Isotypes of autoantibodies against novel differential 4-hydroxy-2-nonenal-modified peptide adducts in serum is associated with rheumatoid arthritis in Taiwanese women

Background Rheumatoid arthritis (RA) is an autoimmune disorder with systemic inflammation and may be induced by oxidative stress that affects an inflamed joint. Our objectives were to examine isotypes of autoantibodies against 4-hydroxy-2-nonenal (HNE) modifications in RA and associate them with increased levels of autoantibodies in RA patients. Methods Serum samples from 155 female patients [60 with RA, 35 with osteoarthritis (OA), and 60 healthy controls (HCs)] were obtained. Four novel differential HNE-modified peptide adducts, complement factor H (CFAH)1211–1230, haptoglobin (HPT)78–108, immunoglobulin (Ig) kappa chain C region (IGKC)2–19, and prothrombin (THRB)328–345, were re-analyzed using tandem mass spectrometric (MS/MS) spectra (ProteomeXchange: PXD004546) from RA patients vs. HCs. Further, we determined serum protein levels of CFAH, HPT, IGKC and THRB, HNE-protein adducts, and autoantibodies against unmodified and HNE-modified peptides. Significant correlations and odds ratios (ORs) were calculated. Results Levels of HPT in RA patients were greatly higher than the levels in HCs. Levels of HNE-protein adducts and autoantibodies in RA patients were significantly greater than those of HCs. IgM anti-HPT78−108 HNE, IgM anti-IGKC2−19, and IgM anti-IGKC2−19 HNE may be considered as diagnostic biomarkers for RA. Importantly, elevated levels of IgM anti-HPT78−108 HNE, IgM anti-IGKC2−19, and IgG anti-THRB328−345 were positively correlated with the disease activity score in 28 joints for C-reactive protein (DAS28-CRP). Further, the ORs of RA development through IgM anti-HPT78−108 HNE (OR 5.235, p < 0.001), IgM anti-IGKC2−19 (OR 12.655, p < 0.001), and IgG anti-THRB328−345 (OR 5.761, p < 0.001) showed an increased risk. Lastly, we incorporated three machine learning models to differentiate RA from HC and OA, and performed feature selection to determine discriminative features. Experimental results showed that our proposed method achieved an area under the receiver operating characteristic curve of 0.92, which demonstrated that our selected autoantibodies combined with machine learning can efficiently detect RA. Conclusions This study discovered that some IgG- and IgM-NAAs and anti-HNE M-NAAs may be correlated with inflammation and disease activity in RA. Moreover, our findings suggested that IgM anti-HPT78−108 HNE, IgM anti-IGKC2−19, and IgG anti-THRB328−345 may play heavy roles in RA development.

