title: Development and multi-center evaluation of a novel immunoadsorption method for anti-DFS70 antibodies.
abstract: OBJECTIVE
  Antinuclear antibodies (ANA) represent a hallmark in the diagnosis of ANA-associated rheumatic diseases (AARD). However, anti-DFS70 antibodies are present in a higher portion of the healthy individuals (HI) than in patients with AARD. Consequently, we developed a novel, highly specific indirect immunofluorescence (IIF) method that blocks anti-DFS70 antibodies from binding to HEp-2 cells and to evaluate the method in a multi-center study.
  
  
  METHODS
  A total of 18 samples from systemic lupus erythematosus patients (SLE, n = 7) and HI (n = 11) were used for the initial development of the immunoadsorption method. For the multi-center evaluation, samples with a dense fine speckled (DFS) pattern (n = 99) were collected at three different sites based on their established IIF screening procedure at the respective laboratories. Additionally, four characterized samples with established clinically relevant IIF patterns (centromere, nucleolar, speckled, homogeneous) were blended in five different ratios (10%, 25%, 50%, 75%, 90%) with a sample positive for anti-DFS70 antibodies, which by itself showed a dense fine speckled (DFS) IIF pattern. All samples were tested by IIF with NOVA Lite HEp-2 ANA and NOVA Lite HEp-2 Select on the NOVA View® instrument, and also tested by QUANTA Flash DFS70 chemiluminescent immunoassay (CIA) for confirmation of anti-DFS70 antibodies (Inova Diagnostics, San Diego, CA, USA).
  
  
  RESULTS
  For the development of the immunoadsorption method, only 1/7 ANA-positive samples from SLE patients, but 8/10 ANA-positive samples from healthy individuals turned negative using the immunoadsorption. Subsequently, 73/99 (73.7%) of the DFS pattern samples were positive by CIA for anti-DFS70 antibodies showing a strong quantitative Spearman's correlation (rho = 0.57 (95% CI, 0.39-0.71, p < 0.0001)) between light intensity units (LIU) measured by NOVA View and CIA. Intensities measured with NOVA Lite HEp-2 and NOVA Lite HEp-2 Select demonstrated significantly lower intensity values after inhibition with DFS70 antigen (p < 0.0001). When samples were processed to mimic samples with mixed patterns (DFS + clinically relevant pattern), the new immunoadsorption method demonstrated that all clinically relevant patterns remained unchanged whereas the LIUs from NOVA View analysis significantly decreased after inhibition (p < 0.0001).
  
  
  CONCLUSIONS
  The data showed that the NOVA Lite HEp-2 Select kit effectively inhibits anti-DFS70 antibody binding to its cellular target antigen.
authors.affiliation: Department of Research, Inova Diagnostics, Inc., San Diego, USA.
authors.forename: C
authors.initials: C
authors.lastname: Bentow
authors.affiliation: Department of Research, Inova Diagnostics, Inc., San Diego, USA.
authors.forename: R
authors.initials: R
authors.lastname: Rosenblum
authors.affiliation: RDL Reference Laboratory, Los Angeles, USA.
authors.forename: P
authors.initials: P
authors.lastname: Correia
authors.affiliation: RDL Reference Laboratory, Los Angeles, USA.
authors.forename: E
authors.initials: E
authors.lastname: Karayev
authors.affiliation: RDL Reference Laboratory, Los Angeles, USA.
authors.forename: D
authors.initials: D
authors.lastname: Karayev
authors.affiliation: Immunology Department, Southampton General Hospital, Southampton, UK.
authors.forename: D
authors.initials: D
authors.lastname: Williams
authors.affiliation: Department of Clinical Immunology and Transplantation, Center of Laboratory Medicine, Medical University of Gdansk, Gdansk, Poland.
authors.forename: J
authors.initials: J
authors.lastname: Kulczycka
authors.affiliation: Cumming School of Medicine, University of Calgary, Calgary, Canada.
authors.forename: M J
authors.initials: MJ
authors.lastname: Fritzler
authors.affiliation: Department of Research, Inova Diagnostics, Inc., San Diego, USA mmahler@inovadx.com m.mahler.job@web.de.
authors.forename: M
authors.initials: M
authors.lastname: Mahler
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000276:immunology*; D000974:Antibodies, Antinuclear / Q000097:blood*; D016022:Case-Control Studies; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods*; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods*; D008180:Lupus Erythematosus, Systemic / Q000276:immunology*; D014157:Transcription Factors / Q000276:immunology*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000974:Antibodies, Antinuclear; C504649:PSIP1 protein, human; D014157:Transcription Factors
keywords: Antinuclear antibodies; DFS; LEDGF; autoantibodies; autoimmune disease