Rheumatoid Factor Assays Research Articles

Diagnostic Performances of Anti-Cyclic Citrullinated Peptides Antibody and Antifilaggrin Antibody in Korean Patients with Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a systemic autoimmune disease of unknown etiology. We studied the diagnostic performances of anti-cyclic citrullinated peptides antibody (anti-CCP) assay and recombinant anti-citrullinated filaggrin antibody (AFA) assay by enzyme linked immunosorbent assay (ELISA) in patients with RA in Korea. Diagnostic performances of the anti-CCP assay and AFA assay were compared with that of rheumatoid factor (RF) latex fixation test. RF, anti-CCP, and AFA assays were performed in 324 RA patients, 251 control patients, and 286 healthy subjects. The optimal cut off values of each assay were determined at the maximal point of area under the curve by receiver-operator characteristics (ROC) curve. Sensitivity (72.8%) and specificity (92.0%) of anti-CCP were better than those of AFA (70.3%, 70.5%), respectively. The diagnostic performance of RF showed a sensitivity of 80.6% and a specificity of 78.5%. Anti-CCP and AFA showed positivity in 23.8% and 17.3% of seronegative RA patients, respectively. In conclusion, we consider that anti-CCP could be very useful serological assay for the diagnosis of RA, because anti-CCP revealed higher diagnostic specificity than RF and AFA at the optimal cut off values and could be performed by easy, convenient ELISA method.

Referral criteria in early rheumatoid arthritis

Dr Suresh (September 2004 JRSM1) highlights the importance of early referral in a patient with suspected rheumatoid arthritis or RA-like polyarthritis. As he indicates, the diagnosis is a clinical one and absence of rheumatoid factor (RF) is not informative. Unfortunately, there is reason to think some general practitioners delay referral when the serology is negative. Sinclair and Hull,2 looked at the reasons general practitioners request RF assays and the effect of the result on their subsequent action. The requests were generally backed by appropriate clinical signs of RA; however, 32% of responders believed that a negative RF excluded RA, even in a patient who fulfilled the American Rheumatism Association4 classification criteria. Referrals were made to a consultant rheumatologist in 52% of patients with a positive RF, but 66% of patients with a negative RF would not be referred. In only 1.2–2.5% of instances were patients referred for specialist review on clinical grounds alone. Seronegative RA should account for about 25% of total cases. With 60% of cases seronegative at presentation,5 the above data suggest that a sizeable number of RA patients are being missed. Should RF be removed from the decision-making process? If general practitioners relied wholly on clinical criteria and the RF test was restricted to rheumatologists only, we could expect a large increase in consultant referrals.

A new polymeric coating for protein microarrays

Despite the increasing interest in arraying proteins in a high-density format, several technical issues still impede the development of protein microarray technology. One of the major problems is the availability of substrates that are able to bind native proteins with high density. In this study, we investigated the suitability of a novel surface as a support for protein microarrays. A polymeric glass coating is obtained by physical adsorption of a N, N-dimethylacrylamide (DMA), N, N-acryloyloxysuccinimide (NAS), and [3-(methacryloyl-oxy)propyl]trimethoxysilyl (MAPS) copolymer. The coating procedure provides a fast and inexpensive method of producing hydrophilic functional surfaces. The slide performance was investigated in a protein–protein interaction experiment and in the assessment of rheumatoid factor (RF) in human serum samples. The results demonstrate that the ligands immobilized on the polymeric surface maintain an active conformation and are easily accessible, providing a detection limit of 54 amol/spot. Moreover, in the RF assay, after hybridization with the sera, the slides have a low background, leading to a detection limit of 900 amol/spot.

Why do general practitioners request rheumatoid factor? A study of symptoms, requesting patterns and patient outcome.