HNE has two reactive electrophilic groups, an aldehyde group and an alkene bond, and can react with residues in amino acid. The C = C double bond in HNE can be targeted via Michael addition and has a mass addition at 156 Da in its non-reduced form [alanine (A), arginine (R), cysteine (C), glutamine (Q), histidine (H), lysine (K) and leucine (L)] or 158 Da in its reduced form (CHKRQ) [11][12][13]. The aldehyde group in HNE can react by forming Schiff base adducts and increase mass of 138 Da in the non-reduced form (CHKAL) or 140 Da in the reduced form (CHKR) [11][12][13]. The non-reduced form of the Schiff base adducts (CHKR) further spontaneously rearranges to form a pyrrole adduct with a mass increase of 120 Da [14,15].
In the present report, four differential novel HNEmodified peptides were re-analyzed via acquired tandem mass spectrometry (MS/MS) data (ProteomeXchange: PXD004546) using PEAKS 7 software (Bioinformatics Solutions, Waterloo, Canada) [6]. Acquired MS/MS spectra were obtained through concanavalin (Con) A affinity chromatography, one-dimensional (1-D) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), in-gel digestion, and nanoliquid chromatography tandem mass spectrometry (nano-LC-MS/MS) in patients with RA versus HCs [6]. We further validated HNE modifications of proteins and examined proteins level in serum and HNE-protein adducts. Moreover, we evaluated the performance of novel diagnostic autoantibodies against unmodified and HNE-modified peptides, which can possibly be used as diagnostic biomarkers for patients with RA, osteoarthritis (OA), and HCs. Herein, we aimed to determine correlations of IgM and IgG autoantibody titers against unmodified and HNE-modified peptide adducts with disease activity and clinical variables in RA patients. Further, the association between higher levels of serum autoantibodies in RA patients with a risk for RA development was assessed compared to HCs. Lastly, to thoroughly evaluate the potential of serum autoantibodies for biomarker development, we incorporated three machine learning algorithms and performed feature selection with WEKA (version 3.8.3) to further classify our subjects.  [30] or 1987 ACR classification criteria [31]. Further, patients with RA received a disease activity score in 28 joints for C-reactive protein (DAS28-CRP) (4.4 ± 1.67) assessment when they were diagnosed as RA. RA patients included in this study had suffered from this disease for a duration of 5.4 ± 6.41 years (Additional file 1: Table S1). OA patients had been diagnosed according to clinical symptoms with assistance from OA criteria by the ACR [32,33]. Therapies were given to patients with OA (65% non-steroidal anti-inflammatory drugs (NSAIDs), 11.4% disease-modifying anti-rheumatic drugs (DMARDs)) and RA (42.9% NSAIDs, 99.3% DMARDs) by clinicians (Additional file 1: Table S1). The institutional review board of the study hospital approved this study, and informed consent was provided by all volunteers before participating. Four novel differential HNE-modified peptide adducts were re-identified using PEAKS 7 software (Bioinformatics Solutions) from previous MS/MS data (ProteomeXchange: PXD004546) [6]. Their protein levels were examined by Western blotting with individual randomly age paired serum from 32 patients with RA and 32 HCs. HNE modifications of HNE-modified peptide adducts were assessed through immunoprecipitation (IP) and Western blotting using the pooled Con A-captured serum samples from above-mentioned 32 pairs of serum samples. Further, serum levels of HNE-protein adducts were determined, and isotypes of autoantibodies against unmodified and HNE-modified peptides were evaluated among individual serum of 60 RA and 35 OA patients and 60 HCs. Clinical and demographic characteristics of patients with RA and OA and HCs are summarized in Additional file 1: Table S1. Serum was stored at -20 °C until analyzed.

Patient samples
Novel differential HNE-modified peptide adducts were re-analyzed using PEAKS 7 software The Peaks PTM module of PEAKS 7 software (Bioinformatics Solutions) was used to identify sequences of HNE-modified peptide from acquired MS/MS spectra against the Universal Protein Resource Knowledgebase, a human protein database (UniProt; http://www.unipr ot.org/) containing 157,433 protein entities (UniProt, 2016/11), and those sequences are shown in Additional file 2: Figure S1A. MS/MS data are available through Pro-teomeXchange with the identifier PXD004546 [6]. S-Pyridylethylation ( Figure S1B. All of the modified MS spectra were identified manually, and fragmented ions were labeled as y, b, y-NH 3 , and b-H 2 O ions. Details are provided in the "Additional file 3: Supplementary Information" section.

Con A affinity chromatography and IP-Western blotting
Serum-derived Con A-captured serum proteins were purified using the protocol of Uen et al. [34]. Protein concentrations were examined using a Pierce ™ Coomassie Plus (Bradford) Assay Kit (Thermo Scientific, Waltham, MA, USA) following to the protocol from manufacturer. Con A-captured proteins were used in IP. IP-Western blotting of pooled Con A-captured proteins was used to confirmed modifications of HNE-modified proteins. We used antibodies in IP including complement factor H (CFAH), haptoglobin (HPT), the Ig kappa chain C region (IGIK), and prothrombin (THRB), following to the protocol of Liao et al. [35]. HNE modifications of proteins were detected using a goat polyclonal anti-HNE antibody. Details of experiments are provided in the "Additional file 3: Supplementary Information" section.

Detection of proteins and HNE-protein adducts
Levels of CFAH, HPT, IGKC, and THRB were detected using Western blotting. HNE-protein adducts were quantified using an enzyme-linked immunosorbent assay (ELISA) [36]. All samples were detected in duplicate. Details of the protocol are provided in the "Additional file 3: Supplementary Information" section.

Measurement of autoantibodies against unmodified and HNE-modified peptides
Polypeptides were synthesized and used in the ELISA [35]. Unmodified peptides are presented as  [37]. Then, CFAH 1211−1230 HNE and HPT 78−108 HNE were reductively stabilized using NaBH 4 [11]. In total, 155 serum samples were evaluated for the presence of IgG and IgM isotypes of anti-unmodified and anti-HNE-modified peptide autoantibodies. All of the samples were detected in duplicate. The protocol details are provided in the "Additional file 3: Supplementary Information" section.

