Informed consent was obtained from all patients and controls. The study protocol was approved by the Ethics Committee of Marseille, France (DC2008-327). The characteristics of patients and controls are shown in Table 1. ACPA were detected using the anti-CCP2 kit from Eurodiagnostica (Malmö, Sweden) for all RA patients and controls. Rheumatoid factor (RF) was detected by ELISA for all RA patients using the Orgentec Kit (Mainz, Germany).

We analyzed the sera of 39 RA patients from the rheumatology unit at Sainte Marguerite Hospital in Marseille. Twenty RA patients had disease duration less than one year and 19 more than five years (time elapsed since first diagnosis by a physician). All RA patients fulfilled the 2010 American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) revised criteria 2. Controls were seven patients with AS, two with SLE and four with SSc, from the rheumatology unit at Sainte Marguerite Hospital in Marseille. Ten healthy controls were recruited among laboratory staff volunteers.

We used Invitrogen (Carlsbad, California, USA) ProtoArrays that contain 8,268 human proteins. All proteins have been expressed as glutathione-S-transferase (GST) fusion proteins, purified under native conditions and spotted on nitrocellulose-coated glass slides 3. Slides were blocked with 1% BSA/phosphate-buffered saline/Tween (PBST). Sera were added to the arrays. After washing, anti-human immunoglobulin G (IgG) conjugated to Alexa Fluor 647 dye was added. Arrays were washed and dried (Partnership, Evry, France). Arrays were scanned with a GenePix 4000B Fluorescent Scanner, GenePix.Molecular Devices, Sunnyvale, California, USA. Data were acquired with GenePix Pro software and processed using ProtoArray Prospector 2.0 (Invitrogen). A panel of values was calculated for each protein array, including the Z-score, the Chebyshev inequality precision (CIP) value and the coefficient of variation (CV) value as previously described 3. A Z-score >3.0, a CIP value <0.05 and a CV <0.5 define a positive spot.

RA patients were chosen from the Rheumatology Ward at Hospital Sainte Marguerite, Marseille and from the Rheumatology Ward at Hospital Jean Minjoz, Besançon. AS patients and PsA patients were chosen from the Rheumatology Ward at Hospital Sainte Marguerite, Marseille. SLE patients were chosen from the Rheumatology Ward at Hospital Sainte Marguerite, Marseille and from the Internal medicine Ward at Hospital Bretonneau, Tours. Patients with SSc were from Hospitals Saint Antoine, Saint Louis, Paris; Hospital Claude Huriez, Lille and Hospital Sainte Marguerite, Marseille. Volunteers from the laboratory staff and the Marseille Blood Transfusion Center staff served as normal controls.

Plates were coated with purified proteins and blocked with PBS containing 5% milk. Sera diluted to 1:100 in PBS were incubated for 3 h. After washing with 0.1% Tween 20, peroxydase-conjugated anti-human IgG (Sigma, Saint-Quentin Fallavier, France) was added. Optical density (OD) was read at 405 nm. Background OD was obtained by adding each serum to a well without protein. Positive sera were defined by an OD value more than twice the background OD 3. Moreover, positive sera were defined by an OD value more than twice the mean OD of the control groups (AS, PsA, SLE, SSC and healthy subjects). Data were similar using both methods. P-values were calculated using the chi squared test.

15-mer peptides encompassing residues from WIBG (locus NM_032345.1) GABARAPL2 (locus NM_007285.5) and ZNF706 (locus NM_016096.1) overlapping on seven amino acids were synthesized using the solid-phase system, and purified (Neosystem, Strasbourg, France).

Plates were coated overnight with 10 μg/well peptides diluted in PBS, pH 7.4. Plates were blocked with 5% milk PBS. Sera, diluted to 1:100 in PBS, were incubated for 2 h. After washing with 0.1% Tween 20, peroxydase-conjugated anti-human IgG, (Sigma, France) was added. OD was read at 405 nm. Background OD was obtained by adding each serum to a well without peptide. Positive sera were defined by an OD value more than twice the background OD. P-values were calculated using the chi squared test.

We selected sera from 20 RA patients with disease duration less than 1 year and compared their reactivity pattern on protein arrays with that of sera from 19 RA patients with disease duration more than 5 years, and with sera from 23 controls. The control group included seven AS patients, two SLE patients, four SSc patients and ten healthy subjects. Autoantibodies were detected by anti-human IgG antibody.

