NSS) 2 hpost-TBI as previously described [29]. Plasma and brainswere collected as described in the “Animals and tissuecollection” section.Studies in APP/PS1 miceAll procedures with APP/PS1 mice were approved bythe University of British Columbia Committee on Ani-mal Care (protocol A14-0003, A14-0057) and were per-formed in strict compliance with the Canadian Councilon Animal Care guidelines. Two APP/PS1 cohorts wereincluded in this investigation. In the first cohort, femaleAPP/PS1 mice and wildtype littermates (Jackson labs,line 85, mixed C57Bl/6 and C3H/HeJ background) wereaged to 3, 6, 9, 12, 18, or 24 months of age. Plasma spec-imens were collected by cardiac puncture as described inButton et al. Alzheimer's Research & Therapy (2021) 13:58 Page 7 of 27the “Animals and tissue collection” section. In the sec-ond cohort, APP/PS1 mice with normal plasma HDLcholesterol (HDL-C) or with plasma HDL-C deficiencywere generated as previously described [30]. Briefly,APP/PS1 mice (Jackson laboratories, B6.Cg-Tg (APPswe,PSEN1dE9)85Dbo/Mmjax, MMRRC stock no: 34832-JAX) on a C57Bl/6 background were first bred with apo-lipoprotein A-I (apoA-I)-deficient mice (Jackson Labora-tories, B6.129P2-Apoa1tm1Unc/J, Stock no: 002055, alsoon a C57Bl/6 background) to produce an F1 generationhemizygous for both apoa1 and the APP/PS1 transgenes.These animals where then backcrossed to apoA-I-deficient mice to produce F2 male and female mice offour genotypes: APP/PS1 mice hemizygous for apoa1(APP/PS1 apoA-IHEM), APP/PS1 mice with completeapoa1 deficiency (APP/PS1 apoA-IKO), nontransgeniclittermates hemizygous for apoa1 (WT apoA-IHEM), andnontransgenic littermates deficient in apoa1 (WT apoA-IKO). ApoA-I is the primary protein component ofplasma HDL; therefore, apoA-IKO mice have drasticallyreduced plasma HDL-C levels, as has been previouslyshown [30]. Plasma specimens were collected from thesemice at 12 months of age as described in the “Animalsand tissue collection” section.Brain biochemistryBrain homogenization and biochemistry were performedby an investigator blinded to animal injury status. Frozenbrains were homogenized using a Tissuemitehomogenizer in 8 volumes of ice-cold radioimmunopre-cipitation (RIPA) buffer (5 mM EDTA, 50 mM NaCl, 10mM Na Pyrophosphate, 50 mM NaF, 1% NP40 alterna-tive, pH 7.4) containing a complete protease inhibitor(11836153001, Millipore Sigma, Burlington, MA), as pre-viously described [22, 30]. RIPA homogenates were cen-trifuged at 9000 rpm for 10min at 4 °C and thesupernatants were stored at − 80 °C until analysis. Inter-leukin 6 (IL-6) was measured using a multiplex ELISAwith samples diluted 1:2 in supplied dilution buffer (V-PLEX Proinflammatory Panel 1 Mouse Kit, K15048D,Meso Scale Discovery, Rockville MD). Analyte concen-trations in the homogenate were normalized to totalprotein concentrations measured using a bicinchoninicacid (BCA) assay (23225, Thermo Fisher, Waltham MA).StatisticsMost statistical analyses were performed with GraphPadPrism 7, and p values < 0.05 were considered statisticallysignificant. One-way ANOVA followed by Dunnett’s orTukey’s multiple comparisons test were used to comparegroups to a control group or to all other groups, respect-ively. One-way ANOVA with repeated measuresfollowed by Dunnett’s multiple comparisons test wasused to compare the effects of hemolysis on assayperformance. Paired t tests were used to compare the ef-fects of serum vs. plasma, route of blood collection, andtime before centrifugation into plasma. Where indicated,data were log transformed before analysis. Differences inplasma GFAP in the proof-of-concept TBI and AD stud-ies were analyzed by two-way ANOVA followed bySidak’s or Tukey’s multiple comparisons test with TBIexposure, time, age, APP/PS1 genotype, or apoA-I geno-type as factors. Coefficients of variation were calculatedas standard deviation divided by mean.Precision data were also analyzed with a mixed modelwith maximum likelihood using SPSS Statistics. Fullmodel syntax and output is available in Additional File 1.Plasma group (low, intermediate, or high concentrationplasma pool) was analyzed as a fixed variable, technicalreplicate (intra-assay variation) as a repeated variableand a random variable, and assay replicate (inter-assayvariation) as a random variable. Three additional modelswere run with each plasma group analyzed separatelyand no fixed variable included.GFAP concentrations were interpolated from an 8-point standard curve by nonlinear regression using a 4parameter logistic (4PL) equation. Standard curve con-centrations and ECL signal were log transformed andcurve fitting was performed using GraphPad Prism 7.ResultsAssay development and analytical validation overviewTo enable plasma GFAP to be quantified in preclinicalspecimens, we used the MSD platform to develop anovel, highly sensitive ELISA assay based on a GFAPcapture-detection antibody pair from Abcam (ab244094,Abcam, Cambridge, UK). The assay requires 12.5 μL ofmurine plasma and can be run in 5 h. We performed apartial validation of this assay using published SOPs forimmunoassay validation endorsed by BIOMARKAPD[23], a consortium of scientists aiming to develop bio-marker assays for AD and Parkinson’s disease. Intra-assay CV is 5.0% and inter-assay CV is 7.2%. The assayhas excellent dynamic range, with an LLOD, LLOQ, andULOQ of 9.0, 24.8, and 16,533.9 pg/mL, respectively.The assay shows dilution linearity over a wide range ofcalibrator and plasma specimen dilutions. Calibrator canbe diluted from 200,000 to 20 pg/mL with 90–123% re-covery, and plasma specimens with high GFAP concen-trations can be diluted up to 32-fold with 96–112%recovery. Spike recovery is 67–100% depending on theconcentration of the spike and the endogenous GFAPconcentration in the specimens with the best recoveryfor low-concentration spikes into high-concentrationspecimens and the worst for low-concentration spikesinto low-concentration specimens. GFAP concentrationsmeasured by the assay remain stable after exposure ofplasma specimens to up to 7 freeze-thaw cycles, 168 h atButton et al. Alzheimer's Research & Therapy (2021) 13:58 Page 8 of 274 °C, 24 h at RT, or 30 days at − 20 °C. GFAP can also bedetected in serum using this assay however measuredconcentrations in serum are on average 1.18-fold higherthan measurements in plasma. Specimen hemolysis cansuppress assay measurements with increased suppressionobserved with increased hemolysis severity. In terms ofpre-analytical factors, storage of blood for up to 4 h onice before centrifugation to plasma does not affect assayperformance but measurements may be biased whenspecimens are collected from the saphenous vein ratherthan cardiac puncture. We were unable to perform val-idation for the trueness and uncertainty of the assay dueto the lack of available certified reference material. De-tailed results for these performance parameters are pro-vided below.Robustness of the assay to small protocol modificationsAssay robustness to small protocol variations wasassessed over 6 assays run in parallel following theprotocol variations described in Table 3. Overall, coeffi-cients of variation (CV) of specimen duplicates allremained < 15% with the exception of conditions wherethe plasma specimen incubation time was modified(Table 3). Specifically, extending specimen incubationtime from 2 h to overnight resulted in a CV of 18.2% forlow-concentration plasma pool duplicates. Capture anti-body incubation time had little effect on duplicate vari-ation when the time was reduced from 1 h to 30 min (<7.5%) or when the time was extended to overnight (<10%). Reduction of the detection antibody incubationtime from 1 h to 30 min also had little effect on dupli-cate variation (< 2.5% CV). Variations in specimen dupli-cates are also illustrated in Fig. 1.The closeness of the measured sample concentrationsbetween different assay protocols was also evaluated bycalculating the percent recovery compared to the stand-ard protocol as detailed in Table 3 and illustrated inFig. 1. Percent recovery ranged from 80 to 119% in allcases with two exceptions. When specimen incubationtime was extended to overnight, the measured concen-tration of the low plasma pool was only 72.7% of thatmeasured with the SOP. When capture antibody incuba-tion time was reduced from 1 h to 30 min, the percentrecovery of the low-concentration plasma pool was76.8%.Overall, all issues with assay robustness were restrictedto measurements of the low-concentration plasma pool.This finding is not surprising given the greater variabilityin measurements of specimens with low concentrationsunder the standard protocol as described below in the"Repeatability and intermediate precision of the assay"section. As the measured concentrations of the low-concentration plasma pool are relatively close to theLLOQ (24 pg/mL as described in the “Assay limits ofquantification and detection” section), higher variation isto be expected compared to measurements of intermedi-ate and high-concentration plasma pools, which haveconcentrations closer to the middle of the quantificationTable 3 Assay protocol modifications and summary of assay robustness resultsAssay Modification Plasma pool Replicate CV (%) %recovery*1 Standard protocol(Capture 1 h,Samples 2 h,Detection 1 h)Low 3.7 –Intermediate 0.7 –High 6.7 –2 Capture 30min Low 6.2 76.8Intermediate 3.7 89.4High 7.4 94.83 Capture overnight Low 9.6 90.7Intermediate 7.1 115.5High 3.9 108.74 Samples 1 h Low 3.4 101.7Intermediate 11.2 85.2High 9.7 97.95 Samples overnight Low 18.2 72.7Intermediate 0.5 81.6High 2.0 80.46 Detection 30 min Low 2.4 100.1Intermediate 2.3 110.8High 0.4 118.4*%recovery = (concentration measured with modified assay protocol/concentration measured with standard assay protocol) × 100%. CV coefficient of variationButton et al. Alzheimer's Research & Therapy (2021) 13:58 Page 9 of 27range of the assay as described in the “Assay limits ofquantification and detection” section. The percent recov-ery of low-concentration specimens was particularly af-fected by two protocol modifications: reducing thecapture antibody incubation duration (protocol 2) andextending the specimen incubation duration (protocol5).In protocol 2, the percent recovery issues for low-concentration specimens may be due to incompletebinding of the capture antibody to the plate. We suspectthat capture antibody binding to the plate may be in-complete by 30min but