Statistical analysis
The significance of blot densitometric differences, and levels of serum proteins and HNE-protein adducts were determined using Student's t-test. A one-way analysis of variance (ANOVA) was used to examine levels of autoantibody isotypes against unmodified and HNE-modified peptides between RA and OA patients and HCs. Scheffe's post-hoc test was applied to evaluate the difference of mean between any two groups, as well as a post-hoc test using the Bonferroni method with a 0.0167 adjusted significance level. We used GraphPad Prism (vers. 5.0; GraphPad Software, San Diego, CA, USA) to evaluate differences in Student's t-test between groups, correlations between measurements, and generated receiver operating characteristic (ROC) curves to evaluate the diagnostic performance of autoantibodies. Pearson's or Spearman's rank correlation coefficients were used to assess correlations among different parameters. To estimate multivariate-adjusted odds ratios (ORs) and their 95% confidence intervals (CIs) for RA risk, Logistic regression models were performed in this study. The positivity of autoantibody isotypes and HNE-protein adducts was decided by ROC curves. The cut-off value for an ROC curve was determined by Youden index, which represents the sum of sensitivity and 1-specificity, and the maximum value of Youden index is the suitable cut-off point for that curve. Pair-wise comparisons of ROC curves were assessed using MedCalc Statistical Software (vers. 15.4; MedCalc Software, Ostend, Belgium). One-way ANOVA and power were determined using SAS (vers. 9.3; SAS Institute, Cary, NC, USA), and power estimations were calculated according to the ROC analysis. The area under the ROC curve (AUC), sensitivity, and specificity were calculated at a 95% confidence level. The significance level of all statistical tests was set to p < 0.05. For feature selection, we first used 'Information Gain' as the attribute evaluator with 'Ranker' as the search method in WEKA (version 3.8) [38] to select discriminative features in identify RA patients. Next, we incorporated ten-fold cross-validation to evaluate our model based on decision trees (DT) [39], random forests [40], and support vector machines (SVM) [41] in scikit-learn (version 0.21.3) [42]. Parameter tuning was performed for each training and validation. During model selection, a forward selection algorithm was used to select the most effective combination of features for classification. In forward selection, a feature was selected in to the optimal feature set if adding the feature into the prediction model improved the AUC. To evaluate the predictive performance, we applied a confusion matrix to calculate the accuracy, precision, sensitivity, specificity, and AUC for assessments.

Validation of HNE modifications on HNE-modified peptide adducts
HNE modifications in four differential HNE-modified peptide adducts were validated by IP-Western blotting with two pooled Con A-captured serum samples from RA and HCs, which detected signals of approximately 184, 43, 25, and 80 kDa, respectively, indicating CFAH, HPT, IGKC, and THRB ( Fig. 1).

Detection of protein levels and HNE-protein adduct levels
Protein levels of CFAH, IGKC, and THRB from 32 randomly paired individual serum samples from RA patients and HCs showed no significant differences (Fig. 2a, c, d). However, levels of HPT in patients with RA were greatly higher than the levels in HCs (1.24-fold, p < 0.041, Fig. 2b). Serum levels of HNE-protein adducts in RA patients were significantly higher than the levels in OA patients (1.16-fold, p = 0.0311) and HCs (1.20-fold, p = 0.0062, Additional file 1: Table S1).

