Study subjects and samples
One hundred ninety-six patients with established RA from the County of Dalarna, Sweden, were included in the cross-sectional, observational, “Secretory antibodies in Rheumatoid Arthritis” (SARA) study with enrolment 2012–2013. RA patients were randomly selected among planned follow-up visits at the Rheumatology Clinic, Falun Hospital, Sweden. Healthy blood donors (n = 101) were recruited as controls from the local blood donor center and referred to the Rheumatology Clinic for blood and saliva sampling. The ethics review board in Uppsala, Sweden, approved of the study, and all participants signed a written informed consent (Uppsala: 211/159). Participants were required to provide at least 0.5 mL of the saliva during the 10-min sampling time; otherwise, they were excluded from the study.
Paired saliva and serum samples were collected at the same visit at the Rheumatology Clinic. Participants were asked to restrain from eating, drinking other liquids than water, brushing teeth, or smoking 1 h before saliva sampling. Passive secretion was used for saliva sampling, i.e., the study participant leaned forward and drooled 10 min into a test tube placed on ice. After the disruption of mucus fibers by pipetting a few times, the saliva samples were centrifuged for 5 min at 5000g. Serum samples were centrifuged 5 min at 5000g. Serum and saliva samples were stored at − 80 °C until further analyses.
Antibody analyses
Commercially available serum IgG-class anti-cyclic citrullinated peptide (anti-CCP) enzyme-linked immunoassays (ELISA) tests (CCPlus® Immunoscan, Euro Diagnostica AB, Malmö, Sweden) were modified to analyze IgA, IgA1, and IgA2 ACPA in the saliva and serum, as well as for serum SC and IgG ACPA. All samples were analyzed in duplicate, and reanalysis was carried out if the coefficient of variation between the duplicate samples was > 20%. The cutoff levels for a positive test of saliva ACPA (IgA, IgA1, and IgA2) were set at the 99th percentile among saliva samples from blood donors, and cutoff levels for serum ACPA (IgA, IgA1, IgA2, and SC) were set at the 99th percentile in serum among blood donors.
Antibody analyses in the saliva
The saliva was thawed at room temperature and centrifuged (15,000g at 4 °C for 10 min) to remove non-soluble material. The supernatant was diluted to a final concentration of 1:20. The secondary antibody in the salivary IgA ACPA assay (Polyclonal Rabbit Anti-Human IgA/HRP, DakoCytomation, Glostrup-Denmark) was diluted 1:200. Secondary antibodies for the subclass analyses (IgA1 antibody No. ABIN135642 and for IgA2 ABIN135642, Antibodies online, Aachen, Germany) were diluted 1:300. Incubation and washing were carried out according to the manufacturer’s instructions. Absorbance was read at 450 nm (Multiscan RC MOD 351, Labsystems, Helsinki, Finland).
To adjust for non-specific IgA adsorption, all samples were tested against a negative control peptide (cyclic arginine peptide, CAP, Euro Diagnostica AB). Anti-CCP and anti-CAP analyses were performed in parallel. Anti-CAP background levels were subtracted from anti-CCP values (delta optical density (OD)-measurements for anti-CCP and anti-CAP), and the cutoff values for a positive test were set to 0.78 for IgA ACPA, 0.70 for IgA1 ACPA, and 0.39 for IgA2 ACPA, corresponding to the 99th percentile of the 101 healthy blood donors.
Total IgA in the saliva was analyzed with enzyme immunoassay tests from the IBL International (IgA Saliva ELISA, IBL International, Hamburg, Germany).
