A highly conserved determinant on human rheumatoid factor idiotypes defined by a mouse monoclonal antibody

Different human IgM rheumatoid factor (IgM RF) idiotypes have been described defined by polyclonal rabbit anti-idiotypic antibodies. These antisera do not allow clear genetic analysis of the idiotypic determinants, be they cross-reactive or private. Therefore, we tried to obtain a set of monoclonal anti-idiotypic antibodies directed against RF idiotypes. Purified IgM RF serum from a patient with classical rheumatoid arthritis was used to immunize BALB/c mice. The spleen cells were fused with Sp 2/0 Ag 14, a nonsecreting mouse myeloma cell line, and a hybrid producing monoclonal anti-idiotypic antibody was selected. The mouse antibody, an IgG1 kappa, reacts with an identical or similar determinant located on (or close to) the binding site of all tested monoclonal or polyclonal IgM RF from totally unrelated patients with Waldenstr?ms's macroglobulinemias or rheumatoid arthritis. The monoclonal antibody also reacts with 2 rheumatoid arthritis patients' IgG RF and with a low proportion of normal polyclonal IgM without detectable RF activity. An hypothesis is proposed to explain the existence of a such highly conserved determinant on RF idiotypes.     

Demonstration of anti-idiotypic antibodies directed against IgM rheumatoid factor in the serum of rheumatoid arthritis patients.

We have identified the presence of anti-idiotypic activity against IgMRF in the sera of RA patients. Only patients seropositive for IgMRF had significant levels of anti-idiotypic activity, while seronegative patients and normal volunteers did not. When this anti-idiotypic activity was affinity-purified from a single RA patient, two separate binding activities were identified. IgG antibodies were pepsin-digested to F(ab')2 fragments before affinity-purification to remove the Fc portion capable of binding to IgMRF. Anti-idiotypic F(ab')2 fragments of IgG were eluted from an IgMRF-Sepharose 4B column. These F(ab')2 bound preferentially to IgMRF bearing an idiotype recognized by the anti-idiotypic murine monoclonal 17.109. A second anti-idiotypic F(ab')2 was affinity purified using rabbit anti-human Fc antibody bound to Sepharose 4B. These eluted antibodies behaved as the internal image of IgG, binding five out of seven IgMRF's tested. The binding of both anti-idiotypic F(ab')2 was inhibited with human IgG. The presence of both IgMRF and anti-idiotypic antibodies directed against it in the sera of RA patients suggests that anti-idiotypic antibodies alone are not capable of inhibiting the production of rheumatoid factor.     

Comparison of idiotypes of rabbit antibodies against Salmonella abortusequi and tobacco mosaic virus. Study of cross reactions between antibody idiotypes against these 2 antigenic materials

Idiotypic determinants of anti-tabacco mosaic virus (TMV) antibodies produced by different rabbits can sometimes show a very strong similarity. The frequency of precipitin reactions between sera against anti-TMV idiotypes and anti-TMV sera which are not used for their preparation (heterologous reactions) is about 4 %. We have compared idiotypy of anti-TMV antibodies and idiotypy of anti-Salmonella abortus-equi (Sae) antibodies produced by different rabbits. We have observed precipitin reactions between anti-Sae sera and sera against anti-TMV idiotypes and vice versa. However, anti-Sae antibodies do not combine with TMV and vice versa. Anti-idiotypic sera which precipitate anti-TMV antibodies in the homologous anti-TMV serum can also precipitate anti-Sae antibodies in a heterologous anti-Sae serum. In the same way, anti-idiotypic sera which precipitate anti-Sae antibodies in the homologous serum can also precipitate anti-TMV antibodies in a heterologous anti-TMV serum. We have observed 3 heterologous reactions during the study of 1260 reactions in which anti-TMV sera and sera against anti-Sae idiotypes are involved. We have observed 3 heterologous reactions during the study of 436 reactions in which anti-Sae sera and sera against anti-TMV idiotypes are involved. Previous absorption of anti-idiotypic serum by the homologous serum causes these heterologous reactions to disappear. We have observed, in the case of two of these heterologous reactions, that addition to the anti-idiotypic serum of an excess of the heterologous serum IgG could cause the homologous reaction to disappear. These heterologous reactions between sera against different antigens do not seem to imply the rabbit rheumatoid factor-like substance. These reactions imply idiotypy of anti-Sae antibodies and idiotypy of anti-TMV antibodies.     