To investigate the reasons why general practitioners (GPs) request rheumatoid factor (RF) assays, we studied 200 consecutive requests for RF from general practice in 1995. By means of an audit questionnaire, we studied 100 negative, 50 positive and 50 borderline RF results and compared these with the presenting symptoms that prompted the request, the GPs' understanding of the significance of the result, the referral intention and behaviour of the GP, and finally, the patient outcome after 5 years. There was an 80% response rate. The presenting symptoms closely matched the American Rheumatism Association revised criteria for the classification of rheumatoid arthritis, indicating that the requests were made on valid clinical grounds, with polyarthralgia, morning stiffness and joint pain being the most common. Most GPs considered a negative or positive result to be meaningful, in that a positive RF meant that a referral was more likely than with a negative or borderline result, even in the presence of appropriate symptoms in all three groups. Seventeen to thirty per cent felt that the test excluded or confirmed RA. The result appeared to influence this decision to a greater extent than it should. A 5-year follow-up on these patients showed that 26/40 patients with positive RF were referred, and that 25 of them developed a rheumatic disease of some kind, with 17 patients eventually being diagnosed with RA. Only 17/80 patients with negative RF were referred, the remainder having no autoimmune problem evident after 5 years, 11 of them developing a rheumatic disease, and only three being diagnosed with RA. Although this is a locally based study, we believe the conclusions would be applicable to all laboratories and GPs undertaking these tests. RF requests are made on valid clinical grounds by GPs, but there may be an over-reliance on the results as regards referral behaviour. If patients were referred on clinical grounds, this would significantly lengthen consultants' waiting lists.

Anti-D Antibody in the Serum of an Rh-Positive Vietnamese Woman

© symptoms.5 It is speculated that the therapeutic benefit of IFN-α is related to its combined antilymphoproliferative and antiviral actions.5 Given the availability of a specific therapy, it is important that HCV-associated type II cryoglobulinemia be recognized and diagnosed as early as possible. It should be part of the differential diagnosis in patients with an unexplained IgM M-protein, signs/symptoms of cryoglobulinemia, a positive cryoglobulin assay, and/or an unexplained positive rheumatoid factor assay.

Diagnostic Accuracy of the Anti-Citrulline Antibody Assay for Rheumatoid Arthritis

Rheumatoid arthritis (RA) is the most common autoimmune rheumatic disease, but specific and practicable tests for its diagnosis are lacking. We evaluated the diagnostic accuracy of a new commercial ELISA in detecting anti-cyclic citrullinated peptide (CCP) antibodies for the diagnosis of RA. Anti-CCP antibodies were determined in 330 serum samples: 98 from RA patients and 232 from controls, including patients with connective tissue diseases, other rheumatic diseases, viral infections, Lyme disease, autoimmune thyroiditis, cancer, and monoclonal gammopathy, and sex- and age-matched healthy subjects. Intra- and interassay CVs were 5-13% and 9-17%, respectively. Rheumatoid factor (RF) was also assayed in every sample, and results were compared to anti-CCP for sensitivity and specificity. At a cutoff value of 50 units, sensitivity was 41% (confidence interval, 31-50%) and specificity was 97.8% (95-100%). Anti-CCP-positive RA patients had a mean antibody concentration of 1100 units (range, 57-3419 units), and anti-CCP-negative RA patients and controls had mean values of 7.6 and 6.8 units, respectively (range, 1-39 units). The area under the ROC curve was 0.71 (95% confidence interval, 0.63-0.78). RF had a higher sensitivity (62%) and a lower specificity (84%) than anti-CCP. When the two antibodies were used together, specificity was 99.6%. Anti-CCP antibody testing may be useful if performed concomitantly with RF assay to diagnose patients with suspected early RA.

An isotype switched and somatically mutated rheumatoid factor clone isolated from a MRL-lpr/lpr mouse exhibits limited intraclonal affinity maturation.

Employing site-directed mutagenesis we have reconstructed and expressed the germ-line precursor of an expanded rheumatoid factor (RF) clone. This RF clone, designated clone F, was isolated from an autoimmune MRL/MpJ-lpr/lpr mouse. Most of the clone members were extensively mutated and isotyped-switched. The predominant isotype of clone F was gamma 3. The RF bound specifically to the MRL gamma 2 a allotype (Igh-1j) but not to the B6 gamma 2a allotype (Igh-1b). The germ-line antibody was also found to bind gamma 2a in an RF assay. The affinities of the germ-line RF and representative members of the clone were measured in an ELISA-based equilibrium binding assay. The dissociation constant (Kd) of the germ-line RF was 2.5 x 10(-6) M. All of the expressed clone members had affinities within a two- to sixfold range of the germ line, indicating that the mechanisms of somatic hypermutation and selection resulted in only limited affinity maturation of this autoantibody clone.

Antiperinuclear factor in juvenile rheumatoid arthritis.

