CoChat
Sign Up
Cited by 8Alzheimer's disease research and treatmentsDementia and Cognitive Impairment ResearchClinical Laboratory Practices and Quality Control

A novel biochemical analysis for ApoE4 quantification in plasma and discrimination of homozygous and heterozygous APOE ε4 carriers

Open access
Rodríguez, Andrés, Calero, Olga, Veiga, Sergio, Menacho-Román, Miriam, Arribas, Ignacio, Cano, Lluís, García-Ribas, Guillermo, Calero, Miguel·2025·Alzheimer's Research & Therapy
DOI 10.1186/s13195-025-01811-wPMC PMC12265242
Open this paper in CoChat — free accountOpen PDF

Abstract

Background: The APOE ε4 allele is the strongest genetic risk factor for late-onset Alzheimer’s disease (AD) and is associated with increased risk of amyloid-related imaging abnormalities (ARIA) during anti-amyloid therapy. Accurate identification of ε4 carriers, particularly APOE ε4/ε4 individuals, is clinically relevant. This study outlines the development and validation of e4Quant, a novel turbidimetric assay for quantifying plasma ApoE4 as a non-genetic alternative to APOE genotyping. Methods: The e4Quant test utilizes a proprietary particle-enhanced immunoturbidimetry method, employing an isoform-specific anti-ApoE4 antibody on standard chemistry analyzers. Plasma samples from 160 individuals of known APOE genotype (35 APOE ε4/ε4 homozygotes, 115 APOE ε4 heterozygotes, and 10 APOE ε4 non-carriers) were analyzed for ApoE4 and total ApoE levels. The test’s discriminatory performance was assessed by ROC analysis and two-threshold classification algorithms. Results: ApoE4 levels ascertained by the e4Quant test exhibited clear genotype-dependent stratification. ROC analysis indicated 100% sensitivity and specificity in distinguishing APOE ε4 carriers from non-carriers, and 88.6% sensitivity and 90.4% specificity for discriminating homozygous from heterozygous carriers. Normalizing ApoE4 to total ApoE improved classification (sensitivity 94.3%, specificity 93.9%). A combined ratio-plus-concentration approach further enhanced discrimination (sensitivity 91.4%, specificity 100%). Three ε4 homozygous samples with low ApoE4/total ApoE ratios were misclassified. Discussion: The e4Quant assay offers a rapid, cost-effective, and highly accurate biochemical alternative to APOE genotyping, suitable for clinical and research settings, particularly in assessing AD risk and optimizing anti-amyloid therapeutic strategies. One subgroup of APOE ε4/ε4 subjects had unexpectedly low ApoE4 levels, raising questions about potential biological heterogeneity and its impact on Alzheimer’s disease biology. Conclusion: The e4Quant assay is a novel alternative for genotyping to determine APOE ε4 carrier status, while also providing quantitative measurements of ApoE4 levels. Its high diagnostic accuracy, ease of use in standard clinical laboratories, and potential utility for personalized medicine in AD treatment highlight its translational value. Further studies are warranted to investigate the clinical significance of APOE ε4 expression variability and its impact on disease progression and treatment response.

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1186/s13195-025-01811-w

Cites

15 of 15 references verified0 citation errors · 0 not checkable

Statistics

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • In the era of disease-modifying anti-amyloid therapies, ApoE ε4 carriers, particularly ε4 homozygotes, exhibit a substantially increased risk of ARIAs.

    “In the era of disease-modifying anti-amyloid therapies, ApoE ε4 status has become directly actionable, as ε4 carriers, particularly ε4 homozygotes, exhibit a substantially increased risk of ARIAs [2, 33].”

