Enrichment and analysis of Alzheimer's Aβ1-42 peptide in human plasma and whole blood

The Aβ1-42 fragment from the Amyloid Precursor Protein (APP) has presented considerable challenges from an analytical perspective. It is present at low levels in the circulation and can bind to proteins which mask its presence in assays. A number of therapeutic strategies target the lowering of this peptide, necessitating more robust and sensitive methods for its measurement. In this study, conditions for extracting and enriching Aβ1-42 using solid-phase extraction (SPE) and reverse-phase HPLC (RP-HPLC) were optimized. The new process provided reproducible recovery of Aβ1-42 of about 80% and allowed for concentration of the peptide prior to assay. Radiolabeled Aβ1-42 and ELISA for Aβ1-42 were used to determine the recovery and distribution of the peptide from whole blood collected in the presence of potassium-EDTA. Endogenous Aβ1-42 yielded a cell pellet:plasma ratio near 40:60 while exogenously added peptide distributed with a ratio of about 27:73. Additionally, the Aβ1-42 in the plasma and cell pellet fractions maintained stability over many hours. Comparing the measurement of Aβ1-42 using a commercial ELISA before and after enrichment demonstrated noticeable improvement of signal in samples enriched for the peptide. The current study also showed that conspicuous amounts of Aβ1-42 partition to the cell pellet but that this fraction can be robustly recovered and measured with SPE and HPLC. The process utilized established chromatographic techniques and is suitable for automation. It is also compatible with other detection methods including mass spectrometry.