title: An immunoassay that distinguishes real neuromyelitis optica signals from a labeling detected in patients receiving natalizumab.
abstract: BACKGROUND
  Cell-based assays for neuromyelitis optica (NMO) diagnosis are the most sensitive and specific methods to detect anti-aquaporin 4 (AQP4) antibodies in serum, but some improvements in their quantitative and specificity capacities would be desirable. Thus the aim of the present work was to develop a sensitive quantitative method for detection of anti-AQP4 antibodies that allows clear diagnosis of NMO and distinction of false labeling produced by natalizumab treatment.
  
  
  METHODS
  Sera from 167 individuals, patients diagnosed with NMO (16), multiple sclerosis (85), optic neuritis (24), idiopathic myelitis (21), or other neurological disorders (13) and healthy controls (8), were used as the primary antibody in an immunofluorescence assay on HEK cells transfected with the M23 isoform of human AQP4 fused with enhanced green fluorescent protein. Cells used were freshly transfected or stored frozen and then thawed just before adding the serum.
  
  
  RESULTS
  Microscopic observation and fluorescence quantification produced similar results in fresh and frozen samples. Serum samples from patients diagnosed with NMO were 100% positive for anti-AQP4 antibodies, while all the other sera were negative. Using serum from patients treated with natalizumab, a small and unspecific fluorescent signal was produced from all HEK cells, regardless of AQP4 expression.
  
  
  CONCLUSIONS
  Our cell-based double-label fluorescence immunoassay protocol significantly increases the signal specificity and reduces false diagnosis of NMO patients, especially in those receiving natalizumab treatment. Frozen pretreated cells allow faster detection of anti-AQP4 antibodies.
authors: Ismael
authors: I
authors: Sánchez Gomar
authors: María
authors: M
authors: Díaz Sánchez
authors: Antonio José
authors: AJ
authors: Uclés Sánchez
authors: José Luis
authors: JL
authors: Casado Chocán
authors: Reposo
authors: R
authors: Ramírez-Lorca
authors: Ana
authors: A
authors: Serna
authors: Javier
authors: J
authors: Villadiego
authors: Juan José
authors: JJ
authors: Toledo-Aral
authors: Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Av, Manuel Siurot s/n, Seville 41013, Spain. irusta@us.es.
authors: Miriam
authors: M
authors: Echevarría
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D061067:Antibodies, Monoclonal, Humanized / Q000097:blood* / Q000627:therapeutic use; D051401:Aquaporin 4 / Q000097:blood*; D001323:Autoantibodies / Q000097:blood*; D001324:Autoantigens / Q000276:immunology; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D008297:Male; D008875:Middle Aged; D009103:Multiple Sclerosis / Q000188:drug therapy; D000069442:Natalizumab; D009471:Neuromyelitis Optica / Q000097:blood / Q000175:diagnosis* / Q000276:immunology; D055815:Young Adult
chemical_list: C496030:AQP4 protein, human; D061067:Antibodies, Monoclonal, Humanized; D051401:Aquaporin 4; D001323:Autoantibodies; D001324:Autoantigens; D000069442:Natalizumab