reaches saturation by 1 h. Inprotocol 5, the issues with variation and percent recov-ery for low-concentration specimens may be due to dif-ferences in standard curve preparation. The standardcurves for protocols 1–4 and protocol 6 were preparedsimultaneously. However, the standard curve for proto-col 5 was prepared separately on the day before theother protocols to allow for overnight specimen incuba-tion. As measurements at the low end of the standardcurve are especially sensitive to changes in standardcurve preparation, this may explain why variation andpercent recovery of the low-concentration pool was rela-tively poor in this case.Repeatability and intermediate precision of the assaySpecimens from low-, intermediate-, and high-concentration plasma pools were repeatedly assayed over5 separate days with 5 replicates each day to evaluateintermediate precision and repeatability of the assay, re-spectively (Fig. 2). Although mean concentrations weresignificantly different between some assay replicates, CVfor repeatability and intermediate precision were all <10% as detailed in Table 4. Overall, specimens from thelow-concentration plasma pool showed the lowest re-peatability and intermediate precision whereas speci-mens from the high-concentration pool showed the bestrepeatability and intermediate precision. Mean repeat-ability (intra-assay variation) across all pools was 5.0%and mean intermediate precision (inter-assay variation)was 7.2%.Precision data were also analyzed with a mixed modelwhereby plasma pool (low, intermediate, or high GFAPconcentration pool) was analyzed as a fixed variable,assay date (inter-assay variation) was analyzed as a ran-dom variable, and technical replicate (intra-assay vari-ation) was analyzed as a nested random variable withinassay date. Detailed results are available in AdditionalFile 1. We found that the greatest source of error wasthe plasma pool, with a standard error of 93.5 pg/mL.Inter-assay and intra-assay variations had standard er-rors of 59.6 pg/mL and 26.4 pg/mL, respectively. Coeffi-cients of variation calculated from variance dataproduced by the mixed model were similar to the CVsreported in Table 4 with a total intra-assay CV of 3.4%and inter-assay CV of 7.7% (Table 5).Assay limits of quantification and detectionTwo methods were used to establish assay limits ofquantification and detection. First, 8 plasma specimenswith low (Fig. 3a) or high (Fig. 3b) GFAP concentrationswere assayed in duplicate and the CV was evaluatedFig. 1 The assay is robust to changes in antibody incubation duration but less robust to changes in samples incubation duration. a Low-, bintermediate-, and c high-concentration plasma pools were assayed using the standard assay protocol (1 h capture antibody incubation, 2 hsample incubation, 1 h detection antibody incubation) and several protocols with modifications to incubation durations. Robustness to protocolmodifications was assessed by observing the variation in sample duplicate measurements and the closeness of the measured sampleconcentrations under each protocol to the measurements under the standard protocol. Mean concentration of duplicates is plotted with eachpoint representing an individual sample replicate and error bars representing ± SD. Differences in measured sample concentration compared tothe standard assay protocol were analyzed by one-way ANOVA with Dunnett’s multiple comparisons test and displayed within the graphs as*p < 0.05. o/n: overnight, SD: standard deviationButton et al. Alzheimer's Research & Therapy (2021) 13:58 Page 10 of 27(Fig. 3c). As all duplicates were within 20% CV, the ac-tual LLOQ may be lower and the actual ULOQ may behigher than reported in Table 6. Second, LLOD andLLOQ were calculated from the variation in ECL signalfrom 16 blank replicates (Fig. 3d) where LLOQ is 10 SDabove the blank measurement and LLOD is 2.5 SDabove the blank measurement (Table 6). The calculatedLLOQ (23.7 pg/mL) closely resembled the estimatedLLOQ from low sample measurement (24.8 pg/mL) sug-gesting that the true LLOQ of the assay is approximately24 pg/mL.Dilution linearity of recombinant GFAP spiked intoplasma and endogenous plasma GFAPDilution linearity of recombinant GFAP calibrator in theassay was determined by spiking calibrator into plasmaspecimens with very low endogenous GFAP. Three spec-imens were spiked at a concentration 50-fold higherthan the highest point in the calibration curve and 121-fold higher than the minimum ULOQ determined in the“Assay limits of quantification and detection” section(Table 6). Serial dilutions were performed to yield an ex-pected concentration lower than the LLOD and speci-mens were assayed in duplicate. The percent recovery ateach dilution factor was plotted (Fig. 4a) and detailed inTable 7. Duplicate variation was < 15% CV, and percentrecovery was between 90 and 123% for dilution factorsfrom 10-fold to 100,000-fold, corresponding to concen-trations of 20 to 200,000 pg/mL. Undiluted specimensand specimens diluted 1,000,000-fold showed poor-mean percent recoveries of 37.6 ± 4.3% and 369.2 ±136.2%, respectively. Dilutions in the 10-fold to 100,000-fold range also showed a linear ECL signal response(Fig. 4b). Overall, these data suggest that very high con-centrations of GFAP can be diluted into the workingrange of the assay with acceptable percent recovery andlevels of variation.Dilution linearity of endogenous GFAP in plasmaspecimens was similarly evaluated by creating serial dilu-tions of 3 plasma specimens with high endogenousGFAP and investigating the percent recovery (Fig. 4c)and ECL signal response linearity (Fig. 4d) across a rangeof dilutions from 1-fold to 64-fold. Average duplicateCV was < 7% at all dilution factors (Table 8). Mean CVfrom neat specimen concentration was less than 20%across all dilution factors and less than 10% up to a 32-fold dilution. Mean percent recovery was between 96and 112% up to a 32-fold dilution but grew to 131.2 ±23.9% at a 64-fold dilution. Overall, these data suggestthat plasma samples with high GFAP concentrations canTable 4 Assay repeatability and intermediate precisionPlasma pool Mean concentration (pg/mL) Repeatability CV (%)(intra-assay) (n = 5)Intermediate precision CV (%)(inter-assay) (n = 5)Low 43.4 7.2 8.5Intermediate 136.6 4.7 6.5High 4979.9 2.9 6.9Mean 1720.0 5.0 7.2CV coefficient of variationFig. 2 High intra-assay and inter-assay precision is observed for low-, intermediate-, and high-concentration samples. Assay precision wasdetermined through the measurement of a low-, b intermediate-, and c high-concentration plasma pools over 5 days with 5 sample replicatesper day. Mean concentration of replicates is plotted with each point representing an individual sample replicate and error bars representing ± SD.Differences in measured sample concentration between assay replicates were analyzed by one-way ANOVA with Tukey’s multiple comparisonstest and displayed within the graph as *p < 0.05, **p < 0.01, and ****p < 0.0001. SD: standard deviationButton et al. Alzheimer's Research & Therapy (2021) 13:58 Page 11 of 27be diluted up to 32-fold with excellent recovery and vari-ation (Table 8).Excellent parallelism between endogenous GFAP inplasma specimens and recombinant GFAP calibratorwas also demonstrated by plotting the ECL signal againstdilution factor and observing the slopes of the responses(Fig. 4d).Recovery of recombinant protein spiked into plasma atvarious concentrationsThe ability of the assay to accurately measure GFAP inplasma spiked with calibrator was tested using 5 plasmaspecimens spiked with 0, 100, 1000, or 10,000 pg/mL ofcalibrator (Fig. 5). The mean percent recovery overallwas 81.2 ± 17.3% (Table 9). Percent recovery wasBlank45505560657075ECL signal (AU)Blank Replicatesa bc d10 100 1000 10000 100000051015Average GFAP concentration (pg/mL)%CVConcentration vs. %CV1 2 3 4 5 6 7 8050100150SampleGFAP (pg/mL)Low Concentration Samples1 2 3 4 5 6 7 805000100001500020000SampleGFAP (pg/mL)High Concentration SamplesfemalemalefemalemaleFig. 3 The assay has a wide quantitative dynamic range from 25 pg/mL to at least 16,533 pg/mL and a lower limit of detection of 9 pg/mL. Eightplasma samples with a very low or b very high expected GFAP concentrations were assayed in duplicate and c CV of duplicates weredetermined. d 16 blank replicates (1% BSA in PBS) were assayed to calculate an estimated lower limit of quantification and lower limit ofdetection as described in Table 6. a,b,d Mean concentration of replicates is plotted with each point representing an individual sample replicateand error bars representing ± SD. c Points represent mean sample concentration and CV of duplicates. a–c Circles represent samples from femalemice, squares represent samples from male mice. AU: arbitrary units, CV: coefficient of variation, SD: standard deviation, BSA: bovine serumalbumin, PBS: phosphate-buffered saline, ECL: electrochemiluminescenceTable 5 Mixed model analysis estimates of fixed effects and covariancePlasma pool Mean concentration(pg/mL)GroupvariationIntra-assay variation Inter-assay variationSE (pg/mL) Variance(pg/mL)SD (pg/mL)SE (pg/mL)%CV Variance(pg/mL)SD (pg/mL)SE (pg/mL)%CVAll (model 1A) 1720.0 93.5 3484.7 59.0 26.4 3.4 17,763.2 133.3 59.6 7.7Low (model 2B) 43.4 – 4.0 2.0 0.9 4.6 3.5 1.9 0.8 4.3Intermediate(model 3C)136.6 – 23.5 4.8 2.2 3.5 24.2 4.9 2.2 3.6High (model 4D) 4979.9 – 0.0 0.0 0.0 0.0 70,844.3 266.2 119.0 5.3AAll groups: plasma pool = fixed effect, technical replicate = random effect nested in assay date, assay date = random effect. BLow plasma pool: technicalreplicate = random effect nested in assay date, assay date = random effect. CIntermediate plasma pool: technical replicate = random effect nested in assay date,assay date = random effect. DHigh plasma pool: technical replicate = random effect nested in assay date, assay date = random effect. SD for intra-assay and inter-assay variation were calculated as follows: SD= √variance). SE for intra-assay and inter-assay variation were calculated as follows: SE = SD/(√n), n = 5. SE standarderror, SD standard deviation, CV coefficient of variationButton et al. Alzheimer's Research & Therapy (2021) 13:58 Page 12 of 27better overall in spiked specimens with higher en-dogenous GFAP concentrations (87.7 ± 19.5%) com-pared to spiked specimens with lower endogenousGFAP concentrations (71.4 ± 6.05). Percent recoverywas best when a low-concentration