    From Plasma ApoE proteotyping for ApoE ε4 stratification in the anti-amyloid therapies era (2026) · cites this paper as [33]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Cites

1 retracted

Flagged first, then by citations

Research works printed but not linked: 54

  1. [2]van Duijn CM, de Knijff P, Cruts M, Wehnert A, Havekes LM, Hofman A, et al. Apolipoprotein E4 allele in a population-based study of early-onset alzheimer’s disease. Nat Genet. 1994;7:74–8. 8075646
  2. [3]Crean S, Ward A, Mercaldi CJ, Collins JM, Cook MN, Baker NL, et al. Apolipoprotein E ε4 prevalence in alzheimer’s disease patients varies across global populations: a systematic literature review and meta-analysis. Dement Geriatr Cogn Disord. 2010;31:20–30. 21124030
  3. [7]Farrer LA, Cupples LA, Haines JL, Hyman B, Kukull WA, Mayeux R, et al. Effects of age, sex, and ethnicity on the association between Apolipoprotein E genotype and alzheimer disease: A Meta-analysis. JAMA. 1997;278:1349–56. 9343467
  4. [8]Craft S, Teri L, Edland SD, Kukull WA, Schellenberg G, McCormick WC, et al. Accelerated decline in Apolipoprotein E-ϵ4 homozygotes with alzheimer’s disease. Neurology. 1998;51:149–53. 9674794
  5. [9]Corder EH, Saunders AM, Strittmatter WJ, Schmechel DE, Gaskell PC, Small GW, et al. Gene dose of Apolipoprotein E type 4 allele and the risk of alzheimer’s disease in late onset families. Science. 1993;261:921–3. 8346443
  6. [10]Salloway S, Chalkias S, Barkhof F, Burkett P, Barakos J, Purcell D, et al. Amyloid-Related imaging abnormalities in 2 phase 3 studies evaluating aducanumab in patients with early alzheimer disease. JAMA Neurol. 2022;79:13–21. 34807243 PMC8609465
  7. [12]Budd Haeberlein S, Aisen PS, Barkhof F, Chalkias S, Chen T, Cohen S, et al. Two randomized phase 3 studies of aducanumab in early alzheimer’s disease. J Prev Alzheimers Dis. 2022;9:197–210. 35542991
  8. [13]Honig LS, Barakos J, Dhadda S, Kanekiyo M, Reyderman L, Irizarry M, et al. ARIA in patients treated with Lecanemab (BAN2401) in a phase 2 study in early alzheimer’s disease. Alzheimer’s Dementia: Translational Res Clin Interventions. 2023;9:e12377. PMC10026083 36949897
  9. [14]Sebastiani P, Gurinovich A, Nygaard M, Sasaki T, Sweigart B, Bae H, et al. APOE alleles and extreme human longevity. J Gerontol Biol Sci Med Sci. 2019;74:44–51. PMC6298189 30060062
  10. [16]Beker N, Sikkes SAM, Hulsman M, Tesi N, van der Lee SJ, Scheltens P, et al. Longitudinal maintenance of cognitive health in centenarians in the 100-plus study. JAMA Netw Open. 2020;3:e200094. 32101309 PMC7137688
  11. [17]Breslow JL, Zannis VI, SanGiacomo TR, Third JL, Tracy T, Glueck CJ. Studies of Familial type III hyperlipoproteinemia using as a genetic marker the ApoE phenotype E2/2. J Lipid Res. 1982;23:1224–35. 7175379
  12. [18]Juul Rasmussen I, Rasmussen KL, Nordestgaard BG, Tybjærg-Hansen A, Frikke-Schmidt R. Impact of cardiovascular risk factors and genetics on 10-year absolute risk of dementia: risk charts for targeted prevention. Eur Heart J. 2020;41:4024–33. 33022702 PMC7672536
  13. [19]Hixson JE, Vernier DT. Restriction isotyping of human Apolipoprotein E by gene amplification and cleavage with HhaI. J Lipid Res. 1990;31:545–8. 2341813
  14. [20]Zivelin A, Rosenberg N, Peretz H, Amit Y, Kornbrot N, Seligsohn U. Improved method for genotyping Apolipoprotein E polymorphisms by a PCR-Based assay simultaneously utilizing two distinct restriction enzymes. Clin Chem. 1997;43:1657–9. 9299950
  15. [21]Calero O, Hortigüela R, Bullido MJ, Calero M. Apolipoprotein E genotyping method by real time PCR, a fast and cost-effective alternative to the TaqMan ® and FRET assays. J Neurosci Methods. 2009;183:238–40. 19583979
  16. [22]Kataoka S, Paidi M, Howard BV. Simplified isoelectric focusing/immunoblotting determination of Apoprotein E phenotype. Clin Chem. 1994;40:11–3. 8287516
  17. [23]Eto M, Watanabe K, Moriyama T, Makino I. Apolipoprotein E phenotyping from plasma by isoelectric focusing and Immunoblotting. Tohoku J Exp Med. 1990;160:301–9. 2382259
