Determination of IgE rheumatoid factor. Comparison of a solid-phase radioimmunoassay and an enzyme immunoassay

A solid-phase radioimmunoassay and an enzyme-linked immunosorbent assay have been developed for the identification of IgE rheumatoid factor (IgE RF). For both, human IgG was used as antigen. Bound IgE RF was detected by means of commercially available rabbit anti-human IgE antiserum and 125I-labelled sheep anti-rabbit IgG as well as monoclonal anti-human-epsilon-chain antibody and horse-radish peroxidase-labelled sheep anti-mouse IgG. The presence of IgM RF did not cause false positive results. Correlation in the results of both assays were significant, the reproducibility was very good. In 50.6% of 79 sera from patients with rheumatoid arthritis IgE RF has been detected with both or one of the methods. Only in 1 out of 12 seronegative rheumatoid arthritis sera IgE RF was identified.     

Enzyme-linked immunosorbent assay for determination of IgM rheumatoid factor.

A semi-automated enzyme-linked immunosorbent assay (ELISA) for determination of IgM rheumatoid factor was established. Human gammaglobulin (Cohn fraction II) was used as an antigen on a solid phase of polystyrene microtitre trays. The results were read on a spectrophotometer. ELISA and the sheep cell agglutination test were compared in a study of sera from 400 blood donors, 53 patients with active rheumatoid arthritis and of 200 sera received for routine determination of rheumatoid factor. The results of the two tests correlated well. The ELISA procedure was precise, objective, inexpensive and well suited for quantitative routine determination of IgM rheumatoid factor.     

Development of a new quantitative rheumatoid factor test

A new simple identification of rheumatoid factor on nitrocellulose was developed that allows quantitative detection. Verification of the results was done by comparison with the established Waaler-Rose-Test and Latextest: Correlation coefficient of linear regression for Latextest rLR = 0.717 and for Waaler-Rose-Test rLR = 0.665; Spearman rank correlation coefficient for Latextest rs = 0.798 and for Waaler-Rose-Test rs = 0.700. The interday and intraday variations showed good results. The histograms for different patient groups showed significant distributions. The nitrocellulose-test satisfies ARA-criterium No. 6.     

Measurement of IgM and IgG antibodies to Yersinia by solid-phase radioimmunoassay

Solid-phase radioimmunoassays for IgM and IgG antibodies to Yersinia enterocolitica serotype 3. Y. enterocolitica serotype 9, and Yersinia pseudotuberculosis IA are described. Bacteria were adsorbed onto polystyrene balls, and serum antibodies bound to the antigen-coated balls were quantitated by their capacity to bind 125I-labeled antibodies to human immunoglobulins.     

A solid-phase radioimmunoassay for detection of IgM antibodies to hepatitis A virus.

The conditions for a sensitive and specific solid-phase radioimmunoassay (RIA) for the detection of IgM antibodies to hepatitis A virus (HAV) were optimized, and the RIA was used to assay sera from patients with hepatitis. IgM antibodies to HAV reached highest concentrations between one and three weeks after onset of icterus and were measurable in follow-up sera for at least 12 months after infection. To prove the specificity, the IgG antibodies were separated from patient sera by sucrose density-gradient centrifugation. The remaining IgM antibodies, after treatment with beta-mercaptoethanol, did not bind in the RIA, and, when the anti-IgM antibody bound to the solid phase was replaced with anti-IgG, a negative result was obtained with incubation of IgM antibody to HAV. Also, the presence of IgG was shown not to interfere with measurement of IgM antibody to HAV. Finally, as a further specificity control, 50 sera positive for rheumatoid factor or from patients infected with hepatitis B virus, cytomegalic inclusion disease, infectious mononucleosis, influenza A virus, rubella, or measles were tested, and all of these sera were negative for IgM antibody to HAV.     

Production of IgM rheumatoid factor by normal lymphocytes after stimulation with preparations containing IgM rheumatoid factor from patients with juvenile arthritis.

Preparations containing IgM rheumatoid factor (RF) and hidden IgM RF were isolated from the serum samples of nine patients with juvenile rheumatoid arthritis. Six of these preparations stimulated lymphocytes from normal donors to produce IgG and IgM, of which up to 11% had IgM RF activity. In contrast, the polyclonal activator pokeweed mitogen also stimulated IgM production, but only 1% had IgM RF activity. A relation between the activator and IgM RF or hidden IgM RF is suggested. This is based on the positive correlation between IgM RF concentration in these preparations and their ability to stimulate lymphocytes to produce IgG, IgM, and IgM RF. These data indicate that preparations from patients with juvenile rheumatoid arthritis containing IgM RF and hidden IgM RF are potent stimulants of lymphocytes from normal donors, with specific production of IgM RF.     

