Comparison of the specificity of rheumatoid factor detected by latex fixation with that of regulatory rheumatoid factor.

BACKGROUND: Rheumatoid factor (RF), originally defined as pathological autoantibodies to IgG that are detected in rheumatoid arthritis, turned out to be multi-specific antibodies, some of which exhibit immunoregulatory properties. Recently, we identified a RF, the production of which confers resistance to experimental autoimmune diseases and is associated with the remission of autoimmune diseases. To differentiate the RF, we discovered from the one associated with rheumatic disease onset or progression and to reflect its immunoregulatory properties, we named it regulatory rheumatoid factor (regRF). Immunization with conformers of Fc fragments that expose regRF neoepitopes reduces collagen-induced arthritis in rats. Certain information about the specificity of classical RF and regRF indicates that these populations may be one and the same. Therefore, the aim of this study was to determine whether there is a difference between the classical RF and regRF. METHODS: Classical RF was measured in diseased blood by the latex fixation method, and regRF was detected by the agglutination of human IgG-loaded tanned erythrocytes. Competitive analysis was used to determine the specificity of rheumatoid factors. RESULTS: It was found that regRF and pathology-associated RF constitute different antibody populations. Pathology-associated RF is specific for lyophilized IgG. RegRF does not interact with IgG. RegRF is specific to conformers of IgG Fc fragments that have a reduced hinge. In latex-positive rheumatoid arthritis sera, regRF may be present in addition to pathology-associated RF. The latex fixation method detects both rheumatoid factor populations. CONCLUSION: RegRF and classical pathology-associated RF have different specificity.

Immunoglobulin G structure and rheumatoid factor epitopes.

Antibodies are important for immunity and exist in several classes (IgM, IgD, IgA, IgG, IgE). They are composed of symmetric dimeric molecules with two antigen binding regions (Fab) and a constant part (Fc), usually depicted as Y-shaped molecules. Rheumatoid factors found in patients with rheumatoid arthritis are autoantibodies binding to IgG and paradoxically appear to circulate in blood alongside with their antigen (IgG) without reacting with it. Here, it is shown that rheumatoid factors do not react with native IgG in solution, and that their epitopes only become accessible upon certain physico-chemical treatments (e.g. heat treatment at 57 °C), by physical adsorption on a hydrophobic surface or by antigen binding. Moreover, chemical cross-linking in combination with mass spectrometry showed that the native state of IgG is a compact (closed) form and that the Fab parts of IgG shield the Fc region and thereby control access of rheumatoid factors and presumably also some effector functions. It can be inferred that antibody binding to pathogen surfaces induces a conformational change, which exposes the Fc part with its effector sites and rheumatoid factor epitopes. This has strong implications for understanding antibody structure and physiology and necessitates a conceptual reformulation of IgG models.

Structure-function analyses of a stereotypic rheumatoid factor unravel the structural basis for germline-encoded antibody autoreactivity.

Rheumatoid factors (RFs) are autoantibodies against the fragment-crystallizable (Fc) region of IgG. In individuals with hematological diseases such as cryoglobulinemia and certain B cell lymphoma forms, the RFs derived from specific heavy- and light-chain germline pairs, so-called "stereotypic RFs," are frequently produced in copious amounts and form immune complexes with IgG in serum. Of note, many structural details of the antigen recognition mechanisms in RFs are unclear. Here we report the crystal structure of the RF YES8c derived from the IGHV1-69/IGKV3-20 germline pair, the most common of the stereotypic RFs, in complex with human IgG1-Fc at 2.8 Å resolution. We observed that YES8c binds to the CH2-CH3 elbow in the canonical antigen-binding manner involving a large antigen-antibody interface. On the basis of this observation, combined with mutational analyses, we propose a recognition mechanism common to IGHV1-69/IGKV3-20 RFs: (1) the interaction of the Leu432-His435 region of Fc enables the highly variable complementarity-determining region (CDR)-H3 to participate in the binding, (2) the hydrophobic tip in the CDR-H2 typical of IGHV1-69 antibodies recognizes the hydrophobic patch on Fc, and (3) the interaction of the highly conserved RF light chain with Fc is important for RF activity. These features may determine the putative epitope common to the IGHV1-69/IGKV3-20 RFs. We also showed that some mutations in the binding site of RF increase the affinity to Fc, which may aggravate hematological diseases. Our findings unravel the structural basis for germline-encoded antibody autoreactivity.

Identification of human IgG1 variant with enhanced FcRn binding and without increased binding to rheumatoid factor autoantibody.

