Circulating immune complexes in experimental filariasis.

Circulating immune complexes have been investigated in jirds (Meriones unguiculatus) infected with the filarial nematode Brugia pahangi. Two-month-old male jirds were inoculated with seventy-five B. pahangi infective larvae into the left groin. At 8 months post-infection, sera of individual animals from a group of seventeen infecteds and seventeen age-matched controls were analysed for immune complexes by (1) a solid-phase C1q binding assay (Clq-SP) and (2) precipitation with 3.5% polyethylene glycol followed by binding of 125I-labelled rabbit anti-jird Ig antiserum (PEG). A significant increase in the level of circulating immune complexes was shown in the infected group as compared with the controls for both assays, with a P value = 0.005 for PEG and P = 0.001 for Clq-SP. Using the mean of the control group +/- 2 s.d. as the upper limit of the normal range, 24% of the infected group had elevated immune complex levels by the PEG assay, and 41% were elevated in the C1q-SP assay. A high degree of variability was noted in the levels of immune complexes among individual animals in the infected group by each test. No correlation between immune complex levels and numbers of circulating microfilariae was found in either assay.     

Characteristics of immune complexes detectable by two independent assays in gynaecological malignancies.

Immune complexes from patients with ovarian and endometrial cancer have been examined using the C1q solid phase (C1qSP) and polyethylene glycol (PEG) precipitation assays. The amount of IgG in the PEG precipitates was inversely correlated with the amount of IgM whilst the IgM values correlated positively with the C1qSP assay values. Separation studies revealed that most of the C1qSP activity was not precipitated from cancer sera by 2% PEG. The immune complexes were fractionated on Sephacryl S-300. Both the PEG precipitation and C1qSP assays detected high molecular weight immune complex like activity (greater than 19S). The C1qSP assay also detected a second peak of activity at approximately 7-8S. Most of the PEG detectable immune complexes from sera dissociated during column chromatography, whereas the C1qSP detectable complexes were relatively stable. Furthermore the IgG from fractionated PEG precipitates emerged as a monomer (7S) component. The PEG assay appeared to be detecting a high molecular weight complex containing mostly IgG rather than IgM with a low affinity for C1q, which was easily dissociated. The C1qSP assay detected a more stable high molecular weight complex containing a relatively high proportion of IgM and a low molecular weight complex, both with a high affinity for C1q.     

A comparative study of complement components in polyethylene glycol precipitated immune complexes from patients with ovarian cancer and patients with rheumatoid arthritis

Circulating immune complexes and complement components C1q, C3 and C4 were measured following polyethylene glycol precipitation of serum from patients with ovarian cancer (OC), patients with rheumatoid arthritis (RA) and control patients (CP). The C3 and C4 content of the precipitated material was significantly greater in those patients with OC in comparison to those with RA or CP, while the C1q content did not differ significantly between the two groups of patients. A statistically significant correlation between IgG, C3 and C4 levels was found in the immune complexes of both groups of patients. C1q levels correlated significantly with IgG immune complex levels in patients with RA and CP, but did not in patients with OC. These data illustrate that immune complexes from patients with OC differ from complexes precipitated from RA sera and normal sera in their complement content. It is suggested that the largely unsuccessful attempts to detect immune complexes in sera from patients with ovarian cancer, using assays dependent on the binding of C1q may be related to differences in solubility of complexes from these patients in comparison to complexes present in non-neoplastic conditions.     

A comparison of two 125I C1q binding tests to detect soluble immune complexes in serum of patients with malignant disease.

The 125I C1q deviation test and the modified 125I C1q PEG precipitation test were compared in their ability to detect soluble immune complexes in serum using a model system of HSA-rabbit-anti-HSA, and were then applied to sera collected from patients with malignant and non-malignant conditions. Despite close agreement in the model system, the two tests gave divergent results for the presence of C1q binding substances in individual serum samples collected from patients. The inherent complexities of interpreting C1q binding in serum, in terms of the presence of soluble immune complexes, makes it questionable whether either test can be relied upon to provide a means of identifying these complexes in the sera from patients with malignant disease.     

Failure to detect circulating DNA--anti-DNA complexes by four radioimmunological methods in patients with systemic lupus erythematosus.

