Restricted accessResearch articleFirst published online 2025-7
High precision and cost-effective multiplex quantification of amyloid-β40,amyloid-β42,p181Tau,p217Tau,neurofilament light chain,and glial fibrillary acidic protein from plasma and serum
Current methods to quantify blood biomarkers for Alzheimer's disease (AD) are expensive and are not widely available.
Objective
To develop a low-cost, sensitive, and accurate multiplex assay to quantify Aβ40, Aβ42, p181Tau, p217Tau, NfL, and GFAP biomarkers in plasma and serum based on a widely available technology.
Methods
We used commercial antibodies to Aβ40, Aβ42, p181Tau, p217Tau, NfL, and GFAP, and xMAP Luminex technology, and developed the multiplex 5ADCSI to quantify these biomarkers from plasma and serum. The utility of 5ADCSI was tested in matched cerebrospinal fluid (CSF) and plasma or serum of a cohort of cognitively normal (CN: n = 35), with mild cognitive impairment (MCI: n = 17), and with AD (n = 11) individuals.
Results
The 5ADCSI demonstrated high specificity and sensitivity, with excellent precision. In clinical samples, moderate to strong correlation is observed between CSF and plasma or serum for Aβ42/40 (r=0.78), p181Tau/Aβ42 (r=0.57), p217Tau/Aβ42 (r=0.72), p181Tau (r=0.59), p217Tau (r=0.75), and GFAP (r=0.59). The AUC of receiver-operator characteristic curve for differentiating CN from AD for plasma/serum and CSF are 0.75, and 0.80 for Aβ42/40, 0.95, 0.91 for p217Tau, 0.76, 0.81 for p181Tau, and 0.73 and 0.78for GFAP, respectively.
Conclusions
The 5ADCSI assay is highly specific, sensitive, and accurate. The wide availability of the base technology of 5ADCSI is an advantage over other similar methods and would allow cost-effective large-scale studies for validation of blood biomarkers for early diagnosis of AD.
PaisMVForlenzaOVDinizBS. Plasma biomarkers of Alzheimer's disease: a review of available assays, recent developments, and implications for clinical practice. J Alzheimers Dis Rep2023; 7: 355–380.
2.
OvodVRamseyKNMawuenyegaKG, et al.Amyloid β concentrations and stable isotope labeling kinetics of human plasma specific to central nervous system amyloidosis. Alzheimers Dement2017; 13: 841–849.
3.
JanelidzeSBaliDAshtonNJ, et al.Head-to-head comparison of 10 plasma phospho-tau assays in prodromal Alzheimer's disease. Brain2023; 146: 1592–1601.
4.
Suárez-CalvetMKarikariTKAshtonNJ, et al.Novel tau biomarkers phosphorylated at T181, T217 or T231 rise in the initial stages of the preclinical Alzheimer's continuum when only subtle changes in Aβ pathology are detected. EMBO Mol Med2020; 12: e12921.
5.
KarikariTKPascoalTAAshtonNJ, et al.Blood phosphorylated tau 181 as a biomarker for Alzheimer's disease: a diagnostic performance and prediction modelling data from four cohorts. Lancet Neurol2020; 19: 422–433.
6.
PalmqvistSTidemanPCullenN, et al.Prediction of future Alzheimer's disease dementia using plasma phospho-tau combined with other accessible measures. Nat Med2021; 27: 1034–1042.
7.
ChatterjeePPedriniSDoeckeJD, et al.Plasma Aβ42/40 ratio, p-tau181, GFAP, and NfL across the Alzheimer's disease continuum: a cross-sectional and longitudinal study in the AIBL cohort. Alzheimers Dement2023; 19: 1117–1134.
8.
Pichet BinetteAPalmqvistSBaliD, et al.Combining plasma phospho-tau and accessible measures to evaluate progression to Alzheimer's dementia in mild cognitive impairment patients. Alzheimers Res Ther2022; 14: 46.
9.
ZhangHLiuJZhangN, et al.Validation of an ultra-sensitive method for quantitation of phospho-Tau 217 (pTau217) in human plasma, serum, and CSF using the ALZpath pTau217 assay on the quanterix HD-X platform. J Prev Alzheimers Dis2024; 11: 1206–1211.
10.
JanelidzeSBarthélemyNRSalvadóG, et al.Plasma phosphorylated Tau 217 and Aβ42/40 to predict early brain Aβ accumulation in people without cognitive impairment. JAMA Neurol2024; 81: 947–957.
11.
NodaKLimYGotoR, et al.Cost-effectiveness comparison between blood biomarkers and conventional tests in Alzheimer's disease diagnosis. Drug Discov Today2024; 29: 103911.
12.
KangJHVandersticheleHTrojanowskiJQ, et al.Simultaneous analysis of cerebrospinal fluid biomarkers using microsphere-based xMAP multiplex technology for early detection of Alzheimer's disease. Methods2012; 56: 484–493.
13.
ParkJCHanSHChoHJ, et al.Chemically treated plasma Aβ is a potential blood-based biomarker for screening cerebral amyloid deposition. Alzheimers Res Ther2017; 9: 20.
14.
LövheimHElghFJohanssonA, et al.Plasma concentrations of free amyloid β cannot predict the development of Alzheimer's disease. Alzheimers Dement2017; 13: 778–782.
15.
R Core Team. R: A language and environment for statistical computing. Vienna: R Foundation for Statistical Computing, 2023.
16.
BachmannSBellMKlimekJ, et al.Differential effects of the six human TAU isoforms: somatic retention of 2N-TAU and increased microtubule number induced by 4R-TAU. Front Neurosci2021; 15: 643115.
17.
