AbstractWe examined Interferon (IFN)-Alpha and -Beta levels in pooled sera and individual bleeds from C57/Bl, Balb/C and CD-1 mice. 20 Individuals from each strain from two independent colonies were evaluated in commercial assays. The IFN-Beta ELISA detected IFN in 1 of the 60 individuals while the IFN-Alpha assay which detects all 14 subtypes measured IFN in 13 of 60. 5 individuals exhibited greater than 4 pg/ml of IFN-Alpha detected. Specificity was demonstrated by knockdown of the signal using non-biotinylated detection antibody. 14 of 54 porcine sera samples from 4 independent sources were positive for IFN alpha by an ELISA under development, and similar knock-down experiments suggest specificity. This is in contrast to human sera/plasma samples where less than 3% of healthy donor samples have quantifiable IFN-Alpha or -Beta above 0.5 pg/ml. This study may raise questions about the regulation of the IFN system in different species and the potential of these differences to affect experimental results in a variety of models.

01 May 2020·The Journal of Immunology

Comparison of Interferons and Other Biomarkers in Influenza and Healthy Donor Samples

Author: Clark, William ; Kisiel, Greggory ; Lavoie, Thomas B ; Schwartz, Barbara ; Panday, Alok ; Skup, Kamil

AbstractToward an understanding of biomarkers for acute viral diseases we have examined Influenza serum in comparison to healthy donors. We determined the levels of Interferon (IFN) Alpha, Beta, Gamma as well as Neopterin and Beta-2 Microglobulin (B2M) by ELISA in the Serum of 38 Influenza and 65 healthy donors using commercial assays. We measured chemokines and proinflammatory cytokines as biomarkers for IFN Activity by electrochemiluminescence. Assays were performed according to the manufacturer’s protocol. IFN-Omega levels were determined using a newly developed ultra-sensitive single molecule assay sensitive to 0.04 pg/ml. Percentile stratification was by Mann-Whitney non-parametric testing and correlations were determined under Spearman conditions. None of the healthy donors exhibited quantifiable IFN-Alpha or Beta and only two had detectable levels of IFN-Omega. Within the Flu cohort, 68.5% of samples contain quantifiable IFN-Alpha and Omega and 39.5% had IFN-Beta. 79% of the Flu samples displayed IFN-Gamma levels above the 95th percentile determined from the healthy donor distribution. Neopterin, B2M, and CXCL-10 were all significantly elevated in the Flu cohort versus the healthy subclass. Other chemokines either showed no correlation or a mild negative correlation. There was a striking concordance between IFN-Alpha and Omega levels. To our knowledge this is the first study to measure IFN-Omega and a broad panel of other analytes in Flu samples. As such this study may provide a beginning to discriminate between different viral diseases.

01 May 2018·The Journal of Immunology

Measuring total IFNα at fg/mL concentrations in human blood

Author: Hanlon, David ; Ferrell, Evan ; Chang, Lei ; Lambert, Jeremy ; Wickman, Jason ; Johnson, Joe ; Kiesel, Gregory

AbstractTotal 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.

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