title: Multiplex cytokine analyses in patients with rheumatoid arthritis require use of agents blocking heterophilic antibody activity.
abstract: OBJECTIVE
  Heterophilic antibodies, such as rheumatoid factor (RF), are known to interfere with enzyme-linked immunosorbent assays (ELISAs). Treatment of rheumatoid arthritis (RA) with tumour necrosis factor (TNF)-α blockers is well established. The aims of this study were to develop a protocol for blocking the interaction of present heterophilic antibodies and to validate this procedure by evaluating the effect on correlations of cytokine levels to clinical response in RA patients treated with adalimumab.
  
  
  METHODS
  Fourteen patients with active RA were evaluated at baseline and 3 months after starting adalimumab treatment. Cytokines were analysed with a commercial 12-plex bead ELISA. To block interference by RF, a commercial blocker (HeteroBlock) was used. To determine the optimal concentration of HeteroBlock, patient sera were analysed with different concentrations of HeteroBlock. Subsequently, baseline and follow-up sera from the 14 patients were analysed and correlated with clinical outcome.
  
  
  RESULTS
  Measured cytokine levels were reduced in the majority of samples when adding the blocker. The optimal concentration of HeteroBlock was 1600 μg/mL of serum. Sera with high RF levels were more prone to produce false positive values, although some RF-negative sera also demonstrated evidence of interference. HeteroBlock did not interfere with the analysis. In RA patients treated with adalimumab, changes in interleukin (IL)-6 levels between baseline and follow-up correlated with changes in erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) in sera with added HeteroBlock.
  
  
  CONCLUSIONS
  When analysing sera from patients with RA with multiplex bead ELISA, the assay should be evaluated for interference by heterophilic antibodies, and if present corrected with, for example, HeteroBlock.
authors.affiliation: a Department of Clinical Sciences, Section of Rheumatology , Lund University , Malmö , Sweden.
authors.forename: P
authors.initials: P
authors.lastname: Olsson
authors.affiliation: a Department of Clinical Sciences, Section of Rheumatology , Lund University , Malmö , Sweden.
authors.forename: E
authors.initials: E
authors.lastname: Theander
authors.affiliation: a Department of Clinical Sciences, Section of Rheumatology , Lund University , Malmö , Sweden.
authors.forename: U
authors.initials: U
authors.lastname: Bergström
authors.affiliation: b Fred and Lena Meijer Heart and Vascular Institute, Spectrum Health , Grand Rapids , MI , USA.|c Van Andel Institute , Grand Rapids , MI , USA.|d Lund Stem Cell Centre , Lund University BMC , Lund , Sweden.
authors.forename: S
authors.initials: S
authors.lastname: Jovinge
authors.affiliation: a Department of Clinical Sciences, Section of Rheumatology , Lund University , Malmö , Sweden.|e Department of Rheumatology and Inflammation Research , Sahlgrenska Academy, Institute of Medicine, University of Gothenburg , Gothenburg , Sweden.
authors.forename: Lth
authors.initials: L
authors.lastname: Jacobsson
authors.affiliation: a Department of Clinical Sciences, Section of Rheumatology , Lund University , Malmö , Sweden.
authors.forename: C
authors.initials: C
authors.lastname: Turesson
mesh_terms: D000068879:Adalimumab / Q000627:therapeutic use; D000368:Aged; D018501:Antirheumatic Agents / Q000627:therapeutic use; D001172:Arthritis, Rheumatoid / Q000097:blood* / Q000188:drug therapy / Q000276:immunology*; D016207:Cytokines / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D017063:Outcome Assessment, Health Care / Q000379:methods; D012217:Rheumatoid Factor
chemical_list: D018501:Antirheumatic Agents; D016207:Cytokines; D012217:Rheumatoid Factor; D000068879:Adalimumab