With this study we introduce the novel Amyblood assays developed on ultrasensitive Simoa technology for detection of the specific N-terminal Abeta peptides Abeta1 − 42 and Abeta1 − 40 in plasma. The validation was successful and the results were highly comparable to technical and clinical validation results obtained for two commercially available assays: the Quanterix triplex and Euroimmun ELISA. The specificity of the Amyblood assays for the specific isoforms was higher compared to the Quanterix assay. Moreover, the Amyblood Abeta1 − 42/1−40 ratio could successfully differentiate AD cases from controls, similar to the Quanterix assay. The ELISA ratio could not differentiate the groups, though the ELISA Abeta1 − 42 concentration by itself could. Our validation data show that the Amyblood assays are suitable for robust measurement of 1–42 and 1–40 amyloid isoforms in plasma.
The development of the Amyblood assays was motivated by the need for an assay that simultaneously offers high specificity, high sensitivity and high throughput. Therefore, we employed the known specificity of the ADx102, ADx103, and ADx101 antibodies as demonstrated earlier in CSF,19,20 together with the sensitivity of the Simoa technology.11 The promising prototype assay results (eFigure 3) urged us to upscale for widespread validation. Upscaling is challenging since production of larger stock volumes can influence the reagent performance and may affect the sensitivity. Therefore, performance of the prototype and upscaled batch were thoroughly tested. Upscaling did not affect the calibrator curve, reproducibility was demonstrated at Amsterdam UMC and ADx (variation < 20%), and lastly, the assay remained successful in differentiating 20 AD patients and 20 controls (p < 0.001). These results indicate that the transformation of our initial prototype to the RTU Amyblood assays was successful.
Full length Abeta1 − 42 and Abeta1 − 40 are the most abundant Abeta proteoforms in AD brain and CSF.19,21 These proteoforms are more soluble and more toxic than N-terminal truncated proteoforms, and are therefore important therapeutic targets.21 This stresses the need for highly specific amyloid assays that can proof target engagement in clinical trials. Our specificity analyses showed that the Amyblood Abeta1 − 42 assay showed minor cross-reactivity for other proteoforms in sample buffer. At 60 pg/ml Abeta1 − 40 in sample diluent, a non-specific concentration of 2.4 pg/mL was measured with the Amyblood Abeta1 − 42 assay. However, this nonspecific signal could be clinically meaningful, being similar to the group difference of also 2.4 pg/mL Abeta1 − 42 as observed between AD and controls. With the Quanterix Abetax−42 assay, a non-specific signal of 0.9 pg/mL was read at 40 pg/mL Abeta1 − 40, which is three times the group difference of 0.3 pg/mL Abetax−42 as measured with this assay. In addition, a minor increase of 0.8 fold the group difference was measured with the Amyblood Abeta1 − 42 assay when Abeta3 − 42 in sample buffer was incubated, where a large increase of 80 fold the group difference was measured with the Quanterix Abetax−42 assay for this proteoform. The difference in assay specificity could be explained by the Amyblood N-terminal ADx102 (21F2) antibody that is specific for the first amino acid of the Abeta peptide, whereas the Quanterix assay detects N-terminal amino acid 4–10. However, our specificity experiments were performed in sample buffer, whereas a wide variety of endogenous proteins are present in plasma which would make low affinity non-specific binding less likely.22 Indeed, our selectivity analyses showed a recovery closer to 100% for Amyblood Abeta1 − 42 for higher Abeta1 − 42 concentrations next to the presence of varying concentrations of Abeta1 − 40 peptide. At physiological concentrations of Abeta1 − 42 and Abeta− 40 in buffer, the %recovery was 125% for Abeta1 − 42. For the Quanterix assay, the Abetax−42 recovery was higher (163%) at physiological concentrations. Our data suggest that the Amyblood Abeta1 − 42 assay had better specificity and selectivity than the Quanterix Abetax−42 assay. Finally, there was no cross reactivity for both Abeta40 assays, indicating the high specificity of the 2G3 antibody for Abeta40 employed in both formats.
The Amyblood and Quanterix assays could successfully differentiate CSF Abeta1 − 42 positive AD patients and Abeta1 − 42 negative controls. The ELISA ratio could not differentiate AD from controls, but the ELISA Abeta1 − 42 concentration alone could. Interestingly, a recent study did show promising results with the Abeta42/40 ratio measured with yet another ELISA assay and similarly showed a reduction in Abeta PET positive cases in a large cohort of controls, MCI and AD. This was against the expectations for ELISA assays being less sensitive towards low protein concentrations.23 Similar to other studies, we found no correlation between CSF Abeta1 − 42 and plasma Abeta42 concentrations, and a weak correlation with the plasma Abeta1 − 42/1−40 ratio.5,6 An explanation could be that amyloid produced peripherally, for example by platelets,24 distorts the association of Abeta measured in plasma with Abeta produced only by the central nervous system, as measured in CSF.
It has been suggested that SCD cases have a higher risk of converting to AD compared to healthy elderly controls.25 We carefully selected controls with normal CSF biomarker values, who are not likely to convert.26 We wish to stress that the focus of our study was to compare analytical performance and clinical samples were included to indicate potential clinical value, since other studies are on their way on early diagnostic use of the Amyblood assays. Our positive results show that the assay is suitable for diagnostic studies.
A next step in the development of these assays is to leverage the multiplexing possibilities of Simoa technology and simultaneously detect multiple biomarkers, to reflect different aspects of AD within one assay run, saving time and resources. In addition, development of Certified Reference Material is critical to calibrate and compare different amyloid assays and to enable clinical implementation. In collaboration with biotechnology companies, the assay will be made available on a global level.