Serodiagnosis of viral hepatitis A by a solid-phase radioimmunoassay specific for IgM antibodies

A solid-phase radioimmunoassay for detecting specific IgM antibodies to hepatitis A virus (HAV) was developed and characterized. The test utilized microtiter plates coated with anti-IgM to specifically absorb the IgM antibodies from the test serum. The anti-hepatitis A IgM antibodies are measured by the specific consecutive binding of hepatitis A antigen and radiolabelled anti-hepatitis A antibodies (anti-HA). In 6 chimpanzees infected with HAV, IgM anti-HA was detected from about the first date of elevated transaminases and was positive for about 3 months. The usefulness of the test was confirmed by testing acute phase sera of 30 patients from a common source outbreak of epidemic hepatitis, and negative sera from 2 control groups. A collection of serum specimens from 190 patients with sporadic HBsAg-negative hepatitis in Brazil was also tested and an etiologic association with HAV was confirmed in the majority of these cases.     

Complement mediated inhibition of immune precipitation in rheumatoid arthritis: studies on interaction of heat aggregated IgG with IgM rheumatoid factor.

Serum samples from patients with seropositive rheumatoid arthritis contain an inhibitor of complement mediated inhibition of immune precipitation (CMIP). This inhibitory effect can be produced by the addition of either purified monoclonal or polyclonal IgM rheumatoid factor (RF) to human serum. The specificity of the rheumatoid factor influences the degree of inhibition, and when precipitation occurs the rheumatoid factor coprecipitates with the antigen-antibody complex. In rheumatoid sera there was a significant positive correlation between IgM RF concentration and inhibitory activity, though the range of inhibitory activity seen for the same concentration of rheumatoid factor was considerable. Small quantities of heat aggregated IgG (HAGG) had a much greater effect on the measurement in an enzyme linked immunosorbent assay (ELISA) of IgM RF than they did on the inhibitory activity of IgM RF in the CMIP assay. Larger quantities of HAGG initiated complement activation and increased the precipitation of immune complexes. IgM RF reduced the complement activating properties of HAGG by reducing the amount of Clq which bound to the aggregate. The mechanisms by which IgM RF overcomes CMIP in rheumatoid sera may involve its inhibitory effects on the binding of Cl to the antigen-antibody complex.     

IgE rheumatoid factor. Occurrence and diagnostic importance in comparison with IgM rheumatoid factor and circulating immune complexes.

The sera of 80 patients suffering from rheumatoid arthritis (RA)--30 of them with extraarticular manifestations (EAM) and 50 patients with articular disease only--of 25 patients with other joint diseases and of 30 normal healthy subjects, were analyzed for the presence of 1) IgE rheumatoid factors (IgE RF) by means of a solid phase radioimmunoassay and an ELISA, 2) IgM rheumatoid factors by using solid phase radioimmune techniques, and 3) circulating immune complexes (CIC) with the C1q binding test (C1q BT) and the solid phase conglutinin binding test (SPCBT). The best technique to discriminate RA patients with EAM from RA patients without EAM and patients with other articular diseases was the determination of IgE RF (73.3%, 38.0%, 0%, resp.) compared to IgM RF (86.7%, 78.0%, and 56.0%) and CIC (C1q BT: 80.0%, 66.0% and 45.0%; SPCBT: 46.7%, 22.0%, and 20.0%). The results suggest a certain role of IgE RF for diagnosis and a possible development of extraarticular manifestations in rheumatoid arthritis.     

Qualitative and quantitative expression of VHI associated cross reactive idiotopes within IgM rheumatoid factor from patients with early synovitis.

Monoclonal rheumatoid factors (RFs) of the major Wa cross reactive idiotype group have been shown to express exclusively VKIII subgroup light chains and VHI subgroup heavy chains. A VKIII associated cross reactive idiotope (CRI) (17-109), however, was shown not to be exclusively expressed on IgM paraproteins having rheumatoid factor activity or to be present at increased levels in the sera of patients with rheumatoid arthritis (RA). Three VHI associated CRIs have been defined with monoclonal antibodies and quantitative studies of their representation are reported, together with VKIII, in IgM and IgM RF isolated from the sera of patients with early synovitis, some of whom progressed to classical RA. The results show (a) the probed CRIs were expressed predominantly on IgM RF rather than on non-RF IgM; (b) 5-10% of IgM RFs from patients with classical RA expressed the CRIs, but this represented a lower proportion of IgM RFs than observed for normal individuals or patients with self limiting synovitis; (c) VKIII light chains were highly associated with IgM RFs rather than non-RF IgM (75% and 25% respectively). It is suggested that the CRIs probed are markers for germline gene encoded antibodies or sequences resulting from minimal mutation of germline genes. The lowered proportion of RFs expressing CRIs in RA may therefore be evidence of polyclonal activation or specific antigenic stimulation, or both, resulting in maturation of the RF response with recruitment of further VH genes or extensive mutation of germline genes. These studies show that monoclonal RFs are relevant models of RF produced in RA and that the repertoire of RF autoantibodies may be encompassed within a small number of CRI expressing families.     