spike (100 pg/mL)was added to specimens with high endogenous GFAPconcentrations (99.9 ± 25.8%) and worst when low-concentration spikes (100 pg/mL) were added to spec-imens with low endogenous GFAP concentrations(67.1 ± 10.5%).Assay stability to plasma temperature variationsThe stability of plasma GFAP to freeze-thaw cyclesand storage at room temperature, 4 °C, or − 20 °C forvarious lengths of time was measured for specimensfrom low-, intermediate-, and high-concentrationpools. Overall, high-concentration pool specimenswere remarkably stable. Percent recovery compared tospecimens with no temperature modulation wasbetween 95 and 97% after up to 7 freeze-thaw cycles(Fig. 6ai), between 92 and 103% after up to 168 h at4 °C (Fig. 6di), between 93 and 96% after up to 24 hat room temperature (Fig. 6di), and between 88 and103% after up to 168 h at room temperature (Fig. 6di).Duplicate variation for specimens from the high-concentration pool were within 4% CV across all con-ditions described above (Fig. 6aii, dii).Plasma GFAP from the low-concentration pool wasless stable yet still showed a percent recovery between84 and 111% (Fig. 6ai, bi) and duplicate CV within 12%for all conditions (Fig. 6aii, bii). Plasma from theintermediate-concentration pool showed percent recov-eries similar to specimens from the high-concentrationpool (between 93 and 115%) (Fig. 6ai, ci); however, du-plicate variation was higher. Specifically, duplicate vari-ation was within 8% in most conditions and reached16.6% in specimens stored at room temperature for 168h (Fig. 6aii, cii).Fig. 4 The assay shows high dilution linearity over a wide range of calibrator and plasma specimen dilutions. Assay calibrator was spiked into 3individual plasma samples with very low endogenous GFAP to a concentration of 2,000,000 pg/mL. Serial dilutions from 10-fold to 1,000,000-foldwere made and samples at each dilution step were assayed in duplicate. a Percent recovery of samples spiked with calibrator plotted againstdilution factor. b ECL signal of samples spiked with calibrator were plotted against the dilution factor. Serial dilutions were performed from 2-foldto 64-fold on 3 plasma samples with high endogenous GFAP concentrations. c Percent recovery of diluted plasma samples plotted againstdilution factor. d ECL signal of diluted plasma samples and calibrator plotted against dilution factor. Points represent mean concentration ofduplicates and error bars represent ±SD. For some points, the error bars are shorter than the height of the symbol and therefore are not shown.AU: arbitrary units, ECL: electrochemiluminescenceTable 6 Limits of quantification and detectionMeasured LLOQ Measured ULOQ Calculated LLOQ Calculated LLOD24.8 pg/mL* 16,533.9 pg/mL* 23.7 pg/mL# 9.0 pg/mL†*All sample replicates < 20% CV; therefore, actual LLOQ may be lower and ULOQ may be higher than reported here. #calculated as 10 SD above blankmeasurement from 16 blank replicates. †calculated as 2.5 SD above blank measurement from 16 blank replicates. LLOQ lower limit of quantification, ULOQ upperlimit of quantification, LLOD lower limit of detection, CV coefficient of variation, SD standard deviationButton et al. Alzheimer's Research & Therapy (2021) 13:58 Page 13 of 27Storage at − 20 °C for 30 days had little impact withpercent recoveries of 97.4%, 93.7%, and 104.4% for low-,intermediate-, and high-concentration pools, respectively(Fig. 6bi, ci, di). Duplicate variations also remained lowafter 30 days at − 20 °C with CV of 4.1%, 2.4%, and 6.9%for low-, intermediate-, and high-concentration pools,respectively (Fig. 6bii, cii, dii).Assay performance in serum and in specimens withhemolysisThe selectivity of the assay was evaluated by investigatingthe ability of the assay to detect GFAP in serum comparedto plasma and the ability to detect GFAP in plasma speci-mens with hemolysis. GFAP concentrations measured inserum were slightly elevated compared to GFAP concen-trations measured in plasma from the same animal. Thisdifference was not significant in sham animals (Fig. 7ai)(1.16-fold. p = 0.4901 by paired t test) but was significantin TBI animals (Fig. 7aii) (1.20-fold, p < 0.0001 by paired ttest). Duplicate variation was similar between plasma andserum measurements (Table 10).The effect of hemolysis on assay performance wasevaluated by spiking red blood cells into sham (Fig. 7bi)and TBI (Fig. 7bii) plasma specimens at various concen-trations then freezing the spiked plasma at − 80 °C for 1h. Hemolysis reduced measured GFAP concentrations,with a non-significant effect observed in plasma fromsham animals (Fig. 7bi) (p = 0.0955 by repeated measuresone-way ANOVA) and a significant effect in plasmafrom TBI animals (Fig. 7bii) (p = 0.0309 by repeatedmeasures one-way ANOVA). Spiking red blood cells intoplasma at 50% by volume reduced the measured concen-tration to 52.3% of the concentration measured in neatplasma overall. Spiking red blood cells into plasma at 5%by volume only reduced the measured concentration to93.0% of the concentration measured in neat plasmaoverall (Table 11). Duplicate variation was similar inspiked and neat plasma specimens (Table 11).Assay stability to modifications in blood collection andprocessing methodAnalyte stability was also evaluated in response to modi-fications in methods of blood collection and processing.Blood was collected via cardiac puncture or saphenousvein bleed from 3 mice exposed to TBI and 3 sham mice6 h after the TBI or sham procedure (Fig. 8a). In shammice, plasma GFAP concentrations from blood collectedby saphenous bleed were only 90.3 ± 44.3% of that col-lected by cardiac puncture (Fig. 8ai) (p = 0.5217 bypaired t test) while in TBI mice, plasma GFAP concen-trations from saphenous blood were 163.4 ± 1.8% of thatfrom cardiac puncture (Fig. 8aii) (p = 0.1314 by paired ttest) (Table 12). We also investigated the effect of delay-ing the centrifugation of blood intro plasma. BloodTable 8 Dilutional linearity of endogenous GFAP in plasma samplesDilution factor Mean duplicate CV (%) Mean CV from neat specimen (%) %recovery*(mean ± SD) (n = 3)1:1 2.7 – –1:2 3.8 2.6 96.5 ± 4.71:4 1.6 2.6 98.2 ± 4.21:8 2.9 3.9 96.6 ± 5.61:16 0.8 3.0 104.4 ± 1.31:32 1.4 7.8 111.8 ± 6.71:64 6.2 18.2 131.2 ± 23.9*%recovery = (measured concentration adjusted for dilution/measured concentration of undiluted sample) × 100%. CV coefficient of variation, SDstandard deviationTable 7 Dilution linearity of plasma spiked with recombinant GFAP proteinDilution factor Spike concentration (pg/mL) Mean duplicate CV (%) %recovery*(mean ± SD) (n = 3)1:1 2,000,000 11.5 37.6 ± 4.31:10 200,000 5.7 105.0 ± 4.91:100 20,000 9.8 116.4 ± 12.11:1000 2000 14.9 123.1 ± 40.91:10,000 200 3.9 114.5 ± 0.51:100,000 20 13.9 90.5 ± 15.01:1,000,000 2 38.1 369.2 ± 136.2*%recovery = (measured concentration adjusted for dilution/theoretical concentration) × 100%Button et al. Alzheimer's Research & Therapy (2021) 13:58 Page 14 of 27collected by cardiac puncture was aliquoted and one ali-quot was centrifuged into plasma within 1 h while theother aliquot was incubated on ice for 4 h before centri-fugation. Overall, delaying centrifugation of blood intoplasma by 4 h had little effect on the concentrations ofGFAP measured in plasma from sham (Fig. 8bi) or TBI(Fig. 8bii) animals compared to plasma centrifugedwithin 1 h of collection (p = 0.9919 and p = 0.1180 bypaired t test for sham and TBI animals, respectively)(Table 12). Blood collection method and processing hadlittle effect on duplicate variation as all duplicates werewithin 10% CV (Table 12).Proof-of-concept analysis of plasma GFAP as a TBI bloodbiomarker in miceIn humans after TBI, blood GFAP levels are high in thefirst 24 h after TBI and decline to baseline or near base-line levels during recovery, with lengthened trajectoriesas injury severity increases [10, 11, 13, 24–26, 31]. Todetermine if comparable kinetics are found in a mouseTBI model, we collected plasma 6 h or 2 days after shamor TBI procedures with a CHIMERA device at an energylevel that we previously determined to result in elevatedplasma total tau and NF-L levels, increased brain cyto-kines levels, blood-brain barrier leakage, microstructuralvascular abnormalities, and microgliosis [22]. PlasmaGFAP was detectable with our novel assay in all miceand was significantly elevated in plasma collected 6 hafter TBI compared to plasma collected 6 h after shamprocedure (49-fold) or 2 days after TBI (31-fold) (Fig. 9a)(injury effect p = 0.0007, time effect p = 0.0009, inter-action effect p = 0.0002 by two-way ANOVA; p < 0.00016 h post-TBI vs. 6 h post-sham and p < 0.0001 6 h post-TBI vs. 2 days post-TBI by Sidak’s multiple comparisonstest). Furthermore, plasma GFAP concentrations werepositively correlated with neurological impairment andneuroinflammation. Neurological severity score (NSS)was evaluated 2 h after injury and correlated with plasmaGFAP concentrations across the entire cohort and spe-cifically in samples collected 6 h after TBI or sham pro-cedures (Fig. 9b) (Spearman r = 0.5262 and p = 0.0099for entire cohort; Spearman r = 0.6177 and p = 0.0468 for6 h samples). Significant correlations were also observedbetween plasma GFAP concentrations and brain IL-6concentrations in the entire cohort, in plasma from TBI-exposed mice, and in plasma collected 6 h after TBI orsham procedures (Fig. 9c) (Spearman r = 0.7225 and p <0.0001 for entire cohort; Spearman r = 0.8187 and p =0.0011 for TBI samples; Spearman r = 0.7790 and p =0.0066 for 6 h samples).Proof-of-concept analysis of plasma GFAP in APP/PS1 miceRecent studies have shown that serum or plasma GFAPmay also be useful candidate blood biomarkers for AD.Serum GFAP was found to be elevated in AD subjectscompared to controls in one study and plasma GFAPwas found to predict amyloid positivity and associatedTable 9 Percent recovery of low-concentration and high-concentration plasma spiked with recombinant GFAPSpike concentration (pg/mL) %recovery* (mean ± SD)All samples Low-concentration samples (n = 2) High-concentration samples (n = 3)100 86.3 ± 25.8 67.1 ± 10.5 99.0 ± 25.81000 77.5 ± 11.8 73.1 ± 3.1 80.4 ± 15.610,000 79.7 ± 13.6 74.1 ± 0.6 83.5 ± 17.8Mean 81.2 ± 17.3 71.4 ± 6.0 87.7 ± 19.5*%recovery = ((measured concentration spiked sample-measured concentration neat sample)/concentration of spike) × 100%. SD standard deviationFig. 