The serological diagnosis of juvenile rheumatoid arthritis (JRA) is difficult, with only 7-10% of patients 19S IgM rheumatoid factor positive. About 60-70% of patients are positive for hidden 19S IgM rheumatoid factor, but this test requires serum separation and is not available in most laboratories. Antiperinuclear factor has been described in both seropositive and seronegative adult patients with rheumatoid arthritis, but has not been thoroughly evaluated in children with JRA. This study determined the diagnostic sensitivity and specificity of antiperinuclear factor in patients with JRA. Serum samples from 64 children with JRA, 24 with systemic lupus erythematosus (SLE), and 24 control subjects were tested for the presence of antiperinuclear factor. A total of 10 (83%) of seropositive, polyarticular onset and six (37%) of seronegative, polyarticular onset patients with JRA were positive for antiperinuclear factor. The occurrence of antiperinuclear factor in five (19%) with pauciarticular onset and one (10%) with systemic onset (JRA) as well as in four (17%) with SLE was not increased compared with the control subjects (1/24 (4%)). These data show an overall diagnostic sensitivity and specificity of 34 and 90% respectively in this group of patients. Although less sensitive than the hidden rheumatoid factor assay, the antiperinuclear factor assay is easier to perform and may contribute to the serological diagnosis of JRA.

A human monoclonal IgA rheumatoid factor using the VkIV light chain gene.

A human hybridoma stably secreting IgA rheumatoid factor (RF) was produced by cell hybridization with peripheral blood lymphocytes of a patient with rheumatoid arthritis. The RF was of the IgA1 isotype with kappa-light chains and was useful for standardization or specificity controls in class-specific RF assays. RF activity was detected only when the IgA molecular were in a polymeric state, and could be measured by enzyme linked immunosorbent assay as well as in conventional agglutination based tests. The RF had the modified Ga fine specificity described previously for several RFs and for protein A. The immunoglobulin V genes used were isolated and sequenced. The light chain was encoded by the VkIV gene rearranged to Jk2; compared to the published VkIV germ line gene there was 90% nucleotide homology. The heavy chain gene used belonged to the VHI family and was rearranged to JH4. Comparisons with published sequences revealed 90% homology with the recently characterized VH gene expressed by RF-TS3, a rheumatoid synovia RF hybridoma.

Focal Scleroderma and Severe Cardiomyopathy

A 21-month-old infant presented with simultaneous localized scleroderma and severe cardiomyopathy with heart failure. Cardiac abnormalities and serological changes (positive rheumatoid factor assay, elevated IgM and IgG levels, and elevated erythrocyte sedimentation rate) reverted to normal with prednisone therapy, and there was substantial, though incomplete, resolution of her skin changes during the same period. To our knowledge, this is the first patient with definite, clinically significant cardiac involvement associated with focal scleroderma. The possibility of internal organ involvement, including cardiac involvement, must be considered with focal scleroderma as well as with progressive systemic sclerosis.

Antibodies to immunoglobulin-G in dog sera, synovial fluids and aqueous humor: a comparative study of rheumatoid factor assays, suitable for routine application

The incidence of anti-IgG antibodies (rheumatoid factors, RF) in body fluids (sera, synovial fluids and aqueous humor) selected from 62 normal and 275 diseased dogs was studied. Fluids were assayed by canine versions of standard agglutinating and/or precipitating RF assays with routine application in human practice. The number of RF detected by dog IgG-coated particles was substantially higher by latex fixation test (LFT) than by modified Rose-Waaler (RW) test (61/144 vs. 14/144). This did not result from false positives by LFT since latex activity was completely inhibited by aggregated dog IgG. Some evidence is presented indicating that results obtained by standard RW in particular, but also those obtained by standard LFT, might be improved by modifying testing conditions currently used. Body fluids were further studied for the presence of precipitins to aggregated dog IgG in 0.6% agarose (gel precipitation test (GPT)). The frequency of RF was higher by GPT than by LFT, both in normal control fluids (for sera 26/52 vs. 19/52) and patient material (for sera 135/197 vs. 95/197). Thus, the canine RF appear to be a serum component with an unexpectedly high frequency in both normal and diseased dogs, but grossly underestimated by the recommended routine RF assays based on agglutination. The GPT, which combines a superior detection rate of theoretically also agglutinating RF with an inability to detect RF quantitatively, seems an ideal RF ‘indicator’ test to dictate improvements to the quantitative LFT/RW assays so as to facilitate RF detection at clinically relevant concentrations. Thus optimized, RW/LFT would provide the optimal detection apparatus for the ultimate isolation of the relevant ‘RF’ repertoire present, for comparative studies aimed ultimately at unraveling the etiopathogenesis of the ‘real’ RF.

