Chicken antibodies: a tool to avoid false positive results by rheumatoid factor in latex fixation tests

19 rheumatoid factor (RF)-positive sera (Waaler-Rose test) and ten control sera were tested in latex agglutination. All RF-positive sera agglutinated latex particles coated with the different mammalian IgGs (cow, horse, human, mouse, sheep and rabbit) and with chicken anti-human C3 (which was used as a positive control), but none of them with non-specific chicken IgG. The control sera were all negative except when tested with chicken anti-human C3. Chicken antibodies should therefore be useful in assays were RF could interfere and give false positive reactions (e.g., nephelometry, latex agglutination or ELISA).     

Rheumatoid factor as a cause of positive reactions in tests for Epstein–Barr virus-specific IgM antibodies

Sera from twenty-eight patients with rheumatoid arthritis (RA) were titrated in indirect immunofluorescence tests for Epstein–Barr virus (EBV) specific antibodies. All had IgG antibodies to viral capsid antigen (VCA), 64% at titres [unk] 320, and 71% reacted also in tests for VCA-specific IgM antibodies at titres ranging from 20 to 640. The reactions observed in the IgM test were not due to VCA-specific IgM antibodies, however, but rather to rheumatoid factor (RF) usually an IgM antibody to the Fc regions of IgG. The titres recorded in the anti-VCA IgM test correlated significantly with the RF titres and both reactivities were abolished by adsorption onto IgG coated latex particles. In addition, they clearly depended upon the height of the IgG antibody titre to VCA, indicating that the more VCA-specific IgG molecules are present the more likely it is that RF will combine with them in sufficient quantity before or after their attachment to VCA-positive test cells so as to become detectable by the fluorescent antibodies to human IgM. Results comparable in every aspect were obtained with those sera from patients with Hodgkin's disease, nasopharyngeal or cervical carcinomas which reacted in the anti-VCA IgM test. Sera from patients with infectious mononucleosis may also contain RF, but in such cases its removal by adsorption onto IgG-coated latex particles did not generally reduce the VCA-specific IgM antibody titre. Removal of RF from any of the sera studied did not affect the titres of VCA-specific IgG and, where applicable, IgA or heterophil antibody titres. These results re-emphasize the pitfall created by RF noted previously in tests for virus-specific IgM antibodies.     

Re-evaluation of ELISA and latex agglutination test for rheumatoid factor detection in the diagnosis of rheumatoid arthritis.

An indirect ELISA for the determination of each isotype (IgM, IgG, IgA, IgD, IgE) of rheumatoid factors (RF) was performed with sera obtained from 77 patients with either classical or definite rheumatoid arthritis (RA) and 319 controls, using rabbit IgG as the antigen. The results were compared with those of a commercial latex agglutination test, using denatured human gamma globulin as the antigen for rheumatoid factor determination. At the cut-off level at which positive results were found in less than 5% of normal controls, ELISA for IgM RF determination had sensitivity, specificity, efficiency, positive predictive value and negative predictive value of 46.75%, 98.12%, 88.13%, 85.71%, 88.41%, while those for IgA RF were 46.75%, 93.42%, 84.34%, 63.16%, 87.91% and for IgG RF were 59.74%, 92.16%, 85.86%, 64.78%, 90.46%, respectively. These indices by latex agglutination test were 83.11%, 93.73%, 91.67%, 76.19% and 95.83%, respectively. IgD RF titre greater than or equal to 1:5 was detected in 19/77 RA patients and 4/200 normal controls while IgE RF titre greater than or equal to 1:5 was detected only in 7/77 RA patients. Thus, ELISA did not appear to have any advantage over latex agglutination test for diagnosis of RA.     

