This study compared the performance of the BioPlex® 2200 anti-CCP kit, a multiplex flow immunoassay, to two ELISA assays for detecting antibodies against cyclic citrullinated peptide (anti-CCP antibodies) in rheumatoid arthritis (RA) patients and controls. The diagnostic performance of the BioPlex® kit was found to be comparable to the ELISA assays in sensitivity, specificity, and area under the curve. The BioPlex® kit also demonstrated advantages over traditional ELISA, including precision, simplicity, reliability, faster turnaround time, and higher throughput. Anti-CCP antibody levels were significantly higher in RA patients compared to controls, and the BioPlex® kit was better than rheumatoid factor

1. Introduction:
Rheumatoid arthritis (RA) is the most commonly occurring form of inflammatory polyarthritis. The diagnosis of RA is primarily based on clinical, radiological and
immunological features. Current laboratory-based serological tests measure factors such as rheumatoid factor (RF) and anti-cyclic citrullinated peptide (CCP)
antibodies. This study compares the performance of the BioPlex® 2200 anti-CCP kit (Bio-Rad lab) a multiplex flow immunoassay intended for the detection of IgG
antibodies CCP to two commercially available second-generation ELISA (anti-CCP2) and defines their diagnostic efficacy on RA.
Results:
• Anti-CCP antibody concentrations (log scale) according to the four categories (BBD, OAD, NAD, and RA) for each assay are shown in Figure 1; using the
cutoff values proposed by the manufacturers . Analysis of three different assays showed that serum anti-CCP antibody concentrations were significantly higher in
RA than non-RA patients (p<0.05).
• Agreement rate between BioPlex® 2200 vs. Aeskulisa® was 97.4 %, BioPlex® 2200 vs. Kallestad™ 98.0% and Aeskulisa® vs. KallestadTM 96.6%; the kappa
coefficients of each assay were 0.95, 0.98, and 0.93 respectively.
• We compared AUCs of all three methods and did not found significant differences (BioPlex®2200 AUC vs. Aeskulisa® AUC p= 0.4351) (BioPlex®2200 AUC
vs. KallestadTM AUC p= 0.4420), (Aeskulisa® AUC vs. KallestadTM AUC p= 1) Figure 2.
• Spearman´s coefficient (rho) BioPlex®2200 vs. Aeskulisa® 0.874, BioPlex® 2200 vs. KallestadTM 0.887, and BioPlex® 2200 vs. RF IMMAGE® 800 0.752 all
with significance level p<0.0001. Rheumatoid factor was positive in 80/114 (70.2%) and C-reactive protein (CRP) was 36(31.6 %) patients with RA .
• Antinuclear antibodies screen was positive in 29 (25.5%) patients with RA and 21 (18%) by IIF.
Conclusions:
•The diagnostic performance of BioPlex® 2200 anti-CCP2 kit was comparable to two ELISA assays evaluated in sensitivity, specificity, PPV, NPV and AUCs.
•It was better than traditional biomarker RF for laboratory diagnosis of RA.
•Additionally BioPlex® anti-CCP2 kit offers advantages over traditional ELISA such as precision, technical simplicity, reliability, short turnaround time and high-
speed throughput.
Methodology:
Serum anti-CCP antibodies from 232 subjects (114 RA and 118 non-RA: 48 non-RA autoimmune diseases, 50 other disease and 20 healthy volunteers) were
simultaneously determined by BioPlex® 2200 Anti-CCP kit, AESKULISA® CCP kit and KallestadTM anti-CCP II ELISA kit (Bio-Rad lab). Rheumatoid Factor
(RF) and C-reactive protein (CRP) were determined by nephelometry IMMAGE® 800 (Beckman-Coulter, Fullerton, CA, USA). Antinuclear antibodies (ANA) by
BioPlexTM2200 ANA screen kit and IIF on Hep-2 cells.
Authors M. Talise, M. Sevilla, E. Sanchez-Zapardiel, M.C. Caballero, A. Delgado, D. Valero-Hervas, S. Lermo, A. Martinez, M.J. Rey, P. Varela, E. Paz-Artal, A. Serrano
Immunology department, University Hospital 12 de Octubre, Madrid, Spain
Evaluation of multiplexed fluorescent microsphere immunoassay for
detection of anti-cyclic citrullinated peptide antibodies in comparison with
commercially available ELISA assays in Rheumatoid Arthritis
Fig.1. Anti-CCP2 antibody (Ab) concentrations in log scale according to the four categories patients for each
assay. BBD= blood bank donors (n=20), OAD = other autoimmune diseases (n=48), NAD= non autoimmune
diseases (n=50), RA= Rheumatoid arthritis (n=114). Fig. 2. ROC curves of BioPlex®2200, KallestadTM and Aeskulisa® anti-CCP2.
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