5 Spike recovery of the assay is optimal for specimens withhigh endogenous GFAP concentrations and sub-optimal forspecimens with low endogenous GFAP concentrations. a Fiveplasma samples were spiked with calibrator at 0, 100, 1000, or10,000 pg/mL. Mean percent recovery is plotted at each spikeconcentration with points representing individual spiked samplepercent recovery and error bars representing ±SD. Point shapesrepresent individual plasma samples. %recovery = ((measuredconcentration spiked sample-measured concentration neat sample)/concentration of spike) × 100%. SD: standard deviationButton et al. Alzheimer's Research & Therapy (2021) 13:58 Page 15 of 27Fig. 6 Assay measurements remain stable in specimens exposed to up to 7 freeze-thaw cycles, 168 h at 4 °C, 24 h at room temperature, or 30days at − 20 °C. a Samples from low-, intermediate-, and high-concentration plasma pools were assayed after a number a freeze-thaw cycles.Samples from b low-, c intermediate-, and d high-concentration plasma pools were also assayed after incubation at 4 °C, room temperature, or −20 °C. (i) Normalized concentrations relative to samples directly stored at − 80 °C and (ii) variation in sample duplicates are plotted. Pointsrepresent (i) mean normalized concentration or (ii) CV of duplicates and (i) error bars represent ±SD. For some points, the error bars are shorterthan the height of the symbol and therefore are not shown. The effect of plasma temperature modification on measured concentration wasanalyzed by repeated measures one-way ANOVA with Dunnett’s multiple comparisons test; no significant differences were found. CV: coefficientof variation, RT: room temperatureButton et al. Alzheimer's Research & Therapy (2021) 13:58 Page 16 of 27with cognitive performance in another [20]. Given thesefindings, we measured plasma GFAP levels over the life-span of APP/PS1 mice from 3 to 24 months of age(Fig. 10a). Overall, plasma GFAP was significantly af-fected by age (p < 0.0001 by two-way ANOVA) but notAPP/PS1 genotype (p = 0.4873 by two-way ANOVA).Levels began to increase at 12 months of age and a sig-nificant increase was observed in 24-month-old micecompared to all other ages across genotypes (p = 0.0003,p = 0.0001, p = 0.0001, p = 0.0002, p = 0.0005 for 3-, 6-,9-, 12-, and 18-month-old mice vs. 24-month-old mice,respectively, by Tukey’s multiple comparisons test)(Fig. 10a). Analysis of the aging effect within genotypesrevealed that plasma GFAP concentrations were onlysignificantly elevated in 24-month-old APP/PS1 miceand not in 24-month-old wildtype littermates (p =0.0015, p = 0.0009, p = 0.0009, p = 0.0013, p = 0.0011 for3, 6, 9, 12, and 18-month-old APP/PS1 mice vs. 24-month-old APP/PS1 mice, respectively, by Tukey’s mul-tiple comparisons test) (Fig. 10a). Although APP/PS1genotype had no significant effect on plasma GFAPlevels, a trend was observed such that plasma GFAPlevels were on average 1.6-fold higher in APP/PS1 micecompared to wildtype littermates across all age groups.We next investigated whether apoA-I deficiency hadany effect on plasma GFAP levels in APP/PS1 mice.Fig. 7 Assay measurements are slightly higher in serum than in plasma and are reduced in the presence of hemolysis. Blood was collected frommice 6 h after isoflurane exposure (sham) (i) or isoflurane exposure and TBI (ii) by cardiac puncture into tubes with (plasma) and without (serum)EDTA. a Plasma and serum were isolated by centrifugation and were assayed in duplicate. Differences in concentrations by matrix were analyzedby paired t test, ****p < 0.0001. b Plasma specimens were spiked with 0%, 5%, 25%, or 50% red blood cells, frozen at − 80 °C for 1 h, thawed, andassayed in duplicate. The effect of hemolysis on measured concentrations was analyzed by one-way ANOVA with repeated measures (exact pvalues are shown below graphs) followed by Dunnett’s multiple comparisons test, no significant differences from neat plasma were found. Meanplasma GFAP concentrations for each mouse are plotted with points representing mean and error bars ±SD of duplicates, in some cases errorbars are smaller than the symbol. TBI: traumatic brain injury, EDTA: ethylenediaminetetraacetic acid, SD: standard deviationTable 10 Duplicate variation in plasma and serum specimensMatrix Duplicate variation (%CV)Sham (n = 6) TBI (n = 7)Plasma 9.3 11.4Serum 2.7 3.6CV coefficient of variation, TBI traumatic brain injuryButton et al. Alzheimer's Research & Therapy (2021) 13:58 Page 17 of 27Although AD is characterized by amyloid beta (Aβ) pla-ques and neurofibrillary tangles, cerebral vessel patholo-gies are also common [32, 33] and may be an early stepin disease progression [34, 35]. Our group recently dem-onstrated that HDL, a class of circulating lipoproteinswith known vasoprotective functions [36], may protectagainst cerebral vessel dysfunction in AD. We showedthat plasma HDL may protect cerebral vessels from Aβdeposition and inflammation in 2D and 3D in vitromodels of cerebral vessels [37–39]. We also previouslystudied the effects of HDL on cerebral vessels in vivo bycrossing mice APP/PS1 mice with mice with loss-of-function mutations in apoA-I that lead to reducedplasma HDL-C levels [30]. We found that the lack ofapoA-I in 12-month-old APP/PS1 mice exacerbated cor-tical amyloid deposition, cerebral vessel amyloid depos-ition, astrogliosis, and cerebral vessel-associatedinflammation. Interestingly, we found that the cerebralvessels in APP/PS1 mice lacking apoA-I were associatedwith significantly more GFAP-positive astrocytes thancerebral vessels in APP/PS1 mice expressing apoA-I[30]. Given these previous observations of increasedcerebral vessel pathology in apoA-I-deficient APP/PS1mice, we investigated whether these mice also have ele-vated plasma GFAP levels. We observed a significant ele-vation in plasma GFAP levels in APP/PS1 micecompared to wildtype littermates overall (p = 0.0126 bytwo-way ANOVA) and in apoA-I-deficient mice com-pared to apoA-I-expressing mice overall (p = 0.0045 bytwo-way ANOVA) (Fig. 10b). Furthermore, mean plasmaGFAP levels in APP/PS1 mice lacking apoA-I were 1.9-fold higher than APP/PS1 mice expressing apoA-I and1.7-fold higher than WT mice lacking apoA-I (p =0.0073 and p = 0.0119 by Sidak’s multiple comparisonstest, respectively). Overall, these observations suggestthat murine AD models develop increased plasma GFAPduring aging and that GFAP may also be affected by cir-culating HDL levels, which have previously been shownto affect cerebral vessel health [30, 37–39].DiscussionGiven the growing evidence for the utility of plasma orserum GFAP as a TBI biomarker with both diagnosticand prognostic contexts of use [4–13, 24–26, 31, 40, 41],we aimed to develop an immunoassay capable of meas-uring GFAP in mouse plasma to facilitate translationalTable 11 Duplicate variation and %concentration of neat plasma vs. plasma spiked with red blood cellsDuplicate variation (%CV) %concentration of neat plasmaRed blood cell spike (% volume) Sham animals(n = 3)TBI animals(n = 3)All animals(n = 6)Sham animals(n = 3)TBI animals(n = 3)All animals(n = 6)0% 8.2 1.1 4.6 – – –4.8 1.5 3.2 94.1 91.9 93.025% 9.6 1.4 5.5 77.1 67.6 72.47.9 4.7 6.3 52.7 51.8 52.3CV coefficient of variationFig. 8 Assay measurements are unaffected by a delay in blood centrifugation to plasma but may be affected by route of blood collection. Bloodwas collected from mice 6 h after isoflurane exposure (sham) or isoflurane exposure and TBI by the saphenous vein and by cardiac puncture.Saphenous blood was immediately centrifuged to plasma. Blood collected by cardiac puncture was separated into two equal aliquots. Onealiquot was immediately centrifuged to plasma (optimal) and one aliquot was left on ice for 4 h before centrifugation to plasma (4 h on ice). Aftercentrifugation to plasma, samples were immediately stored at − 80 °C. Samples were assayed in duplicate. Mean plasma GFAP concentrations foreach mouse are plotted with points representing mean and error bars ±SD of duplicates. The effect of blood collection and processing methodon measured concentration was analyzed by paired t test; no significant differences were found. SD: standard deviation, TBI: traumatic brain injuryButton et al. Alzheimer's Research & Therapy (2021) 13:58 Page 18 of 27TBI studies using blood GFAP levels as an outcome. Ro-dent models remain critical tools in understanding bio-mechanical, histological, and biochemical outcomes afterTBI, as they enable precise control over a wide variety ofexperimental injury conditions, have a clearly definedtime of injury, and yield brain tissue specimens impos-sible to obtain in most studies of human TBI. Many as-says of human GFAP have been developed with theQuanterix assay becoming an industry leader in thequantification of serum or plasma GFAP in humans [7,12–14, 19, 42]. However, the Quanterix GFAP assaydoes not cross-react with murine GFAP [43]. Further-more, commercial sandwich ELISAs for murine GFAPcannot reliably detect GFAP in plasma or serum andhave a limited dynamic range (as described in Table 14).These are critical limitations as serum and plasma GFAPmay be expected to have a large dynamic range inmouse models of TBI given the expected large dynamicrange of serum and plasma GFAP observed in healthyhumans and in humans after TBI [7–9, 24, 31, 44].The novel GFAP immunoassay described here was de-veloped for the Meso Scale Discovery platform, whichuses ECL technology to provide high sensitivity, lowbackground, and a broad dynamic range. We first ob-tained a commercial GFAP antibody pair from Abcamwith established mouse, rat, and human cross-reactivity.The capture antibody was biotinylated to bind to strep-tavidin coated plates for increased sensitivity and the de-tection antibody was labeled with the MSD proprietarydetection label. During plate reading, electrodes in theplate are activated resulting in a chemical reaction withbound detection label that produces light measured bythe instrument [45].We validated the analytical performance of our novelassay following the SOP suggested by Andreasson et al.