Which antigen to use in the detection of rheumatoid factors? Comparison of patients with rheumatoid arthritis and subjects with 'false positive' rheumatoid factor reactions

In the search for differences between rheumatoid factors (RF) in patients with rheumatoid arthritis (RA) and in non-rheumatoid subjects, the reactivity of IgM- and IgA-class RF with rabbit IgG (rIgG) and Fc fragments of rabbit and human IgG (rFc, hFc) was studied by enzyme immunoassay. From a community-based cohort (n = 7124) representing the adult population of Finland, RA patients (n = 130), other subjects positive in the Waaler-Rose (WaRo, sensitized sheep cell agglutination) test (n = 137), and controls matched for age, sex and living area were selected for further study. In RA sera there was a good correlation between the results in the WaRo test and in the IgM-RF ELISA (rIgG, rFc and hFc). A considerable number of 'false positive' sera, though positive in the WaRo test and the rIgG-ELISA, were negative in the rFc-ELISA. Twenty-two per cent of the false positive sera reacted with either hFc or rFc, both types being equally common. IgA-RF reacted more frequently with hFc than with rFc in both the RA and the 'false positive' sera. In some sera, IgM- and IgA-RFs reacted differently with human and rabbit Fc, e.g. IgM-RF reacted only with human Fc, and IgA-RF reacted with both hFc and rFc, thus suggesting different regulation of their formation.     

The laboratory identification of serum rheumatoid factor in the dog

A modified Rose-Waaler test with sheep red blood cells coated with canine IgG was found satisfactory for detecting rheumatoid (antiglobulin) factor (RF) in dog serum. Low titres of RF were found in some normal dogs. Most dogs with rheumatoid arthritis were positive for RF at titres of 1 in 40 or greater. A small proportion of dogs with diseases other than polyarthritis were also positive for RF. A commercial slide agglutination test used for detecting human RF was unsatisfactory for the dog, giving false positive and negative reactions. A latex tube agglutination test with latex particles coated with dog IgG was developed but non-specific agglutination was a constant technical problem.     

Clinico-diagnostic significance of rheumatoid factors of different classes in rheumatoid arthritis

Enzyme immunoassay was used to measure the content of rheumatoid factors (RF) of the M, A, and G classes in the blood sera of 111 patients with rheumatoid arthritis, 30 patients with osteoarthrosis deformans, and 60 donors. The ranges of normal values for each RF class were defined. The levels of all the three RF classes were significantly higher in the patients with rheumatoid arthritis vs. the reference groups. IgM, IgA, and IgG RF were regularly elevated in the patients with rapidly progressing rheumatoid arthritis and with visceritis, whereas IgM and IgG RF levels were increased in those with the maximal activity of the disease. RF of various classes are more often detectable by the enzyme immunoassay than by the Vaaler-Roset's or latex agglutination tests.     

Value of anti-cyclic citrullinated peptides antibodies in comparison with rheumatoid factor for rheumatoid arthritis diagnosis

The detection of anti-cyclic citrullinated peptides (CCP) antibodies is a new marker for diagnosis of rheumatoid arthritis. We screened 100 patients suffering from rheumatic pathologies or from other affections where rheumatoid factor is frequently detected. The screening was assessed by a second generation ELISA (Immunoscan RA) in comparison with agglutination assay (Latex and Waaler-Rose) and specific ELISA (IgM, IgG and IgA). The sensitivity of the anti-CCP is good (>70%) with an excellent specificity (98%). In our study the predictive value of the Immunoscan RA reached 71% and more among patients with joints symptoms. Anti-CCP antibody test could serve as a better diagnosis marker than rheumatoid factor.     

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.     

MRSPAH: A simple microtitre plate test for rheumatoid factors of different classes

A simple and inexpensive microtitre plate test (the mixed reverse (solid-phase) passive antiglobulin haemadherence test, or MRSPAH) has been developed for the measurement of antiglobulins (RFs) of different classes. Results obtained for IgM RF by this test have been compared with results of latex and Rose-Waaler (DAT) tests on rheumatoid arthritis (RA) sera. Levels of RFs of IgM, IgG and IgA classes have been measured by MRSPAH using rabbit IgG as antigen in RAs and normal people. 94% of RA sera tested were above the upper limit of normal for IgM and/or IgA RF. There was a considerable overlap between IgG RF levels in RAs and normals, although the means of the two groups were significantly different.     