Antibody analyses in the serum
Serum samples were analyzed for IgG ACPA according to the manufacturer’s instruction (Euro Diagnostica AB), with cutoff set at 25 U/mL. Serum IgA and SC ACPA analyses were performed as previously described [5, 18]. In brief, for SC ACPA analyses, serum was diluted 1:25 and for IgA ACPA 1:100. As secondary antibodies, a polyclonal goat IgG anti-human secretory component (GAHU/SC/PO, NordicBiosite, Täby, Sweden) diluted 1:2000 in kit buffer was used in the SC ACPA assay (this secondary antibody does not differentiate between secretory IgA or secretory IgM, as it binds to the secretory component), and for the IgA ACPA assay, a polyclonal anti-human antibody diluted 1:2000 was used (Polyclonal Rabbit Anti-Human IgA/HRP, DakoCytomation, Glostrup, Denmark). High-leveled serum samples of IgA and SC ACPA were used to create standard curves. Absorbance was read at 450 nm (Multiscan RC MOD 351, Labsystems). Cutoff levels for positive serum tests of IgA ACPA were set to 25 arbitrary units (AU)/mL [5] and corresponding to the 99th percentile of the 101 healthy blood donors cutoff for SC ACPA was set to 72 AU/mL.
For IgA1 and IgA2 ACPA analyses, serum was diluted 1:50 and the subclasses were detected using anti-IgA1 and anti-IgA2 polyclonal mouse IgG anti-human Fc-region-specific antibodies, conjugated with HRP (IgA1/IgA2 Antibody, No.ABIN135642/ABIN135642, Antibodies online, Aachen, Germany). Secondary antibodies were diluted 1:500 in the IgA1 assay and 1:400 in the IgA2 assay. Seven-step serial dilutions of high-leveled serum samples were used as a calibrator, and the results were expressed as AU/mL. Incubation and washing were carried out as instructed by the manufacturer. Absorbance was read at 450 nm (Multiscan RC MOD 351, Labsystems). Cutoff limits for IgA1 ACPA were set to 27 AU/mL and for IgA2 ACPA 179 AU/mL.
Total IgA in serum was analyzed with a PEG-enhanced immunoturbidimetric method, using Siemens Atellica CH930 (Atellica CH Analyzer, Siemens Healthcare Diagnostics Inc. Tarrytown, NY, USA).
Disease characteristics and risk factors
At inclusion, the participants answered a questionnaire regarding smoking habits [24]. At the time of sampling, the functional ability of patients was assessed using the Swedish version of the health assessment questionnaire (HAQ) [25], and disease activity was assessed using the 28-joint disease activity score (DAS28) [26]. RF status refers to the time point of RA diagnosis and was retrospectively obtained from medical records.
The prevalence of radiographic erosions was assessed from written reports by experienced radiologists in routine clinical care between 1982 and 2013. Results were dichotomized into having erosions or not.
Genetic analyses
Genotyping of HLA-DRB1 was performed by Sanger sequencing (BGI Clinical Laboratories, Shenzhen, China). Shared epitope (SE) was defined as HLA-DRB1*01, *0401, *0404, *0405, *0408, *0409, *0410, *0413, *0416, *0419, *0421, or *10.
Statistics
Statistical analyses were performed using SPSS v. 26.0 (SPSS, Chicago, USA), and two-tailed p values < 0.05 were considered significant. Antibody test results below the reference curve were given a value corresponding to half of the lowest detected value among patients and controls. Regarding saliva samples, negative delta values (i.e., when OD values for anti-CAP were greater than OD values for anti-CCP) were set to zero.
As IgG ACPA has been known to associate with poorer prognosis [27], comparisons of disease activity, radiographic outcome, smoking habits, and SE status, between IgA/IgA1/IgA2/SC ACPA-positive and ACPA-negative patients were performed only within the subgroup of IgG ACPA-positive patients, in order to avoid the confounding effect of IgG ACPA. Fisher’s exact test was used to test differences in proportions regarding smoking status, positive tests regarding different salivary, and circulating ACPAs (IgA, IgA1, IgA2, SC, and IgG) and SE. The Mann-Whitney U test was used to relate antibody status in the serum and saliva versus clinical variables, inflammatory markers, HAQ, and levels of other ACPAs. Spearman’s rho correlation coefficient (r) was used to test correlations between antibody levels in the saliva and serum. Linear regression analysis was used to evaluate associations between disease activity and levels of salivary IgA ACPA adjusting for levels of serum IgA ACPA, disease duration, and treatment. By including 196 RA patients of which 12% tested positive for IgA ACPA in the saliva, we achieved 80% power at alpha = 0.05 to detect a difference in DAS28 of 0.9 units between patients positive vs. negative for IgA ACPA in saliva.