Functional affinity of IgM rheumatoid factor in patients with rheumatoid arthritis and other autoimmune diseases.

The functional affinity of IgM rheumatoid factors (RF) was measured in 31 patients with rheumatoid arthritis (RA), 24 with systemic lupus erythematosus (SLE), 13 with Sjögren''s syndrome (SS), and in 13 seropositive healthy individuals. The functional affinity of IgM RF from patients with RA was significantly lower than in the other clinical groups studied. In addition, there was a significant inverse correlation between functional affinity and titre of IgM RF in all the groups. These results suggest that the usual mechanisms of affinity based selective pressure (somatic diversification and antigen selection) may operate differently for autoantibodies to serum antigens such as IgG.     

Inhibitory effect of IgM rheumatoid factor on immune complex solubilization capacity and inhibition of immune precipitation

Purified IgM rheumatoid factors (RF; 3 monoclonal and 2 polyclonal) were shown to inhibit, in a dose-dependent manner, 2 complement-mediated functions, i.e., the immune complex solubilization capacity and the inhibition of immune precipitation. Inhibition of immune complex solubilization capacity occurred only if RF was added at the same time as, but not after, addition of the complement source. Experimental evidence suggests that the effects of RFs were not related to their anticomplementary activity, but rather required the attachment of RF to the Fc region of the IgG molecule. Although no clinical data are available so far, it might be plausible that these newly described properties of RF have biologic relevance.     

Comparison of a slide agglutination test, LeptoTek Dri-Dot, and IgM-ELISA with microscopic agglutination test for Leptospira antibody detection

A slide agglutination test (SAT), LeptoTek Dri-Dot and IgM-ELISA were compared with a microscopic agglutination test (MAT) for the detection of Leptospira antibodies. Paired sera from 10 patients whose leptospirosis was clinically suspected and diagnosed by MAT, were evaluated in this study. Our data, especially from acute samples, demonstrate the SAT and Dri-Dot were more sensitive as initial screening tests than MAT. IgM-ELISA has an advantage over MAT, SAT, and Dri-Dot since the results can be interpreted from a single serum testing if the results of the test are positive. Eight of the ten cases could be diagnosed by IgM-ELISA. Our data suggest that IgM-ELISA may be used for the diagnosis of leptospirosis. However, the agglutination test is useful for screening and for secondary infection cases for which IgM antibodies may be undetectable. MAT can be performed as a reference test and when information regarding the causative serovar is required.     

Relationship of serum IgG rheumatoid factor to IgM rheumatoid factor and disease activity in rheumatoid arthritis

Rheumatoid factors (RF) constitute the major autoantibodies in rheumatoid arthritis (RA). RF are directed against IgG Fc, are polyclonal, and are predominantly of the IgG and IgM classes. RF may participate in both synovial and extraarticular inflammation in RA, although the precise roles of serum IgG and IgM RF are unclear. The purpose of our study was to correlate serum IgG RF with serum IgM RF levels measured by radioimmunoassay and with clinical disease activity in 42 prospectively evaluated seropositive RA patients. IgM RF correlated with IgG RF levels and articular disease activity. IgG RF correlated with IgM RF but not with articular disease activity when adjusted for IgM RF.     

IgA rheumatoid factor synthesis by dissociated synovial cells. Characterization and relationship to IgM rheumatoid factor synthesis

We examined patterns of IgA rheumatoid factor (RF) and IgM-RF synthesis by dissociated synovial cells obtained from 27 patients with seropositive rheumatoid arthritis. Synthesis of IgA-RF was observed in 19 of 34 synovial cell preparations from these patients and constituted a mean of 16% of the total IgA produced. IgA-RF expression correlated only weakly with IgM-RF production (r = 0.385) and could be dissociated from production of IgA-RF (and IgM-RF) exhibited by simultaneously obtained peripheral blood plasma cells. While wide variations were observed in the ratio of IgA-RF:IgM-RF produced by synovial B cells in the patient sample studied, remarkable consistency in the relationship of IgA-RF to IgM-RF synthesis was observed over time in different joints of the same patient. IgA-RF synthesized by dissociated synovial cells was predominantly of the IgA1 subclass and existed in both monomeric and polymeric forms. Our results are compatible with the view that local production of IgA-RF and IgM-RF are regulated independently of each other.     