[23] and compared its performance to that of GFAP as-says for human blood and commercial ELISAs availablefor mouse GFAP. The most commonly used assays formeasuring GFAP in human blood are the Banyan BTIassay and the Quanterix Neurology 4-plex assay. TheTable 12 Impact of blood collection and processing method on assay performanceSample handling Mean duplicate CV (%) %recovery*(mean ± SD) (n = 3)Sham mice TBI mice Sham mice TBI miceCardiac puncture, centrifuged < 1 h after collection 7.8 4.7 – –Saphenous collection 5.1 9.4 90.3 ± 44.3 163.4 ± 1.84 h delay before centrifugation 5.4 3.3 100.5 ± 15.4 111.5 ± 1.5*%recovery = (concentration measured with modified collection or processing/concentration measured with optimal collection and processing) × 100%. CVcoefficient of variation, SD standard deviationFig. 9 Murine plasma GFAP concentrations are acutely elevated after a closed-head TBI. Male and female C57Bl/6 mice aged 3.5–5 months wereanesthetized with isoflurane then exposed to a 2.5 J closed-head TBI with a CHIMERA device or to isoflurane without TBI (sham). Blood wascollected by cardiac puncture 6 h or 2 d after the sham or TBI procedure and centrifuged to plasma. a Mean plasma GFAP concentrations ofeach group are plotted with points representing individual mouse plasma samples and error bars representing ±SD. Circles represent femalemice and triangles represent male mice. The effects of injury and time after injury on plasma GFAP concentrations were analyzed by two-wayANOVA (exact p values below graph) followed by Sidak’s multiple comparisons test (detailed within graph) where ****p < 0.0001. Correlationsbetween plasma GFAP concentrations and b neurological severity score 2 h after sham or TBI procedure and c brain IL-6 concentrations wereanalyzed by Spearman correlation. Significant correlations are displayed below graphs with exact p values. TBI: traumatic brain injury, SD: standarddeviation, IL-6: interleukin 6, CHIMERA: closed-head injury model of engineered rotational accelerationButton et al. Alzheimer's Research & Therapy (2021) 13:58 Page 19 of 27Banyan assay was foundational for its use in studies lead-ing to the FDA approval of GFAP, along with UCH-L1,as a biomarker for concussion [4]. In 2016, Quanterix li-censed the GFAP antibody from the Banyan assay foruse in their highly sensitive Simoa system [46], which isnow the mostly commonly used assay for clinical studies[7, 12–14, 26, 31, 44]. Earlier GFAP biomarker studiesused commercially available or prototype ELISAs fromvarious companies [6, 11, 15, 16, 18, 41, 47].Performance data for precision, limits of quantifica-tion, dilutional linearity, and recovery have been re-ported for some human and mouse assays and aresummarized in Table 13 and Table 14. Data have notbeen routinely reported for robustness, parallelism, se-lectivity, and analyte stability. In terms of precision, therepeatability of our assay (5.0%) was very similar to thatof the Banyan (3.6%) and Quanterix (4.2%) assays al-though the intermediate precision was considerablyhigher (7.2%) than the reported between-day CV of thehuman assays (0.8% and 0.0% for Banyan and Quanterix,respectively). Both precision parameters were very simi-lar to those reported for commercial mouse ELISAs.The limits of quantification of our assay have some ad-vantages and some disadvantages compared to other as-says. Although our assay is less sensitive than theQuanterix assay and some commercial mouse ELISAs, ithas a large detection range and a similar LLOQ to theBanyan assay. Importantly, all plasma specimens assayedin this study, including those from uninjured wildtypemice, were detectable by our assay with the lowestconcentration measured at 24.8 pg/mL in plasma from afemale 2.5-month-old C57Bl/6 mouse, well above thecalculated LLOD of 9 pg/mL.We assessed the dilution linearity of plasma specimenswith high endogenous GFAP concentration up to a 64-fold dilution. Dilution linearity up to a 16-fold dilutionwas similar or better than that reported for the Quan-terix and Banyan assays, although the Quanterix assayshowed better linearity over a larger dilution range of upto 132-fold. By this metric, our assay also performed bet-ter than the commercial mouse ELISAs. However, lin-earity of the commercial mouse ELISAs was mainlyestablished by spiking plasma, serum, or media sampleswith recombinant GFAP and diluting the spiked sam-ples. Using this approach, the dilution linearity of ourassay was far superior to the commercial ELISAs with91–123% linearity up to a 100,000-fold dilution.To assess recovery, we spiked specimens with low andhigh endogenous GFAP with recombinant GFAP at 3different concentrations. Mean spike recovery of ourassay was superior to that reported for the Quanterixassay. Spike recovery performance has not been reportedfor the Banyan assay. Although spike recovery for low-concentration specimens was poor compared to datafrom commercial mouse ELISAs, spike recovery for highconcentration specimens was comparable.Having validated important analytical parameters ofthis novel assay, we next examined the ability of ourassay to detect GFAP in mouse plasma with kinetics thatresemble that of the human GFAP response after TBI.Fig. 10 Plasma GFAP levels are elevated in aged APP/PS1 mice and APP/PS1 mice lacking apoA-I. a Female APP/PS1 mice (on a mixed C3H/Bl6background) were aged to 3, 6, 9, 12, 18, or 24 months old then plasma specimens were collected by cardiac puncture. Mean plasma GFAPconcentrations of each group are plotted with points representing individual mouse plasma samples and error bars representing ±SD. The effectsof age and APP/PS1 genotype on plasma GFAP concentration were analyzed by two-way ANOVA (exact p values below graph) followed byTukey’s multiple comparisons test across genotypes (exact p values below graph) or Sidak’s multiple comparison’s test within each genotype(detailed within graph, **p < 0.01, ***p < 0.001). b Male and female apoA-I hemizygous (apoA-IHEM) or knockout (apoA-IKO) mice with or withoutAPP/PS1 transgenes (on a C57Bl/6 background) were aged to 12 months old then plasma specimens were collected by cardiac puncture. Meanplasma GFAP concentrations of each group are plotted with points representing individual mouse plasma samples and error bars representing ±SD. The effects APP/PS1 genotype and apoA-I genotype on plasma GFAP concentration were analyzed by two-way ANOVA (exact p values belowgraph) followed by Sidak’s multiple comparisons test (detailed within graph *p < 0.05, **p < 0.01). apoA-I: apolipoprotein A-I, SD:standard deviationButton et al. Alzheimer's Research & Therapy (2021) 13:58 Page 20 of 27Table 13 Assay characteristics compared to human GFAP assaysSupplier Quanterix# Banyan# BioVendor Randox Elecsys Novel MSD assayCatalog number N4PA BC-2209 RD192072200R StrokeArrayVolume of sample/well (μL)152 250 35 N/A 50 12.5LLOD(pg/mL)Outcome 0.221 N/A 45 N/A *50 †30 9Protocol 2.5 SD abovebackground signalof blank. 2 reagentlots and 3instruments, 12 runstotal.N/A 3 SD abovebackgroundsignal.N/A *2 SD above loweststandard point.†N/A2.5 SD above backgroundsignal of 16 blank replicates.LLOQ(pg/mL)Outcome 0.467 10 N/A 160 N/A < 24.8Protocol Serial dilutions ofcalibrator assayed intriplicate. 2 reagentlots and 3instruments, 12 runstotal.Lowest measuredconcentration ofserum pools (n = 7,some spiked) withduplicate CV < 15%.N/A N/A N/A 10 SD above backgroundsignal of 16 blank replicates.Lowest measuredconcentration of plasmaspecimens (n = 8) withduplicate CV < 20%.ULOQ(pg/mL)Outcome 4000 320 25,000 100,000 *150,000†100,000> 16,534Protocol N/A Highest measuredconcentration ofserum pools (n = 7,some spiked) withduplicate CV < 15%.N/A N/A N/A Highest measuredconcentration of plasmaspecimens (n = 8) withduplicate CV < 20%.Intra-assay CV(%)Outcome 4.2 3.6 5.1 3–4 *1.1–1.9†0.8–0.625.0Protocol Within-run CV from5 days, 2 runs/day, 1plasma pool/run, 3replicates/pool.Within-run CV from5 days, 1 run/day, 5serum pools/run, 4replicates/pool.Within-run CVfrom 8replicates of 2serumspecimens.N/A *Within-run CV from1 run, 4 serumspecimens, 21replicates/specimen.†Within-run CV from1 run, 5 serumspecimens, 21replicates/specimen.Within-run CV from 5 days, 1run/day, 3 plasma pools/run, 5replicates/pool.Inter-assay CV(%)Outcome 0.0 0.8 5.7 N/A *2.7–4.2†1.8–5.97.2Protocol Between-day CVfrom 5 days, 2 runs/day, 1 plasma pool/run, 3 replicates/pool.Between-day CVfrom 5 days, 1 run/day, 5 serum pools/run, 4 replicates/pool.Between-runCV from 7runs, 2 serumspecimens/run.N/A *Between-day CVfrom 10 runs, 4serum specimens/run, 21 replicates/specimen†Between-day CVfrom 10 runs,maximum 2 runs/day,5 serum specimens/run, 6 replicates/specimen.Between-day CV from 5 days,1 run/day, 3 plasma pools/run,5 replicates/poolRecovery Outcome 50–120% N/A 83–115% N/A N/A 62–97%A86–105%BProtocol Spike 80 and 800pg/mL into serum(n = 2) and plasma(n = 2).N/A Spike 500,1000, and5000 pg/mLinto serum(n = 3).N/A N/A Spike 100, 1000, and 10,000pg/mL into plasma (n = 5).DilutionLinearityOutcome 102–121% 89–100% 97–121% N/A N/A 93–105%Protocol Spiked serumdiluted 1:4 to 1:128.Combined clinicalspecimens with alow-concentrationserum pool inSerum (n = 3)diluted 1:2 to1:8.N/A N/A Plasma (n = 3) diluted 1:2 to 1:16.Button et al. Alzheimer's Research & Therapy (2021) 13:58 Page 21 of 27We used the non-surgical CHIMERA model of TBI forthis proof-of-concept experiment. Plasma GFAP was de-tectable in all injured and uninjured specimens and weobserved an acute, robust increase in plasma GFAP afterTBI. Specifically, GFAP levels were 49-fold higher inplasma collected 6 h after TBI compared to plasma col-lected 6 h after the sham procedure. By 2 days after in-jury, GFAP levels did not differ significantly betweenTBI and sham mice. This acute response of plasmaGFAP to TBI resembles the acute response of GFAP instudies of human TBI where GFAP levels are found topeak within 24 h of injury [11, 24–26]. Longer follow-upstudies have suggested that the long-term response ofGFAP may be biphasic with an initial reduction after in-jury followed by an increase after 6 months [31]. Add-itional future studies will be needed to determine theprecise temporal profiles of plasma GFAP after TBI orother neurological insults in mice both at acute andchronic timepoints.We also performed preliminary analyses of the associa-tions between plasma GFAP and TBI outcomes in mice.Plasma GFAP correlated with neurological severityscores evaluated 2 h after the TBI or sham procedureacross the entire cohort and specifically in plasma speci-mens collected 6 h after sham or TBI. As previously dis-cussed, blood GFAP levels have been shown to associatewith several outcomes after TBI in many human studies.For example, serum GFAP levels were higher after con-cussion in subjects experiencing loss of