Detection of antibodies against streptococcal peptidoglycan and the peptide subunit (synthetic tetra-D-alanyl-bovine serum albumin complex) in rheumatic-diseases.

Serum antibodies reactive with streptococcal cell wall peptidoglycan (PG) and its peptide subunit (synthetic tetra-D-alanine) were measured by enzyme-linked immunosorbent assay (ELISA) in patients with rheumatoid arthritis (RA), juvenile rheumatoid arthritis (JRA), osteoarthritis and acute rheumatic fever (RF) compared with healthy subjects. Using 'checkerboard' titrations, anti-PG antibody in human serum was detected at a concentration of PG antigen at 10 micrograms per well with serum dilutions of 1:1,000. For measurement of anti-tetra-D-alanine antibody, the antigen, (D-Ala4)31 was used at 0.5 micrograms per well and sera were diluted to 1:200. When the IgG antibody levels to the PG and the tetra-D-alanine of the sera of patients with RA, JRA and RF were compared with sera from healthy subjects, the sera of the patients had significantly higher levels than did healthy subjects. Antibody that reacted with the PG in serum was absorbed with purified group-specific C-carbohydrate (A-CHO), but A-CHO was not capable of absorbing anti-(D-Ala4)31 antibodies. Therefore, the peptide subunit should be used as antigen in order to measure the specific antibody to PG. Both anti-PG and anti-tetra-D-alanine antibody in human sera primarily belonged to the IgG2 subclass.     

Kinetic measurement of the interaction of rheumatoid factor with IgG-coated latex particles and the influence of the first component of human complement

Interaction between isolated rheumatoid factor (RF) of the IgM class or sera from patients with rheumatoid arthritis (RA) and IgG-coated latex particles has been studied kinetically by means of standard aggregometer equipment. The agglutination of particles mediated by isolated RF or RA-sera is inhibited by fresh normal human serum (NHS). The RF-inhibiting principle is heat-labile and recovered in the high molecular weight fractions of NHS separated on a G-200 column. Partially purified first component of complement, C1, also inhibits RF-mediated particle agglutination and disintegrates preformed RF-IgG-latex particle agglutinates. Addition of C1 to heated (56 degrees C, 30 min) NHS restores its RF-inhibiting activity. The most probable basis of this serum activity is competition between C1 with higher affinity for IgG bound to particles and RF. After about 5 min of incubation of NHS with IgG-latex particles the RF-inhibiting activity is gradually lost and interpreted to mean that C1 during the activation of the complement system is discharged from IgG bound to particles. The RF-inhibiting activity of NHS gradually decreases by incubation of serum with increasing doses of activators of the classical complement pathway probably due to the inability of activated C1 to hinder RF-interaction with IgG-particles. The presence in certain RA-sera of C1 in mainly activated form explains why such sera, even if they are fresh are able to agglutinate IgG-particles.     

The interference of IgM rheumatoid factor in enzyme-linked immunosorbent assays of rubella IgM and IgG antibodies

The interference of IgM rheumatoid factor (RF) in the indirect enzyme-linked immunosorbent assay (ELISA) for rubella IgM and IgG antibodies was evaluated quantitatively. In an ELISA for rubella IgM antibodies IgM RF produced false positive results in tests of sera with a rubella IgG concentration exceeding approximately 30 I.U./ml and an IgM RF concentration higher than 3.5 I.U./ml as determined by ELISA. Sera from 3 of 70 patients with recent rubella and sera from a similar proportion of blood donors contained IgM RF in a concentration exceeding this level.The false positive results were reproduced when an IgM RF preparation was added to sera containing rubella IgG. The rubella IgG values in ELISA, however, decreased after the addition of IgM RF . RF was absorbed by incubation of sera with a suspension of latex particles coated with aggregated human IgG. This method prevented the false positive results of the rubella IgM assay and increased the rubella IgM values of rubella convalescent sera. The activity in the rubella IgG assay also increased after absorption of RF. The interference of RF has been explained by a secondary binding of RF to rubella IgG-antigen complexes. The RF might subsequently be detected by the anti-IgM conjugate or compete with the anti-IgG conjugate.The common occurrence of IgM RF in concentrations sufficient to produce false positive results of the ELISA for specific IgM antibodies necessitates routine testing of IgM antibody positive sera for RF by a sensitive method and/or absorption of RF from such sera.     

