Multi Epitope-Targeting Immunoprecipitation Using F(ab′) Fragments with High Affinity and Specificity for the Enhanced Detection of a Peptide with Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry

Human plasma has been frequently studied using mass spectrometry for new biomarker discovery, although detection of low-abundance biological molecules can be challenging due to sample complexity and dynamic protein concentration ranges of plasma proteins. While immunoprecipitation coupled with mass spectrometric analysis is an essential method for overcoming this difficulty, its sensitivity can be insufficient to detect clinically relevant circulating biomarkers because of limited antibody affinity or specificity. To increase antibody affinity, we developed a strategy using a F(ab′) fragment coupled to polyethylene glycol. We produced hetero-F(ab′)-(PEG)24 beads composed of two monoclonal antiamyloid β antibodies (6E10 and 4G8) that are specific for different epitopes of amyloid β and assessed the detection limit of amyloid β1–28-spiked human plasma. In human plasma, the detection limit of amyloid β1–28 was 6.14 pM, which was 25- to 50-fold more sensitive than single IgG-protein G beads. In addition, an i...