Erroneous augmentation of multiplex assay measurements in patients with rheumatoid arthritis due to heterophilic binding by serum rheumatoid factor

Arthritis Rheum. 2011 Apr;63(4):894-903. doi: 10.1002/art.30213.


Objective: Serum rheumatoid factor (RF) and other heterophilic antibodies potentially interfere with antibody-based immunoassays by nonspecifically binding detection reagents. The purpose of this study was to assess whether these factors confound multiplex-based immunoassays, which are used with increasing frequency to measure cytokine and chemokine analytes in patients with rheumatoid arthritis (RA).

Methods: We performed multiplex immunoassays using different platforms to measure analyte concentrations in RA patient samples. Samples were depleted of RF by column-based affinity absorption or were exposed to agents that block heterophilic binding activity.

Results: In RA patients with high-titer RF, 69% of analytes demonstrated at least a 2-fold stronger multiplex signal in non-RF-depleted samples as compared to RF-depleted samples. This degree of erroneous signal amplification was less frequent in low-titer RF samples (17% of analytes; P < 0.0000001). Signal amplification by heterophilic antibodies was blocked effectively by HeteroBlock (≥ 150 μg/ml). In 35 RA patients, multiplex signals for 14 of 22 analytes were amplified erroneously in unblocked samples as compared to blocked samples (some >100-fold), but only in patients with high-titer RF (P < 0.002). Two other blocking agents, heterophilic blocking reagent and immunoglobulin-inhibiting reagent, also blocked heterophilic activity.

Conclusion: All multiplex protein detection platforms we tested exhibited significant confounding by RF or other heterophilic antibodies. These findings have broad-reaching implications in the acquisition and interpretation of data derived from multiplex immunoassay testing of RA patient serum and possibly also in other conditions in which RF or other heterophilic antibodies may be present. Several available blocking agents effectively suppressed this erroneous signal amplification in the multiplex platforms tested.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Heterophile / blood*
  • Arthritis, Rheumatoid / blood
  • Arthritis, Rheumatoid / diagnosis*
  • Arthritis, Rheumatoid / immunology*
  • Biomarkers / blood
  • Chemokines / blood
  • Cytokines / blood
  • Diagnostic Errors
  • Humans
  • Immunoassay / methods*
  • Protein Binding
  • Rheumatoid Factor / blood*


  • Antibodies, Heterophile
  • Biomarkers
  • Chemokines
  • Cytokines
  • Rheumatoid Factor