consciousness orposttraumatic amnesia than in concussion subjects with-out these conditions [26]. In CT-positive subjects, sub-jects with acute unfavorable outcomes, including deathor craniotomy, had increased serum GFAP compared toCT-positive subjects with stable, improving, or transientneurological dysfunction [9]. Blood GFAP also associateswith injury severity measured with Glasgow ComacScale [10, 40, 41] although associations may not existwhen investigating exclusively mild TBI [6]. Many TBIoutcomes in human studies are evaluated 6 to 12 monthsafter injury [5, 6, 11, 13]; therefore, future studies willneed to be performed to determine whether blood GFAPlevels also predict long-term outcomes after TBI inmice.TBI is only one of many neurological indications forwhich blood biomarkers may be useful. Advances inassay technology have also aided the development ofblood biomarkers for AD including Aβ42/40, phosphor-ylated tau, and neurofilament light [48]. GFAP has thusfar been less studied as a blood biomarker for AD butrecent investigations have found that it is elevated in ADsubjects [19], can predict brain amyloid positivity [20],and associates with cognitive function [20]. Therefore,we aimed to provide preliminary data on GFAP in theblood of AD model mice using our novel assay. In APP/PS1 mice, a common AD model, we observed an age-dependent increase in plasma GFAP starting at 12months of age, around the time at which amyloid pla-ques are well-established [49]. By 24months of age,plasma GFAP levels were significantly higher than levelsat 3, 6, 9, 12, or 18 months of age. Although plasmaGFAP concentrations tended to be higher in APP/PS1mice compared to wildtype littermates at most ages, thedifference was not significant.Intriguingly, we observe elevated plasma GFAP levelsin 12-month-old APP/PS1 mice with apoA-I-deficiency.ApoA-I is the primary protein component of plasmaHDL therefore apoA-I-deficient mice have drastically re-duced plasma HDL-C levels [50]. We previously showedthat plasma HDL has vasoprotective functions onin vitro models of cerebral vessels [37–39] and apoA-Ideficiency exacerbates cortical amyloidosis, astrogliosis,and vascular inflammation in APP/PS1 mice [30]. Micehave relatively high vascular resilience compared tohumans due to their high plasma HDL to low-densitylipoprotein ratios [51] and lack of exposure to modifiablerisk factors, which account for up to 40% of dementiarisk in humans [52]. Therefore, our data suggest that al-though amyloidosis on its own may not cause elevatedplasma GFAP in mice, combining amyloidosis with fac-tors that reduce vascular resilience, such as apoA-I defi-ciency, may better recapitulate changes in plasma GFAPobserved in human AD subjects.Table 13 Assay characteristics compared to human GFAP assays (Continued)Supplier Quanterix# Banyan# BioVendor Randox Elecsys Novel MSD assaydifferent proportions(n = 11)Performance dataretrieved from:Supplier Supplier Supplier Postiet al2017[47]*Tichy et al [15]†Luger et al [18]References [7, 11–14, 26, 31, 44] [4, 8, 9, 17, 24, 25, 40] [6, 10, 11] [16, 41,47][15, 18]#Same antibody pair used between assays. A Recovery data for samples with low endogenous GFAP. B Recovery data for samples with high endogenous GFAP. N/A data not available, LLOD lower limit of detection, LLOQ lower limit of quantification, ULOQ upper limit of quantification, CV coefficient of variation, SDstandard deviationButton et al. Alzheimer's Research & Therapy (2021) 13:58 Page 22 of 27Table14AssaycharacteristicscomparedtoexistingcommercialELISAassaysformiceSupplierMyBioSourceAbcam*ELISAGenieMilliporeCusabioNovelMSDassayCatalognumberMBS723651MBS2515511MBS2018967MBS763852MBS2701012ab233621MOFI00191NS830E08603mCost/plate$640USD$490USD$625USD$415USD$350USD$593CAD€599$693CAD$600USD$150USDVolumeofsample(μL)501001001001005010010050–10012.5Detectionrange(pg/mL)50–100015.63–100015.6–100015.6–100062.5–4000125–800015.6–10001500–100,0003.12–20024.8–16,533.9LLOD(pg/mL)Outcome19.38<6.39.3823.38.79.37515000.789ProtocolN/AN/A2SDabovebackgroundof20blanksreplicates.N/A2SDabovebackgroundof20blankreplicates.N/AN/AN/A2.5SDabovebackground16blankreplicatesIntra-assayCV(%)Outcome<94.6<10<8<104.4<86.8<85.0ProtocolN/AWithin-runCVfrom1run,3specimens,20replicates/specimen.Within-runCVfrom1run,3specimens,20replicates/specimen.Within-runCVfrom1run,3specimens,20replicates/specimen.Within-runCVfrom1run,3specimens,20replicates/specimen.Within-runCVfrom1run,8replicates.Within-runCVfrom1run,3specimens,20replicates/specimen.Within-runCVfrom1run,3specimens,16replicates/specimen.Within-runCVfrom1run,3specimens,20replicates/specimen.Within-dayCVfrom5days,1run/day,3plasmapools/run,5replicates/pool.Inter-assayCV(%)Outcome<105.1<12<10<125.8<105.2<107.2ProtocolN/ABetween-runCVfrom3runs,3specimens/run,20replicates/specimen.Between-runCVfrom3runs,3specimens/run,8replicates/specimen.Between-runCVfrom3runs,3specimens/run,8replicates/specimen.Between-runCVfrom3runs,3specimens/run,8replicates/specimen.Between-runCVfrom3runs.Between-runCVfrom3runs,3specimens/run,8replicates/specimen.Between-runCVfrom3runs,6specimens/run,2replicates/specimen.Between-runCVfrom20runs,3specimens/run.Between-dayCVfrom5runs,3plasmapools/run,5replicates/pool.RecoveryOutcome94–103%89–103%78–99%85–105%80–98%95–100%88–105%95–109%90–104%62–97%A86–105%BProtocolN/ASpikedserum,plasma,culturemedia(n=5each)at3concentrations.Spikedplasma(n=10)andserum(n=5).Spikedplasma(n=10)andserum(n=5).Spikedplasma(n=10)andserum(n=5).Spikedbrainextract.Spikedplasma(n=10)andserum(n=5).Spikebrain(n=1)orcell(n=2)lysatewith1.56–50ngGFAP.Spikedserum(n=5)orplasma(n=5).Spikeplasma(n=5)with100,1000,and10,000pg/mLGFAP.DilutionlinearityOutcome92–108%80–109%80–108%81–104%78–105%87–100%81–104%77–122%85–108%93–105%Protocol2×to16×Spikedserum,plasma,culturemedia(n=5each),diluted1:2to1:16.Spikedplasma(n=10)andserum(n=5),diluted1:2to1:16.Spikedplasma(n=10)andserum(n=5),diluted1:2to1:16.Spikedplasma(n=10)andserum(n=5),diluted1:2to1:16.Brainextract(n=2)diluted1:2to1:16.Spikedplasma(n=10)andserum(n=5),diluted1:2to1:16.Spikedbrainlysate(n=3)1:3to1:27.Spikedserum(n=4),diluted1:1to1:8.Plasma(n=3)diluted1:2to1:16.* Sameantibodypairusedbetweenassays.ARecoverydataforsampleswithlowendogenousGFAP.BRecoverydataforsampleswithhighendogenousGFAP.LLODlowerlimitofdetection,CVcoefficientofvariation,SDstandarddeviation.DataretrievedfromsupplierproductwebsitesButton et al. Alzheimer's Research & Therapy (2021) 13:58 Page 23 of 27Notably, our study compares apoA-I-deficient mice tomice with apoA-I haploinsufficiency rather than micewith wildtype apoA-I expression levels due to survival is-sues with apoA-I-deficient mice that have previouslybeen thoroughly explained [30]. Previous studies havefound a greater magnitude difference in plasma apoA-Ilevels between mice with total apoA-I deficiency andapoA-I haploinsufficiency compared to the differencebetween mice with wildtype apoA-I levels and mice withhaploinsufficiency [50]. Furthermore, we have previouslyshown a robust reduction in plasma HDL-C levels inapoA-I-deficient mice compared to mice with apoA-Ihaploinsufficiency [30].The ability to complement the many exceptional on-going clinical biomarker studies with preclinical datafrom mouse models is a considerable advance for thefield. Studying TBI exclusively in humans is challengedby considerable heterogeneity in TBI severity, mechan-ism of injury, age at injury, and many other variables. Al-though the majority of TBI cases are classified as mild,many of these cases may not seek medical attention orbe admitted to hospital for treatment or enrollment intoa clinical research study [53]. TBI studies in animalmodels allow for a level of control over injury severityand sample collection not possible in human studies.Studying TBI in mouse models also allows for more ex-tensive analysis of how blood biomarkers relate tochanges in brain tissue through histological and bio-chemical analyses. We began to make use of this advan-tage in the current study in our analysis of braincytokine concentrations 6 h and 2 days after sham orTBI procedures. We found that plasma GFAP concen-trations correlated with brain IL-6 concentrations inmice exposed to TBI and sham injuries. Mouse modelsalso have important roles in preclinical drug testing andfor experimental investigation of the interactions of TBIwith co-morbid conditions, such as Alzheimer’s disease[54, 55] and anabolic steroid use [56]. We are encour-aged by the potential ability to measure TBI blood bio-markers in serial mouse plasma specimens, as this maycatalyze profound advancements and reduce animalnumbers needed for a preclinical study by shifting tolongitudinal analyses that include within-subjectoutcomes.Although GFAP has shown great promise as a bloodbiomarker for TBI, some limitations remain. A 2017study by Posti et al. measured serum GFAP in peoplewith orthopedic injuries without CNS involvement andfound levels higher than or the same as levels in CT-negative mild TBI patients [47]. The authors thereforeurged caution in the use of GFAP in diagnosing mildTBI in orthopedic trauma patients. The use of GFAP asa diagnostic biomarker may also be hindered by the rela-tively narrow time window after injury during which it iselevated. Studies have consistently shown that bloodGFAP levels peak within the first 24 h after injury andthen decline [11, 24–26]. Future studies will need to beperformed to determine whether the long-term temporalprofile of blood GFAP after TBI in mice reflects that ofhumans and the specificity to CNS injury. One potentiallimitation in performing these studies is the need for in-terim blood collection. We performed preliminary testscomparing GFAP levels in blood collected by cardiacpuncture compared to blood collected from the saphe-nous vein in the same mouse in order to address thisquestion. Although only 12.5 μL of plasma is required tomeasure GFAP in our novel assay, technical difficultiesin obtaining blood via the saphenous vein may still limitserial GFAP measurement, as occurred in our prelimin-ary study presented here. Our preliminary study alsosuggested a potential bias in the measured concentrationbased on blood collection method. No significant differ-ences were found between blood collection or process-ing methods by paired t tests; however, variations in themeasurements were substantial and further studiesassessing these pre-analytical factors will be