In vitro synthesis of IgM and IgM rheumatoid factor in seronegative arthritides

Summary In vitro patterns of IgM and IgM rheumatoid factor (RF) synthesis exhibited by peripheral blood B cells (MNL) obtained from healthy individuals as well as patients with seropositive and seronegative rheumatoid arthritis (RA) have been previously examined. The present study was performed in a group of patients with non-rheumatoid seronegative arthritis (SNA) in order to compare patterns of in vitro IgM and IgM RF synthesis to that previously observed with seropositive and seronegative RA. Eighteen patients with SNA (4 ankylosing spondylitis, 10 psoriatic arthritis, and 4 unclassified variant disease) were studied as well as 18 healthy adult controls.Spontaneous release of IgM RF by MNL was not observed in SNA or controls. In contrast, IgM RF was detected in pokeweed mitogen (PWM)-stimulated MNL culture supernatants from 9/18 SNA (mean±SD=17.0±9.9 ng/10⁶ cells), and 10/18 normal controls (16.7±9.9 ng). Synthesis of IgM by PWM-stimulated MNL from SNA (2 062±1 200 ng) was significantly less than observed with MNL from controls (4 093±1 896 ng) (P<0.001). There were no differences among the various SNA subsets with regards to the levels of IgM and IgM RF produced either spontaneously or after PWM stimulation. IgM RF constituted a small fraction of the total IgM in SNA and normals (0.9% and 0.5%, respectively). This is clearly distinct from seropositive RA in which we have previously established that IgM RF constitutes a substantial fraction of the total IgM (10.7%) (P<0.01). IgM and IgM RF production did not appear to be significantly influenced by immunosuppressive medication.The results suggest that regulatory mechanisms governing the expression of RF are intact in patients with SNA and that lack of in vivo RF production in these patients cannot be related to an absolute deficiency of PWM-inducible mature RF committed B cells.     

Hidden and classical 19S IgM rheumatoid factor in a juvenile rheumatoid arthritis patient

Hidden 19S IgM rheumatoid factors (RF), i.e., 19S IgM RF which can be detected in the IgM containing fraction after acid gel filtration of serum, are found in 59-68% of patients with juvenile rheumatoid arthritis (JRA). Their presence generally correlates with disease activity. We describe a 12 year-old female with a polyarticular onset of JRA who, during her first 15 months of disease, was seronegative but had hidden RF titers of 1:128----1:256. Inhibition studies on her hidden RF showed specificity for HIgG greater than RIgG and equal specificity for the human IgG subclasses (IgG1 = IgG3). In the second and third year of disease, she became seropositive with RF titers varying from 1:40 to 1:320 while her hemolytic titers on her IgM fractions were decreased from 1:128 to 1:32. Inhibition studies now demonstrated a higher avidity for RIgG; and HIgG3 inhibited more than HIgG1. These studies documented for the first time a JRA patient who early in the disease was negative for RF and positive for hidden RF, and who later became seropositive.     

The functional affinity of IgM rheumatoid factor is related to the disease duration in patients with rheumatoid arthritis

OBJECTIVE—To determine the relevance of the functional affinity of IgM rheumatoid factor (RF) to the clinical and serological characteristics of patients with rheumatoid arthritis.
METHODS—The functional affinity of IgM RF of 57 seropositive rheumatoid arthritis patients was evaluated by an enzyme linked immunosorbent assay based on the use of a chaotropic agent. The inhibition index was taken as an estimate of functional affinity. The patient group was divided into high functional affinity subgroup 1 (functional affinity < 0.5, n = 37) and low functional affinity subgroup 2 (functional affinity > 0.5, n = 20). The medical records of all patients were reviewed with a particular note of the disease activity and the articular damage score.
RESULTS—The disease duration was shorter (P < 0.01) in subgroup 1 patients [7.9 (SD 6.4) years] than in subgroup 2 patients [13.4 (11.29) years], so that Ritchie's, Lee's, and Steinbrocker's indices were lower in the former than in the latter (P < 0.01, 0.001, and 0.01, respectively). In contrast, erythrocyte sedimentation rates, C reactive protein concentrations, antinuclear antibody, and HLA DR4 prevalences were similar in the two subgroups.
CONCLUSIONS—Different forms of RF are present during progression of the disease.