Newly Discovered Autoantibodies In Rheumatoid Arthritis: Detection And Significance

BackgroundNew autoantigen and autoantibody systems have been discovered with the aid of protein microarray, in particular, immunochip. It is indicated that peptidylarginine deiminase 4 (PADI4) and the catalytic domain of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) have been recently indentified as two autoantigens related to rheumatoid arthritis (RA). Further studies on antoantibodies to these newly discovered autoantigens will provide new insights into the diagnosis and therapy of RA.Our studies on natural bispecific antibodies in animal models indicate that a group of natural bispecific antibodies might be readily produced in vivo when two unrelated antigens immunized simultaneously. Autoimmune diseases share some common characteristics with the animal models, such as more frequently occurred in women, and the body stimulated with various autoantigens during long-term disease duration. Thus, we propose that a group of bispecific autoantibodies against two different autoantigens might exist in patients with autoimmune diseases.Purposes1. To determine the prevalence of IgG anti-PADI4 antibodies, IgG anti-BRAF's catalytic domain antibodies and anti-peptide P25 antibodies in patients and controls, and investigate the possible associations of these autoantibodies with clinical and laboratory indicators in RA patients.2. To determine the subclass distribution of IgG anti-PADI4 antibodies in patients with RA, and investigate the associations with the disease.3. To identify a group of bispecific autoantibodies against immunoglobulin G (IgG) and cyclic citrullinated peptide (CCP) (anti-IgG/CCP) in the sera from RA patients.Methods1. The recombinant PADI4 was produced by soluble expression in Escherichia coli expression system via the prokaryotic plasmid pDEST17-PADI4, and purified by Ni2+affinity chromatography.2. The prevalence of IgG anti-PADI4 antibodies was determined by an indirect enzyme-linked immunosorbent assay (ELISA) in patients with RA, systemic lupus erythematosus (SLE), primary Sjogren syndrome (pSS), systemic sclerosis (SS) and healthy controls by using recombinant PADI4 as antigens, and the possible associations between anti-PADI4 antibodies and disease indicators were investigated in RA.3. The subclass distribution of IgG anti-PADI4 antibodies was determined by establishing the "equipotency" curves for 4 horseradish peroxidase conjugated anti-IgG subclass monoclonal antibodies.4. The expression vector pET28b-BRAF was constructed by cloning the sequence of wild type BRAF's catalytic domain into the plasmid pET28b after PCR amplification.5. The recombinant BRAF's catalytic domain was expressed in Escherichia coli expression system via the prokaryotic plasmid pET28b-BRAF, and purified by separation of the inclusion bodies and the Ni2+affinity chromatography.6. The prevalence of IgG anti-BRAF's catalytic domain antibodies and anti-peptide P25 antibodies were determined by indirect ELISAs in patients with RA, SLE, pSS and healthy controls by using recombinant BRAF's catalytic domian and the synthesized peptide P25, respectively, and the possible associations between these antibodies and disease indicators were investigated in RA.7. Bispecific autoantibodies to IgG and CCP were identified in sera from RA patients using a double antigen sandwich ELISA with CCP as the coating antigen and HRP conjugated rabbit IgG as the detection antigen.Results1. Purified recombinant PADI4 and BRAF's catalytic domain were obtained after a series of purification steps.2. The prevalence of IgG anti-PADI4 antibodies were significantly higher in patients with RA (32.0%) than in patients with SLE (6.0%; p<0.001), pSS (8.6%; p=0.003), SSc (5.0%; p=0.017) and healthy controls (2.0%; p<0.001); whereas the prevalence of IgG anti-BRAF's catalytic domain antibodies or anti-peptide P25 antibodies were higher in patients with RA (20.8%and 18.8%, respectively) than in healthy controls (6.4%and 2.2%, respectively), but with no significant difference when compared to SLE (20.3%and 21.2%, respectively) and pSS (20.5%and 18.2%, respectively).3. IgGl and IgG3 were the predominant subclasses of IgG anti-PADI4 antibodies in patients with RA.4. Statistical analysis showed that IgG anti-PADI4 antibodies were associated with disease status and other autoantibodies such as anti-CCP, and its subclasses were differentially distributing between patients with active disease and those without. IgG anti-BRAF's catalytic domain antibodies and anti-peptide P25 antibodies were found to be associated with the inflammatory indicator such as ESR and the disease status of the patients.5. A group of bispecific autoantibodies to IgG and CCP were identified in sera from RA patients.Conclusions1. IgG anti-PADI4 antibodies is a specific marker for RA. This study is the first to determine the IgG subclass distribution of anti-PADI4 in RA. Our results show that IgG1 and IgG3 are the predominant subclasses, enriching the context of the research for IgG anti-PADI4 antibodies in RA.2. This study is the first to determine both of IgG anti-BRAF's catalytic domain antibodies and anti-peptide P25 antibodies in larger cohorts of RA and patients with other autoimmune diseases. Our results indicate that they were not specific markers for RA, but related to RA due to their associations with inflammatory indicators of RA.3. We report a group of natural bispecific autoantibodies to rabbit IgG and CCP in sera from RA patients, for the first time, laying the foundation of further studies on the relationship between the bispecific autoantibodies and RA.