IgM anti-idiotypes that block anti-HLA antibodies: naturally occurring or immune antibodies?

Using dithiothreitol (DTT) technique, IgM anti-HLA anti-idiotypic antibodies were detected in a multiparous multitransfused woman. These antibodies were able to inhibit the binding of specific IgG anti-HLA antibodies on their corresponding antigen. The recognized determinants were cross-reactive determinants since they were partially found on anti-HLA antibodies from unrelated individuals. By studying the patient's sera over a period of 2 years, no IgM-IgG switch was observed but the presence of these antibodies was stable in time, despite the disappearance of the idiotypes (anti-HLA antibodies). However, when looking at the patient's earlier serum, it was shown that these IgM anti-idiotypic antibodies were absent from the first available serum. Thus, these anti-idiotypic antibodies seem to behave both like natural and immune antibodies. The incidence of such antibodies in pretransplant patients is discussed.     

A study of idiotypic suppression in adult rabbits immunized with Salmonella abortus-equi.

It is possible to induce idiotypic suppression in adult rabbits immunized with Salmonella abortus-equi (S.a.e.). Ten months after priming we injected the rabbit with anti-idiotypic serum prepared against its own antibodies to S.a.e. and, 3 weeks later, gave it a booster injection of bacteria. A new anti-idiotypic serum was preparedwith the serum to S.a.e. collected after this boost and was used for the following isiotypic suppression attempt made 10 months after the first one. Using this procedurewe succeeded in two successive idiotypic suppression attempts in the same rabbit. Inthe three attempts we carried out, idiotypic suppression was totally effective, i.e. idiotypes detected by the serum used for the suppression totally disappeared after thesuppression, and the suppression lasted during the life of the rabbits (maximum 10 months). This observation is consistent with a suppression resulti-g from an interaction of anti-idiotypic antibodies with the complementary receptors at the surface of memory cells. This suppression was without effect on antibody to S.a.e. titre and on IgG concentration. Idiotypes detected by the anti-idiotypic serum prepared withthe serum to S.a.e. collected after the suppression. These idiotypes were different from those detected by the anti-idiotypic serum used for the suppression. This observation confirms that idiotypic recognition is confined to a limited number of clonal products, despite the fact that a very heterogeneous antibody population was used forthe anti-idiotypic immunization. Thus we did not observe the appearance of new idiotypes produced previously silent cell clones. All the different idiotypes we detected during the successive idiotypic suppression attempts carried determinants shich remained peculiar to each individual rabbit.     

Occurrence of two germline-related rheumatoid factor idiotypes in rheumatoid arthritis and in non-rheumatoid seropositive individuals.

