B-267 Development of an Ultra-Sensitive Anti-TNF-a ELISA Using BOLD Technology

Abstract Background Tumour necrosis factor alpha (TNF-a), a cytokine involved in inflammation and immune regulation, is found in both transmembrane and soluble forms. It triggers pathways that can lead to cell death or survival (1). TNF-a levels are usually 0-2 pg/mL in plasma (2, 3) and 20-50 pg/mL in serum (4) but can increase in various diseases such as autoimmune disorders, severe infections, chronic inflammation and cancer. Conventional immunoassays typically have a sensitivity of 1-15 pg/mL, and enhancing this sensitivity could improve the detection of small changes and enable testing with limited sample volumes. Methods Cavidi has developed a new technology, BOLD, which is based on a molecular biology approach to bring ultra-sensitive detection to virtually any immunodiagnostic assay. BOLD stands for Binding Oligo Ladder Detection and is a process that can be added to any immunoassay. Integration of BOLD into an immunoassay will improve the sensitivity and the limit of detection by up to 50x. To integrate BOLD into the immunoassay, an oligo-dT Primer is conjugated, using click chemistry, to an unmodified detector antibody of the standard immunoassay. The conjugation is performed with the Exazym® ClickChem Conjugation Kit. This oligo-dT Primer detector antibody conjugate serves as a crucial bridge for the addition of the BOLD amplification steps to the standard immunoassay. The Exazym® Polymerase Reaction Kit includes the polymerase and essential building blocks for DNA/RNA hybrid strand synthesis. This facilitates enhanced detection through the binding of multiple biotinylated anti-DNA/RNA tertiary antibodies from the Exazym® Biotin Detection Kit to each analyte. Subsequently, a streptavidin-conjugate (or other detection label) is used for final detection. Results BOLD amplification of the standard ELISA demonstrated a mean LoD of 0.24 pg/mL, representing a 6-fold improvement over the standard ELISA’s mean LoD of 1.38 pg/mL. The repeatability (triplicate) and intermediate precision (three runs) were evaluated for the high, medium and low samples (Figure 5). The BOLD amplified ELISA consistently delivers reliable results across a broad range of sample concentrations, both within and between runs. In contrast, the standard ELISA showed lower precision at the lower end of the curve, where it failed to reliably detect TNF-a. Conclusion The integration of BOLD technology into the standard ELISA protocol has significantly enhanced the sensitivity of TNF-a detection, achieving a six-fold lower LoD compared to the standard ELISA. This improvement allows for more accurate detection of low-abundance analytes, which is particularly critical in applications where sample concentrations or volumes are minimal or where detecting small changes is crucial. Additionally, the BOLD amplified ELISA maintained low background signals and demonstrated consistent reliability and precision across various sample concentrations, outperforming the standard ELISA at the lower detection levels.