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. 2018 Dec 8;10(1):121.
doi: 10.1186/s13195-018-0448-x.

A two-step immunoassay for the simultaneous assessment of Aβ38, Aβ40 and Aβ42 in human blood plasma supports the Aβ42/Aβ40 ratio as a promising biomarker candidate of Alzheimer's disease

Hedieh Shahpasand-Kroner  1   2   3 , Hans-W Klafki  1 , Chris Bauer  4 , Johannes Schuchhardt  4 , Melanie Hüttenrauch  1 , Martina Stazi  1 , Caroline Bouter  5 , Oliver Wirths  1 , Jonathan Vogelgsang  1 , Jens Wiltfang  6   7   8
Affiliations

A two-step immunoassay for the simultaneous assessment of Aβ38, Aβ40 and Aβ42 in human blood plasma supports the Aβ42/Aβ40 ratio as a promising biomarker candidate of Alzheimer's disease

Hedieh Shahpasand-Kroner et al. Alzheimers Res Ther. .

Abstract

Background: The quantification of amyloid-beta (Aβ) peptides in blood plasma as potential biomarkers of Alzheimer's disease (AD) is hampered by very low Aβ concentrations and the presence of matrix components that may interfere with the measurements.

Methods: We developed a two-step immunoassay for the simultaneous measurement of the relative levels of Aβ38, Aβ40 and Aβ42 in human EDTA plasma. The assay was employed for the study of 23 patients with dementia of the Alzheimer's type (AD-D) and 17 patients with dementia due to other reasons (OD). We examined relationships with the clinical diagnosis, cerebral Aβ load as quantified by amyloid-positron emission tomography, apolipoprotein E genotype, Aβ levels and Tau protein in cerebrospinal fluid.

Results: Preconcentration of plasma Aβ peptides by immunoprecipitation substantially facilitated their immunological measurements. The Aβ42/Aβ40 and Aβ42/Aβ38 ratios were statistically significantly lower in the AD-D patients than in the OD group. The areas under the receiver operating characteristic curves reached 0.87 for the Aβ42/Aβ40 ratio and 0.80 for the Aβ42/Aβ38 ratio.

Conclusions: The measurement of plasma Aβ peptides with an immunological assay can be improved by preconcentration via immunoprecipitation with an antibody against the Aβ amino-terminus and elution of the captured peptides by heating in a mild detergent-containing buffer. Our findings support the Aβ42/Aβ40 ratio in blood plasma as a promising AD biomarker candidate which correlates significantly with the validated core biomarkers of AD. Further studies will be needed for technical advancement of the assay and validation of the biomarker findings.

Keywords: Alzheimer’s disease; Amyloid beta; Biomarker; Blood plasma; Cerebrospinal fluid; Dementia; Immunoprecipitation.

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Conflict of interest statement

Ethics approval and consent to participate

The collection and archiving of biological samples and clinical data in strictly pseudonymous form in a local biobank and their use in biomarker research were approved by the ethics committee of the University Goettingen (9/2/16). Written informed consent was obtained prior to the inclusion in the biobank from all participants or their legal representatives. All procedures involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

Consent for publication

All authors read and approved the final manuscript.

Competing interests

JW is a member of the European Advisory Board, Eli Lilly, the National Advisory Board, MSD SHARP & DOHME, and advisor for Boehringer-Ingelheim and Hofmann-La-Roche.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Preconcentration of Aβ peptides from EDTA plasma facilitates semi-quantitative measurement of a Aβ42, b Aβ40 and c Aβ38 in human blood plasma. Bars indicate calculated concentrations in 4-fold diluted EDTA-plasma samples (black bars) and in 10-fold or 20-fold diluted IP eluates (light and dark gray bars) obtained by magnetic bead immunoprecipitation from 800 μl of plasma. Signals above LLOQ of the assay were observed after immunoprecipitation in five out of five tested samples for Aβ42 and Aβ40 and in four out of five samples for Aβ38. Indicated LLOQs were taken from lot-specific certificates of analysis provided with assay kits. Mean ± SD of duplicate reads shown. Aβ amyloid beta, IP immunoprecipitation, LLOQ lower limit of quantification
Fig. 2
Fig. 2
Dichotomized comparison of levels of Aβ38, Aβ40 and Aβ42 and concentration ratios Aβ42/Aβ40, Aβ42/Aβ38 and Aβ38/Aβ40 in diluted IP eluates. No statistically significant differences in levels of a Aβ42, b Aβ40 and c Aβ38 between diagnostic groups. In contrast, significantly lower d Aβ42/Aβ40 (t test p < 0.001) and e Aβ42/Aβ38 (t test p = 0.0031) ratios observed in AD-D group compared to OD group. f Aβ38/Aβ40 ratio did not differ significantly between diagnostic groups (p = 0.0785). Statistical tests were done after log transformation. Accordingly, scaling of y axes is logarithmic. Aβ amyloid beta, AD-D dementia of the Alzheimer type, OD dementia due to other reasons
Fig. 3
Fig. 3
Biomarker performance of Aβ42/Aβ40 and Aβ42/Aβ38 ratios in plasma. ROC analyses for Aβ42/Aβ40 and Aβ42/Aβ38 ratios for classification of OD vs AD-D. For both ROC curves, null hypothesis that AUCs were equal to 0.5 could be rejected (p < 0.001 for Aβ42/Aβ40 ratio and p = 0.0013 for Aβ42/Aβ38 ratio). Aβ amyloid beta, AUC area under the curve
Fig. 4
Fig. 4
Correlations between Aβ measures in CSF and plasma. No statistically significant correlations observed between a CSF Aβ1–42 and plasma Aβ42 or b CSF Aβ1–40 and plasma Aβ40. c Aβ1–42/Aβ1–40 ratio in CSF statistically significantly correlated with plasma Aβ42/Aβ40 ratio. Scatterplots of indicated Aβ variables in blood plasma and CSF are shown. Pearson correlation coefficients annotated and Deming regressions shown as dashed lines. Statistical tests were done after log transformation. Accordingly, scaling of x and y axes is logarithmic. Aβ amyloid beta, CSF cerebrospinal fluid, IP immunoprecipitation
Fig. 5
Fig. 5
Effect of APOE ε4 on Aβ measures in IP eluates and on frequency of dichotomized diagnostic classifications. No statistically significant effects of number of APOE ε4 alleles on plasma levels of a Aβ42, b Aβ40 and c Aβ38. In contrast, APOE genotype had a statistically significant effect on d Aβ42/Aβ40 (F test p < 0.001) and e Aβ42/Aβ38 (F test p < 0.001) ratios. f Proportion of AD-D patients seemed to increase with increasing number of APOE ε4 alleles. However, in the small sample size studied, this effect did not reach statistical significance (Fisher p value = 0.1). Statistical test were done after log transformation. Accordingly, scaling of y axes is logarithmic in (a)–(e). Aβ amyloid beta, AD-D dementia of the Alzheimer type, APOE apolipoprotein E, OD dementia due to other reasons
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