The presence of DNA-anti-DNA complexes in sera from patients with systemic lupus erythematosus (SLE) was investigated by two new radioimmunoassays (RIA) developed for this purpose and by measuring the C1q and DNA binding activity of serum before and after treatment with DNAse. Two direct RIA developed in this study were based on the reactivity of [3H]actinomycin D ([3H]ACT-D) or solid-phase methylated bovine serum albumin (mBSA) with DNA-anti-DNA complexes. DNA-anti-DNA complexes prepared in vitro could be efficiently detected at various antigen--antibody ratios by these two RIA. Increased levels of circulating immune complexes as indicated by the C1q binding test were found in 52% of SLE sera. However, the frequency of specific DNA-anti-DNA complexes detected in SLE sera was very low. Only 6% of sera exhibited an increased value deviating by more than three s.d. from the normal mean when tested with the [3H]ACT-D binding RIA or the solidphase mBSA RIA. On the other hand, there was no significant difference in the serum C1q or DNA binding activity after treatment with DNAse. These results suggest that DNA-anti-DNA complexes do not occur frequently in circulating blood and represent only a very small portion of the immune complexes detected in serum from patients with SLE.     

Characterization of immune complexes detected by the 125I-C1q binding assay in breast cancer

The aim of the present study was to isolate and characterize immune complexes measured by the 125I-C1q binding assay in breast cancer sera. The C1q binding assay detects immune complexes by their binding to 125I-C1q and subsequent precipitation with PEG. We purified the C1q binding material from these precipitates by superose 6 gel filtration and cation exchange chromatography. The isolation steps were monitored by the C1q binding assay and the purified material was analyzed by SDS--PAGE and immunodiffusion. The C1q binding material isolated from two breast cancer sera contained complexes of IgM, C4b binding protein (C4-bp), and C1q. The C4-bp fraction showed significant C1q binding activity which increased after the addition of IgM. This C4-bp was not different from C4-bp isolated from normal serum. The IgM fraction, however, differed from normal IgM by its binding to C4-bp and C1q and its stronger affinity to the cation exchange column. These unique properties were not due to rheumatoid factor activity. They might be caused by a different glycosylation pattern or by complex formation with an as yet unknown polysaccharide antigen.     

Detection of Hepatitis B Antigen in Circulating Immune Complexes in Acute and Chronic Hepatitis

By using polyethylene glycol precipitation at low concentration (PEG test) and the radiolabeled C1q binding test, immune complexes were detected sera from acute (23/28) and chronic (28/32) hepatitis patients, hemodialyzed patients with chronic hepatitis B surface (HBs) antigenemia (7/19), and asymptomatic HBs antigen carriers (2/11). After treatment of PEG precipitates with acidic pH, heating, or proteolytic enzyme (protease), electroimmunodiffusion or radioimmunoassay revealed the presence of HBs antigen or antibody in dissociated immune complexes in sera from several acute and chronic hepatitis patients. Electron microscopy showed immune complexes of HB virus in 9 of 12 PEG precipitates obtained from PEG-test-positive sera; these 9 precipitates were from patients with acute or chronic hepatitis and the other three from chronic HBs Ag carriers. Free HB virus particles were observed after protease digestion of PEG precipitates. Neither immune complexes nor virus particles were seen in precipitates from PEG-test-negative but HBs-Ag-positive sera from chronic carriers.     

Isolation and partial characterization of circulating immune complexes in sera of children with HBV-mediated glomerulonephritis.

Circulating immune complexes (CIC) containing HBsAg and HBeAg were identified in sera of 5 out of 6 children with hepatitis B mediated membranous glomerulonephritis. CIC were precipitated from sera by 3.5% PEG, washed and subsequently analysed after acid dissociation and trypsin digestion. HBsAg, anti-HBs and albumin; HBeAg, anti-HBe and anti-HBc were recovered from the isolated complexes and these findings are discussed. Analysis of 3.5% PEG mediated precipitate of human serum proteins showed the relatively high content of IgG classical pathway complement components: C1q, C4 and C3.     

