Ultra-Sensitive Cytokine Quantification Using Binding Oligo Ladder Detection: A Universal Signal-Amplification Strategy for Standard Immunoassays 2308422

Abstract

Abstract Introduction Accurate quantification of low-abundance cytokines is essential for resolving early immune activation, subtle inflammatory shifts, & cytokine dynamics in limited-volume samples. However, many cytokines circulate at concentrations near or below the analytical limits of conventional assays. Here we describe the application of Binding Oligo Ladder Detection (BOLD)–a molecular amplification chemistry that integrates directly into existing immunoassay workflows to achieve sub-pg/mL sensitivity across multiple cytokines. Methods BOLD functions by conjugating an oligo-dT primer to the detector antibody, enabling enzymatic generation of a DNA/RNA hybrid strand that recruits multiple anti-hybrid antibodies for amplified signal generation. The method requires no specialized instrumentation & is compatible with standard detection methods such as colorimetry, fluorescence and ECL. Results Across four cytokine systems, BOLD consistently improved sensitivity as compared to standard assays. For IL-4, the limit of detection (LOD) improved to 0.040 pg/mL (∼50×) under optimized conditions. TNF-α detection improved ∼6×, with LOD reduced to 0.24 pg/mL and with reproducible performance across replicate runs. Newly developed BOLD-enhanced assays for IL-6 and IL-23 demonstrated 7× and 8× improvements in sensitivity, respectively. Across all cytokines, the concentration of primer-modified detectors was significantly reduced compared to standard ELISA detector levels, while maintaining low background & favorable signal-to-noise ratios. Assay robustness was further supported by minimal dependence on tertiary antibody concentration & stable performance across polymerase reaction conditions. Conclusion These results demonstrate that BOLD is a versatile, platform-agnostic technology capable of transforming standard assays into ultra-sensitive detection systems. Its applicability across diverse cytokines highlights its potential for advancing immune monitoring, translational research, & high-sensitivity biomarker discovery. Funding Source n/a Topic Categories Technological Innovations in Immunology (TECH)