Various studies have demonstrated that Fc engineering to enhance neonatal Fc receptor (FcRn) binding is effective for elongating half-life or increasing cellular uptake of IgG. A previous study has shown that a N434H mutation to enhance FcRn binding resulted in increased binding to rheumatoid factor (RF) autoantibody, which is not desirable for therapeutic use in autoimmune disease. In this study, we first showed that all the existing Fc variants with enhanced FcRn binding also show increased RF binding, and then identified specific mutations that could be introduced to those Fc variants to reduce the RF binding. Furthermore, we generated novel Fc variants that do not increase RF binding and show half-lives of 45 d in cynomolgus monkey, which is longer than those of previously reported Fc variants. In addition, we generated novel Fc variants with antigen sweeping activity that do not increase RF binding. We expect that these novel Fc variants will be useful as antibody therapeutics against autoimmune diseases.

The idiotypic network in the regulation of autoimmunity: Theoretical and experimental studies.

The regulation of autoimmunity is a key issue in fundamental immunology. Despite outstanding achievements on this front, we currently have more questions than answers. The idea of an immune network as a regulatory mechanism is quite attractive, since it enables us to explain the selectivity (specificity), and moreover the clonality, of the regulation. Nevertheless it remains unclear how this mysterious network of immune cells is organized, how it operates, and how it exerts control over autoimmunity. This article presents an attempt to understand how the immune network functions and how it controls autoreactivity. We present a mathematical model of the immune network that is based on principles of immune network organization and function that we arrived at from a survey of the available literature. To test the principles on which the mathematical model is based, we studied the model and compared the different responses to antigen that it generated with the results obtained from experimental studies of immune response. The modeled kinetics of idiotype and anti-idiotype in response to the administration of antigen are in good agreement with the experimental kinetics of idiotypic and anti-idiotypic antibodies. To obtain evidence of the existence of idiotypic mechanisms for regulating autoimmunity, we studied a mathematical model containing autoclones and compared the model results with data from experimental studies in a model of autoimmune hemolytic anemia in mice. Because the results from the theoretical and the experimental studies coincide, there is justification to conclude that autoreactive lymphocytes are normal components of the immune network within which they are regulated. We discuss a possible molecular/cellular mechanism for negative control of autoreactive cells as affected by anti-idiotypic antibodies.

Data mining of supersecondary structure homology between light chains of immunogloblins and MHC molecules: absence of the common conformational fragment in the human IgM rheumatoid factor.

It is shown that fuzzy search and data mining techniques of supersecondary structure homology for subunits of proteins using conformational code patterns of α-helix-type (3ß5α4ß) and ß-sheet-type (6α4ß4ß) fragments can be used to extract correlations between fragments of MHC class I molecules and the light chain of immunoglobulins. The new method of conformational pattern analysis with fuzzy search of structural code homology reflects well the shape of main chain rather than secondary structure in comparison with the DSSP method. Further, the data mining technique using the combination of h- and s-fragment patterns can quantify the supersecondary structure homology between any subunits of proteins with different amino acid sequences. Characteristic fragment patterns (string "shhshss"), which were sandwiched between two identical amino acid sequences His and Pro, were found in light chains of various types of immunogloblins, α-chain and ß-2 microglobulin of MHC class I and α-chain and ß-chain of MHC class II, but not in heavy chains of Fab immunoglobulin fragments and T cell receptors (TCR). Leukocyte immunoglobulin-like receptors (LILR) are related by the conformational fragment (string "shhshss") to ß-2 microglobulins as a type of pair forms (string "sohsss"). Further, human IgM rheumatoid factor, one of the immunogloblins, did not strongly exhibit the conformational fragment pattern. Nonclassic MHC class I molecules CD1D, MIC-A, and MIC-B, which have functions to activate NKT, NK, and T cells, did not also clearly show the patterns. These code-driven mining techniques can be utilized as a metadata-generating tool for systems biology to elucidate the biological function of such conformational fragments of MHC I and II molecules, which come in contact with various signal ligands on the surface of T cells and natural killer cells.

Cytokine measurements and possible interference from heterophilic antibodies--problems and solutions experienced with rheumatoid factor.