The Use of TNP-Conjugated Polyacrylamide Beads in a C1q Binding Inhibition Test for Circulating Immune Complexes

A new assay for the detection of circulating immune complexes is described. It is based on the same principle as the C1q deviation test: the binding of radiolabelled C1q to a solid phase is inhibited by immune complexes. Trinitrophenylated polyacrylamide beads are used as a stable C1q-reactive solid phase in our test. Aggregated IgG in normal serum could be detected by this method to a minimum concentration of about 10 µg/ml. The test was used to quantitate circulating immune complexes in sera of patients with glomerulonephritis, liver diseases, lymphoma, and myeloid leukemia. The results are compared and correlated with those obtained by the C1 q binding assay for the same sera.     

Circulating immune complexes in patients with acquired immune deficiency syndrome contain the AIDS-associated retrovirus

Using the polyethylene glycol (PEG) precipitation technique, circulating immune complexes were demonstrated in 88% of patients with acquired immune deficiency syndrome (AIDS) and AIDS-related disorders. The finding of immune complexes in these individuals was confirmed by using a second assay, the commercially available Clq-IgG enzyme-linked immunosorbent assay kit. The material precipitated by PEG contained elevated levels of IgG, IgM, Clq, and C3. There was no substantial correlation between the level of IgG in the complexes and the serum IgG concentration, although there was a significant association between complexed IgG and the titer of anti-AIDS-associated retrovirus (ARV) antibody in the serum. In some precipitates, ARV antigens were detected by immunoblot analysis. Coculture of other precipitates with normal lymphocytes revealed the presence of infectious virus. These data confirm the existence of immune complexes in the sera of AIDS patients and indicate that in some cases they may comprise ARV and anti-ARV antibodies.     

Polyethylene Glycol Precipitates of Serum Contain a Large Proportion of Uncomplexed Immunoglobulins and C3

High pressure liquid chromatography (HPLC) was used to fractionate redissolved polyethylene glycol (PEG) precipitates isolated from the sera of normal volunteers and from patients with IgA nephropathy (IgAN) and systemic lupus erythematosus (SLE), 2 diseases characterized by elevated levels of circulating immune complexes. The individual fractions were analyzed by solid phase ELISA for IgA, IgM, C3, IgG, and complexes of IgG-IgA and IgG-C3. Although PEG precipitates were enriched for high molecular weight IgA and IgG (presumably bound within CIC), significant amounts of IgM, unbound IgG and C3 were also present. The quantities of the PEG-precipitable proteins did not correlate with their serum concentrations. IgG-IgA and IgG-C3 complexes were found in all precipitates examined, but the levels of complexes were higher in both patient groups. These results indicate that PEG precipitates a considerable quantity of proteins not bound in immune complexes. There appeared to be greater protein precipitation from sera of the patient groups compared to the amount precipitated from the normal sera. These results suggest that an understanding of the mechanism of PEG precipitation may be important in defining abnormalities in IgAN, SLE and perhaps other diseases characterized by elevated levels of CIC. In addition, the possibility of undetected CIC in PEG precipitable material must be considered.     

Polyethylene Glycol Precipitates of Serum Contain a Large Proportion of Uncomplexed Immunoglobulins and C3

High pressure liquid chromatography (HPLC) was used to fractionate redissolved polyethylene glycol (PEG) precipitates isolated from the sera of normal volunteers and from patients with IgA nephropathy (IgAN) and systemic lupus erythematosus (SLE), 2 diseases characterized by elevated levels of circulating immune complexes. The individual fractions were analyzed by solid phase ELISA for IgA, IgM, C3, IgG, and complexes of IgG-IgA and IgG-C3. Although PEG precipitates were enriched for high molecular weight IgA and IgG (presumably bound within CIC), significant amounts of IgM, unbound IgG and C3 were also present. The quantities of the PEG-precipitable proteins did not correlate with their serum concentrations. IgG-IgA and IgG-C3 complexes were found in all precipitates examined, but the levels of complexes were higher in both patient groups. These results indicate that PEG precipitates a considerable quantity of proteins not bound in immune complexes. There appeared to be greater protein precipitation from sera of the patient groups compared to the amount precipitated from the normal sera. These results suggest that an understanding of the mechanism of PEG precipitation may be important in defining abnormalities in IgAN, SLE and perhaps other diseases characterized by elevated levels of CIC. In addition, the possibility of undetected CIC in PEG precipitable material must be considered.     

Detection of circulating immune complexes in patients with glomerulonephritis.