Symposium on the immunodiagnosis of rheumatic and related diseases, Part II. Rheumatoid factors

Human rheumatoid factors are antibodies of IgG, IgA, or IgM class that show reactions with antigenic determinants present on other immunoglobulin molecules. The most commonly measured rheumatoid factor relates to the 19S IgM type, which reacts by agglutination of latex particles coated with 7S IgG and is often measured in the standard latex fixation test. Approximately 65 to 70 per cent of patients with rheumatoid arthritis show positive serologic tests for rheumatoid factor; however, a number of other chronic disease conditions are also associated with positive rheumatoid factor reactions, including infective endocarditis, sarcoidosis, leprosy, and other hyperglobulinemic conditions. Although extensive serologic and immunochemical studies have identified a number of specific antigenic structural sites on immunoglobulin molecules that react with rheumatoid factors, recent studies have shown that a certain proportion of such antibodies may show cross-reactivity with DNA-histone complexes as well. It is still not entirely clear how rheumatoid factors fit into the pathogenesis of rheumatoid arthritis itself.     

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.     

A simple method for detection of IgG rheumatoid factor

A simple method for detection of IgG rheumatoid factor (RF) in sera and joint fluid is described. The technique is based on the action of 0.1 M 2-mercaptoethanol mixed directly with the material. After 2 h of incubation determinations of human anti-IgG were performed by latex agglutination test. Comparison with data obtained by using the conventional method, sequential 24 h treatment with 0.1 M 2-mercaptoethanol and 0.01 M iodoacetamide, shows similar results for both methods.A correlation was observed between the presence of IgG_RF in synovial fluid and a severe clinical course with invalidating forms in patients suffering rheumatoid arthritis.     

The Distribution of Class-Specific Rheumatoid Factors Is Similar in Rheumatoid and Pre-Illness Sera

The occurrence of IgM, IgG, and IgA class rheumatoid factors (RF) was compared by means of ELISA in 21 sera from patients with established rheumatoid arthritis (RA) and in 22 pre-illness sera from subjects who 4 months to 5 years later developed seropositive rheumatoid arthritis. The positive cases were virtually confined to three groups of about equal size: positive for all three RF classes, positive for IgM-RF and IgG-RF, and positive for IgM-RF only. Thus, the distribution of the RF isotypes was similar in rheumatoid and pre-rheumatoid sera.     

Studies of Antibody to Herpes Simplex Virus Fcγ-Binding Protein gE in Patients with Rheumatoid Arthritis, Juvenile Rheumatoid Arthritis and Normal Controls

IgG antibody to gE, the Fc gamma-binding herpes simplex 1 (HSV-1) viral glycoprotein, was studied in 49 rheumatoid arthritis (RA) patients and 43 normal controls. Antibody to gD, another important HSV-1 antigen, was assayed in parallel. No difference between RA patients and normal controls was found in levels of anti-gE antibody measured by reactivity of IgG F(ab')2 fragments reacting with gE coated to ELISA plates. No difference in anti-gD antibody was recorded between normals and patients with RA. Levels of IgG anti-IgE antibody did not correlate with quantitative elevations of serum rheumatoid factor (RF) in RA patients. When IgG anti-gE and anti-gD were assayed in 20 patients with juvenile rheumatoid arthritis and 22 children controls, no significant differences were noted. However, when individual RFs from patients with RA were tested for reactivity against a panel of affinity-isolated F(ab')2 antibodies to gE, some evidence for individual autospecificity was obtained. Four of 20 monoclonal IgM RFs produced from RA patients' B cells showed marked elevations of reactivity with some RA patients' F(ab')2 antibodies to gE. All four of the monoclonal RFs showing this specificity were derived from RA synovial tissue B cells. These findings may provide support for the concept that some RFs in patients with RA show individual specificity for internal image determinants of IgG antibodies to viral Fc gamma-binding proteins.     

