title: Enrichment and analysis of Alzheimer's Abeta1-42 peptide in human plasma and whole blood.
abstract: The Abeta1-42 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 Abeta1-42 using solid-phase extraction (SPE) and reverse-phase HPLC (RP-HPLC) were optimized. The new process provided reproducible recovery of Abeta1-42 of about 80% and allowed for concentration of the peptide prior to assay. Radiolabeled Abeta1-42 and ELISA for Abeta1-42 were used to determine the recovery and distribution of the peptide from whole blood collected in the presence of potassium-EDTA. Endogenous Abeta1-42 yielded a cell pellet:plasma ratio near 40:60 while exogenously added peptide distributed with a ratio of about 27:73. Additionally, the Abeta1-42 in the plasma and cell pellet fractions maintained stability over many hours. Comparing the measurement of Abeta1-42 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 Abeta1-42 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.
authors.affiliation: Bristol-Myers Squibb Company, Discovery Medicine and Clinical Pharmacology, Clinical Biomarkers, Pennington, NJ 08534, United States. noah.ditto@bms.com
authors.forename: Noah T
authors.initials: NT
authors.lastname: Ditto
authors.forename: Tiffany R
authors.initials: TR
authors.lastname: Kline
authors.forename: Peter D
authors.initials: PD
authors.lastname: Alfinito
authors.forename: J Randall
authors.initials: JR
authors.lastname: Slemmon
mesh_terms: D000544:Alzheimer Disease / Q000097:blood*; D016229:Amyloid beta-Peptides / Q000097:blood*; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008297:Male; D008875:Middle Aged; D018832:Molecular Chaperones / Q000378:metabolism; D010446:Peptide Fragments / Q000097:blood*; D010949:Plasma / Q000737:chemistry; D011485:Protein Binding; D052616:Solid Phase Extraction
chemical_list: D016229:Amyloid beta-Peptides; D018832:Molecular Chaperones; D010446:Peptide Fragments; C075222:amyloid beta-protein (1-42)