important.GFAP concentration in blood collected via the saphe-nous vein of TBI-exposed mice was on average 163% ofthat measured in blood collected via cardiac puncturefrom the same animal. As blood was collected from thesaphenous vein on average 92 min before collection viacardiac puncture, it is possible that the differences inGFAP concentrations could be due to the rapid changesin circulating GFAP levels that occur in the first fewhours after TBI [24, 25]. Differences may also be the re-sult of differences in the materials and techniques usedto collect blood via cardiac puncture vs. saphenous vein.An additional concern with respect to saphenous veinblood collection is the increased risk of specimenhemolysis as we found that hemolysis can suppress mea-sured GFAP concentrations in our assay.LimitationsThis study has several limitations. First, this study mustbe considered a partial validation. Although this studyevaluated most of the immunoassay validation parame-ters proposed by Andreasson et al., we did not evaluatetrueness or uncertainty and performed only a limitedevaluation of selectivity [23]. Protocols for the validationof assay trueness and uncertainty provided by Andreas-son et al. require comparison to a certified reference ma-terial [23]. As certified reference materials for mouseplasma GFAP are not available, validation of these pa-rameters was not possible. However, if this assay is to beapplied to human specimens in the future, validation ofassay trueness and uncertainty should be considered.Our analysis of selectivity was limited to investigatingserum and plasma specimens and to investigating theButton et al. Alzheimer's Research & Therapy (2021) 13:58 Page 24 of 27effects of specimen hemolysis. Future studies will furtherevaluate selectivity by testing the ability of the assay todetect GFAP in additional matrices and in the presenceof confounding factors. Furthermore, validation proto-cols were only performed in a single laboratory; there-fore, future cross-laboratory validation experiments willbe necessary to fully understand the performance of thisassay. The validation of the assay was also limited in thatonly a single monoclonal antibody pair was tested. Theantibody pair used was selected for its advertised speciesreactivity, the buffer in which the antibodies were dis-solved, and the relatively large quantities available forpurchase. However, alternative antibody pairs could beinvestigated in the future and may also be effective inthis assay.In addition to limitations with respect to assay valid-ation parameters, this study is also limited in the typesof specimens tested. Future studies will be needed to as-sess whether this assay can detect GFAP in fluids or tis-sues from other species. Studies to assess the ability ofthis assay to detect GFAP in human, rat, pig, and ferretblood specimens are ongoing and preliminary experi-ments show promise for the utility of this assay in thesespecies. Finally, although we described how our assayperforms compared to existing human and mouse GFAPin Table 13 and Table 14, we have not directly comparedany of these assays to our assay on the same samples inour laboratory.ConclusionsWe developed the first known immunoassay capable ofquantifying GFAP in murine plasma. Having establishedits analytical rigor, we used this assay to reveal a murineplasma GFAP response to CHIMERA TBI that is similarto the GFAP response to TBI in humans. We also pro-vide preliminary data using this assay to study plasmaGFAP in AD model mice. Future studies with this assayhave the potential to bridge the gap between clinical andpreclinical TBI research and accelerate the understand-ing of TBI pathophysiology and development of TBItherapeutics.AbbreviationsGFAP: Glial fibrillary acidic protein; TBI: Traumatic brain injury; CT: Computedtomography; MRI: Magnetic resonance imaging; CHIMERA: Closed-headimpact model of engineered rotational acceleration; AD: Alzheimer’s disease;CSF: Cerebrospinal fluid; UCH-L1: Ubiquitin carboxy-terminal hydrolase L1;FDA: Food and Drug Administration; ELISA: Enzyme-linked immunosorbentassay; MS: Multiple sclerosis; HDL: High-density lipoprotein; RT: RoomTemperature; MSD: Meso Scale Discovery; EDTA: Ethylenediaminetetraaceticacid; PBS: Phosphate-buffered saline; BIOMARKAPD: Biomarkers forAlzheimer’s disease and Parkinson’s disease; SOP: Standard operatingprocedure; rpm: Revolutions per minute; CV: Coefficient of variation;SD: Standard deviation; ECL: Electrochemiluminescence; LLOD: Lower limit ofdetection; RIPA: Radioimmunoprecipitation; BCA: Bicinchoninic acid;LLOQ: Lower limit of quantification; ULOQ: Upper limit of quantification;HDL-C: High-density lipoprotein cholesterol; apoA-I: Apolipoprotein A-ISupplementary InformationThe online version contains supplementary material available at https://doi.org/10.1186/s13195-021-00793-9.Additional file 1. GFAP assay precision mixed model output. ExportedSPSS Statistics output analyzing precision data (“Repeatability andintermediate precision of the assay” section, Fig. 2, Table 5). (XLSM 57 kb)AcknowledgementsThe authors thank Dr. Gabriel Alfaro, Dr. Eric Sandberg, and Mr. Ken Dickfrom Meso Scale Discovery for their assistance and advice in the initialdevelopment of this assay. The authors also thank Dr. Mike Williams atAblabs for his advice on obtaining an optimal antibody pair and for hisservices conjugating the detection antibody. Finally, the authors thank thelaboratory of Dr. Lisa Galea for the use of their Meso Scale instrument for ourassay measurements.Authors’ contributionsEBB was responsible for study conception, designing experiments,performing all immunoassay experiments, analyzing and interpreting results,and preparing the manuscript. CHIMERA experiments were performed byWHC, CB, HC, AB, and DCB. WHC and CB assisted in the design of CHIMERAexperiments. Experiments assessing the effects of blood collection methodsand specimen processing were designed and performed by EBB, WHC, andCB. JC, JG, and SS provided guidance on assay development and validationprocedures. EBB, CB, SS, and JR collected mouse specimens for non-CHIMERA experiments. EMR assisted in the specimen management for stabilitytesting. PAC and CLW are the supervisory authors on the project. CLW wasinvolved in the concept formation and manuscript preparation. JR, TC, andDCB were involved in manuscript edits. The author(s) read and approved thefinal manuscript.FundingOperating funding for this work was provided by the Weston Brain Institute,Transformational Grants TR140070 and TR192003. Cure Alzheimer Fundsupported the development of the APOE3 targeted replacement mice usedin this study.Availability of data and materialsThe data sets used and analyzed during the current study are available fromthe corresponding author on reasonable request.DeclarationsEthics approval and consent to participateAll procedures were approved by the University of British ColumbiaCommittee on Animal Care (protocol A15-0096, A19-0040, A14-0057, A14-0003) and were performed in strict compliance with the Canadian Councilon Animal Care guidelines.Consent for publicationNot applicable.Competing interestsThe authors declare that they have no competing interests.Author details1Djavad Mowafaghian Centre for Brain Health, University of British Columbia,2215 Wesbrook Mall, Vancouver, British Columbia V6T 1Z3, Canada.2Department of Pathology and Laboratory Medicine, University of BritishColumbia, Vancouver, British Columbia V6T 1Z3, Canada. 3InternationalCollaboration on Repair Discoveries, University of British Columbia,Vancouver, British Columbia V5Z 1M9, Canada. 4School of BiomedicalEngineering, University of British Columbia, Vancouver, British Columbia V6T1Z3, Canada.Button et al. Alzheimer's Research & Therapy (2021) 13:58 Page 25 of 27Received: 4 November 2020 Accepted: 17 February 2021References1. Hampel H, O’Bryant SE, Molinuevo JL, Zetterberg H, Masters CL, Lista S, et al.Blood-based biomarkers for Alzheimer disease: mapping the road to theclinic. Nat Rev Neurol. 2018;14(11):639–52.2. Zetterberg H, Blennow K. Fluid biomarkers for mild traumatic brain injuryand related conditions. Nat Rev Neurol. 2016;12(10):563–74.3. FDA authorizes marketing of first blood test to aid in the evaluation ofconcussion in adults. 2018. Available from: https://www.fda.gov/news-events/press-announcements/fda-authorizes-marketing-first-blood-test-aid-evaluation-concussion-adults. Accessed 18 Sep 2020.4. Bazarian JJ, Biberthaler P, Welch RD, Lewis LM, Barzo P, Bogner-Flatz V, et al.Serum GFAP and UCH-L1 for prediction of absence of intracranial injurieson head CT (ALERT-TBI): a multicentre observational study. Lancet Neurol.2018;17(9):782–9.5. Anderson TN, Hwang J, Munar M, Papa L, Hinson HE, Vaughan A, et al.Blood-based biomarkers for prediction of intracranial hemorrhage andoutcome in patients with moderate or severe traumatic brain injury. JTrauma Acute Care Surg. 2020;89(1):80–6.6. Metting Z, Wilczak N, Rodiger LA, Schaaf JM, Van Der Naalt J. GFAP andS100B in the acute phase of mild traumatic brain injury. Neurology. 2012;78(18):1428–33.7. Gill J, Latour L, Diaz-Arrastia R, Motamedi V, Turtzo C, Shahim P, et al. Glialfibrillary acidic protein elevations relate to neuroimaging abnormalities aftermild TBI. Neurology. 2018;91(15):E1385–9.8. Yue JK, Yuh EL, Korley FK, Winkler EA, Sun X, Puffer RC, et al. Associationbetween plasma GFAP concentrations and MRI abnormalities in patientswith CT-negative traumatic brain injury in the TRACK-TBI cohort: aprospective multicentre study. Lancet Neurol. 2019;18(10):953–61.9. Lewis LM, Papa L, Bazarian J, Weber A, Howard R, Welch RD. Biomarkersmay predict unfavorable neurological outcome after mild traumatic braininjury. J Neurotrauma. 2020;37(24):2624–31.10. Žurek J, Fedora M. The usefulness of S100B, NSE, GFAP, NF-H, secretagoginand Hsp70 as a predictive biomarker of outcome in children with traumaticbrain injury. Acta Neurochir. 2012;154(1):93–103.11. Lei J, Gao G, Feng J, Jin Y, Wang C, Mao Q, et al. Glial fibrillary acidic proteinas a biomarker in severe traumatic brain injury patients: a prospectivecohort study. Crit Care. 2015;19(1):362.12. Pattinson CL, Meier TB, Guedes VA, Lai C, Devoto C, Haight T, et al. Plasmabiomarker concentrations associated with return to sport following sport-related concussion in collegiate athletes-a concussion assessment, research,and education (CARE) consortium study. JAMA Netw Open. 2020;3(8):e2013191.13. Thelin E, Al Nimer F, Frostell A, Zetterberg H, Blennow K, Nyström H, et al. Aserum protein biomarker panel improves outcome prediction in humantraumatic brain injury. J Neurotrauma. 