Human rheumatoid factor (RF) paraproteins express two distinct light chain cross-reactive idiotypes defined by the monoclonal antibodies 17.109 and 6B6.6. These germline gene-related cross-reactive idiotypes are both carried on VK3 light chains and are each present on about one-third of IgM RF paraproteins. We assessed the degree to which these idiotypes are represented in polyclonal RFs. We used rheumatoid arthritis (RA) and non-RA RF-positive sera selected from a large cross-sectional population study (the Mini-Finland Health Survey), and sera from a community-based follow-up study of recent-onset RA patients from Heinola, Finland. In the Mini-Finland Health Survey, elevated levels of the 17.109 RF idiotype were seen in sera of 13% of the RA and 19% of the non-RA group; 6B6.6 RF was seen in 26% of the RA and 28% of the non-RA group. In sera of the Heinola follow-up study, 17.109 RF was seen in 12% initially, but in only 3% at 8 years. Similarly, 6B6.6 RF was detected in 25% initially, but in only 7% at 8 years. Ten sera positive for RF prior to the onset of clinical RA were identified from individuals of a second large population study from Finland (North Karelia project); two of these sera exhibited the 6B6.6 idiotype; none exhibited the 17.109 idiotype. The data are consistent with the concept that these germline gene-related cross-reactive RF idiotypes occur frequently in the polyclonal RF of non-RA as well as RA sera, and that in RA the idiotypes may sometimes be reduced or lost as a consequence of somatic diversification of the RF through somatic mutation, usage of new germline genes, or both.     

Novel autoantibody markers for early and seronegative rheumatoid arthritis

Approximately one-third of rheumatoid arthritis (RA) patients are seronegative for the 2 serological RA markers, rheumatoid factor (RF) and antibodies against cyclic citrullinated peptides (ACCP). Moreover, the sensitivities of both markers are lower in the diagnostically important early disease phase. The aim of this study was to identify additional autoantibody markers for early RA and for RF-negative, ACCP-negative (seronegative) RA.We screened an RA synovium cDNA phage display library with autoantibodies in plasma from 10 early (symptoms of maximum 1 year) and 10 seronegative (RF-negative, ACCP-negative) RA patients with validation in 72 additional RA patients and 121 controls (38 healthy controls, 43 patients with other inflammatory rheumatic diseases, 20 osteoarthritis patients and 20 subjects with mechanical joint complaints). Fourteen novel autoantibodies were identified that showed a 54% sensitivity and 90% specificity for RA. For 11 of these autoantibodies, an exclusive presence was demonstrated in RA patients (100% specificity, 37% sensitivity) as compared to controls. All early RA patients were positive for at least one of the identified autoantibodies and antibody-positivity was associated with a shorter disease duration (P = 0.0087). 52% of RA patients who initially tested negative for RF and ACCP, tested positive for at least one of the 14 novel autoantibodies, resulting in a 19% increase in sensitivity compared to current serological testing. Moreover, 5 identified autoantibodies were detected more frequently in seronegative RA patients, indicating that these autoantibodies constitute novel candidate markers for this RA subtype. We demonstrated that the targets of 3 of these 5 autoantibodies had an increased expression in RA synovial tissue compared to control synovial tissue, pointing towards a biological rationale for these auto antibody targets in RA.In conclusion, we identified novel candidate autoantibody markers for RA that can be detected in early and seronegative RA patients indicating the potential added value for RA diagnostics.     

Idiotypes of rabbit anti-Salmonella abortus-equi antibodies (recent results)

Anti-idiotypic sera which precipitate the anti-Salmonella abortus-equi (Sae) serum used for their preparation (homologous reactions) sometimes precipitate anti-Sae sera other than the one used for their preparation (heterologous reactions). The frequency of these heterologous reactions is about 3 % for 5036 studied reactions. We have detected heterologous idiotypes in several different individuals, which apparently were not distinguished from homologous ones in the reaction in gel medium with the same anti-idiotypic serum. However, the quantity of heterologous anti-Sae serum necessary for inhibiting the homologous reaction is always larger than that necessary for inhibiting the heterologous reaction observed with the same anti-idiotypic serum. Homologous and heterologous reactions indicate that antibodies produced by rabbits submitted to the same anti-idiotypic immunization were sometimes directed against different idiotypes and even against different idiotypic determinants of the same idiotype. We have separated two different IgG idiotypes by the isoelectrofocusing technique. These two idiotypes seem to have different idiotypic specificities.     

Idiotypic determinants on human thyroglobulin autoantibodies derived from the serum of Hashimoto patients and EB virus transformed cell lines.

