Enzyme immunoassay detection of circulating immune complexes by monoclonal antibodies to C3 neoepitopes with special reference to IgG concentration and to interfering anti-immunoglobulin antibodies

A sensitive solid-phase anti-C3 enzyme immunoassay for detection of circulating immune complexes (CIC) is described. A mixture of the monoclonal antibodies (MoAbs) bH6 and Clone 9 specific for neoepitopes on C3 activation products was used as capture reagent. MoAb bH6 recognized C3b, iC3b and C3c, and Clone 9 recognized iC3b and C3dg. Detection antibody was a polyclonal peroxidase-conjugated rabbit anti-human Ig antiserum. A quantitative assay was constructed using serum incubated with heat aggregated IgG (HAG) as standard. The lower detection limit was 5 micrograms/ml of HAG. Interassay and intra-assay coefficient of variation was 15% and 5%, respectively. Anti-animal immunoglobulin antibodies were detected both in normal and pathological sera. This activity was efficiently absorbed by nonimmune immunoglobulins added to the samples. The present assay was compared with a polyethylene glycol precipitation assay for CIC determination. The latter assay was strongly influenced by the IgG concentration (rs = 0.78; P = 0.006), whereas no such correlation was seen for the anti-C3 immune complex assay (rs = -0.30; P = 0.20).     

Correlation of C3d fixing circulating immune complexes with disease activity and clinical parameters in patients with systemic lupus erythematosus

Using anti-C3d as a solid phase reagent, C3d fixing circulating immune complexes (CIC) were detected in sera from patients with systemic lupus erythematosus (SLE), rheumatoid arthritis, membranous nephropathy and IgA nephropathy. Particularly, sera from SLE showed the highest CIC levels and highest incidence of positivity among these diseases. In the 51 serum samples from 48 patients with SLE we studied, the CIC detected by the anti-C3d assay correlated well (P less than 0.01) with the CIC detected by the solid phase C1q assay, but not with those detected by the conglutinin assay. In addition, the CIC detected by the anti-C3d assay correlated more significantly (P less than 0.001) with disease activity, as well as some clinical parameters (serum anti-dsDNA antibodies, CH50 and C3 levels) than CIC detected by the other two assays of SLE sera. The anti-C3d binding materials were found to be of intermediate (8-19S) and small (7S) sizes in a small number of SLE sera which we analysed.     

A method to differentiate between anti-C1q antibodies and C1q-binding immune complexes using collagenase-digested solid phase C1q.

A method for the detection of circulating immune complexes in the presence of autoantibodies to C1q is described. Solid phase C1q-digestion with bacterial collagenase results in the elimination of the collagen-like region of C1q. Binding of model immune complexes to this modified solid phase C1q is practically unaltered, while reactivity of anti-C1q antibodies is abolished by this procedure. In conjunction with an ELISA using the collagen-like region of C1q as antigen this modified C1q solid phase assay may be used to determine immune complexes and anti-C1q antibodies in the sera of patients with autoimmune rheumatic diseases.     

Immunofluorescence studies on the subcomponents of the first component of complement (C1): Detection of C1q and C1s in different cells of biopsy material and on Human as well as on guinea pig peritoneal macrophages

The first component of complement (C1) ist a macromolecule consisting of three distinct subcomponents, C1q, C1r, and C1s. In regard to its production site and its role in phagocytic processes it was of interest to find out whether these different subcomponents could be detected in human biopsy material only as a complex in individual cells or whether C1 subcomponents could be found on different cells. To study this question, monospecific fluorescein-labelled anti-human-C1q IgG and monospecific rhodamine-labelled anti-human C1s IgG were used. Biopsy material from human rectum was stained with fluoresceinated antisera, either by use of one antiserum or by double staining. Using this technique, these oberservations were made: C1q as well as C1s were detectable in individual cells in the subepithelial area of the gut. Furthermore, C1q and C1s could be found together in the same cell or separately in different cells.These findings were supported by experiments with cultured peritoneal macrophages either from human or from guinea pig. The examination of the cultured cells with the two antisera revealed that individual cells were stained either by anti-C1q or by anti-C1s antibodies. The specificity of the detection of the individual subcomponents was also proven by the peroxidase technique and by using fluoresceinated anti-human C1q F(ab')₂. The membrane immunofluorescent staining revealed the presence of C1q on the membrane of the macrophage.     

