A reappraisal of the monoclonal rheumatoid factor test for circulating immune complexes: a comparison of two monoclonal rheumatoid factor reagents

Assays involving monoclonal rheumatoid factor (mRF) reagents are frequently used for the detection of circulating immune complexes, particularly in the rheumatic diseases. A study has been performed to investigate the interaction of two purified mRF reagents with normal sera, sera and synovial fluid from patients with rheumatic diseases and with heat-aggregated human IgG used as a model of immune complexes. Interaction has been measured by a simple laser nephelometric technique and a sensitive radiolabelled mRF precipitation assay. Both mRF reagents showed little reactivity with normal sera but reacted strongly with many of the pathological specimens. Similarly both mRFs reacted with large molecular sized heat aggregates of IgG while a variable reactivity was found with uncomplexed or monomeric IgG. However in pathological sera, both mRFs reacted predominantly with monomeric IgG and a significant correlation was found between the two reagents. This reactivity with monomeric IgG remained after separation of pathological sera in low pH sucrose gradients suggesting it was not due to the presence of small immune complexes of the classical type. In addition no reactivity was found with either reagent with IgG-RF intermediate complexes. It is concluded that mRF reagents are not specific for IgG containing immune complexes. They also react with monomeric IgG and this reactivity is particularly prominent in certain pathological sera. The possible nature of this reactive monomeric IgG is discussed.     

Flow-cytometric detection of circulating immune complexes

In an effort to develop an assay which would rapidly detect and analyze circulating immune complexes, we have adapted the Raji cell radioimmunoassay (RIA) to a flow cytometric (FCM) analysis. The advantages of immune complex analysis by FCM are many. Foremost is the effectiveness and efficiency of the FCM method relative to the radioimmunoassay (RIA) method. The data demonstrated that FCM detection is three times more sensitive than RIA detection. Only populations of viable Raji cells bearing immune complexes are analyzed because parameters of the FCM analysis permitted the elimination (gating out) of dead cells. The determinations are rapid and the data are immediately available for several additional analyses. Because of the availability of many fluorescent monoclonal antibodies to complement components, viral antigens, light chains and immunoglobulin isotypes, it is possible to detect many components that might be present in the Raji cell bound complexes. Finally, the Raji cells can be characterized in different stages of their cell cycle to generate information about the state of the cells and the density of the receptors involved in binding the complexes.     

The detection of heat-aggregated IgG (as a model for immune complexes) by reverse passive haemagglutination using a human monoclonal rheumatoid factor coupled to erythrocytes

A human monoclonal IgM rheumatoid factor (RF) produced in vitro by an Epstein-Barr virus (EBV)-immortalized cell line was purified by protein A-Sepharose adsorption and coupled by the chromic chloride method to human erythrocytes. The RF-coupled cells were incorporated in reverse passive haemagglutination (RPH) assays to detect immune complexes (IC) using heat-aggregated human IgG as a model system. The sensitivity of the RPH was comparable to an enzyme-linked immunosorbent assay (ELISA) using sheep C1q for the detection of ICs.     

Influence of serum complement and rheumatoid factor on detection of immune complexes by the C1q and monoclonal rheumatoid factor solid-phase assay

Soluble purified monoclonal and polyclonal rheumatoid factor, total serum complement, and soluble C1q all inhibit the detection of model tetanus toxoid/anti-toxoid immune complexes in the solid-phase C1q assay. The binding of these immune complexes to solid-phase monoclonal rheumatoid factor is less inhibited by soluble C1q and by total serum complement, but clearly decreased by soluble monoclonal or polyclonal rheumatoid factor. Serum complement does not reduce the size of these model complexes. We recommend the use of low ionic strength EDTA (10 mM) to partly neutralize the complement-mediated inhibition. This procedure is shown to be superior to currently used higher EDTA concentrations and to the use of IgG-Sepharose.     

A simple radiolabelled rheumatoid factor binding assay for the measurement of circulating immune complexes.

A simple assay for circulating immune complexes has been developed and the optimal conditions for reaction defined. Partially purified radiolabelled polyclonal or monoclonal rheumatoid factor was incubated with aggregated IgG or soluble immune complexes and the resultant macromolecule precipitated with 3% polyethylene glycol. Monoclonal rheumatoid factor was much the better reagent, allowing the detection of approx. 0.35microgram/ml or more of aggregated IgG. Soluble immune complexes formed in vitro at between x2--x20 antigen excess were detectable over a wide range of molecular size. Clinical studies indicated that the assay is a useful addition to the currently available techniques for measuring circulating immune complexes.     

An enzyme-immunoassay method for detecting circulating immune complexes by inhibition of polyclonal rheumatoid factor.