Rabbit anti-idiotypic reagents were prepared against three different human serum autoantibodies to thyroglobulin (Tg). Two of the rabbit antisera recognized private idiotypes (IdI) whilst a third antiserum recognized both an IdI and a cross-reactive idiotype (IdX) which was expressed in 50% of Hashimoto patients tested. A fourth anti-idiotype was produced against an IgM anti-Tg secreted by a patient's Epstein-Barr (EB) virus transformed lymphocytes. This antiserum only reacted with the immunizing IgM anti-Tg and therefore also recognized a private idiotype. Both the private and the public idiotypes appear to be restricted to the anti-Tg set of antibodies; this would favour the view that the IdX represents a collection of idotopes with the ability to bind Tg as their common feature, rather than a common structure based upon closely similar germ line derived amino acid sequences.     

The common occurrence of internal image type anti-idiotypic antibodies in rabbits immunized with monoclonal and polyclonal human IgM rheumatoid factors.

We have previously reported that rabbits immunized with a polyclonal human rheumatoid factor (RF) autoantibody preparation could induce anti-idiotypic antibodies bearing the 'internal image' of the Fc fragments of human IgG. The 'internal image' anti-idiotype have been shown to react with both the RF molecules as well as with the RF receptors on B lymphocytes. Under what conditions these anti-idiotypes occur is not known. Presently, we report that these anti-idiotypic antibodies occur more frequently than previously thought and could be isolated in sera of rabbits immunized with either monoclonal paraproteins with RF activity or other purified human polyclonal serum RFs. Immunization of rabbits with a peptide corresponding to the second complementarity-determining regions of a monoclonal RF did not induce this anti-idiotype. Immunization of goats with human RF similarly did not result in induction of such anti-idiotype. Induction of these anti-idiotypes thus depended upon immunization with the intact RF antigen as well as the species of animal immunized. The repeated isolation of 'internal image' anti-idiotypic antibodies from RF immunized rabbits suggests that the antigenic conformations recognized by human RF autoantibodies are restricted, and that 'internal image' anti-idiotypic species to RF may pre-exist within the rabbit immune network. Such broadly cross-reactive anti-idiotypic reagents provide unique reagents for studying the regulation of RF autoantibody synthesis.     

The Major Rheumatoid Factor Cross-Reactive Idiotype in Rheumatic Disease

The major rheumatoid factor cross-reactive idiotype (RCRI), a tertiary structure formed by both light and heavy chains, is found on 60% of all monoclonal IgM kappa RFs. To determine if the RCRI is expressed in patients with rheumatic disease, we used polyclonal rabbit anti-idiotypic antibodies to detect RCRI in sera and in pokeweed mitogen cultures of blood mononuclear cells (PBM) from patients with rheumatoid arthritis (RA) and juvenile rheumatoid arthritis (JRA). We detected increased expression of RCRI + plasma cells in PWM cultures, and in sera from these patients. We have determined that some 7S IgM molecules from RF+ RA patients are RCRI +, and can bind IgG in a sensitive RF ELISA. We have also observed that the CD5+ B cell subset, which is responsible for autoantibody production, generates RCRI+ antibodies. We review these data and discuss the relationship of the idiotypic network of interacting antibodies with rheumatic disease.     

Antibody to streptococcal cell wall peptidoglycan-polysaccharide polymers in seropositive and seronegative rheumatic disease

An ELISA has been developed for serum antibodies to streptococcal cell wall peptidoglycan-polysaccharide polymers (PG-GSP). A significantly increased prevalence of serum anti-PG-GSP antibody was found in juvenile chronic arthritis and both seropositive and seronegative rheumatoid arthritis (RA), compared with ankylosing spondylitis, systemic lupus erythematosus, myeloma and healthy controls. Anti-PG-GSP antibody was always of the IgG class and there was no correlation of anti-PG-GSP levels with C reactive protein, rheumatoid factor (RF) or anti-streptolysin O titres. There was no direct cross-reaction of RF with PG-GSP, nor did the presence of IgM-RF significantly interfere with the assay. Examination of paired serum and synovial fluid samples offered no evidence for local production of anti-PG-GSP antibody in synovial tissue. These data are compatible with an increased systemic immunization by bacterial fragments in RA.     