Cytokines are important in the understanding of the immune process in health and disease and are valuable indicators in diagnostics. Measurements of cytokines are based on immunometric methods, and it is important to understand possible pitfalls in these methods to produce reliable cytokine data. This paper focuses on obtaining optimal measurements when applying enzyme-linked immunosorbent assay (ELISA) or multiplex immunoassays (MIA). Cytokines are measured in serum or plasma, as well as in various other body fluids, all containing a series of antibodies and the possibility of interference from these. Some antibodies, such as heterophilic and human anti-animal antibodies, are able to interfere with all immunoassays, but the immunometric techniques are most prone to serious interference from this source. Another type, rheumatoid factor (RF) is a composite of different autoimmune antibodies which can be present in both blood and synovial fluid. RF is present in some arthritic diseases as well as in some other medical conditions. When present, especially RF IgM is known to interfere with the immunometric measurements. A possible and affordable solution to diminish this interference is PEG precipitation, but other efficient, but more expensive, methods, such as precipitation using Protein L or commercially available blocking agents, are also available. Interference of RF is at present not tested in all cytokine assays, but degree of interference from RF, human anti-animal and heterophilic antibodies, as well as from other possible disease-specific antibodies, must always be considered when developing and applying new assays for cytokine measurements.

Isolation, characterization, and molecular modeling of a rheumatoid factor from a Hepatitis C virus infected patient with Sjögren's syndrome.

We have previously isolated several IgG rheumatoid factors (RFs) from patients with both rheumatoid arthritis and idiopathic thrombocytopenia purpura using phage display system. To study IgG RFs in patients with other autoimmune diseases, phage display antibody libraries from a hepatitis C virus infected patient with Sjögren's syndrome were constructed. After panning, a specific clone RFL11 was isolated for characterization in advance. The binding activity and specificity of RFL11 to IgG Fc fragment were comparable to those of RFs previously isolated. The analysis with existed RF-Fc complex structures indicated the homology model of RFL11 is similar to IgM RF61 complex with high binding affinity of about 6 × 10⁻8 M. This effect resulted from longer complementarity-determining region (CDR) combining key somatic mutations. In the RFL11-Fc interfaces, the CDR-H3 loop forms a finger-like structure extending into the bottom of Fc pocket and resulting in strong ion and cation-pi interactions. Moreover, a process of antigen-driven maturation was proven by somatically mutated VH residues on H2 and H3 CDR loops in the interfaces. Taken together, these results suggested that high affinity IgG RFs can be generated in patients with Sjögren's syndrome and may play an important role in the pathogenesis of this autoimmune disease.

Sialylation determines the nephritogenicity of IgG3 cryoglobulins.

Monoclonal 6-19 IgG3 anti-IgG2a rheumatoid factor derived from lupus-prone MRL-Fas(lpr) mice can induce GN and cryoglobulinemia, but the features that confer nephritogenic potential are not completely understood. Asparagine-linked oligosaccharide chains of 6-19 IgG3 mAb are poorly galactosylated and hardly sialylated, possibly contributing to the pathogenic potential of 6-19 IgG3 rheumatoid factors. Here, we used the 6-19 model of cryoglobulin-associated GN to define the relative contributions of galactosylation and sialylation, in relation to cryoglobulin activity, to the nephritogenic potential of IgG3 antibodies. We generated one highly sialylated and two distinct more galactosylated 6-19 IgG3 rheumatoid factor variants. Although the mere extent of galactosylation had no effect on either the cryogenic and nephritogenic activities of 6-19 IgG3 rheumatoid factor, terminal sialylation attenuated the nephritogenic potential of 6-19 IgG3 by limiting its cryoglobulin activity. These data suggest a protective role of IgG sialylation against the development of cryoglobulin-mediated GN, highlighting the anti-inflammatory activity of sialylated IgG antibodies.

Preferential targeting of somatic hypermutation to hotspot motifs and hypermutable sites and generation of mutational clusters in the IgVH alleles of a rheumatoid factor producing lymphoblastoid cell line.