An enzyme-immunoassay for circulating immune complexes (IC-EIA is described which depends on inhibition of binding of rheumatoid factor (RF) to aggregated IgG. The assay involves 3 steps: (1) sera are pretreated with an aggregated IgG sorbent in order to remove any endogenous RF; (2) absorbed sera are than incubated with a polyclonal RF of known titre and an aggregated IgG sorbent; (3) after washing, the amount of RF on the solid phase is revealed by an IgG-glucose oxidase conjugate.Results are expressed as per cent inhibition of RF fixation. The assay range extends from 1 to 100 mg/l. Intra- and inter-assay coefficients were 6 and 9% respectively.Specificity has been assessed by various tests: dose-response curve with aggregated monomeric or denatured IgG and human albumin localization of the inhibitory fraction of sera after Sephadex G-200 chromatography, absence of interference of IgG, and use of artificial IC.Poor correlation was obtained between IC-EIA and other techniques.The method demonstrated the occurence of IC in a large proportion of patients with viral hepatitis, schistosomiasis and rheumatoid arthritis and their absence in normal subjects.     

IgG rheumatoid factor in subacute bacterial endocarditis: relationship to IgM rheumatoid factor and circulating immune complexes.

With recently developed radioimmunoassays, we have been able to study the levels and properties of IgG rheumatoid factor (IgG RF) and IgM rheumatoid factor (IgM RF) in patients with subacute bacterial endocarditis (SBE), as well as the relationship of these autoantibodies to circulating immune complexes. We found significantly elevated amounts of IgG RF and IgM RF in SBE sera. The IgG RF chromatographed on Sepharose 6B as an intermediate complex, indistinguishable from the pattern seen in rheumatoid arthritis. RF levels peaked later in the course of SBE than did levels of circulating immune complexes. With antibiotic treatment RF levels declined, although not as fast nor as completely as circulating immune complexes. These results suggest that both IgG RF and IgM RF in SBE may be part of a polyvalent antibody response to elevated levels of circulating immune complexes which do not themselves contain RF.     

HIV antigen detection in circulating immune complexes

Various methods were evaluated for their effectiveness in releasing HIV antigen (Ag) from artificial immune complexes (IC) and from IC present in serum from HIV antibody (Ab) positive subjects. The most effective methods for recovering HIV Ag from IC were those which included a denaturation step to prevent reassociation of Ag with Ab. IC precipitation in 2.5% polyethylene glycol followed by acid treatment with 1 M glycine.HCl (pH 2) for 10 min at 70 degrees C in the presence of 0.05% SDS gave very satisfactory results. With this method, IC were detected in sera from HIV antibody positive Caucasian subjects at all stages of infection. After HIV IC dissociation, HIV Ag was detected in a significant number (8/17 or 47%) of asymptomatic subjects. IC were most prevalent during the late stages of infection. A substantial increase in HIV Ag positivity was also observed in 20 Senegalese HIV Ab positive sera. After HIV IC dissociation HIV antigen detection increased from 2/20 to 12/20. The relevance of IC detection is discussed.     

Serum and blister fluid immune complexes in bullous pemphigoid: detection with C1q and monoclonal rheumatoid factor.

Eighty serum samples and 24 blister fluids from 51 patients with active bullous pemphigoid were tested for the presence of immune complexes by both a monoclonal rheumatoid factor (mRF) inhibition radioassay and a C1q-binding radioassay. Forty-two of the 80 serum samples were positive by the mRF assay, while 27 were positive by the C1q-binding assay. Antibody titres to the basement membrane zone did not correlate with levels of circulating immune complexes. Thirteen of 24 blister fluids had detectable immune complexes by the C1q assay, while only seven of 24 blister fluids were positive by the mRF assay. Sucrose density-gradient ultracentrifugation studies suggest that the mRF- and C1q-reactive substances in both bullous pemphigoid sera and blister fluids are of a size compatible with immune complexes. Although immune complexes are detectable in a high percentage of bullous pemphigoid patients, their role in this disease may be epiphenomenal rather than pathogenetic, merely reflecting the presence of autoantibody and soluble antigen.     

Solid-phase Clq-binding fluorescence immunoassay for detection of circulating immune complexes.

