| Summary: | 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.
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