Rheumatoid factor tests in the diagnosis and prediction of rheumatoid arthritis.

Four assays of rheumatoid factor (RF) have been measured on serum from 213 individuals from 13 families containing at least two sufferers from classical or definite rheumatoid arthritis (RA). Families were not uniformly RF positive or negative, and there was no evidence that non-RA RF positivity was inherited. Four individuals developed definite RA over a two year period, showing that the family members were at increased risk of RA. IgG RF and latex RF assays predicted the RA in the four cases. An association of RF positivity in RA with DR4 was observed, but this may be related to disease severity.

Cross-reactivity between solid-phase immunoassay plates and intermediate filaments demonstrated by human monoclonal antibodies

While screening supernatants of human-human hybridomas for rheumatoid factor and anti-cellular activity we found that a significant number of supernatants which react with the Falcon polyvinyl chloride immunoassay plate used in an enzyme-linked immunosorbent rheumatoid factor assay also react with intracellular intermediate filaments.

Circulating immune complexes in Hashimoto's thyroiditis. Correlation to HLA and autoantibodies.

Circulating immune complexes (IC) were determined in sera from 41 patients with Hashimoto's thyroiditis by a polyclonal rheumatoid factor (pRF) assay based on the inhibition of the agglutination of IgG-coated latex particles. Elevated levels of IC were found in 63% (26/41) of the sera. There was a significant correlation (Rho = 0.91, P less than 0.001) between results obtained before and after treatment of sera with dithiothreitol (DTT). By precipitation with 2.5% polyethylene glycol (PEG) before pRF inhibition assay, the activity of IC was found in only 7% (3/41) of the sera. Size chromatography studies of the sera showed the inhibitory activity predominantly in the intermediary region. When found in the IgM-region the activity was not reduced by DTT. By use of a polyethylene glycol complement consumption test (PEG-CC) the occurrence of IC was 10% (4/41). It was not possible to find any correlation between the detectable IC and the presence of microsomal, thyroglobulin, or thyroid-stimulating antibodies. Based on our studies the sizes of IC seemed to be heterogeneously distributed and the majority were not precipitated by PEG (2.5%), final concentration). The antibodies involved in the formation of complexes seemed to be of IgG or IgA classes. HLA-D typing of the patients showed a non-significant association between HLA-Dw5 and low levels of IC while the presence of HLA-Dw4 was significantly associated with a high level of IC (P less than 0.05).

False positive results in class-specific rheumatoid factor (RF) assays due to interaction between RF and Fc fragments of anti-immunoglobulin indicator reagents

A recently developed, simple immunological method, diffusion-in-gel ELISA, has been adapted for class-specific determination of rheumatoid factor (RF). Evidence was obtained of an analytical error due to interaction between RF and antigenic determinants on the Fc part of the indicator immunoglobulin (Ig) molecules used. This was eliminated by replacing the usual indicator reagent, complete Ig molecules, by conjugated Fab or F(ab′) 2 fragments. The findings imply that unless the RF-Fc interaction mentioned is avoided in the assaying technique, false positive results may be obtained for, e.g. IgG RF and IgA RF in sera containing IgM RF.

Performance of a new rate-nephelometric assay for rheumatoid factor, and its correlation with tube-titer results for human sera and synovial fluid.