Epstein-Barr virus transforms human peripheral B cells into lymphoblastoid cell lines (LCL) that secrete specific antibodies. Our previous studies showed that a monoclonal LCL that secretes a rheumatoid factor expressed activation-induced cytidine deaminase (AID) and displayed an ongoing process of somatic hypermutation (SHM) at a frequency of 1.7×10⁻³ mut/bp in its productively rearranged IgVH gene. The present work shows that SHM similarly affects the nonproductive IgVH allele of the same culture. Sequencing of multiple cDNA clones derived from cellular subclones of the parental culture, showed that both alleles exhibited an ongoing mutational process with mutation rates of 2-3×10⁻5 mut/bp×generation with a high preference for C/G transition mutations and lack of a significant strand bias. About 50% of the mutations were targeted to the underlined C/G bases in the WRCH/DGYW and RCY/RGY hotspot motifs, indicating that they were due to the initial phase of AID activity. Mutations were targeted to the VH alleles and not to the Cµ or to the GAPDH genes. Genealogical trees showed a stepwise accumulation of only 1-3 mutations per branch of the tree. Unexpectedly, 27% of all the mutations in the two alleles occurred repeatedly and independently within certain sites (not necessarily the canonical hotspot motifs) in cellular clones belonging to different branches of the lineage tree. Furthermore, some of the mutations seem to arise as recurrent mutational clusters, independently generated in different cellular clones. Statistical analysis showed that it is very unlikely that these clusters were due to random targeting of equally accessible hotspots, indicating the presence of 'hypermutable sites' that generate recurring mutational clusters in the IgVH alleles. Intrinsic hypermutable sites may enhance affinity maturation and generation of effective mutated antibody repertoires against invading pathogens.

Rheumatoid factor interference in immunogenicity assays for human monoclonal antibody therapeutics.

Rheumatoid factors (RFs) are endogenous human antibodies that bind to human gamma globulins. RFs demonstrate preferential binding to aggregated gamma globulins and are involved in the clearing mechanism of immune complexes. Immunoassays designed to measure human anti-human antibodies (HAHA) after administration of monoclonal antibody therapeutics are thus vulnerable to interference from RFs. When using a sensitive electrochemiluminescent (ECL) bridging immunoassay, samples from subjects with rheumatoid arthritis demonstrated much higher baseline reactivity than healthy subjects. Interference was found to be dependent on the aggregation state of the therapeutic antibody that had been conjugated with the detection reagent (ruthenium). Size exclusion high performance liquid chromatography (SE-HPLC) demonstrated that of the total integrated peaks, as little as 0.55% high molecular weight aggregates (>600kDa) were sufficient to cause increased reactivity. Stability studies of the ruthenium and biotin conjugated therapeutic antibody indicated that storage time, temperature and buffer formulation were critical in maintaining the integrity of the reagents. Through careful SE-HPLC monitoring we were able to choose appropriate storage and buffer conditions which led to a reduction in the false reactivity rate in therapeutic-naïve serum from a rheumatoid arthritis population.

Ongoing somatic hypermutation of the rearranged VH but not of the V-lambda gene in EBV-transformed rheumatoid factor-producing lymphoblastoid cell line.

Epstein-Barr virus (EBV) transforms human peripheral B cells into lymphoblastoid cell lines (LCLs) that secrete specific antibodies. In contrast to peripheral blood B cells, LCLs express the activation-induced cytidine deaminase (AID) gene, a key enzyme in the generation of somatic hypermutation (SHM) in immunoglobulin variable genes. We have previously studied an LCL that secretes a rheumatoid factor (RF: an IgM(lambda) anti-IgG antibody) and identified the accumulation of SHM at a frequency of 1.5 x 10(-3)mut/bp in the rearranged variable region heavy chain gene (VH) of its RF sub-culture (i.e., RF-2004). The aim of the present work was to find out whether SHM was initiated as an early event following EBV transformation. Our results show that already the earliest RF-culture (RF-1983) mutates its VH at a somewhat higher frequency of 1.9 x 10(-3). Overall, we detected 17 point mutations in the RF-2004 culture and in 26 cellular clones derived from the RF-1983 and RF-2004 cultures. Most of the mutations were due to C to T or G to A transitions, with preferential targeting to WRCH/DGYW hotspot motifs, indicating that they were due to the initial phase of AID-directed mutations. A genealogical tree demonstrates that mutations were accumulated in a stepwise manner with 1-2 mutations per cell division. However, no mutations were found in the rearranged V-lambda (Vlambda) gene in the same RF-cultures and their subclones (i.e., <1.2 x 10(-4)mut/bp). To our knowledge this is the first reported clonal cell line that generates SHM in the VH, but not in the Vlambda. It may be due to abrogation of a cis-regulatory element(s) in the Vlambda or to a lack of a specific trans-acting factor which differentially direct the SHM machinery to this gene. Out of the 17 point mutations detected in both cell lines there were, 1 stop codon, 3 mutations which obliterated the binding of the RF antibody to its IgG antigen and 1 or 2 mutations which enhanced antigen-binding affinity. These results show that the evolutionary developed germline encoded antibody combining site is highly sensitive to amino acid replacements. Our combined findings that the RF cells accumulate in a stepwise manner up to 1-2 point mutations/sequence per cell division and the generation of high percentage of functionally deleterious mutations, are in accord with the 'multiphase-recycling model' of SHM, which states that B cells in the germinal center are subjected to multiple rounds of somatic mutations interchanged with periods of antigenic selection.

