title: A rapid, accurate and robust particle-based assay for the simultaneous screening of plasma samples for the presence of five different anti-cytokine autoantibodies.
abstract: OBJECTIVE
  To establish and validate a rapid, cost-effective and accurate screening assay for the simultaneous testing of human naturally occurring anti-cytokine autoantibodies (c-aAb) targeting interleukin-1α (IL-1α), interleukin-6 (IL-6), interleukin-10 (IL-10), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon α (IFNα). Because the c-aAbs can be transferred to patients through blood transfusion, the assay was used to assess c-aAb levels in a cohort of patients who were receiving blood transfusions and subsequently presented with or without febrile reactions.
  
  
  METHODS
  The microsphere-based Luminex platform was used. Recombinant forms of human IL-1α, IL-6, IL-10, GM-CSF, and IFNα were gently coupled to MAG-PLEX beads. Plasma IgG binding was measured with phycoerythrin (PE)-labeled secondary antibodies. Previously confirmed c-aAb positive and negative donor plasma samples and pooled normal immunoglobulin preparations were used to validate the assay. Plasma samples from 98 transfusion recipients, half of whom presented with febrile reactions, were tested by the assay.
  
  
  RESULTS
  The assay detected specific and saturable immunoglobulin G (IgG) binding to each of the tested cytokines in previously confirmed c-aAb positive plasmas and in preparations of pooled normal immunoglobulin. Confirmed c-aAb negative plasmas gave no saturable binding. The detection limit of the cytokine autoantibodies was estimated to be between 1 pM and 10 pM. The recovery of confirmed cytokine autoantibodies quantities in the negative plasma samples ranged between 80% and 125%. The analytical intra- and inter-assay variations were 4% and 11%, respectively. Varying c-aAb levels were detectable in the transfusion recipients. There was no difference in c-aAb frequency between the patients with or without febrile transfusion reactions. The c-aAb level before and after the blood transfusions varied only slightly and in an irregular manner.
  
  
  CONCLUSIONS
  This assay simultaneously detected up to five different c-aAbs in pooled human IgG and in plasma from individual blood donors, and it was deemed suitable for larger screenings. Based on confirmed antibody binding characteristics and the resultant reactivity in this multiplex assay, a classification of the c-aAb levels was suggested. The screening results of the recipients who received blood transfusions indicate that more studies are needed to clarify the role of antibodies, if any, in transfusion medicine and in high-dose immunoglobulin treatment.
authors.affiliation: Department of Clinical Immunology and Blood Bank, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark.
authors.forename: Daniel Kring Rasmussen
authors.initials: DKR
authors.lastname: Guldager
authors.affiliation: Department of Clinical Immunology and Blood Bank, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark.
authors.forename: Jakob Hjorth
authors.initials: JH
authors.lastname: von Stemann
authors.affiliation: Department of Clinical Immunology and Blood Bank, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark.
authors.forename: Rune
authors.initials: R
authors.lastname: Larsen
authors.affiliation: Department of Clinical Immunology and Blood Bank, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark.
authors.forename: Jakob Thaning
authors.initials: JT
authors.lastname: Bay
authors.affiliation: Department of Clinical Immunology and Blood Bank, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark.
authors.forename: Pia Søndergaard
authors.initials: PS
authors.lastname: Galle
authors.affiliation: Institute for Inflammation Research, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark.
authors.forename: Morten
authors.initials: M
authors.lastname: Svenson
authors.affiliation: Department of Clinical Immunology and Blood Bank, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark.
authors.forename: Henrik
authors.initials: H
authors.lastname: Ullum
authors.affiliation: Department of Clinical Immunology and Blood Bank, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark. Electronic address: bagge@dadlnet.dk.
authors.forename: Morten Bagge
authors.initials: MB
authors.lastname: Hansen
mesh_terms: D001323:Autoantibodies / Q000276:immunology*; D001681:Biological Assay / Q000379:methods*; D016207:Cytokines / Q000097:blood* / Q000276:immunology*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D016898:Interferon-alpha / Q000276:immunology; D016753:Interleukin-10 / Q000276:immunology; D053582:Interleukin-1alpha / Q000276:immunology; D015850:Interleukin-6 / Q000276:immunology; D010949:Plasma / Q000276:immunology*
chemical_list: D001323:Autoantibodies; D016207:Cytokines; C508609:IL10 protein, human; C508584:IL1A protein, human; C508600:IL6 protein, human; D007074:Immunoglobulin G; D016898:Interferon-alpha; D053582:Interleukin-1alpha; D015850:Interleukin-6; D016753:Interleukin-10; D016178:Granulocyte-Macrophage Colony-Stimulating Factor
keywords: Antibody detection; Autoantibodies; Blood transfusion; Cytokine; Multiplex assay