title: [Flow cytometry model for the detection of neutralizing antibodies against of IFN-β].
abstract: BACKGROUND
  Interferon beta (IFN-β) is a treatment for relapsing remitting multiple sclerosis. However, the therapeutic use of recombinant proteins induces a humoral immunologic response resulting in the induction of binding (BAb) or neutralizing (NAb) antibodies against the biological product. The presence of neutralizing antibodies has been associated with decreased IFN-β treatment efficacy.
  
  
  METHODS
  Two tumor cell lines (K562 and U937) were cultivated with human recombinant IFN-β1a at different concentrations and lengths of time in order to measure the expression of intracellular ISG15, an inducible molecule in the IFN-β1a signaling cascade. Blood was obtained from non-immunized and IFN-β1a immunized (100,000 IU) New Zealand rabbits. The presence of BAb was evaluated by ELISA. For NAb detection, sera 1:20 dilution were added to the IFN-β1a-stimulated cell lines, and ISG15 expression was evaluated by flow cytometry.
  
  
  RESULTS
  K562 cells provided the better cell line for the assay, stimulated with a dose of 1,000 IU of IFN-β1a, and a 1:100 dilution for the primary antibody and a 1:200 dilution for the secondary antibody. ISG15 expression was compared between cells alone or cultivated with IFN-β1a. Mean fluorescence intensity (MFI) for ISG-15 expression median was 198 arbitrary units (AU) with interquartile ranges of 173-231 AU for non-stimulated cells and 430 AU with interquartile ranges of 316-611.5 AU for IFN-β1a stimulated cells (p<0.01). Immunized rabbit sera decreased the expression of ISG-15 in K562 cells stimulated with IFN-β1a, whereas non-immunized rabbit sera did not.
  
  
  CONCLUSIONS
  This rabbit model demonstrates that ISG15 expression evaluated with flow cytometry can be used as a detection assay for NAb.
authors.affiliation: Grupo de Ciencias Básicas Médicas, Facultad de Medicina, Universidad de los Andes, Bogotá, D.C., Colombia.
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Villa-Camacho
authors.forename: Juan Camilo
authors.initials: JC
authors.lastname: Vargas-Zambrano
authors.forename: John Mario
authors.initials: JM
authors.lastname: González
mesh_terms: D000818:Animals; D000910:Antibodies, Heterophile / Q000097:blood*; D000937:Antigen-Antibody Reactions; D016207:Cytokines / Q000096:biosynthesis* / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007114:Immunization; D016899:Interferon-beta / Q000276:immunology* / Q000494:pharmacology; D020014:K562 Cells / Q000378:metabolism; D008297:Male; D009500:Neutralization Tests; D011817:Rabbits; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D020298:U937 Cells / Q000378:metabolism; D014452:Ubiquitins / Q000096:biosynthesis* / Q000235:genetics; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D000910:Antibodies, Heterophile; D016207:Cytokines; D011994:Recombinant Proteins; D014452:Ubiquitins; C074034:ISG15 protein, human; D016899:Interferon-beta