143 patients were evaluated clinically on the basis of the renal biopsy. Three methods for detecting circulating immune complexes (CIC) were employed: complement consumption test, inhibition of erythrocyte antibody IgG-EA rosette forming test and optical density of 4% PEG precipitated sera. CIC were present in the sera of all patients with acute poststreptococcal glomerulonephritis (2 weeks after streptococcal infection of the throat). In a group of patients with chronic glomerulonephritis the highest values in positive results were observed in lupus nephritis, chronic proliferative glomerulonephritis and chronic submicroscopic glomerulonephritis. These results were compared with levels of total hemolytic complement, C3, C4 components and serum immunoglobulins (IgA, IgG, IgM).     

Detection of circulating immune complexes with polyethylene glycol precipitation F(ab')2 anti-C3 ELISA

A polyethylene glycol (PEG) precipitation F(ab')2 anti-C3 ELISA for the detection of complement-fixing IgG circulating immune complexes (CIC) is described. For this assay, test sera were treated with 3.5% PEG and then measured with F(ab')2 anti-C3 ELISA. The lower detection limit was 4 micrograms/ml of heat aggregated human IgG (HAHG). Intra-assay coefficient of variation (CV) was 4.9-8.3%. High levels of CIC are found in the sera of patients with systemic lupus erythematosus (SLE), hepatitis B and stomach cancer.     

Comparison of some procedures detecting circulating immune complexes

Tests for circulating immune complexes were performed by means of 1) plain polyethylene glycol (PEG) precipitation (PEGprec), 2) immunoelectrophoresis of PEG precipitates (IEpp), 3) anti-antibody (AA) inhibition test with sera (AA-Is), and 4) AA inhibition test with PEG precipitates (AA-Ipp). The tests were performed with 156 pathological sera from patients with myasthenia gravis, syphilis, adenocarcinomas of the gastrointestinal tract, rheumatoid arthritis and systemic lupus erythematosus, and 51 normal sera from blood donors. PEGprec was positive with 76 sera, IEpp with 84 sera, AA-Is with 64 sera, and AA-Ipp with 74 sera. Comparison of results in all four tests showed high degree of correlation; all p values were below 0.005. The lower sensitivity of AA inhibition tests was due to the fact that these tests detect only complexes formed by IgG but not by IgM, whereas the remaining two tests detect complexes formed by antibodies of both these immunoglobulin classes. When sera of patients with rheumatoid arthritis and SLE were removed from the material studied, the four tests showed about equal sensitivity. PEGprec gave positive results with two normal sera and the remaining tests were negative with all these sera. It appears that the simultaneous application of PEGprec, IEpp, AA-Is and AA-Ipp will give sensitive and reliable procedure for detecting circulating immune complexes.     

Comparison of some procedures detecting circulating immune complexes

Tests for circulating immune complexes were performed by means of 1) plain polyethylene glycol (PEG) precipitation (PEGprec), 2) immunoelectrophoresis of PEG precipitates (IEpp), 3) anti-antibody (AA) inhibition test with sera (AA-Is), and 4) AA inhibition test with PEG precipitates (AA-Ipp). The tests were performed with 156 pathological sera from patients with myasthenia gravis, syphilis, adenocarcinomas of the gastrointestinal tract, rheumatoid arthritis and systemic lupus erythematosus, and 51 normal sera from blood donors. PEGprec was positive with 76 sera, IEpp with 84 sera, AA-Is with 64 sera, and AA-Ipp with 74 sera. Comparison of results in all four tests showed high degree of correlation; all p values were below 0.005. The lower sensitivity of AA inhibition tests was due to the fact that these tests detect only complexes formed by IgG but not by IgM, whereas the remaining two tests detect complexes formed by antibodies of both these immunoglobulin classes. When sera of patients with rheumatoid arthritis and SLE were removed from the material studied, the four tests showed about equal sensitivity. PEGprec gave positive results with two normal sera and the remaining tests were negative with all these sera. It appears that the simultaneous application of PEGprec, IEpp, AA-Is and AA-Ipp will give sensitive and reliable procedure for detecting circulating immune complexes.     

