Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-α

Autor: Llibre, Alba, Bondet, Vincent, Rodéro, Mathieu, Hunt, David, Crow, Yanick, Duffy, Darragh
Přispěvatelé: Immunobiologie des Cellules dendritiques, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Imagine - Institut des maladies génétiques (IMAGINE - U1163), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), University of Edinburgh, University of Manchester [Manchester], DD and YJC acknowledge support from the ANR (Project IFNX, no. CE17001002) and ImmunoQure AG for providing monoclonal antibodies. YJC acknowledges the European Research Council (GA 309449: Fellowship to YJC), and a state subsidy managed by the National Research Agency (France) under the 'Investments for the Future' program bearing the reference ANR-10-IAHU-01., We thank Brigitte Bader-Meunier, Nathalia Bellon, Christine Bodemer, Alex Belot, Isabelle Melki, and Pierre Quartier for providing clinical samples., ANR-10-IAHU-0001,Imagine,Institut Hospitalo-Universitaire Imagine(2010), ANR-16-CE17-0010,IFNX,Investigation des interferonopathies type I humaine(2016), Vougny, Marie-Christine, Instituts Hospitalo-Universitaires - Institut Hospitalo-Universitaire Imagine - - Imagine2010 - ANR-10-IAHU-0001 - IAHU - VALID, Investigation des interferonopathies type I humaine - - IFNX2016 - ANR-16-CE17-0010 - AAPG2016 - VALID, Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris]
Rok vydání: 2018
Předmět:
Zdroj: Journal of Visualized Experiments : JoVE
Llibre, A, Bondet, V, Rodero, M P, Hunt, D, Crow, Y J & Duffy, D 2018, ' Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-α ', Journal of Visualized Experiments (JoVE), no. 136 . https://doi.org/10.3791/57421
Journal of visualized experiments : JoVE
Journal of visualized experiments : JoVE, 2018, 136, pp.e57421. ⟨10.3791/57421⟩
Journal of visualized experiments : JoVE, JoVE, 2018, pp.e57421. ⟨10.3791/57421⟩
ISSN: 1940-087X
Popis: International audience; The main aim of this protocol is to describe the development and validation of an interferon (IFN)-α single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-α protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN. The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-α single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-α subtypes. This new methodology allows the detection and quantification of IFN-α protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.
Databáze: OpenAIRE
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