Anti-CCP antibodies have more diagnostic impact than rheumatoid factor (RF) in a population tested for RF.

To compare the diagnostic powers of rheumatoid factor (RF) and anti-cyclic citrullinated peptide (CCP) in a population selected for its high statistical relevance, over a 6-month period, an informed consent to test for anti-CCP was obtained from 1,025 consecutive patients for whom RF was ordered at a University laboratory. Within 1 year, a diagnosis was obtained without informing the physician about the anti-CCP result. Extensive statistical analyses were performed. A total of 768 patients satisfied the inclusion criteria, and 132 were classified as having RA, yielding a pre-test probability of RA of 17%. The sensitivities for anti-CCP and RF were 62 and 64% (P = 0.83), and the specificities were 97 and 90% (P < 0.001), respectively. The positive predictive value (PPV) was 79% for anti-CCP and 56% for RF (P < 0.001), whereas the negative predictive value was 92% for both. The likelihood ratio (LR) was 17.9 for anti-CCP and 6.2 for RF (P < 0.005). Forty RA patients were diagnosed with RA of less than 2 years length, and the same significant statistic differences between anti-CCP and RF were observed. Placing the results of both tests together, or using different cutoff points, increased the diagnostic utility of the tests. The anti-CCP test has statistically shown significant higher specificity, PPV, and LR for RA than the RF test in a clinically diverse population. If new criteria are to be devised to help diagnose early RA, anti-CCP should be included because it has a greater diagnostic impact than RF.

Crystal structure of a human autoimmune complex between IgM rheumatoid factor RF61 and IgG1 Fc reveals a novel epitope and evidence for affinity maturation.

Rheumatoid factors (RF) are autoantibodies that recognize epitopes in the Fc region of immunoglobulin (Ig) G and that correlate with the clinical severity of rheumatoid arthritis (RA). Here we report the X-ray crystallographic structure, at 3 A resolution, of a complex between the Fc region of human IgG1 and the Fab fragment of a monoclonal IgM RF (RF61), derived from an RA patient and with a relatively high affinity for IgG Fc. In the complex, two Fab fragments bind to each Fc at epitopes close to the C terminus, and each epitope comprises residues from both Cgamma3 domains. A central role in the unusually hydrophilic epitope is played by the side-chain of Arg355, accounting for the subclass specificity of RF61, which recognizes IgG1,-2, and -3 in preference to IgG4, in which the corresponding residue is Gln355. Compared with a previously determined complex of a lower affinity RF (RF-AN) bound to IgG4 Fc, in which only residues at the very edge of the antibody combining site were involved in binding, the epitope bound by RF61 is centered in classic fashion on the axis of the V(H):V(L) beta-barrel. The complementarity determining region-H3 loop plays a key role, forming a pocket in which Arg355 is bound by two salt-bridges. The antibody contacts also involve two somatically mutated V(H) residues, reinforcing the suggestion of a process of antigen-driven maturation and selection for IgG Fc during the generation of this RF autoantibody.

Rheumatoid factor interference in the determination of carbohydrate antigen 19-9 (CA 19-9).

BACKGROUND: Investigation of a 61-year-old Caucasian male suffering from fatigue and weight loss led to the finding of a carbohydrate antigen 19-9 (CA 19-9) concentration of 80 kU/L using an ADVIA Centaur analyser. Determination of CA 19-9 on Vidas, AxSYM and Architect i2000 systems gave normal results. His rheumatoid factor concentration was very high (900 kIU/L) and assay interference was suspected. METHODS: Besides using several laboratory procedures to show the cause of the interference, we tried to estimate the frequency of the suspected interference. Therefore, two studies were performed. The first was carried out in a multicentre setting using four different CA 19-9 methods on 51 randomly selected samples with high rheumatoid factor concentrations and ten samples containing no or very low rheumatoid factor. In the second study we used heterophilic blocking tubes for 68 routinely analysed samples with CA 19-9 concentrations ranging between 37 and 250 kU/L using an ADVIA Centaur analyser. RESULTS: In the multicentre study we found eight discrepant CA 19-9 results, but only one was clearly due to interference. We showed that the interference detected, just as in the index case, was caused by rheumatoid factor. The other discrepancies could not be explained, but are probably related to method-dependent differences. In the 68 routinely analysed samples, no interference could be shown using the heterophilic blocking tubes. CONCLUSIONS: Although interferences in the CA 19-9 assay are not frequent, the ADVIA Centaur system appears to be more sensitive to rheumatoid factor interference. The lack of standardisation remains an important issue for this assay. The determination of CA 19-9 during the follow-up of patients should be performed using a single method. If, however, there is any clinical doubt about a result, CA 19-9 should be determined using another method to exclude possible interferences.