Prevalence of anti-Fab antibodies in patients with autoimmune and infectious diseases.

Sensitive ELISA were devised to examine the specificity of circulating IgM and IgA autoantibodies for whole human IgG, Fc and Fab fragments of human IgG. Sera from patients with autoimmune and infectious conditions such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), tuberculosis (TB), infectious mononucleosis (IM) and cystic fibrosis (CF) were studied. Results of the ELISA assays using whole human IgG as antigen revealed that a proportion of patients in each of the groups studied had circulating IgM and IgA rheumatoid factors (RF). Fifteen normal individuals studied were negative. In the latex positive RA group, IgM RF and IgA RF had primarily anti-Fc reactivity (100% and 93% respectively), although 3/15 patients also showed IgM anti-Fab reactivity and one patient had high IgA anti-Fab activity. Patients with SLE and TB who had detectable RF levels also revealed predominantly anti-Fc specificity. In contrast, examination of 25 patients with IM showed positivity for IgM RF activity in 8% of patients using whole IgG as antigen, 24% positivity using purified Fc fragments as antigen and 45% positivity when plates were coated with Fab fragments. Similarly, a large number of CF patients (54%) also showed predominantly IgM anti-Fab activity. Of interest, 69% of the CF patients who were all studied at the time of bacterial infection had detectable IgA RF levels, with 46% of these patients showing both IgA anti-Fc and anti-Fab activity. These findings suggest that autoantibody specificities in autoimmune and infectious diseases are different.     

Determination of human IgG and IgM class antibodies to West Nile virus by enzyme linked immunosorbent assay (ELISA)

An enzyme-linked immunosorbent assay (ELISA) was developed and used for the detection of IgG and IgM antibodies to West Nile virus in human sera. Thirteen paired sera of clinical cases and 24 control sera taken randomly from a blood bank repository were tested. The sera were reacted in microtiter plates coated with PEG-treated WNV antigen. IgG or IgM antibodies were quantitated by the use of alkaline-phosphatase-conjugated anti-human IgG or IgM antibodies. Of the 24 randomly collected serum samples, 7 were positive in the IgG-ELISA test. One positive by the IgM-ELISA was found to contain rheumatoid factor. In 12 of 13 paired sera of clinical cases, IgM as well as IgG antibodies were detected in the second serum sample taken about 3 wk after the onset of clinical signs. The IgM positive sera were screened for rheumatoid factor (RF) on IgG-coated plates. None of them contained RF. Antibody titers obtained by ELISA showed a good correlation with titers obtained by hemagglutination inhibition, complement fixation, and neutralization tests. The ELISA tests for detection of IgM and IgG antibodies to WNV therefore can replace the other serological methods for epidemiological surveillance and diagnostic purposes.     

Enzyme Immunoassay for IgG Rheumatoid Factor Combining with Homologous IgG

An enzyme immunoassay (EIA) was developed to detect, in human sera, IgG rheumatoid factor (RF) combining with human IgG. Wells of the EIA plate were coated with the Fc fragment of human IgG. Binding of RF was detected by goat antibodies to F(ab')₂ of human IgG followed by rabbit anti-goat IgG conjugated with alkaline phosphatase and finally by the substrate. Using this procedure, RF was detected in 35%-85% of various pathological human sera, but only in 7% of normal human sera. An analogous procedure was also described for detection, in rabbit sera, of an RF combining with rabbit IgG.