A fluorescence immunoassay for detection of immune complexes bound to solid-phase C1q was developed. The method was standardized by using human aggregated immunoglobulin G (IgG) to simulate immune complexes. A linear relationship existed between the concentrations of the aggregated IgG standards and the resulting fluorescent intensity. The method was found to be reproducible and capable of detecting as little as 10 micrograms of aggregated IgG per ml of heat-inactivated human serum. Antigen-antibody complexes prepared in vitro were detectable from equivalence to moderate antigen excess. Endogenous serum C1q inhibited the binding of aggregated IgG to solid-phase C1q. Pretreatment of test sera with EDTA was ineffective in eliminating this competitive effect. Heating the sera at 56 degrees C alleviated, but did not abolish, interference of endogenous C1q. Elevated levels of immune complexes were detectable in sera fro seven of nine patients wit systemic lupus erythematosus, provided the samples were heat inactivated before testing. Heparin and DNA were also found to interfere with the detection of aggregated IgG added to human serum. Assay values were falsely decreased due to competitive inhibition by these anions. Lipopolysaccharides from a variety of bacterial preparations produced no detectable interference. A comparative study was conducted on samples that had previously been tested by fluid-phase C1q-binding radioimmunoassay. The two methods were concordant in assigning normal or elevated levels of immune complexes in 70% of the samples tested. This solid-phase fluorescence immunoassay is proposed as a possible alternative to radioimmunoassay for the detection of circulating immune complexes.     

A rapid solid phase assay for the detection of circulating immune complexes

A simplified process, which we have termed Enzyme Immune Complex Assay (EICATM) for the detection of circulating immune complexes (CICs), is described herein. The method utilizes readily available reagents, small quantities of serum, and can be performed quickly with a minimal amount of equipment. Serum from 38 normal controls, 98 burn patients, 36 frostbite injury patients, and 21 patients with elevated rheumatoid factor (RF) were tested for CICs in an immune function study. Elevated immune complex levels were found in the group of patients with frostbite injury, and in the group with elevated RF. Serum from thermally injured patients had slightly depressed yet normal CIC levels. The detection of elevated CICs by the EICATM method compared favorably with the more cumbersome Raji cell method, with the added advantage of simplicity, speed, and the ability to detect non-IgG immune complexes.     

Circular dichroism of immune complexes with rheumatoid factor activity

The circular dichroism (CD) of IgG complexes with RF activity and that of monomeric IgG without this activity, and isolated from the same individual, were compared. The IgG complexes had a significantly deviant CD spectrum in the near u.v. region, both before and after dissociation, whereas the monomeric IgG had a normal spectrum. Immunoglobulins were isolated from the same serum with the use of specific antiserum against unique determinants in some IgG complexes with RF activity. Both before and after dissociation the CD spectrum in the far u.v. region of these immunoglobulins differed significantly from that of the above-mentioned preparations. The results confirmed that the structure of IgG in the RF-active complexes differed from that of normal IgG. The immunoglobulins with the unique determinants had, in turn, a structure that was not found in the pool of the RF-active IgG molecules or in normal IgG.     

HIV Antigens as complement fixing circulating immune complexes

To detect HIV antigens in circulating complement fixing immune complexes (ICs) we assessed an ELISA using wells of microtitre plates coated with F(ab)2 anti-C3b and monoclonal antibodies anti-HIV gp120 and anti-HIV p24. We tested 24 anti-HIV positive subjects (Group A), 10 anti-HIV negative subjects at risk of acquiring HIV infection (Group B), 20 normal controls (Group C) and 2 seroconversion panels. We found HIV antigens in ICs in all sera from seroconversion panels, in 25.5% of sera from subjects in Group A, in 28.6% of sera from subjects in Group B and in no serum from subjects in Group C. A subject in Group B acquired HIV infection during the observation. HIV antigens in ICs by our assay were detected 8 months before Anti-HIV and Ag by commercial ELISA.     

Soluble FcgammaRIIa inhibits rheumatoid factor binding to immune complexes

Soluble low-affinity receptors for IgG are known to inhibit immune complex (IC)-mediated inflammation, and expression by leukocytes is elevated in several inflammatory diseases. Immunoglobulin M (IgM) rheumatoid factors (RF), anti-Fc autoantibodies, are found in autoimmune diseases, such as rheumatoid arthritis (RA), as well as in normal immune responses. This study demonstrated that soluble FcgammaRIIa inhibits the interaction of rheumatoid factors with ICs. The recombinant soluble low-affinity FcgammaR, rsFcgammaRIIa, partially inhibited (30-70%) the rate of precipitation of soluble ICs by RF-positive RA sera. This required the normal interaction of FcgammaRIIa with Fc as the effect could be abrogated with the Fab fragment of the blocking mAb IV-3. Furthermore, rsFcgammaRIIa partially inhibited (40%) the binding of a monoclonal IgM RF (RF-AN) to an IC formed by IgG2 antibody binding to an antigen-coated biosensor chip. Since RF-AN has been characterized by crystallography to bind to the CH2/CH3 interface of the IgG-Fc, and leukocyte FcgammaRIIa binds to a distinct site centred on the lower hinge, this inhibition is uncompetitive. Some inhibition (15%) of staphylococcal protein A binding to IC was also observed. As soluble FcgammaRIIa disrupts Fc:Fc interactions in IgG-ICs, we propose that this alteration of the IC also reduces the accessibility of Fc portions in the IC, resulting in the partial inhibition of ligands, particularly IgM RF, which bind Fc. We propose that the high concentrations of soluble FcgammaR found during inflammation can affect the properties of ICs and their interaction with the immune system.     