[A comparative structural analysis of Fab-fragments of monoclonal rheumatoid and non-rheumatoid immunoglobulins M].

The spin-labeling method was used to study the Fab- and Fab-RF-fragments of IgM and IgM-RF, respectively. The spin-label 2,2,6,6-tetramethyl-4-dichloro-sym-triazinyl-aminopiperidine-1-oxyl was introduced into the peptide moiety of the proteins. The rotational correlation time t of the spin-label carrier was determined based on the temperature-viscosity dependence of the EPR spectra parameters of the spin-labeled proteins. The tau values for Fab- and Fab-RF-fragments were 21 +/- 2 and 12 +/- 1 ns, respectively. The data strongly suggest that the significantly lower tau value for the Fab-RF-fragment may be due to the local structural flexibility of the fragment, which in turn may explain the peculiarities of IgM-RF as an autoantibody.

Among B cell non-Hodgkin's lymphomas, MALT lymphomas express a unique antibody repertoire with frequent rheumatoid factor reactivity.

We analyzed the structure of antigen receptors of a comprehensive panel of mature B non-Hodgkin's lymphomas (B-NHLs) by comparing, at the amino acid level, their immunoglobulin (Ig)V(H)-CDR3s with CDR3 sequences present in GenBank. Follicular lymphomas, diffuse large B cell lymphomas, Burkitt's lymphomas, and myelomas expressed a CDR3 repertoire comparable to that of normal B cells. Mantle cell lymphomas and B cell chronic lymphocytic leukemias (B-CLLs) expressed clearly restricted albeit different CDR3 repertoires. Lymphomas of mucosa-associated lymphoid tissues (MALTs) were unique as 8 out of 45 (18%) of gastric- and 13 out of 32 (41%) of salivary gland-MALT lymphomas expressed B cell antigen receptors with strong CDR3 homology to rheumatoid factors (RFs). Of note, the RF-CDR3 homology without exception included N-region-encoded residues in the hypermutated IgV(H) genes, indicating that they were stringently selected for reactivity with auto-IgG. By in vitro binding studies with 10 MALT lymphoma-derived antibodies, we showed that seven of these cases, of which four with RF-CDR3 homology, indeed possessed strong RF reactivity. Of one MALT lymphoma, functional proof for selection of subclones with high RF affinity was obtained. Interestingly, RF-CDR3 homology and t(11;18) appeared to be mutually exclusive features and RF-CDR3 homology was not encountered in any of the 19 pulmonary MALT lymphomas studied.

Inmunoanálisis positivos falsos debido a la presencia de factor reumatoide / False-positive immunoassays caused by rheumatoid factor

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Reactivity of agalactosyl IgG with rheumatoid factor.

BACKGROUND: The difference in the reactivity of rheumatoid factor (RF) with agalactosyl IgG and galactosyl or intact IgG is controversial. METHODS: ELISAs for IgM-RF, using agalactosyl IgG and galactosyl IgG as a coated antigen, were developed. The sera of patients with RA, systemic lupus erythematosus (SLE), chronic liver diseases (CLD) and healthy subjects were measured for IgM-RF. An absorption test was performed using these IgGs with or without heat-pretreatment to determine any differences in the antigenicity between agalactosyl IgG and intact IgG. RESULTS: IgM-RF titers were higher against agalactosyl IgG than those against both heated-intact and intact IgG in three of nine RA patients' sera. Three other serum samples showed similar titers against all three forms of IgG and the remaining three serum samples showed varied results. IgM-RF titers were higher in sera of RA patients than those of SLE, CLD and healthy subjects. Absorptions of RA sera showed that RF was completely absorbed by 5x of absorptions with agalactosyl IgG as well as intact IgG. CONCLUSIONS: The reactivity of IgM-RF is higher with agalactosyl IgG than with heated-intact or intact IgG. The antigenic epitopes of agalactosyl and intact IgG were shown to be closely similar by the absorption test.