title: Incorporation of Slow Off-Rate Modified Aptamers Reagents in Single Molecule Array Assays for Cytokine Detection with Ultrahigh Sensitivity.
abstract: Slow off-rate modified aptamers (SOMAmers) are attractive protein recognition reagents due to their high binding affinities, stable chemical structures, easy production, and established selection process. Here, biotinylated SOMAmer reagents were incorporated into single molecule array (Simoa)-based assays in place of traditional detection antibodies for six cytokine targets. Optimization and validation were conducted for TNF-α as a demonstration using a capture antibody/detection-SOMAmer detection scheme to highlight the performance of this approach. The optimized assay has a broad dynamic range (>4 log10 units) and an ultralow detection limit of 0.67 fM (0.012 pg/mL). These results show comparable sensitivity to our antibody pair-based Simoa assays, and tens to thousands-fold enhancement in sensitivity compared with conventional ELISAs. High recovery percentages were observed in a spike-recovery test using human sera, demonstrating the feasibility of this novel Simoa assay in detecting TNF-α in clinically relevant samples. Detection SOMAmers were also used to detect other cytokines, such as IFN-γ, IL-1β, IL-2, IL-6, and IL-10, in human samples. Although not yet demonstrated, in principle it should be possible to eventually replace both the capture and detector antibodies with corresponding SOMAmer pairs in sandwich immunoassays. The combination of the ultrasensitive Simoa platform with the higher reliability of SOMAmer binding reagents will greatly benefit both biomarker discovery and disease diagnostic fields.
authors.affiliation: Department of Chemistry, Tufts University , 62 Talbot Avenue, Medford, Massachusetts 02155, United States.
authors.forename: Danlu
authors.initials: D
authors.lastname: Wu
authors.affiliation: SomaLogic, Inc. , 2945 Wilderness Place, Boulder, Colorado 80301, United States.
authors.forename: Evaldas
authors.initials: E
authors.lastname: Katilius
authors.affiliation: SomaLogic, Inc. , 2945 Wilderness Place, Boulder, Colorado 80301, United States.
authors.forename: Edgar
authors.initials: E
authors.lastname: Olivas
authors.affiliation: Department of Chemistry, Tufts University , 62 Talbot Avenue, Medford, Massachusetts 02155, United States.
authors.forename: Milena
authors.initials: M
authors.lastname: Dumont Milutinovic
authors.affiliation: Department of Chemistry, Tufts University , 62 Talbot Avenue, Medford, Massachusetts 02155, United States.
authors.forename: David R
authors.initials: DR
authors.lastname: Walt
mesh_terms: D000906:Antibodies / Q000276:immunology; D052157:Aptamers, Nucleotide / Q000737:chemistry*; D016207:Cytokines / Q000097:blood* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry*; D007700:Kinetics
chemical_list: D000906:Antibodies; D052157:Aptamers, Nucleotide; D016207:Cytokines; D007202:Indicators and Reagents