Diagnostic value and clinical laboratory associations of antibodies against recombinant ribosomal P0, P1 and P2 proteins and their native heterocomplex in a Caucasian cohort with systemic lupus erythematosus

Altogether 479 serum samples were obtained from the following groups: (1) patients with SLE (n = 163), who fulfilled the American College of Rheumatology (ACR) 1982 revised criteria for the classification of SLE [4], (2) patients with systemic sclerosis (SSc, n = 66) who met the ACR 1980 criteria for scleroderma [33], (3) patients with primary Sjögren's syndrome (pSS, n = 54) who fulfilled the preliminary European League Against Rheumatism criteria of Vitali et al. [34], (4) patients with rheumatoid arthritis (RA, n = 90) who met the ACR 1987 revised criteria for the classification of rheumatoid arthritis [35] and (5) healthy donors (HD, n = 100).

Disease activity of SLE patients was defined based on the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI 2000) [36‐38] in 101 patients: 6 of them had no activity (SLEDAI score 0), 35 were mildly active (0 < SLEDAI ≤ 5), 41 had moderate disease activity (5 < SLEDAI ≤ 10), 14 were highly active (10 < SLEDAI ≤ 20), and 5 had very high activity (SLEDAI > 20). Juvenile onset was diagnosed when the age at diagnosis was 18 years or younger according to the Pediatric Rheumatology International Trials Organization [39]. Twenty-four (14.7%) patients with juvenile onset SLE and 139 (85.3%) patients with adult onset SLE were studied. Disease damage was measured according to the criteria of the Systemic Lupus International Collaborative Clinics (SLICC) [40, 41] and the weighted damage score (WDS) [40]. All patients were recruited from the outpatient and inpatient facilities of the Department of Rheumatology and Clinical Immunology, Charité University Hospital, Berlin, Germany. The Ethics Committee of the Medical Faculty of Charité University Hospital approved the study, and written informed consent was obtained from all subjects. Sera from healthy donors were used in cooperation with University of Lübeck, Germany. Written informed consent was obtained from all healthy subjects.

Microtiter plates (Nunc, Roskilde, Denmark) were coated with 1 μg/ml full-length recombinant ribosomal protein P0, P1 or P2 expressed in insect cells (DIARECT, Freiburg, Germany). Sera diluted 1:201 in phosphate-buffered saline (PBS) and 0.1% (wt/vol) casein were added and allowed to react for 30 minutes, followed by three washing cycles with PBS 0.05% (vol/vol) and Tween 20. For detection of bound antibodies, the plates were incubated with antihuman immunoglobulin (IgG) peroxidase conjugate (EUROIMMUN, Lübeck, Germany) for 30 minutes, washed three times and allowed to react with tetramethylbenzidine (EUROIMMUN) for 15 minutes. After addition of acidic stopping solution (EUROIMMUN), the optical density (OD) was read at 450 nm using an automated spectrophotometer (Spectra Mini; Tecan, Crailsheim, Germany). All steps were performed at room temperature. A highly positive index patient serum was used to generate a standard curve consisting of three calibrators (2, 20 and 200 relative units (RU)/ml). Relative units per milliliter were calculated for all samples using this three-point standard curve. The analytical reproducibility of all aRibP assays was evaluated by repeated testing of two serum samples (10 determinations each) in the same run, giving intraassay coefficients of variation (CV) of 2.4% (aRibP_R0), 2.1% (aRibP_R1) and 2.7% (aRibP_R2), respectively. Relationships between sensitivity and specificity at different cutoff values were examined for all assays by receiver-operating characteristics (ROC) curve analyses, allowing also for the determination of test characteristics at predefined specificities.

The anti-RibP_NH enzyme-linked immunosorbent assay (ELISA) (IgG, CV 2.6%), anti-Sm ELISA, anti-dsDNA radioimmunoassay (RIA) (Farr assay) and anti-dsDNA ELISA are commercially available assays from EUROIMMIUN and were performed following the manufacturer's instructions.

Statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software, La Jolla, CA, USA). The diagnostic significance of antiribosomal proteins N, P0, P1 and P2 antibodies was assessed and areas under the curve (AUCs) were created using ROC analysis. To determine associations, the Mann-Whitney U test (for comparing medians between groups; MWT), Fisher's exact test (FET) and Spearman's rank test (SRT) were used. Two-tailed t-tests were used throughout with an α set at 0.05.