Anti-C1q autoantibodies from active lupus nephritis patients could inhibit the clearance of apoptotic cells and complement classical pathway activation mediated by C1q in vitro

Anti-C1q antibodies are prevalent in patients with active lupus nephritis and were found to be closely associated with renal involvement and predictive for a flare of nephritis. However, the pathogenesis of anti-C1q antibodies involved in human lupus nephritis remains unclear. C1q, which plays a key role in apoptotic cell and immune complex removal, is a very important functional molecule in the pathogenesis of SLE. The aim of this study was to investigate the influence of anti-C1q autoantibodies from active lupus nephritis patients on the bio-functions of C1q in vitro. We purified IgG autoantibodies against C1q from lupus nephritis patients, and found that they could recognize C1q bound on early apoptotic cells at 30 μg/ml, and could significantly decrease the phagocytosis by macrophages of early apoptotic cells opsonized by 50 μg/ml C1q in comparison with normal IgG. Levels of circulating immune complexes of the ten patients were measured by a circulating immune complexes (CIC)-C1q Enzyme Immunoassay Kit. Anti-C1q autoantibodies affinity purified by microtiter plates could significantly inhibit the deposition of C3c on CIC-C1q in a dose dependent manner in comparison with IgG from 10 healthy blood donors. The binding of opsonized immune complexes to RBCs was significantly inhibited by anti-C1q autoantibodies purified by microtiter plates in a dose dependent manner. Our observations suggest that serum anti-C1q autoantibodies from active lupus nephritis patients could interfere with some biological function of C1q in vitro.     

Human Autoantibodies Against C1q: Lack of Cross-Reactivity with the Collectins Mannan-Binding Protein, Lung Surfactant Protein A and Bovine Conglutinin

The collectins, a group of humoral C-type lectins, have globular and collagen-like regions and share structural features with the complement protein C1q. The question was asked if autoantibodies to the collagen-like region of C1q (anti-C1qCLR) might cross-react with collectins, such as mannan-binding protein (MBP), lung surfactant protein A (SP-A) and bovine conglutinin (BK). Anti-C1qCLR antibodies of the systemic lupus erythematosus (SLE) type and anti-C1qCLR antibodies of the hypocomplementemic urticarial vasculitis syndrome (HUVS) type were investigated. Cross-absorption and elution experiments combined with antibody detection by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis gave no evidence of cross-reactive anti-C1qCLR antibodies. However, one serum with HUVS type anti-C1qCLR antibodies contained anti-MBP antibodies that were cross-reactive with SP-A. Judging from results of ELISA inhibition experiments and immunoblot analysis, four SLE sera contained antibodies to native BK, while two sera with HUVS type anti-C1qCLR antibodies contained antibodies to epitopes of denatured BK. This might imply that autoimmunity to collagen-like structures is not restricted to C1qCLR in HUVS and HUVS/SLE overlap syndromes.     