  18. [24]Steinmetz A. Phenotyping of human Apolipoprotein E from whole blood plasma by Immunoblotting. J Lipid Res. 1987;28:1364–70. 3430065
  19. [25]McDowell IF, Wisdom GB, Trimble ER. Apolipoprotein E phenotype determined by agarose gel electrofocusing and Immunoblotting. Clin Chem. 1989;35:2070–3. 2791274
  20. [26]Dittrich A, Blennow K, Tan K, Benedet AL, Skoog I, Höglund K, et al. Evaluation of two plasma-based proteotyping assays against APOE ε4 genotyping in a memory clinic setting: the Gothenburg H70 clinical studies. Alzheimers Dement. 2025;21:e14610. 39988952 PMC11847986
  21. [27]Calero O, García-Albert L, Rodríguez-Martín A, Veiga S, Calero M. A fast and cost-effective method for apolipoprotein E isotyping as an alternative to APOE genotyping for patient screening and stratification. Scientific Reports [Internet]. 2018 [cited 2018 Jun 15];8. Available from: http://www.nature.com/articles/s41598-018-24320-3. PMC5899170 29654261
  22. [28]Veiga S, Rodríguez-Martín A, Garcia-Ribas G, Arribas I, Menacho-Román M, Calero M. Validation of a novel and accurate ApoE4 assay for automated chemistry analyzers. Sci Rep. 2020;10:2138. 32034174 PMC7005722
  23. [29]Rezeli M, Zetterberg H, Blennow K, Brinkmalm A, Laurell T, Hansson O, et al. Quantification of total Apolipoprotein E and its specific isoforms in cerebrospinal fluid and blood in alzheimer’s disease and other neurodegenerative diseases. EuPA Open Proteom. 2015;8:137–43.
  24. [30]Rezeli M, Sjödin K, Lindberg H, Gidlöf O, Lindahl B, Jernberg T, et al. Quantitation of 87 proteins by nLC-MRM/MS in human plasma: workflow for Large-Scale analysis of biobank samples. J Proteome Res. 2017;16:3242–54. 28738677
  25. [32]Giannisis A, Al-Grety A, Carlsson H, Patra K, Twohig D, Sando SB, et al. Plasma Apolipoprotein E levels in longitudinally followed patients with mild cognitive impairment and alzheimer’s disease. Alz Res Therapy. 2022;14:115. PMC9400269 36002891
  26. [33]Rasmussen KL, Tybjaerg-Hansen A, Nordestgaard BG, Frikke-Schmidt R. Plasma levels of Apolipoprotein E and risk of dementia in the general population. Ann Neurol. 2015;77:301–11. 25469919
  27. [34]Rasmussen KL, Tybjærg-Hansen A, Nordestgaard BG, Frikke-Schmidt R. Plasma Apolipoprotein E levels and risk of dementia: A Mendelian randomization study of 106,562 individuals. Alzheimer’s Dement. 2018;14:71–80. 28774656
  28. [38]Elliott DA, Tsoi K, Holinkova S, Chan SL, Kim WS, Halliday GM, et al. Isoform-specific proteolysis of apolipoprotein-E in the brain. Neurobiol Aging. 2011;32:257–71. 19278755
  29. [40]Vance JE, Hayashi H. Formation and function of Apolipoprotein E-containing lipoproteins in the nervous system. Biochim Biophys Acta. 2010;1801:806–18. 20170744
  30. [42]Sevigny J, Chiao P, Bussière T, Weinreb PH, Williams L, Maier M, et al. The antibody aducanumab reduces Aβ plaques in alzheimer’s disease. Nature. 2016;537:50–6. 27582220
  31. [44]van Dyck CH, Swanson CJ, Aisen P, Bateman RJ, Chen C, Gee M, et al. Lecanemab in early alzheimer’s disease. N Engl J Med. 2023;388:9–21. 36449413
  32. [45]Sims JR, Zimmer JA, Evans CD, Lu M, Ardayfio P, Sparks J, et al. Donanemab in early symptomatic alzheimer disease. JAMA. 2023;330:512–27. 37459141 PMC10352931
  33. [47]Shah FA, Qadir H, Khan JZ, Faheem M, Alanazi FE, Khalid T. A review: from old drugs to new solutions: the role of repositioning in alzheimer’s disease treatment. Neuroscience. 2025;S0306–4522(25):00266–0. 40164279
  34. [48]Sperling RA, Jack CR, Black SE, Frosch MP, Greenberg SM, Hyman BT, et al. Amyloid related imaging abnormalities (ARIA) in amyloid modifying therapeutic trials: recommendations from the alzheimer’s association research roundtable workgroup. Alzheimers Dement. 2011;7:367–85. 21784348 PMC3693547
  35. [49]Cummings J. Anti-Amyloid monoclonal antibodies are transformative treatments that redefine alzheimer’s disease therapeutics. Drugs. 2023;83:569–76. 37060386 PMC10195708
  36. [50]Liu C-C, Kanekiyo T, Xu H, Bu G. Apolipoprotein E and alzheimer disease: risk, mechanisms and therapy. Nat Reviews Neurol. 2013;9:106–18. PMC3726719 23296339