We evaluated a rate-nephelometric assay for rheumatoid factor (RF), as developed for use with the Beckman Immunochemistry System (ICS). Within- run precision (CV) for low-, mid-, and high-dilution samples (60-400 kilo-int. units/L) was 3.5, 1.5, and 1.6%; between-run precision was 3.4%. Analytical linearity was excellent. Biological interference resulted in some degree of nonlinearity in more than 70% of the patients' samples tested. Sensitivities were 96.5 and 94.1% and specificities were 98.5 and 95.3% for the ICS RF and Wampole slide methods, respectively, for a clinically defined population of 170 patients. Results for 100 ICS RF-positive samples correlated well with concomitantly measured Calbiochem-Behring tube-titers. Weekly measurement of Calbiochem-Behring. Hyland Diagnostics, and ICL Scientific tube-titer values for RF along with the ICS RF values on samples from the same patients indicated stable ICS RF values but showed at least +/- 1 tube dilution variances both within and between the tube-titer methods. Therefore, it may not be appropriate to compare a precise new method with a relatively imprecise comparison method. Treatment of RF-positive samples with dithiothreitol, which disrupts the pentameric character of immunoglobulin M, rendered the samples negative by repeat ICS RF assay and confirmed the method's specificity for pentameric immunoglobulin M-RF. Serum RF concentration as determined by ICS paralleled changes in clinical symptoms in a patient treated with both effective and relatively ineffective regimens, which suggests a useful role for the assay in monitoring efficacy of clinical treatment.

Gonococcal arthritis-dermatitis syndrome. Study of serum and synovial fluid immune complex levels.

Immune complexes from serum and synovial fluid were detected by the C1q binding assay in 12 patients with disseminated gonococcal infection. Since immune complexes were regularly higher in synovial fluids than in paired sera and were not detected by the monoclonal rheumatoid factor radioimmunoassay, we suggest that IgM may be present in these complexes and that this represents a primary immune response. In contrast, only 2 of 10 patients with local gonococcal infection were slightly positive both with the C1q assay and the monoclonal rheumatoid factor assay. In patients with disseminated gonococcal infection, immune complexes closely paralleled the disease activity and negatively correlated with complement levels. Synovial fluid immune complexes seem to occur in the early and aseptic phase of polyarthritis and to aid the entrance of circulating gonococcal organisms. From the results of our study, it seems that immunologic processes initiate and/or sustain inflammation in disseminated gonococcal infections that appear, at least in part, as a form of reactive arthritis.

A quantitative assay for IgA rheumatoid factor

We have developed a solid-phase radioimmunoassay capable of detecting nanogram quantities of human IgA rheumatoid factor (RF) in biological fluids. Human IgM RF, IgG RF, IgG, IgA, IgM and whole serum did not significantly interfere with the IgA RF assay. Patients with sero-positive rheumatoid arthritis (RA) had significantly higher concentrations of IgA RF than sero-negative RA patients or healthy adult controls. Concentrations of IgA RF in paired sera and synovial fluids from sero-positive RA patients were comparable. Levels of IgA RF demonstrated a moderately good correlation with levels of IgM RF in sero-positive RA sera ( r = 0.673). However, the ratio of IgA RF concentration to IgM RF concentration in sero-positive RA sera varied widely.

Association of anti-F (ab')2 antibodies (pepsin agglutinators) with immune complexes as determined by enzyme-linked immunosorbent assays.

A solid-phase assay for the detection of anti-F(ab')2 antibodies is described. Wells of microtiter plates are coated with F(ab')2, dilutions of sera are added, and IgG bound to the solid phase is detected using peroxidase-labeled Protein A. Anti-F(ab')2 antibodies were found in 61% of sera from patients with rheumatoid arthritis, 77% with subacute bacterial endocarditis , and 34% with systemic lupus erythematosus. Simultaneous analysis of these sera for immune complexes (IC), using modified Clq and monoclonal rheumatoid factor assays, showed that a correlation existed between anti-F(ab')2 antibodies and the levels of IC. Characterization of anti-F(ab')2 antibodies by inhibition and absorption experiments and by immunological and physical means indicated that they were similar to serum proteins described in the 1960s and designated pepsin agglutinators.

A solid-phase assay for circulating immune complexes using monoclonal rheumatoid factors and peroxidase-linked protein A

A solid-phase monoclonal rheumatoid factor (mRF) assay for circulating immune complexes (IC) is described. Microtiter plates are coated with mRF, serum diluted 1 : 4 is added, and complexed IgG is detected with protein A coupled to peroxidase. Selective binding was obtained with aggregated human gamma-globulin and soluble IC prepared in vitro. Sucrose density gradient ultracentrifugation studies indicate that mRF binds intermediate-size IC (between 7S and 19S). Abnormal levels of IC were found in 78% of sera from patients with rheumatoid arthritis, 67% with systemic lupus erythematosus, and 87% with subacute bacterial endocarditis.