抗 C1q 抗体及补体 C3、C4 在神经精神性狼疮诊断中的应用价值

目的:探讨抗C1q 抗体及补体C3、C4 在神经精神性狼疮诊断中的应用价值。方法:对22 例狼疮脑病及66例SLE 患者进行横断面研究。抗C1q 抗体采用ELISA 方法,C3 及C4 采用免疫比浊法检测。分析补体与狼疮脑病的临床表现相关性,采用Logistic 回归分析狼疮脑病的危险因素。结果:NPSLE 患者抗C1q 抗体水平明显高于非NPSLE 患者,补体C4明显低于非NPSLE 患者,采用ROC 曲线分析抗C1q 抗体诊断NPSLE 敏感性及特异性分别为63.6%、66.7%。单因素分析后发现抗C1q 抗体、抗核糖体P 蛋白抗体、抗核小体抗体与神经精神性狼疮相关,但多因素Logistic 回归分析并未发现其与狼疮脑病发生有关。补体C4 降低与SLE 患者脑血管意外发生有关。结论:抗C1q 抗体在狼疮脑病诊断中具有一定的诊断价值,且补体C4 可能参与狼疮脑病脑血管意外的发生。     

Anti-C1q antibodies in hepatitis C virus infection

Autoantibodies against C1q have been described in many immune-complex diseases including hypocomplementaemic urticarial vasculitis and systemic lupus erythematosus (SLE). No study has focused on the role of anti-C1q antibodies in hepatitis C virus (HCV) infection. The aim of this study was (i) to evaluate the prevalence of anti-C1q antibodies in HCV infection; and (ii) to analyse the association of anti-C1q antibodies with clinical and biological features of HCV-mixed cryoglobulinaemia (MC) vasculitis. We searched for anti-C1q antibodies using an enzyme-linked immunosorbent assay (ELISA) test in 111 HCV patients (75 had cryoglobulin and 23 systemic vasculitis), 60 SLE patients and 109 blood donors. Anti-C1q antibodies were detected in 26% of HCV patients compared to 10% of healthy donors (P < 0.01), and 38% in patients with SLE. Although there was a higher prevalence of anti-C1q antibodies among HCV patients with type III cryoglobulin (50%, P < 0.01), the overall prevalence of anti-C1q antibodies was similar in HCV patients being cryoglobulin-positive or cryoglobulin-negative (26%versus 25%, P = 0.98). A significant association was found between anti-C1q antibodies and low C4 fraction of complement (P < 0.05). No association was found between anti-C1q antibodies and HCV genotype, severity of liver disease or with specific clinical signs of HCV-MC vasculitis. This study shows an increased prevalence of anti-C1q antibodies in HCV-infected patients. Anti-C1q antibodies were associated with low C4 levels. No association was found between anti-C1q antibodies and HCV-MC vasculitis, nor between anti-C1q antibodies and cryoglobulinaemia.     

Immune deposition of C1q and anti-C1q antibodies in the kidney is dependent on the presence of glomerular IgG

Anti-C1q autoantibodies are found frequently in patients with Systemic Lupus Erythematosus (SLE) and several studies indicate that these autoantibodies are associated with renal involvement. We have shown earlier that administration of anti-C1q antibodies to normal BALB/c mice results in the deposition of these antibodies and C1q in the kidney. In the present study we have investigated which factors are essential for this C1q-anti-C1q deposition. Injection of anti-C1q antibodies in C57BL/6 mice results in deposition of both C1q and anti-C1q in glomeruli, while administration of equal concentrations of anti-C1q to immunoglobulin deficient Rag2-/- mice did not result in deposition of anti-C1q antibodies. Analysis of renal sections of naive Rag2-/- mice revealed absence of mouse IgG and C1q in the glomeruli, while circulating C1q was within normal levels. Reconstitution of Rag2-/- mice with IgG, either by injection with purified mouse IgG or by splenocyte transfer, resulted in restored localization of mouse IgG together with C1q in the kidney. Subsequent injection of anti-C1q antibodies in these IgG reconstituted mice resulted in clear deposition of C1q together with anti-C1q in the kidneys comparable to that found in C57BL/6 mice receiving anti-C1q. We propose that the continuous presence of serum derived non-immune IgG in the glomerulus serves as a target for low affinity interactions with C1q, which then can serve as antigen for anti-C1q antibodies. Therefore we hypothesize that high and fluctuating levels of IgG as observed in patients with SLE may contribute to flares of renal inflammation in those patients with anti-C1q autoantibodies.     

