Two hundred and thirty six consecutive serum samples from RA patients, who fulfilled the 1987 American College of Rheumatology (ACR) classification criteria (17), were screened, and those positive for CCP2 were used in the subsequent analysis (n=180). Subjects with other autoimmune diseases (Sjogren's syndrome, n=11; systemic lupus erythematosus (SLE), n=8; chronic thyroiditis, n=4, mixed connective tissue disease (MCTD), n= 7; CREST syndrome, n=12) were also included for the analysis. To define the cutoff levels of the antibodies, 40 healthy control subjects were recruited. Serum was isolated and stored at −20°C until use. This study complies with the Declaration of Helsinki, and the study protocol was approved by the Regional Committee of Ethics for Human Research at the Faculty of Medicine of the Kyushu University (24-174). All subjects provided written informed consent before participating in the study.
Enzyme-linked immunosorbent assay(ELISA)
To detect antibodies specifically bound to CCP1, citrullinated fibrinogen (cFib), citrullinated a-enolase (cEno), and citrullinated vimentin (cVim) peptides, each pairs of citrullinated and control non-citrullinated peptides were used (Supplementary Table 1). All peptides were produced through amino acid synthesis and conjugated with biotin at the C-terminal. Ninety-six well avidin-coated plates (Avidin Plate, Sumitomo Bakelite Co., Ltd., Tokyo, Japan) were incubated with the peptides (2 mg/mL) overnight at 4°C, and after blocking non-specific binding by an incubation with Blocking One solution (Nacalai Tesque, Kyoto, Japan), the plates were incubated with the serum diluted at 1:30 by LowCross-Buffer (CANDOR Bioscience GmbH, Wangen, Germany), for 2 h at 37°C. Peroxidase-conjugated F(ab)2 goat anti-human IgG (Rockland Immunochemicals, Limerick, PA, USA) was used for the detection. Antibody binding was visualized using tetramethylbenzidine substrate solution (Interchim, Montluçon, France), and the absorbance was measured at 450 nm. The antibody level was expressed as DOD between citrullinated and native peptides. Sera with DOD higher than the mean plus 2SD of the healthy control sera were considered positive. To measure the levels of IgG against CCP2, we used a commercially available ELISA kit (Euro-Diagnostica, Malmö, Sweden). For the detection of anti-influenza, and anti-diphtheria toxin antibodies, 96-well plates were coated with 1 μg/ml of A/H1N1 subunit (Bio-Rad, Hercules, CA, USA) and diphtheria toxin (List Biological Laboratories, Campbell, CA, USA), respectively. We utilized precoated commercial kits to measure the avidity of anti-SS-A/Ro, anti-dsDNA, anti-centromere, and anti-U1-RNP antibodies (all from IBL INTERNATIONAL, Hamburg, Germany).
Measuring avidity of antibodies
Avidity of antibodies was determined by an elution assay using sodium thiocyanate (NaSCN) according to a previous report by Suwannalai et al. (16) with minor modifications. Serum was serially diluted (1:25, 1:50, 1:100, 1:200, 1:400) with Low cross buffer (CANDOR Bioscience GmbH) to define the dilution at which the antibody response was in the linear part of the curve. Sera with OD value of native peptide higher than 0.5 were excluded from the analysis in order to ensure data quality. Antigen-coated plates were incubated with the diluted serum for 2 h at 37°C. After washing, the plate wells were incubated with NaSCN at the concentration of 0, 0.125, 0.25, 0.5, 1.0, 2.0 or 4.0 M for 15 min at 37°C. The plates were washed, and remaining bound antibodies were detected using peroxidase-conjugated goat anti-human IgG. We calculated the avidity index (AI) at each concentration of NaSCN as follows:
(OD value of serum treated with given M of NaSCN – OD value of serum treated with 4M of NaSCN) / (OD value of serum treated with 0 M of NaSCN – OD value of serum treated with 4 M of NaSCN) × 100.
The strength of avidity is depicted as AIAUC, the area under the curve of AI at seven reference points, in order to minimize the bias in the avidity of antibodies with different concentrations (dilutions). AIAUC was calculated according to Perciani et al. with minor modifications (Supplementary Figure 1) (18).
The extent of cross-reactivity between different ACPAs was examined by a peptide inhibition assay as reported previously with minor modification (7). In brief, serum in appropriate dilution at which the antibody response was in the linear part of the curve as defined above, were incubated with 100 mg/ml of competitor peptide (non-biotinylated peptide) overnight at 4°C with continuous mixing. Then the serum samples were incubated in the plates precoated with the biotinylated test peptides, and subsequent ELISA steps were performed as described above. Only sera with OD value lower than 0.5 for the native peptides and those with DOD value larger than 0.2 were analyzed. The inhibition rate of antibody binding was calculated as follows:
(OD value of untreated serum – OD value of serum pretreated with competitor peptide / OD value of untreated serum) × 100.
All statistical analyses were performed with EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan) (19), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria). Difference between groups was analyzed by Steel-Dwass test, and correlation was determined by Spearman’s correlation coefficient. P values less than 0.05 were considered significant.