Measuring total IFNα at fg/mL concentrations in human blood

• Published in: The Journal of immunology (1950) Vol. 200; no. 1_Supplement; pp. 174 - 174.15
• Main Authors: Lambert, Jeremy, Ferrell, Evan, Wickman, Jason, Chang, Lei, Hanlon, David, Johnson, Joe, Kiesel, Gregory
• Format: Journal Article
• Language: English
• Published: 01.05.2018
• ISSN: 0022-1767; 1550-6606
• DOI: 10.4049/jimmunol.200.Supp.174.15

Abstract Total IFNα has been challenging to quantify in normal human plasma and serum due to its low abundance and variety of subtypes. Here we describe the development of a digital immunoassay capable of measuring all 12 subtypes of human IFNα in plasma and serum using Quanterix’s Simoa technology. The Simoa digital ELISA technology counts signal generated from single immunocomplexes formed on superparamagnetic beads confined in arrays of femtoliter-sized wells in which fluorescent molecules are highly concentrated. The Simoa IFNα multi-subtype assay has a dynamic range of approximately 3.5 orders of magnitude and a sensitivity below 100 fg/mL. The median LOD (2.5 SD) over 12 runs was approximately 30 fg/mL and the LOQ was approximately 80 fg/mL. This represents a significant improvement over commercial ELISAs, which claim IFNα sensitivities in the pg/mL range. In repeated screens of 30 normal samples, up to 60% measured above LOD and testing of serum from Lupus patients and stimulated PBMCs resulted in concentrations within the dynamic range of the assay. Depletion experiments conducted by incubating unlabeled capture or detection antibody with these same samples demonstrated that the assay is specific for IFNα. IFNα has been useful in several applications including as a therapeutic agent, as a biomarker for monitoring response to immunotherapy, and as a marker of disease progression in human clinical and preclinical studies. In each example, increased sensitivity for detection of all subtypes of IFNα provides researchers and clinicians with the ability to measure the biomarker in both normal and acute samples with robustness required to advance precision medicine programs.