The Simoa pan-IFNα assay was developed using the Quanterix Homebrew kit according to the manufacturer’s instructions and using two autoantibodies specific for IFNα isolated and cloned from two APS1/APECED patients. The 8H1 antibody clone was used as a capture antibody after coating on paramagnetic beads (0.3 mg/mL), and the 12H5 was biotinylated (biotin/antibody ratio = 30/1) and used as the detector at a concentration of 0.3ug/mL. The SBG revelation enzyme concentration was 150 pM. Recombinant IFNα17 was used as calibrator. The limit of detection was calculated by the mean value of all blank runs + 2SD after log conversion. The assay is fully described in Rodero et al. [13 (link)] and a video presenting all the steps is shown in Llibre et al [19 (link)].
Other organizations :
Université Paris Cité, Centre National de la Recherche Scientifique, Sorbonne Université, University of Tartu, St Mary's Hospital, St. Mary's Hospital, University of Manchester, Institut Pasteur du Cambodge, Manchester University NHS Foundation Trust, Manchester Academic Health Science Centre, NIHR Manchester Biomedical Research Centre, University of Birmingham, Sandwell & West Birmingham Hospitals NHS Trust, University College London, MRC Institute of Genetics and Molecular Medicine, Medical Research Council, University of Edinburgh, Bicêtre Hospital, Assistance Publique – Hôpitaux de Paris, Inserm, Université Paris-Saclay, Hôpital Cochin, Department of Virology
Antibody clone 12H5 biotinylated and used as detector
dependent variables
IFNα concentration
control variables
Paramagnetic beads coated with capture antibody at 0.3 mg/mL
Detector antibody concentration of 0.3 μg/mL
SBG revelation enzyme concentration of 150 pM
Recombinant IFNα17 used as calibrator
controls
Blank runs used to calculate the limit of detection
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