Antibodies against ribosomal P_NH, P_R0, P_R1 and P_R2 proteins (Figure 1), Sm and dsDNA (ELISA and Farr assays) were measured in sera from 163 SLE patients, 210 disease controls and 100 healthy donors to define and compare the sensitivity and specificity in ROC curve analysis (Table 1). For aRibP_NH, a sensitivity of 5.5% and a specificity of 100% were calculated using the manufacturer's cutoff (20 RU/ml). At a predefined specificity of 98% among 210 patients with other rheumatic diseases (SSc, pSS and RA), only five (2.4%), four (1.9%), four (1.9%) and four (1.9%) had elevated aRibP_NH, aRibP_R0, aRibP_R1 and aRibP_R2 titers, respectively. At the same specificity among 100 healthy donors, only zero (0%), one (1.0%), two (2.0%) and two (2.0%) patients had high titers of aRibP_NH, aRibP_R0, aRibP_R1 and aRibP_R2. Among antiribosomal P protein antibodies, aRibP_R0 had the highest performance with regard to criteria such as AUC and maximum sum of sensitivity and specificity, followed by aRibP_NH (Table 1). All test criteria of aRibP_R0 were inferior to those of the anti-dsDNA ELISA or the Farr assay, but were almost equal to those of the anti-Sm ELISA.

Although the native heterocomplex of ribosomal P contains all immunological domains of the subunits P0, P1 and P2, there were considerable differences in the cutoffs and in sensitivities for the detection of aRibP_NH, aRibP_R0, aRibP_R1 and aRibP_R2 (Table 1), with outstanding results for aRibP_R0.

Thus, we further investigated whether there were patients negative for aRibP_NH but positive for aRibP_R0, aRibP_R1 or aRibP_R2 (Figure 2). Sera fulfilling these criteria would point out that there are some epitopes of ribosomal P proteins that are not accessible to autoantibodies because of the spatial conformation of the native heterocomplex.

At 99% specificity, among 141 aRibP_NH^- patients there were 19 (13.5%) positive for aRibP_R0, six (4.3%) positive for aRibP_R1 and 11 (7.8%) positive for aRibP_R2. Some of those sera were further exclusively positive for one of the recombinant aRibPs and showed an increased titer up to twofold of the corresponding cutoff (Figure 2b).

To investigate the auxiliary diagnostic value of antiribosomal P protein antibodies in SLE, we searched for patients who were negative for antibodies against dsDNA and Sm, but positive for aRibP_NH, aRibP_R0, aRibP_R1 or aRibP_R2 at a specificity of 100% (Figure 3). This analysis was performed twice, taking either the results of the anti-dsDNA ELISA (Figure 3a) or those of the Farr assay (Figure 3b).

Among 163 SLE patients, there were 11 (6.7%) individuals who could be diagnosed only by detection of aRibPs, while 63 (38.7%) patients were regularly diagnosed by the presence of anti-dsDNA or anti-Sm antibodies. Considering the excellent Farr assay, these relations adjusted to 89 (54.6%) individuals with regular diagnosis and five (3.1%) individuals with additional diagnosis only by the presence of aRibP.

To determine the special characteristics of lupus patients with elevated aRibPs, we compared medical records, including ACR criteria, SLEDAI 2000 items and laboratory parameters, including autoantibodies, immunosuppressants and antimalarials, with those of aRibP^- lupus patients. All clinical laboratory results and detailed demographic information about the study cohort are shown in Table 2.

ARibP_NH⁺ patients fulfilled significantly more ACR criteria and more often had photosensitivity. Moreover, the frequency of patients with decreased C3 levels was higher among aRibP_NH⁺ patients. Lymphocytopenia was associated with the presence of aRibP_R0, and a higher γ-glutamyl transpeptidase (GGT) level was found in aRibP_R1⁺ patients. Anti-Sm, anti-dsDNA and anti-U₁-ribonucleoprotein (anti-U₁-RNP) antibodies were much more frequent in all aRibP⁺ patients.

To study the prognostic role of ribosomal P protein antibodies, SLICC scores and WDS were assessed in aRibP⁺ patients and in an age-, sex- and nephritis-matched group of aRibP^- patients at the time of blood sampling and 3 years later. Changes in damage scores (ΔSLICC, ΔWDS) were calculated, and both groups were separately compared. Damage scores from 41 of all 58 aRibP⁺ patients were completely assessable at the time of blood sampling and 3 years later. There were 22 aRibP_NH⁺, 27 aRibP_R0⁺, 18 aRibP_R1⁺ and 23 aRibP_R2⁺ patients. SLICC and WDS correlated significantly with disease duration and the ages of patients, but not with ACR scores or with anti-dsDNA, anti-Sm or any antiribosomal P protein antibodies. Total disease damage and damage to every organ system separately was not significantly higher in aRibP⁺ patients than in their aRibP^- counterparts within these 3 years. Thus, we found no prognostic role for aRibP.