Measuring autoantibodies against unmodified and HNE-modified peptides
The AUC, sensitivity, and specificity were used to assess clinical performances of IgG and IgM that against unmodified and HNE-modified peptides. The ANOVA analysis indicated that differences in all autoantibodies against unmodified and HNE-modified peptides were significant among patients with RA, OA, and HCs (Fig. 3, Table 1).
Levels of IgG against CFAH 1211−1230 in patients with RA were greatly higher than those in patients with OA  . Proteins were immunoprecipitated from pooled concanavalin (Con) A-captured serum samples (32 healthy controls (HCs) and 32 patients with rheumatoid arthritis (RA)) using anti-complement factor H (CFAH), anti-haptoglobin (HPT), anti-immunoglobulin kappa chain C region (IGKC), and anti-prothrombin (THRB) antibodies and then subjected to Western blotting with anti-HNE antibodies (upper panel). Individually selected random serum samples (HCs and RA patients) were used as controls; these were simultaneously used for Western blotting with anti-HNE antibodies. Percentages of the SDS-PAGE gel and IP loading amounts of Con A-captured serum proteins were 8% and 20 µg, 10% and 20 µg, 12% and 5 µg, and 8% and 20 µg for CFAH, HPT, IGKC, and THRB, respectively. A duplicate gel was stained with Coomassie brilliant blue (CBB) as a loading control, including CFAH (b), HPT (c), IGKC (d), and THRB (e). Red arrows indicate immunoprecipitated proteins 60.0% sensitivity and 60.0% specificity) for detecting patients with RA and OA ( Table 2). Levels of IgG against CFAH 1211−1230 HNE in RA patients were greatly higher than the levels in OA patients by 1.32-fold (p = 0.0006), and HCs by 1.32-fold higher (p = 0.0001) (Fig. 3b, left panel), and AUC values were 0.71 (with 76.7% sensitivity and 55.0% specificity) and 0.52 (with 51.4% sensitivity and 50.0% specificity) for detecting patients with RA and OA (Table 2). HC-RA versus HC-OA showed a statistically significant difference (p = 0.0135, Fig. 3b, right panel, Table 2) in pair-wise comparisons of ROC curves. Further, levels of IgM against CFAH 1211−1230 in RA patients were greatly higher than the levels in HCs by 1.21-fold (p = 0.0103) (Fig. 3c, left panel), and AUC values were 0.68 (with 81.7% sensitivity and 53.3% specificity) and 0.68 (with 77.1% sensitivity and 60.0% specificity) for detecting patients with RA and OA ( Table 2). Levels of IgM against CFAH 1211−1230 HNE in RA patients were greatly higher than the levels in HCs by 1.40-fold (p = 0.0002, Fig. 3d, left panel), and AUC values were 0.70 (with 85.0% sensitivity and 41.7% specificity) and 0.59 (with 80.0% sensitivity and 36.7% specificity) for detecting patients with OA and RA (Table 2). Further, HC-OA versus HC-RA showed a significant difference (p = 0.0421, Fig. 3d, right panel, Table 2) in pair-wise comparisons of ROC curves.

Correlations of serum anti-unmodified and anti-HNE-modified peptide autoantibodies with clinical variables in RA patients
A correlation analysis was conducted of autoantibody reactivities against unmodified and HNE-modified peptides with DAS28-CRP measurements and serum clinical variables (RF, anti-CCP, CRP, ESR, and the HNE-protein adduct) in patients with RA. In Table 3 Table 3).

Associations of serum anti-unmodified and anti-HNE-modified peptide autoantibodies with RA patients compared to HCs
As shown in   achieved an AUC of 0.92, and support vector machine achieved an AUC of 0.88. The ROC plots of these algorithms were presented in Additional file 4: Figure S2. Our results demonstrated that random forest performed better than the other algorithms for predicting RA from HC or OA.