Antibodies against C1q in anti-glomerular basement membrane nephritis.

The prevalence of antibodies against the collagen-like region of the subcomponent of the first component of complement, C1q, was investigated in 11 patients with anti-glomerular basement membrane (GBM) nephritis. Anti-C1q antibodies (anti-C1qAb) were detected in seven patients. IgG anti-C1qAb were found in four and IgA anti-C1qAb in five patients. During follow up of the patients a relationship was observed between the levels of IgG anti-C1qAb and the levels of anti-GBM antibodies (anti-GBMAb). Gelfiltration experiments indicated that both IgG anti-C1qAb as well as IgG anti-GBMAb were monomeric and that binding also occurred with the F(ab')2 fragments of the antibodies. Although anti-C1qAb and anti-GBMAb are both directed against a collagen-like structure, it was demonstrated by means of inhibition experiments that anti-C1qAb and anti-GBMAb are directed against different antigenic sites. Comparison of patients with anti-GBM nephritis with and without anti-C1qAb revealed that there were no differences in disease activity or disease severity. Therefore, the results of this study suggest that anti-C1qAb do not play a direct pathogenetic role in anti-GBM nephritis.     

Detection of autoantibodies against the globular domain of human C1q in the sera of systemic lupus erythematosus patients

The anti-C1q antibodies present in systemic lupus erythematosus (SLE) patients' sera are associated with renal involvement and the titer of these autoantibodies correlates with the clinical activity of the disease. It has previously been shown that anti-C1q antibodies bind neo-epitopes within the collagen region of human C1q. Evidence that these polyclonal autoantibodies recognize epitopes within the globular domain (gC1q) of the molecule has not been documented. In this study, we screened, using ELISA, a number of sera from SLE patients for the presence of anti-gC1q autoantibodies using recombinant globular head regions of individual A (ghA), B (ghB) and C (ghC) chains of human C1q. The recombinant proteins were used as test antigens to determine the levels of autoantibodies directed against ghA, ghB and ghC. SLE sera, containing high levels of anti-C1q antibodies, showed differentially increased binding towards ghA and ghB, which suggested that the gC1q domain can also be target of anti-C1q antibodies generated in SLE patients. Such antibodies can have severe pathophysiological consequences since these are likely to further impair the ability of C1q to clear immune complexes.     

Evaluation of anti-C1q capture assay for detecting circulating immune complexes and comparison with polyethylene glycol-immunoglobulin G, C1q-binding, and Raji cell methods.

An anti-C1q capture method kit (C1q-immunoglobulin G [IgG]) (Ortho Diagnostics, Inc., Raritan, N.J.) for measuring circulating immune complexes (CIC) was evaluated. The kit showed poor diagnostic sensitivity (P less than 0.005) for identifying CIC in patients with systemic lupus, rheumatoid arthritis, and bacterial endocarditis, as compared with polyethylene glycol-IgG and Raji cell tests (12, 24, and 24 positive, respectively, of 31 patients). Of the patients who were positive with the C1q-IgG test, 25% showed discrepancies when their results were compared with the polyethylene glycol-IgG and C1q-binding test results. Gel filtration chromatography of two of these discrepant sera showed the only peak of C1q-IgG activity to be associated with monomeric IgG (molecular weight, less than 200,000). We concluded that the kit method may be measuring substances other than CIC in some sera, because molecules of C1q attached to IgG should exhibit a molecular weight of greater than 500,000.     