The sera of RA patients with disease duration more than 5 years bound, on average, 101 proteins. AS sera bound 112 proteins, SLE sera bound 91 proteins, SSc sera bound 103 proteins and healthy controls' sera bound 89 proteins (data not shown). Sera from patients with early RA bound, on average, 58 proteins. Among these proteins, we identified 25 that were recognized by 30% to 60% of RA patients with disease duration less than 1 year and less than 10% of controls (Table 2). These proteins were recognized by 0% to 37% of RA patients with disease duration more than 5 years.

To confirm the validity of protein array detection, we developed ELISAs using the 25 purified proteins. We tested sera from 124 RA patients with disease duration less than 1 year, 40 RA patients with disease duration more than 5 years, 81 AS patients, 30 PsA patients, 19 SLE patients, 16 SSC patients and 40 healthy subjects. Four proteins were significantly recognized by early RA patients and less than 10% of controls (Figure 1A,B). These proteins are WIBG, GABARAPL2, ZNF706 and PAD4 (peptidyl arginine deiminase 4).

Autoantibodies to GABARAPL2, ZNF706 and PAD4 were less sensitive and specific for early RA than autoantibodies to WIBG. Indeed, autoantibodies to PAD4 identified 38% of RA patients with disease duration more than 5 years. Moreover, autoantibodies to GABARAPL2, ZNF706 and PAD4 were also found in SLE and SSC patients. Autoantibodies to WIBG were strongly associated with early RA. Indeed, 33% of RA patients' sera recognized WIBG versus 5% of RA patients with disease duration more than 5 years (P = 0.0004), 0% of AS patients (P <10^-7), 0% of PsA patients (P = 0.0002), 10% of SLE patients (P = 0.05), 6% of SSc patients (P = 0.03) and 0% of healthy individuals (P = 0.00003).

In combination, WIBG, GABARAPL2 and ZNF706 proteins identified 48% of patients with early RA versus 10% of RA patients with disease duration more than 5 years and 6% of controls (Table 3). Among patients with early AS, PsA, SLE and SSC with disease duration less than one year, only 2/34 were positive for GABARAPL2 and WIBG (data not shown). Of interest, the combination of WIBG, GABARAPL2 and ZNF706 proteins identified 43% of early RA patients negative for anti-CCP, 44% of early RA patients negative for RF and 39% of early RA patients negative for anti-CCP and RF.

To identify B cell epitopes on ZNF706, GABARAPL2 and WIBG, we synthesized overlapping 15-mer peptides encompassing the entire sequence of these proteins. As a first step, we screened these peptides with the sera of 15 RA patients known to contain autoantibodies to ZNF706, GABARAPL2 and WIBG and 20 controls. Among the ten peptides from ZNF706, eight were recognized by at least one of the tested sera (Figure 2). One peptide, P1, was specifically recognized by sera from RA patients. Indeed, 8/15 RA sera recognized P1 versus 0/20 controls (Figure 2A).

Among the 15 peptides from GABARAPL2, 8 were recognized by at least one of the tested sera (Figure 2B). One peptide, P15, was specifically recognized by sera from RA patients. Indeed, 7/15 RA sera recognized P15 versus 0/20 controls.

Among the 26 peptides from WIBG, 18 were recognized by at least one of the tested sera (Figure 2C). One peptide, P22, was preferentially recognized by sera from RA patients. Indeed, 7/15 RA sera recognized P22 versus 4/20 controls.

To confirm these reactivities, we tested by ELISA sera from 60 patients with early RA and 106 controls (20 AS, 24 PsA and 22 SLE patients, and 40 healthy subjects) on ZNF706 P1, GABARAPL2 P15 and WIBG P22 (Table 4).

Autoantibodies to ZNF706 P1, GABARAPL2 P15 and WIBG P22 were respectively found in 35%, 37% and 30% of patients with early RA, with a specificity ranging from 95% to 100%. In combination, these three peptides identified 45% of patients with early RA versus 8% of controls (P <10^-7 for 60 patients with early RA versus 106 controls). Of interest, the combination of these peptides identified 41% of early RA patients negative for anti-CCP, 35% of early RA patients negative for RF and 42% of early RA patients negative for anti CCP and RF.