Surface Assay for Specific Detection of Soluble Amyloid Oligomers Utilizing Pronucleon Peptides Instead of Antibodies

Immunoassays such as enzyme-linked immunosorbent assays (ELISAs) are widely used for diagnostics; however, antibodies as detection reagents may be insufficiently selective and have other shortcomings. We present a novel non-antibody-based detection method based on binding target molecules to peptides (used as recognition molecules): a surface assay for A-beta oligomers employing a peptide comprising amino acid residues of the human beta-amyloid protein (Pronucleon peptide) as the capture agent. For the sake of convenience, we term this the Pronucleon peptide linked immunosorbent assay, or PLISA. Pronucleon peptides are amino acid sequences matched to target amyloids of interest, in particular soluble A beta-1-42 amyloid protein oligomers, which are widely considered as an early biomarker for Alzheimer's disease in body fluids. The Pronucleon peptide in a PLISA is immobilized on the surface and substitutes for the capture antibody used in an ELISA for binding the A beta-1-42 oligomers present in the sample. We present data comparing synthetic oligomer PLISAs in spiked buffer and body fluids (such as cerebrospinal fluid, brain extracts, or whole blood) to those from an ELISA and demonstrate better selectivity of the PLISA for amyloid beta-42 oligomers versus monomers and fibrils. The detection limit, calculated as the mean (blank) plus three standard deviations, was in the range of 0.35-1.5 pM (32-135 ng/L) (oligomers contained approximately 20 monomers on average).