Idiotypes on antibodies to the La (SS-B) antigen are restricted and associated with the antigen binding site.

Rabbit anti-idiotypic antibodies were prepared against affinity purified autoantibodies to the ribonucleoprotein La (SS-B) antigen from the sera of three unrelated patients. Each anti-idiotype recognized private idiotypes expressed only on the immunizing anti-La antibody. In each case they were conformationally dependent and related to the antigen binding site. This demonstration of immunodominant private idiotypes on human autoantibodies to ribonucleoproteins is in direct contrast to the cross-reactive idiotypes described on rheumatoid factors and autoantibodies to DNA. We discuss the possibility that anti-La antibodies, unlike anti-DNA, arise as a result of autoantigenic stimulation.     

The B cell repertoire in rheumatoid arthritis. I. Frequency of EBV-inducible circulating precursors producing autoantibodies.

The frequency of B cell precursors producing antibodies against various autoantigens (Fc fragment of IgG, F(ab')2 fragment of IgG, type II collagen, cytoskeleton filaments and insulin) was determined in patients with rheumatoid arthritis (RA) using immortalization of peripheral blood B cells by the Epstein-Barr virus (EBV) and limiting dilution analysis. Equally large numbers of B cell precursors producing IgM-rheumatoid factors (RFs) were present in the peripheral blood of seronegative and seropositive RA patients and of controls. On average, 1 out of 15,000 B cells could be induced by EBV to secrete IgM-RFs, which represents 0.5-1% of the EBV-induced proliferating clones. By cloning or somatic hetero-hybridization of EBV cell lines derived from patients and controls, we obtained two types of monoclonal RFs: one polyreactive, reacting with Fc but also with the other autoantigens tested, and the other monoreactive, reacting with Fc only and that previously had only been found in the RA B cell repertoire. Moreover, patients and controls had similar numbers of circulating B cell precursors secreting IgM antibodies against other autoantigens that might be regarded as specific targets of RA (F(ab')2 fragment of IgG and type II collagen), and against cytoskeleton filaments that are targets of natural autoantibodies, increased in RA. The frequencies of EBV-induced B cells producing antibodies against all these autoantigens were of the same order of magnitude as the frequency of EBV-induced B cells producing RFs. The patients also possessed a similar number of precursors producing antibodies against insulin, an autoantigen irrelevant to the pathogenesis of the disease, taken as control. These data tend to demonstrate no abnormality in the autoantibody repertoire of B cells activable by EBV in RA, especially those secreting RFs. In vitro spontaneous RF secretion by circulating B cells was observed in seropositive RA patients but not in seronegative patients and in the controls tested. We enumerated the number of B cells spontaneously secreting RFs in seropositive RA patients and found that it correlated with the serum RF titer, but not with the number of RF-secreting B cells activated by EBV. The mean frequency values of B cells secreting RFs either spontaneously or after EBV infection were of the same order of magnitude, showing that the expanded population of in vivo-activated B cells was not (at least partially) infectable by EBV. This raised the possibility that EBV triggers a repertoire which may not reflect the status of B cells secreting autoantibodies in autoimmune diseases.     

