Multiplexing immunoassays for cytokine detection in the serum of patients with rheumatoid arthritis: lack of sensitivity and interference by rheumatoid factor.
OBJECTIVE
Accurately measuring cytokines in clinical material remains an important challenge in the development of biomarkers. Enzyme-linked immunoabsorbent assays (ELISAs) are considered 'gold standard'; however, their use is limited by the relatively large sample volume required for multiple analyte testing. Several alternatives (including membrane or bead-ELISA) have been developed particularly to enable multiplexing. Concerns were raised regarding their use in rheumatology due to interference by heterophilic antibodies, notably rheumatoid factor (RF). In this report, we compared several multiplex assays using serum from rheumatoid arthritis (RA) patients with respect to the presence of residual RF following attempted removal employing commonly used procedures.


METHODS
Healthy control and RF-positive/negative RA sera were used to compare 4 multiplex assays with ELISA: bead-based 'Luminex' immunoassay, cytometric bead assays (CBAs), membrane-based and Mosaic™ ELISAs. Sera were tested following Ig blockade (mixed species serum) or removal (using PEG6000 or sepharose-L).


RESULTS
Ig removal was only partially efficient and residual RF was detected in most sera. RF had no impact on cytokine measurement by ELISA. In single and multiplex Luminex, cytokine levels associated with false positive results correlated directly with RF titres. Following Ig-blockade/removal, these relationship remained suggesting false positivity was still associated with the presence of residual RF. Conversely, detection of cytokines in multiplex membrane-based or Mosaic- ELISA were not affected by the presence of RF; however, levels of cytokines readily detected by ELISA were often below the detection threshold of these assays. CBA assays were also low on sensitivity but unaffected by RF.


CONCLUSIONS
False positivity, due to the presence of heterophilic antibodies, mainly affected Luminex assays. Other assays however remained limited in their sensitivity. Multiplexing of cytokine measurement remains a challenge, particularly in rheumatological pathologies, until assays of adequate sensitivity are developed. ELISA remains the gold standard.
Leeds Institute of Molecular Medicine, University of Leeds, Leeds, UK. s.m.churchman@leeds.ac.uk
Sarah M
SM
Churchman
Janina
J
Geiler
Rekha
R
Parmar
Elizabeth A
EA
Horner
Leigh D
LD
Church
Paul
P
Emery
Maya H
MH
Buch
Michael F
MF
McDermott
Frederique
F
Ponchel
D001172:Arthritis, Rheumatoid / Q000097:blood / Q000175:diagnosis* / Q000276:immunology; D015415:Biomarkers / Q000097:blood; D016207:Cytokines / Q000097:blood* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards*; D005189:False Positive Reactions; D006801:Humans; D008863:Microspheres; D012015:Reference Standards; D015203:Reproducibility of Results; D012217:Rheumatoid Factor / Q000097:blood* / Q000276:immunology; D012680:Sensitivity and Specificity
D015415:Biomarkers; D016207:Cytokines; D012217:Rheumatoid Factor