In vivo reduction of circulating C1q binding immune complexes by intravenous gammaglobulin administration

Six patients with systemic lupus erythematosus were treated with high-dose intravenous gammaglobulin. Immunological parameters were studied and included solid-phase immune complex determinations, quantitative immunoglobulins G, A, and M, as well as C3 and C4 concentrations. Pretreatment values of circulating immune complex concentrations as measured by either C1q binding or anti-C3 binding assays were elevated in all patients. Posttreatment values showed reductions in all C1q binding immune complexes (p less than 0.01) and anti-C3 binding immune complexes also decreased in 5 out of 6 patients. These assays are described in detail and were also used to define in vitro interactions between the intravenous gammaglobulin preparation and heat-aggregated IgG or sera containing elevated circulating immune complexes. No reduction of immune complex levels were observed when IgG was incubated in vitro with either heat-aggregated IgG or sera with elevated immune complex concentrations. The duration of the in vivo effect and the patients' clinical responses are described. These findings show that high-dose intravenous gammaglobulin administration can reduce certain types of immune complexes in patients with elevated levels of these substances.     

Differential precipitation of the Clq subcomponent of the first complement component (C1) by polyethylene glycol from normal human serum and sera of patients with collagen diseases.

Sera were, under strictly standardized conditions, centrifuged in the presence of 0-5% PEG and the Clq concentration in the precipitates was measured by radial immunodiffusion. The presence of circulating immune complexes and rheumatoid factor(s) resulted in a shift of Clq precipitation to lower PEG concentrations. Clq precipitation at 1.5% PEG was shown to be specific for sera containing immune complexes. Under similar conditions addition of aggregated IgG to normal human serum gave rise to Clq precipitation directly proportional to the amount of aggregated IgG. Precipitation of endogenous Clq at 1.5% PEG and assay by radial immunodiffusion may therefore be useful for the detection and quantitation of immune complexes in human serum.     

An analysis of the fluid phase C1q binding assay. The effect of endogenous C1q on the precipitation and detection of an immune complex model

We examined the effect of endogenous C1q on the sensitivity of the fluid-phase C1q binding assay (C1qBA) in detecting an immune complex (IC) model, heat-aggregated IgG (HAIgG), at concentrations of 10-10,000 micrograms/ml sample. Results in normal human serum (NHS) or plasma (NHP) were compared with those in heat-inactivated NHS (NHS/56) in which most endogenous C1q was depleted by heat denaturation. Higher HAIgG concentrations were required in NHP and NHS to produce the same 125I-C1q precipitation seen in NHS/56. This decreased sensitivity varied from 70% at low HAIgG concentrations to 0% at high concentrations, as predicted for a large pool of endogenous C1q, in equilibrium with 125I-C1q, but in excess of that which could bind to all but the highest concentrations of IC model. In serum depleted of functional C1q on an immunoadsorbant of HAIgG, the precipitation of radiolabeled HAIgG under C1qBA conditions was concentration dependent and generated a saturation curve, showing that only a fraction of IC are usually precipitated in this assay. HAIgG precipitation was enhanced 1.4-fold in NHS/56 (8 micrograms C1q/ml) and three-fold in NHS (67 micrograms C1q/ml) suggesting that IC size is increased by endogenous C1q. In dual label experiments using 131I-HAIgG, the precipitation of 125I-C1q in NHS/56 was directly proportional to IC model precipitation, but markedly discordant in NHP, showing the measurement of IC in heat-inactivated sera superior to that in native serum. A comparison of the C1q:HAIgG ratio in PEG precipitates with that in samples, indicated that equilibrium was established between C1q and IC model. Thus the precipitation of 125I-C1q in the C1qBA represents (1) the fraction of total C1q bound to IC, and (2) the fraction of IC precipitated by PEG.     

Determination of circulating immune complexes: comparison between agglutination inhibition and C1Q precipitation in polyethylene-glycol-EDTA (author's transl)]

Three different methods for the determination of circulating immune complexes were compared : precipitation of radioiodinated C1q in polyethyleneglycol-EDTA, inhibition of IgG-coated latex particles agglutination by C1q or polyclonal rheumatoid factor. Normal values and their upper limits as well as the reproducibility of each technique are presented. The possible effect of several factors was investigated : age, sex, anticoagulants, freezing and thawing, contamination by DNA or endotoxin. Comparison of the results obtained with normal or pathological sera revealed a positive correlation between the three techniques. However, the discrepancies noted with some sera make the use of at least two different methods highly recommendable for the study of circulating immune complexes in disease.