Discussion
To the best of our knowledge, this is the first study to investigate autoantibodies isotypes against unmodified and HNE-modified peptides, its correlation with activity of disease in Taiwanese women with RA, and associations of risks for RA compared to HCs. However, a critical limitation should be noted due to our samples used in this study were not strictly selected during the disease progression of RA. Therefore, the efficacy of this test may be affected. Levels of HNE-protein adducts in RA patients were greater than the levels in HCs (Additional file 1: Table S1), which is consistent with results from a previous study [24]. Barrera et al. suggested that HNE-protein adducts also featured a pathogenic contribution of oxidative stress [43]. HNE-protein adducts, OSEs, are recognized as danger signals by innate immune receptors, such as the lectin-like oxidized LDL receptor 1 (LOX1) [44,45]. HNE-protein adducts (OR 2.413) showed a risk for RA development (Table 4). Chronic inflammation can be triggered by the accumulation of OSEs, an important target of innate immunity, which increases the risk of developing chronic inflammation [5]. Binder et al. indicated that IgM isotypes against OSEs can enhance the clearance and neutralization of proinflammatory effect [5,16,17]. If OSEs cannot be efficiently cleared, OSEs would act as damage-associated molecular patterns (DAMPs) that trigger sterile inflammation [46]. Pattern recognition receptors (PRRs) can recognize DAMPs and activate the innate immune response to trigger sterile inflammation [47]. LOX1, a cellular PRR, can recognize and bind to HNE to mediate its uptake and inflammatory effect in atherosclerosis [48]. Macrophages can take up the IgM-NAA-HNE complex by C1q-calreticulin-CD91dependent or mannose-binding lectin (MBL) and MBL receptor-dependent mechanisms in chronic inflammatory diseases and atherosclerosis [49]. Siloşi et al.
reported that B-1 cells produce NAAs (IgM > IgG > IgA) and pathogenic autoantibodies (IgG > IgM > IgA) [50]. However, the boundary line between natural immunity and pathogenic autoimmunity is unclear [50]. Further, IgM-NAAs can control IgG-NAA activity and regulate expression of natural IgG autoreactive repertoire by F(ab')2 fragments of IgG-NAAs in human and mice serum [51,52]. Chen et al. proved that inhibition of Tolllike receptor (TLR) and IgG-immune complex-mediated inflammatory responses mediate anti-inflammatory features of IgM-NAAs [53]. Moreover, IgG-NAAs may be involved in autoimmune disease pathogenesis, including SLE, Sjögren's syndrome, and Graves' disease [50,54], and anti-OSE NAAs themselves may have a protective effect. Further, we deduced that elevated levels of anti-OSE NAAs may be a risk index of RA development based on protein function and disease activity when oxidative stress occurs over a sustained period in patients with RA (Additional file 1: Table S1,  Fig. 1). The biological function of CFAH, a soluble inhibitor of the alternative complement pathway, is to inhibit the inflammatory response through oxidative stress and to protect host tissues from complement-mediated damage [55][56][57]. Okroj et al. reported that complement activation  contributes to the pathological process of RA [58]. The complement system is a central innate immune system that participates in eliminating pathogens and promotes inflammatory responses [55]. Weismann et al. indicated that CFAH is able to bind MDA, and as an MDA-binding protein, it blocks the proinflammatory effects that induced by MDA in vivo in mice [59]. Moreover, it was reported that the HNE modification was unbound by CFAH [59]. However, we found that CFAH was modified with HNE at K1230 in RA patients (Table 1, Additional file 2: Figure S1C). Trojnár et al. identified three linear epitopes on serum CFAH (CFAH 1157−1171 , CFAH 1177−1191 , and CFAH 1207−1226 ) in atypical hemolytic uremic syndrome (aHUS) [60]. Interestingly, CFAH 1211−1230 is also an autoantigen in RA (Fig. 3a, c). The HNE-modified CFAH 1211−1230 peptide can enhance autoantibody levels in patients with RA compared to patients with OA and HCs (Fig. 3b, d). Several studies demonstrated that aHUS was associated with the presence of autoantibodies against CFAH [56,57]. Autoantibodies against CFAH are also present in significant ratios in RA [57]. Insufficient inhibition of CFAH activity may be caused by pathologyassociated autoantibodies [56,58] (Table 4).