  37. [51]Lindberg O, Kern S, Skoog J, Machado A, Pereira JB, Sacuiu SF, et al. Effects of amyloid pathology and the APOE ε4 allele on the association between cerebrospinal fluid Aβ38 and Aβ40 and brain morphology in cognitively normal 70-years-olds. Neurobiol Aging. 2021;101:1–12. 33548794
  38. [52]Heneka MT, Golenbock DT, Latz E. Innate immunity in alzheimer’s disease. Nat Immunol. 2015;16:229–36. 25689443
  39. [53]Abushakra S, Porsteinsson AP, Sabbagh M, Watson D, Power A, Liang E, et al. APOLLOE4 phase 3 study of oral ALZ-801/valiltramiprosate in APOE ε4/ε4 homozygotes with early alzheimer’s disease: trial design and baseline characteristics. Alzheimers Dement (N Y). 2024;10:e12498. 39144121 PMC11322500
  40. [55]van de Rest O, Berendsen AA, Haveman-Nies A, de Groot LC. Dietary patterns, cognitive decline, and dementia: A systematic review. Adv Nutr. 2015;6:154–68. 25770254 PMC4352174
  41. [56]Wei B-Z, Li L, Dong C-W, Tan C-C, Xu W. The relationship of Omega-3 fatty acids with dementia and cognitive decline: evidence from prospective cohort studies of supplementation, dietary intake, and blood markers. Am J Clin Nutr. 2023;117:1096–109. 37028557 PMC10447496
  42. [57]Keum M, Lee BC, Choe YM, Suh G-H, Kim SG, Kim HS, et al. Protein intake and episodic memory: the moderating role of the Apolipoprotein E ε4 status. Alzheimer’s Res Therapy. 2024;16:181. PMC11318328 39135146
  43. [58]Urich TJ, Tsiknia AA, Ali N, Park J, Mack WJ, Cortessis VK, Dinalo JE, Yassine HN. APOE ε4 and Dietary Patterns in Relation to Cognitive Function: An Umbrella Review of Systematic Reviews. Nutr Rev. 2025;83(7):e2113–e2126. PMC12166175 39499795
  44. [59]Shinto LH, Murchison CF, Silbert LC, Dodge HH, Lahna D, Rooney W, et al. ω-3 PUFA for secondary prevention of white matter lesions and neuronal integrity breakdown in older adults: A randomized clinical trial. JAMA Netw Open. 2024;7:e2426872. 39088212 PMC11294966
  45. [60]Rasmussen KL, Frikke-Schmidt R. The current state of Apolipoprotein E in dyslipidemia. Curr Opin Lipidol. 2024;35:78–84. 38054895
  46. [61]Eichner JE, Dunn ST, Perveen G, Thompson DM, Stewart KE, Stroehla BC. Apolipoprotein E polymorphism and cardiovascular disease: A huge review. Am J Epidemiol. 2002;155:487–95. 11882522
  47. [62]Mahley RW. Central nervous system lipoproteins. Arterioscler Thromb Vasc Biol. 2016;36:1305–15. 27174096 PMC4942259
  48. [63]Sudlow C, Martínez González NA, Kim J, Clark C. Does Apolipoprotein E genotype influence the risk of ischemic stroke, intracerebral hemorrhage, or subarachnoid hemorrhage?? Stroke. 2006;37:364–70. 16385096 PMC2577180
  49. [64]Paternoster L, González NAM, Lewis S, Sudlow C. Association between Apolipoprotein E genotype and carotid Intima-Media thickness May suggest a specific effect on large artery atherothrombotic stroke. Stroke. 2008;39:48–54. 18063831 PMC2577179
  50. [65]Lagging C, Lorentzen E, Stanne TM, Pedersen A, Söderholm M, Cole JW, et al. APOE ε4 is associated with younger age at ischemic stroke onset but not with stroke outcome. Neurology. 2019;93:849–53. 31619479 PMC6946482
  51. [66]Crous-Bou M, Minguillón C, Gramunt N, Molinuevo JL. Alzheimer’s disease prevention: from risk factors to early intervention. Alzheimers Res Ther. 2017;9:71. 28899416 PMC5596480
  52. [67]Risk Reduction of Cognitive Decline and Dementia: WHO Guidelines [Internet]. Geneva: World Health Organization. 2019 [cited 2025 Apr 1]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK542796/ 31219687
  53. [69]Yost OC, Downey A, Powell T, editors. Preventing and Treating Dementia: Research Priorities to Accelerate Progress [Internet]. Washington (DC): National Academies Press (US); 2025 [cited 2025 Mar 30]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK610426/ 39693470
  54. [70]Deza-Lougovski YI, Weiss LM, Horton HM, Sun A, Borbye‐Lorenzen N, Skogstrand K, et al. Circulating apoE4 protein levels from dried blood spots predict cognitive function in a large population‐based survey setting. Alzheimers Dement. 2024;20:7613–23. 39234633 PMC11567846