2019;36(20):2850–62.14. Abdelhak A, Huss A, Kassubek J, Tumani H, Otto M. Serum GFAP as abiomarker for disease severity in multiple sclerosis. Sci Rep. 2018;8(1):1–7.15. Tichy J, Spechtmeyer S, Mittelbronn M, Hattingen E, Rieger J, Senft C, et al.Prospective evaluation of serum glial fibrillary acidic protein (GFAP) as adiagnostic marker for glioblastoma. J Neuro-Oncol. 2015;126(2):361–9.16. Katsanos AH, Makris K, Stefani D, Koniari K, Gialouri E, Lelekis M, et al. Plasmaglial Fibrillary acidic protein in the differential diagnosis of Intracerebralhemorrhage. Stroke. 2017;48(9):2586–8.17. Ren C, Kobeissy F, Alawieh A, Li N, Li N, Zibara K, et al. Assessment of serumUCH-L1 and GFAP in acute stroke patients. Sci Rep. 2016;14(6):1–9.18. Luger S, Witsch J, Dietz A, Hamann GF, Minnerup J, Schneider H, et al. Glialfibrillary acidic protein serum levels distinguish between intracerebralhemorrhage and cerebral ischemia in the early phase of stroke. Clin Chem.2017;63(1):377–85.19. Oeckl P, Halbgebauer S, Anderl-Straub S, Steinacker P, Hussa AM,Neugebauer H, et al. Glial fibrillary acidic protein in serum is increased inAlzheimer’s disease and correlates with cognitive impairment. J AlzheimersDis. 2019;67(2):481–8.20. Verberk IMW, Thijssen E, Koelewijn J, Mauroo K, Vanbrabant J, de Wilde A,et al. Combination of plasma amyloid beta (1-42/1-40) and glial fibrillaryacidic protein strongly associates with cerebral amyloid pathology.Alzheimers Res Ther. 2020;12(1):118.21. McNamara EH, Grillakis AA, Tucker LB, McCabe JT. The closed-head impactmodel of engineered rotational acceleration (CHIMERA) as an applicationfor traumatic brain injury pre-clinical research: a status report. Exp Neurol.2020;333:113409.22. Bashir A, Abebe ZA, McInnes KA, Button EB, Tatarnikov I, Cheng WH, et al.Increased severity of the CHIMERA model induces acute vascular injury, sub-acute deficits in memory recall, and chronic white matter gliosis. ExpNeurol. 2020;324:113116.23. Andreasson U, Perret-Liaudet A, van Waalwijk van Doorn LJC, Blennow K,Chiasserini D, Engelborghs S, et al. A practical guide to immunoassaymethod validation. Front Neurol 2015;6:179.24. Papa L, Brophy GM, Welch RD, Lewis LM, Braga CF, Tan CN, et al. Timecourse and diagnostic accuracy of glial and neuronal blood biomarkersGFAP and UCH-L1 in a large cohort of trauma patients with and withoutmild traumatic brain injury. JAMA Neurol. 2016;73(5):551–60.25. Papa L, Zonfrillo MR, Welch RD, Lewis LM, Braga CF, Tan CN, et al.Evaluating glial and neuronal blood biomarkers GFAP and UCH-L1 asgradients of brain injury in concussive, subconcussive and non-concussivetrauma: a prospective cohort study. BMJ Paediatr Open. 2019;3(1):1–13.26. McCrea M, Broglio SP, McAllister TW, Gill J, Giza CC, Huber DL, et al.Association of Blood Biomarkers with Acute Sport-Related Concussion incollegiate athletes: findings from the NCAA and Department of DefenseCARE consortium. JAMA Netw Open. 2020;3(1):e1919771.27. Huynh TPV, Wang C, Tran AC, Tabor GT, Mahan TE, Francis CM, et al. Lack ofhepatic apoE does not influence early Aβ deposition: observations from anew APOE knock-in model. Mol Neurodegener. 2019;14(1):37.28. Sweeney MD, Sagare AP, Pachicano M, Harrington MG, Joe E, Chui HC, et al.A novel sensitive assay for detection of a biomarker of pericyte injury incerebrospinal fluid. Alzheimers Dement. 2020;16(6):821–30.29. Flierl MA, Stahel PF, Beauchamp KM, Morgan SJ, Smith WR, Shohami E.Mouse closed head injury model induced by a weight-drop device. NatProtoc. 2009;4(9):1328–37.30. Button EB, Boyce GK, Wilkinson A, Stukas S, Hayat A, Fan J, et al. ApoA-Ideficiency increases cortical amyloid deposition, cerebral amyloidangiopathy, cortical and hippocampal astrogliosis, and amyloid-associatedastrocyte reactivity in APP/PS1 mice. Alzheimers Res Ther. 2019;11(1):44.31. Shahim P, Politis A, van der Merwe A, Moore B, Ekanayake V, Lippa SM, et al.Time course and diagnostic utility of NfL, tau, GFAP, and UCH-L1 insubacute and chronic TBI. Neurology. 2020;95(6):e623–36.32. Sweeney MD, Montagne A, Sagare AP, Nation DA, Schneider LS, Chui HC,et al. Vascular dysfunction—the disregarded partner of Alzheimer’s disease.Alzheimers Dement. 2019;15(1):158–67.33. Attems J, Jellinger KA. The overlap between vascular disease andAlzheimer’s disease – lessons from pathology. BMC Med. 2014;12(1):206.34. Iturria-Medina Y, Sotero R, Toussaint P, Mateos-Pérez J, Evans A. Alzheimer’sDisease Neuroimaging Initiative. Early role of vascular dysregulation on late-onset Alzheimer’s disease based on multifactorial data-driven analysis. NatCommun. 2016;7:11934.35. Nation D, Sweeney MD, Montagne A, Sagare AP, D’Orazio L, Pachicano M,et al. Blood-brain barrier breakdown is an early biomarker of humancognitive dysfunction. Nat Med. 2019;25(2):270–6.36. Riwanto M, Landmesser U. High density lipoproteins and endothelialfunctions : mechanistic insights and alterations in cardiovascular disease. JLipid Res. 2013;54(12):3227–43.37. Robert J, Button EB, Stukas S, Boyce GK, Gibbs E, Cowan CM, et al. High-density lipoproteins suppress Aβ-induced PBMC adhesion to humanendothelial cells in bioengineered vessels and in monoculture. MolNeurodegener. 2017;12(1):60.38. Robert J, Button EB, Martin E, McAlary L, Gidden Z, Gilmour M, et al.Cerebrovascular amyloid Angiopathy in bioengineered vessels is reduced byhigh-density lipoprotein particles enriched in Apolipoprotein E. MolNeurodegener. 2020;15(1):23.39. Robert J, Button EB, Yuen B, Gilmour M, Kang K, Bahrabadi A, et al. Clearance ofbeta-amyloid is facilitated by apolipoprotein E and circulating high- densitylipoproteins in bioengineered human vessels. Elife. 2017;6:e29595.40. Diaz-Arrastia R, Wang KKW, Papa L, Sorani MD, Yue JK, Puccio AM, et al.Acute biomarkers of traumatic brain injury: relationship between plasmalevels of ubiquitin C-terminal hydrolase-l1 and glial fibrillary acidic protein. JNeurotrauma. 2014;31(1):19–25.41. Posti JP, Takala RSK, Runtti H, Newcombe VF, Outtrim J, Katila AJ, et al. Thelevels of glial Fibrillary acidic protein and ubiquitin C-terminal hydrolase-L1Button et al. Alzheimer's Research & Therapy (2021) 13:58 Page 26 of 27during the first week after a traumatic brain injury: correlations with clinicaland imaging findings. Neurosurgery. 2016;79(3):456–63.42. Elahi FM, Casaletto KB, La Joie R, Walters SM, Harvey D, Wolf A, et al. Plasmabiomarkers of astrocytic and neuronal dysfunction in early- and late-onsetAlzheimer’s disease. Alzheimers Dement. 2020;16(4):681–95.43. Species Cross-Reactivity: N4PA, N3PA. 2019. Available from: https://www.quanterix.com/resources/whitepapers-app-notes/species-cross-reactivity-n4pa-n3pa-mouse-rat. Accessed 25 Sep 2020.44. Asken BM, Yang Z, Xu H, Weber AG, Hayes RL, Bauer RM, et al. Acute effectsof sport-related concussion on serum glial Fibrillary acidic protein, ubiquitinC-terminal hydrolase L1, Total tau, and Neurofilament light measured by amultiplex assay. J Neurotrauma. 2020;37(13):1537–45.45. MesoscaleDiscovery. Electrochemiluminescence. Available from: https://www.mesoscale.com/en/technical_resources/our_technology/ecl. Accessed25 Sep 2020.46. Quanterix. Quanterix and Banyan Biomarkers Join Together to AddressTraumatic Brain Injury Head On. Lexington, MA; 2016. Available from:https://www.quanterix.com/resources/press-releases/quanterix-and-banyan-biomarkers-join-together-address-traumatic-brain. Accessed 25 Sep 2020.47. Posti JP, Hossain I, Takala RSK, Liedes H, Newcombe V, Outtrim J, et al. GlialFibrillary acidic protein and ubiquitin C-terminal hydrolase-L1 are notspecific biomarkers for mild CT-negative traumatic brain injury. JNeurotrauma. 2017;34(7):1427–38.48. Zetterberg H, Bendlin BB. Biomarkers for Alzheimer’s disease—preparing for anew era of disease-modifying therapies. Mol Psychiatry. 2020;26(1):296–308.49. Garcia-Alloza M, Robbins EM, Zhang-Nunes SX, Purcell SM, Betensky RA, RajuS, et al. Characterization of amyloid deposition in the APPswe/PS1dE9mouse model of Alzheimer disease. Neurobiol Dis. 2006;24(3):516–24.50. Plump AS, Azrolan N, Odaka H, Wu L, Jiang X, Tall A, et al. ApoA-I knockoutmice : characterization of HDL metabolism in homozygotes andidentification of a post-RNA mechanism of apoA-l up-regulation inheterozygotes. J Lipid Res. 1997;38(5):1033–47.51. Gordon SSM, Li H, Zhu X, Shah AAS, Lu LJ, Davidson WS. A comparison ofthe mouse and human Lipoproteome: suitability of the mouse model forstudies of human lipoproteins. J Proteome Res. 2015;14(6):2686–95.52. Livingston G, Huntley J, Sommerlad A, Ames D, Ballard C, Banerjee S, et al.Dementia prevention, intervention, and care: 2020 report of the lancetcommission. Lancet. 2020;396(10248):413–46.53. James SL, Bannick MS, Montjoy-Venning WC, Lucchesi LR, Dandona L,Dandona R, et al. Global, regional, and national burden of traumatic braininjury and spinal cord injury, 1990-2016: a systematic analysis for the globalburden of disease study 2016. Lancet Neurol. 2019;18(1):56–87.54. Cheng WH, Stukas S, Martens KM, Namjoshi DR, Button EB, Wilkinson A,et al. Age at injury and genotype modify acute inflammatory andneurofilament-light responses to mild CHIMERA traumatic brain injury inwild-type and APP/PS1 mice. Exp Neurol. 2018;301(Pt A):26–38.55. Cheng WH, Martens KM, Bashir A, Cheung H, Stukas S, Gibbs E, et al. CHIMERA repetitive mild traumatic brain injury induces chronic behavioural andneuropathological phenotypes in wild-type and APP/PS1 mice. AlzheimersRes Ther 2019;11(1):1–21.56. Namjoshi DR, Cheng WH, Carr M, Martens KM, Zareyan S, Wilkinson A, et al.Chronic exposure to androgenic-anabolic steroids exacerbates axonal injuryand microgliosis in the CHIMERA mouse model of repetitive concussion.PLoS One. 2016;11(1):e0146540.Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.Button et al. Alzheimer's Research & Therapy (2021) 13:58 Page 27 of 27
Article
eng
Reviewed
Vancouver : University of British Columbia Library
BioMed Central
Attribution 4.0 International (CC BY 4.0)
Faculty
GFAP
Plasma biomarker
TBI
CHIMERA
Immunoassay
Development of a novel, sensitive translational immunoassay to detect plasma glial fibrillary acidic protein (GFAP) after murine traumatic brain injury
Text