HPT is a hemoglobin-binding protein that can prevent oxidative damage to organs and participates in activating innate and adaptive immune responses [61]. Increased synovial fluid (SF) and serum HPT levels found in RA patients were associated with inflammation and tissue destruction [62]. Yildirim et al. indicated that serum HPT was an acute-phase protein and significantly correlated with disease activity in patients with RA [63]. In this study, we identified one novel HNE modification at C92 on HPT 78-108 in RA patients (Table 1, Additional file 2: Figure S1D). Korngold indicated that the HPTanti-HPT reaction can block HPT-hemoglobin-binding action [64]. Muta et al. reported that the level of the anti-HPT antibody in serum increased and the level of HPT decreased after febrile non-hemolytic transfusion reactions (FNHTRs) [3]. In this study, higher levels of HPT in serum were greatly 1.24-fold higher (p = 0.041) in RA patients than in HCs (Fig. 2b). IgG and IgM against HPT 78−108 and HPT 78−108 HNE were greatly higher in RA patients than in HCs (Fig. 3e-h). Thus, we inferred that high levels of autoantibodies against HPT may inhibit HPT's function and play a role in the risk of developing  Table S2). RFs are autoantibodies against the fragment crystallizable (Fc) region of IgG that, via antigenic stimulation, acts against an abnormal immune response from the host's natural antibody repertoire [65]. The IgM RF is commonly measured in clinical practice and serves as a marker of RA, other rheumatic diseases, and chronic infections [66]. Sidorov et al. found that the human regulatory RF (regRF) can be induced by the hinge region of Fc fragments of homologous IgG and can prevent rheumatic diseases [2]. RF production can also be stimulated by modified IgG, including agalactosyl IgG, or advanced glycated end-product (AGE)-damaged IgG that are associated with more-severe RA and can play a meaningful role in pathogenesis of RA [67,68]. In this study, we identified two novel HNE modifications at A4 and A5 on IGKC 2−19 , which is located on the IgG light chain in RA patients (Table 1, Additional file 2: Figure S1E). IgG and IgM against IGKC 2−19 and IGKC 2−19 HNE were greatly higher in patients with RA than in HCs (Fig. 3il). Thus, we inferred that high levels of autoantibodies against IGKC may promote the risk of developing RA. Indeed, IgM anti-IGKC 2−19 was greatly positively correlated with DAS28-CRP (r = 0.2816), RF (r = 0.5674), ESR (r = 0.2692), and HNE-protein adducts (r = 0.2667). However, IgM anti-IGKC 2−19 HNE was significantly correlated with RF (r = 0.5404), ESR (r = 0.2985), and HNEprotein adducts (r = 0.2709) ( Table 3). Interestingly, IgG anti-IGKC 2−19 (r = − 0.3538) and IgG anti-IGKC 2−19 HNE (r = − 0.3432) were significantly negatively correlated with DAS28-CRP (  (Table 4).
RA is characterized by activation of both inflammatory and coagulation processes resulting in erosion of the joints [69]. THRB is transformed into thrombin by a prothrombinase when injury occurs to tissues and then changes via fibrinogen to form fibrin in a coagulation process [70]. Ohba et al. suggested that high levels of thrombin activity in SF via strong mitogenic activity toward synovial fibroblast-like cells play a significant role in the RA pathogenesis [71]. Yang et al. reported that anti-THRB autoantibodies can display prothrombinase activity and contribute to thrombosis in antiphospholipid syndrome (APS) and SLE [72]. In this study, novel HNE modifications at K344 on THRB 328-345 in RA patients were identified (Table 1, Additional file 2: Figure S1F). Next, levels of IgG and IgM against THRB 328−345 and THRB 328−345 HNE were greatly higher in patients with RA than in HCs (Fig. 3m-p). Thus, high levels of anti-THRB autoantibodies may be considered as a risk factor for RA. Interestingly, IgG anti-THRB 328−345 was greatly positively correlated with DAS28-CRP (r = 0.2703) and RF (r = 0.6140), IgG anti-THRB 328−345 HNE was significantly positively correlated with RF (r = 0.3072) and anti-CCP (r = 0.2549), IgM anti-THRB 328−345 HNE was greatly positively correlated with RF (r = 0.2845) and ESR (r = 0.2597), but IgM anti-THRB 328−345 was significantly negatively correlated with the HNE-protein adduct (r = − 0.2796) as shown in Table 3. Further, IgG anti-THRB 328−345 (OR 5.761), IgG anti-THRB 328−345 HNE (OR 9.542), and IgM anti-THRB 328−345 HNE (OR 5.043) exhibited risks for RA development (Table 4).
Several previous studies showed the feasibility in early diagnosis of autoimmune diseases using a machine learning application for RA. Rodrigo Torres et al. suggested that feature selection can be a powerful tool in biomarker discovery [73]. Therefore, we believe that with appropriate proteomic data and machine learning algorithms, the biomarker candidates we developed can be optimized into a set of highly accurate features. In our experiment, we incorporated feature ranking and a forward selection method to identify IgM anti-HPT 78−108 and HNE-protein adducts that can identify RA from HC combined with random forest algorithm. We then compared our results with the accuracy of anti-CCP which was considered a well performance biomarker in RA. Other studies reported that the sensitivity and specificity of anti-CCP were 63% ~ 91.4% and 69.7% ~ 97.6%, respectively [74][75][76][77]. However, we found only 50% of positive anti-CCP in RA samples. Moreover, consistency was observed between statistical analyses of odds ratio and prediction results of classification. Our results from statistical models, feature selection, and machine learning classifiers supported that IgM anti-HPT 78−108 HNE, IgM anti-IGKC 2−19 , and IgM anti-IGKC 2−19 HNE showed potential to be developed as biomarkers for RA.