A basic study of determination matrix metalloproteinase-3 and carbohydrate in rheumatoid factor in serum for diagnosis of rheumatoid arthritis

The examination of rheumatoid factor (RF), one of the diagnostic marker of rheumatoid arthritis (RA), showed negative about 25% of patients with RA. We analyzed a matrix metalloproteinase-3 (MMP-3) and a carbohydrate in rheumatoid factor (CA.RF) for diagnosis of RA: the former is used the kit "Panaclear MMP-3[Plate]" and the latter is used the kit "Picolumi CA.RF". The basic study of these reagents showed satisfactory results. In 73.3% of seronegative RA showed positive on both MMP-3 and CA.RF levels in serum, respectively. We found that these examinations might be useful for diagnosis of RA, especially during seronegative RA.     

Localization of Circulating Immune Complexes from Patients with Rheumatoid Arthritis in Murine Spleen Germinal Centres

In previous studies we have demonstrated high levels of rheumatoid factor (RF) and large-size (greater than 22S) circulating immune complexes (CIC) in the serum of rheumatoid arthritis (RA) patients with extra-articular disease. These findings were paralleled by a concurrent increase in the level of RF-associated cross-reactive idiotypes (CRI) and an apparent diversification of the RF repertoire detected in the serum of the same patients. In the present study we examine the ability of CICs to activate the complement system in vivo, and its possible influence on expanding the RF repertoire in RA patients with extra-articular disease. Activation of complement by CICs is the key for germinal centre localization and long-term retention of such complexes on the surface of follicular dendritic cells (FDC), and so provides a source for the selection of cells with high affinity receptors for IgG and leads to the establishment of immunological memory. CICs containing different immunoglobulin isotypes and from different patients localized in mouse spleen germinal centres. However, intense localization was mainly seen for IgG-containing complexes from the serum of patients with large-size (greater than 22S) IgG-IgM RF complexes. The ability of these complexes to localize in mouse spleen germinal centres was related to activation of the complement system via the classical pathway in the patients' sera. Localization of IgG complexes was significantly (P less than 0.05) higher in sera from RA patients with extra-articular disease than those with articular disease alone. This study demonstrates the ability of large-size (greater than 22S) IgG-IgM RF complexes to activate complement, and suggests a possible role for such complexes in modulating the immune response to IgG in RA patients with extra-articular disease.     

Analysis and modulation of the immune response of mice to acetylcholine receptor by anti-idiotypes

Anti-idiotypes were raised in mice against three well-characterized anti-acetylcholine receptor (AChR) monoclonal antibodies (mcAbs), as well as against polyclonal mouse anti-AChR antibodies. In binding experiments, the anti-idiotypic antibodies inhibited the binding of AChR only to the immunizing idiotype. However, a less restricted specificity was found in in vivo experiments. Mice producing anti-idiotypes were challenged with AChR and the idiotypic composition of their anti-AChR response was analysed using specific rabbit anti-idiotypic antibodies. It was found that preimmunization with a certain idiotype leads to the preferential suppression of this particular idiotype in the polyclonal response to AChR. However, preimmunization with either polyclonal or monoclonal anti-AChR antibodies resulted in a reduction of the overall anti-Torpedo AChR and anti-muscle AChR titers. This reduction was greater than would be expected from the representation of each of the respective idiotypes in the polyclonal anti-AChR serum, and may imply that in addition to the immunizing idiotype other anti-AChR idiotypes are also suppressed. Our results suggest that anti-idiotypes may have a potential for the modulation of the autoimmune response directed against AChR in myasthenia.     

A high incidence of rheumatoid factor idiotypes in monoclonal proteins in the serum and in lymphoma cells in patients with Sj?gren's syndrome