Cited by

Sorted by citations

Authors
Rodríguez, A., Calero, O., Veiga, S., Menacho-Román, M., Arribas, I., Cano, L., García-Ribas, G., & Calero, M.
Affiliations
  • Instituto de Salud Carlos III (ES)
  • Instituto Ramón y Cajal de Investigación Sanitaria (ES)
Corresponding author
Calero, Miguel
ORCID
Venue
Alzheimer's Research & Therapy
Publisher
Springer Science and Business Media LLC
Published
2025-07-15
Volume
17
Issue
1
Pages
163-163
ISSN
1758-9193 (ISSN-L)
DOI
10.1186/s13195-025-01811-w
PMC
PMC12265242
OpenAlex
W4412467821
Cited by
8
References
15
Citations by year
2026: 7 · 2025: 1
FWCI
4.49
Type
journal article
Language
en
Open access
Licence
Locations
  • Alzheimer s Research & Therapy (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Biocross, S.L.
  • Servicio Gallego de Salud (SERGAS) and Fondo Europeo de Desarrollo Regional (AB-SER1-19-025)
  • CIBER - Consorcio Centro de Investigación Biomédica en Red (CB06/05/0085)
Topics
  • Health Sciences › Medicine › Physiology › Alzheimer's disease research and treatments
  • Health Sciences › Medicine › Psychiatry and Mental health › Dementia and Cognitive Impairment Research
  • Health Sciences › Medicine › Physiology › Clinical Laboratory Practices and Quality Control
Keywords
APOE ε4, apolipoprotein E, turbidimetric immunoassay, Alzheimer's disease risk, anti-amyloid therapy, amyloid-related imaging abnormalities
MeSH terms
Aged, Aged, 80 and over, Alzheimer Disease, Female, Genotype, Heterozygote, Homozygote, Humans, Male, Middle Aged, ROC Curve, Apolipoprotein E4

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review