ShawGMadorskyILiY, et al.Uman-type neurofilament light antibodies are effective reagents for the imaging of neurodegeneration. Brain Commun2023; 5: fcad067.
18.
CipollariESzaparyHJPicataggiA, et al.Correlates and predictors of cerebrospinal fluid cholesterol efflux capacity from neural cells, a family of biomarkers for cholesterol epidemiology in Alzheimer's disease. J Alzheimers Dis2020; 74: 563–578.
19.
FazeliBHussAGómez de San JoséN, et al.Development of an ultrasensitive microfluidic assay for the analysis of glial fibrillary acidic protein (GFAP) in blood. Front Mol Biosci2023; 10: 1175230.
20.
ArmbrusterDAPryT. Limit of blank, limit of detection and limit of quantitation. Clin Biochem Rev2008; 29(Suppl 1): S49–S52.
21.
Suarez-CalvetMKarikariTKAshtonNJ, et al.Novel tau biomarkers phosphorylated at T181, T217 or T231 rise in the initial stages of the preclinical Alzheimer's continuum when only subtle changes in A beta pathology are detected. EMBO Mol Med2020; 12: e12921.
22.
IinoTWatanabeSYamashitaK, et al.Quantification of amyloid-β in plasma by simple and highly sensitive immunoaffinity enrichment and LC-MS/MS assay. J Appl Lab Med2021; 6: 834–845.
23.
WestTKirmessKMMeyerMR, et al.A blood-based diagnostic test incorporating plasma A beta 42/40 ratio, ApoE proteotype, and age accurately identifies brain amyloid status: findings from a multi cohort validity analysis. Mol Neurodegeneration2021; 16: 30.
24.
PlancheVBouteloupVPellegrinI, et al.Validity and performance of blood biomarkers for Alzheimer disease to predict dementia risk in a large clinic-based cohort. Neurology2023; 100: e473–e484.
25.
FischerT. Automation solutions for Luminex systems, (2023, 2025).
26.
MandelkowEMMandelkowE. Biochemistry and cell biology of tau protein in neurofibrillary degeneration. Cold Spring Harb Perspect Med2012; 2: a006247.
27.
AlquezarCAryaSKaoAW. Tau post-translational modifications: dynamic transformers of tau function, degradation, and aggregation. Front Neurol2020; 11: 595532.
28.
BudelierMMHeYBarthelemyNR, et al.A map of neurofilament light chain species in brain and cerebrospinal fluid and alterations in Alzheimer's disease. Brain Commun2022; 4: fcac045.
29.
CoultonJBHeYBarthélemyNR, et al.Multi-peptide characterization of plasma neurofilament light chain in preclinical and mild Alzheimer's disease. Brain Commun2024; 6: fcae247.
30.
AshtonNJSuarez-CalvetMKarikariTK, et al.Effects of pre-analytical procedures on blood biomarkers for Alzheimer's pathophysiology, glial activation, and neurodegeneration. Alzheimers Dement (Amst)2021; 13: e12168.
31.
JanelidzeSTeunissenCEZetterbergH, et al.Head-to-head comparison of 8 plasma amyloid-β 42/40 assays in Alzheimer disease. JAMA Neurol2021; 78: 1375–1382.
32.
BarthélemyNRSalvadóGSchindlerSE, et al.Highly accurate blood test for Alzheimer's disease is similar or superior to clinical cerebrospinal fluid tests. Nat Med2024; 30: 1085–1095.
33.
SchindlerSEBollingerJGOvodV, et al.High-precision plasma β-amyloid 42/40 predicts current and future brain amyloidosis. Neurology2019; 93: e1647–e1659.
34.
PereiraJBJanelidzeSSmithR, et al.Plasma GFAP is an early marker of amyloid-β but not tau pathology in Alzheimer's disease. Brain2021; 144: 3505–3516.
35.
HanssonOLehmannSOttoM, et al.Advantages and disadvantages of the use of the CSF amyloid β (Aβ) 42/40 ratio in the diagnosis of Alzheimer's disease. Alzheimers Res Ther2019; 11: 34.
36.
AmftMOrtnerMEichenlaubU, et al.The cerebrospinal fluid biomarker ratio Aβ42/40 identifies amyloid positron emission tomography positivity better than Aβ42 alone in a heterogeneous memory clinic cohort. Alzheimers Res Ther2022; 14: 60.
37.
MendesAJRibaldiFLathuiliereA, et al.Head-to-head study of diagnostic accuracy of plasma and cerebrospinal fluid p-tau217 versus p-tau181 and p-tau231 in a memory clinic cohort. J Neurol2024; 271: 2053–2066.
38.
Montoliu-GayaLBenedetALTissotC, et al.Mass spectrometric simultaneous quantification of tau species in plasma shows differential associations with amyloid and tau pathologies. Nat Aging2023; 3: 661–669.
39.
ChengYPereiraMRaukarN, et al.Potential biomarkers to detect traumatic brain injury by the profiling of salivary extracellular vesicles. J Cell Physiol2019; 234: 14377–14388.
40.
ZetterbergHBozzettaEFavoleA, et al.Neurofilaments in blood is a new promising preclinical biomarker for the screening of natural scrapie in sheep. PLoS One2019; 14: e0226697.
41.
MattssonNZetterbergHNielsenN, et al.Serum tau and neurological outcome in cardiac arrest. Ann Neurol2017; 82: 665–675.
42.
BarroCChitnisTWeinerHL. Blood neurofilament light: a critical review of its application to neurologic disease. Ann Clin Transl Neurol2020; 7: 2508–2523.
43.
DelabyCVialaretJHirtzC, et al.Analytical comparison of ELISA and mass spectrometry for quantification of serum hepcidin in critically ill patients. Bioanalysis2021; 13: 1029–1035.
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