Patients with Sj?gren's syndrome (SS) develop lymphoproliferative disorders such as monoclonal gammopathies and non-Hodgkin's lymphomas. Cross-reactive idiotypes (CRI) were studied in 22 serum monoclonal immunoglobulins (Igs) and in cytoplasmic Ig in four B-cell lymphoma cells in patients with SS. This was done by utilizing monoclonal anti-idiotypic antibodies which were produced against monoclonal rheumatoid factors (RF) derived from three patients with SS and one patient with Waldenstr?m's macroglobulinemia. By the Western blotting or dot immunobinding technique, CRI was detected not only in monoclonal RFs but in monoclonal Igs which had different heavy- or light-chains from the original monoclonal RF used for immunization. A higher incidence of CRI was found in 22 monoclonal Igs associated with SS than in 27 monoclonal Igs in patients with Waldenstr?m's macroglobulinemia, multiple myeloma or malignant lymphoma. In four patients with malignant lymphoma associated with SS, three showed one or three CRI in the lymphoma cells, whereas only two out of 20 patients with other malignant lymphoma showed CRI, demonstrating a significant difference between two groups. These data indicate that monoclonal proliferation of B-cell lineage in patients with SS, benign or malignant, takes place more often among RF-producing clones than other B-cell disorders.     

Demonstration of idiotypes expressed on basophil-bound IgE antibodies by using anti-idiotype-induced histamine release in grass pollen-allergic patients.

This study was designed to analyse further the idiotypic cross-reactivity between anti-Lol p I murine monoclonal antibodies of IgG isotype and basophil-bound human IgE antibodies from grass pollen-sensitive patients. It was also designed to determine the expression frequency of the idiotypes present on cell-bound IgE. Rabbit anti-idiotypic antisera were produced against idiotypes of three anti-Lol p I monoclonal antibodies (290A-167, 539A-6 and 348A-6) of different specificities. Basophils from 19 patients reacting to Lol p I allergen, as shown by positive skin test reactions and by the presence of serum-specific IgE antibodies (measured by RAST), were challenged with these rabbit anti-idiotypic antibodies and the histamine released was measured. Our data indicate that IgE-borne idiotypes were expressed as follows: (i) co-expression of the three idiotypes in 15% of patients; (ii) co-expression of two idiotypes in 21% of patients; and (iii) expression of a unique idiotype (290A-167) in 42% of patients. Among the three idiotypes, 290A-167 was shown to be a public idiotype since it was expressed in 80% of patients. Fab fragments of anti-idiotypic antibodies could inhibit anti-idiotype-induced histamine release, but optimal conditions varied from one patient to another.     

Anti-Citrullinated Protein Antibodies in Rheumatoid Arthritis: As Good as it Gets?

Anti-citrullinated protein antibodies (ACPAs) have recently emerged as sensitive and specific serological markers of rheumatoid arthritis (RA), providing superior alternative of the rheumatoid factor (RF) test in the laboratory diagnostics of RA. The first members of this autoantibody family were anti-perinuclear factor (APF) and anti-keratin antibodies (AKA). It became evident that both APF and AKA recognize citrullinated epitopes of filaggrin. Citrullination is a post-translational modification of arginine by deimination, physiologically occurring during apoptosis, inflammation or keratinization. The presence of several citrullinated proteins has been demonstrated in the RA synovium. The identification of citrullinated epitopes as targets for anti-filaggrin antibodies led to the development of the first and later second generation anti-cyclic citrullinated peptide (anti-CCP) antibody assays. The widely used anti-CCP2 assays have high diagnostic sensitivity and specificity, and they also show important predictive and prognostic value in RA. The anti-Sa antibody has been identified a decade ago; however, recent studies confirmed that anti-Sa is directed against citrullinated vimentin, hence it is a new member of the family of ACPAs. The newly developed anti-mutated citrullinated vimentin (anti-MCV) assay has similar diagnostic performance than the anti-CCP2 ELISA; however, the diagnostic spectrum of the anti-MCV test is somewhat different from that of anti-CCP2. It’s especially useful in the diagnosis of RA in RF and anti-CCP2 seronegative patients. The combined application of anti-CCP2 and anti-MCV assays can improve the laboratory diagnostics of RA. The family of ACPAs is expected to expand; there is an increasing need for developing new diagnostic strategies after careful evaluation of the characteristics of the available assays. Zoltán Szekanecz and Lilla Soós with equal contribution.