Biomarkers

The analysis of plasma biomarkers for Alzheimer's disease is non-invasive and suitable for large-scale screening. We have reported that the combination of three amyloid peptides in human plasma (Aβ1-40, Aβ1-42, APP669-711) shows a good correlation with amyloid accumulation in human brain. (Nakamura et al., Nature 2018) Here, we report the results of the analytical validation of our plasma amyloid-β assay using immunoprecipitation-matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (IP-MALDI-MS) with a benchtop MALDI-TOF mass spectrometer, instead of a conventional floor-standing model previously reported (Yoda et al., AAIC 2021).

Using magnetic beads covalently bound with anti-amyloid β antibody (clone 6E10), immunoprecipitation was performed on commercial plasma and/or synthetic peptide mixtures. The samples obtained from immunoprecipitation were measured by the benchtop MALDI-TOF MS (Shimadzu/Kratos, UK). The peak intensities from amyloid-related peptides were normalized using stable isotope-labeled (SIL) Aβ1-38 as internal standards. The biomarkers Aβ1-40/Aβ1-42 and APP669-711/Aβ1-42 were evaluated for intra-assay and inter-assay reproducibility. For dynamic range, spike recovery, and dilution linearity, the normalized intensities of each peptide were assessed.

Each biomarker values showed a coefficient of variation (CV) of less than 5% for both intra-assay and inter-assay reproducibility, which was better than the previous data with the CV of < 5% in some cases. Trueness of 100±20% was confirmed over the range from 15 to 150 pM for Aβ1-40, and from 1.5 to 15 pM for Aβ1-42 and APP669-711, which was superior to the previous results showing the trueness of > 130% at the lowest concentration. In the spike recovery test, recovery rates ranged from 87% to 113% across two concentration levels (low and high). In the dilution linearity test, linearity ranged from 88% to 111% for 2-fold and 4-fold dilutions.

These results indicate that immunoprecipitation-mass spectrometry system using the benchtop instrument possesses sufficient stability and reliability for the measurement of amyloid-β peptides in plasma, with improvements in reproducibility and trueness.