A technique for the purification of immune complexes using rheumatoid factor.

A method for the purification of immune complexes (IC) from human serum is described, using as a model complexes of tetanus toxoid with human antitoxoid antibodies (TAT). The technique is based on the ability of IC to bind to tubes coated with rheumatoid factor (RF). Small amounts of TAT IC were added to human serum and the mixtures were rotated overnight in tubes coated with RF. The tubes were then washed and the bound material was eluted with sodium dodecyl sulphate and iodinated with 125I. Analysis of the labeled preparation by electrophoresis in polyacrylamide gels revealed the presence of the toxoid component. The technique should be useful in isolating small amounts of IC for analytical purposes.     

Binding of in vivo formed immune complexes by monoclonal mouse rheumatoid factors

Mouse rheumatoid factors (RF) have previously been shown to bind immune complexes formed in vitro with greater affinity than monomeric IgG. The present report extends this observation and shows that this property of mouse RF is retained with immune complexes formed in vivo. This observation strengthens the idea that the interaction of RF with immune complexes could play a physiological role.     

Evaluation of a Cordia-IC enzyme-linked immunosorbent assay kit for the detection of circulating immune complexes.

A commercial kit (Cordia-IC) from Cordis Laboratory, Miami, Fla., was compared with the Raji cell radioimmunoassay for its ability to detect circulating immune complexes (CIC) in sera from 30 control subjects and 118 patients with infectious diseases. The 118 patients were categorized into the following groups: (i) 23 patients with bacterial endocarditis, (ii) 41 patients with bacteremia from an infected intravascular catheter or access device, and (iii) 54 patients with Staphylococcus aureus bacteremia related to a deep tissue infection. The Cordia-IC was comparable to the Raji cell radioimmunoassay in intraassay variability (4.0 versus 8.0%) and interassay reproducibility (8.7 versus 20.0%). Neither assay found CIC amounts above 12.5 micrograms equivalents (eq) of aggregated human gamma globulin (AHG) per ml in any of the 30 control individuals. In group 1, Cordia-IC detected 19 of 23 positives (mean, 73.6 micrograms eq of AHG per ml), whereas the Raji cell detected 16 of 23 positives (mean, 54.8 micrograms eq of AHG per ml). In group 2, Cordia-IC detected 19 of 41 positives (mean, 20.6 micrograms eq of AHG per ml), whereas the Raji cell detected 16 of 41 positives (mean, 15.1 micrograms eq of AHG per ml). In group 3, Cordia-IC found 38 of 54 positives (mean, 28.0 micrograms eq of AHG per ml), whereas the Raji cell found 32 of 54 positives (mean, 23.9 micrograms eq of AHG per ml). Statistically, these findings were not significantly different in any of the three patient groups (P> 0.15), and there was an overall good correlation between the results obtained by the two assays (r+0.64, P<0.001). The Cordia-IC provided a suitable assay for the detection of CIC and might find application in routine clinical laboratories.     

Evidence that a putative anti-idiotypic monoclonal antibody may actually be recognizing circulating immune complexes.

Murine monoclonal antibodies (mAb) reacting with affinity-purified antihistone antibodies (AHA) from serum of a patient with systemic lupus erythematosus (SLE) were obtained. One of them, 8B3, was initially considered to recognize idiotypic (Id) determinants in AHA since (a) it reacted with AHA but not with control IgG; (b) this reactivity could be inhibited using affinity-purified AHA, but not with control IgG or whole serum; (c) affinity-isolated 8B3+ antibodies showed antihistone activity and not other activities tested so far; (d) antihistone activity due to 8B3+, but not that of 8B3- from the same serum, could be fully inhibited by the presence of 8B3 mAb in the antihistone assay and (e) serum levels of 8B3 reactivity were higher than normal in SLE patients with AHA (56%), in contrast with SLE patients without AHA (6%). From these results it was deduced that 8B3 defined a cross-reactive Id shared by a subset of AHA in SLE patients. However, the present results suggest that (a) 8B3 mAb did not recognize AHA or Ig, but did recognize a 55 kDa histone-binding protein; (b) this 55 kDa protein was present free at low concentration in all human sera, but also associated with IgG in 8B3+ SLE sera and (c) these complexes are responsible for the false positive results in the antihistone assay as shown for DNA/anti-DNA complexes. Thus, mAbs recognizing the non-Ig moiety of circulating immune complexes may resemble anti-Id antibodies with features of the so-called epibodies. These immune complexes may be responsible for false positive results and caution should be exercised in the interpretation of results.