Current situation and problems in the estimation of circulating immune complexes]

Some methods employing murine monoclonal antibodies have been developed for the estimation of circulating immune complexes (ICs). In the assays using monoclonal antibodies to C1q and C3d, ICs attached by reaction of C1q or C3d with the corresponding antibodies are detected by enzyme-labelled anti-IgG antibody. The murine monoclonal rheumatoid factor (RF) of IgG class is employed in the assay for detection of ICs. ICs reacted with the RF on the solid phase are further detected by the reaction with the second anti-IgG antibody labelled with the enzyme. The anti-C1q antibody in the sera as well as ICs produces positive reactions in the solid phase C1q assay, the assays using monoclonal antibodies are recommended for use in the detection of circulating ICs. In the pretreatment of serum samples, heating at 56 degrees C induces aggregation of IgG to produce a positive reaction by these sensitive assays, and the addition of EDTA-Na2 increases free C1q detached from C1 to induce increased binding to IgG. Reactions of aggregated IgG with RF and C1q in the fluid phase inhibit the following binding of monoclonal RF and anti-C1q antibody on the solid phase. Sera of patients with SLE were examined for CH50, anti-DNA antibody and ICs. The levels of ICs determined by the anti-C1q and C3d antibody assay did not correlate with other parameters. Positivity of ICs was unexpectedly lower in SLE sera. To evaluate the significance of the estimation of ICs, more data must be analyzed by these methods.     

Anti-C1q column: ligand specific purification of immune complexes from human serum or plasma. Analysis of the interaction between C1q and immune complexes

An efficient and reproducible procedure has been developed for the specific isolation of immune complexes. PEG precipitation of EDTA serum or plasma was an essential preliminary step to separate complex-bound from free C1q. PEG had no discernible effect on the molecular weight size of the extracted complexes. Redissolved complexes were incubated with a Sepharose-4B column coated with anti-human C1q antibodies and following removal of unbound material the bound complexes were sequentially eluted with 0.02 M EDTA, 0.5 M NaCl and 1 M propionic acid. Characteristics of the affinity column were established by the purification of 125I-labelled BSA-anti-BSA complexes and heat-aggregated IgG (HAGG) incubated in normal human serum (NHS). EDTA and NaCl eluted complexes were of similar molecular size and contained antigen, specific antibody, as well as human IgM, IgG, albumin, C3, C3c, C3d and C1q. Acid eluted complexes contained the highest yield of specific antigen and antibody and comprised in addition human C1q and C3d. Activation of complement components after C1q made the bond between C1q and immune complexes resistant to 0.5 M NaCl and interfered with the binding between solid phase anti-C1q and complex bound C1q. Using BSA-anti-BSA complexes and HAGG activated in NHS it was apparent that only a minority of the complexed material was isolated via the C1q ligand and this probably applies to the C1q binding assay. Most complexed material could be isolated using an anti-C3 affinity column.     

Glomerular deposition of C1q and anti-C1q antibodies in mice following injection of antimouse C1q antibodies

Anti-C1q autoantibodies are present in the serum of patients with different autoimmune diseases such as systemic lupus erythematosus (SLE). The occurrence of these autoantibodies correlates with renal involvement. In the present study we examined whether injection of rabbit antimouse C1q antibodies in mice leads to deposition in kidneys. Injection of healthy mice with a single dose of rabbit IgG antimouse C1q antibodies resulted in deposition of both C1q and IgG anti-C1q in glomeruli. The pattern of deposition observed in the glomeruli of mice injected with antimouse C1q antibodies both at 24 h and 2 weeks was both glomerular basement membrane (GBM)-associated and mesangial. Injection of control IgG did not have a detectable effect on circulating C1q levels, and no deposition of either C1q or rabbit IgG was seen at 24 h. The deposition of rabbit antimouse C1q and C1q in glomeruli resulted in complement activation, as assessed by C3 deposition, and influx of leucocytes associated with albuminuria in some, but not all mice. In none of the control mice was albuminuria observed. This report is the first to show that anti-C1q antibodies deposit in the healthy glomerulus together with autologous C1q. This deposition is stable for at least 2 weeks, causes complement activation, leucocyte influx and can lead to mild albuminuria.     

抗人C1q McAb轻链可变区基因结构特点

运用计算机进行基因序列分析在分子生物学研究中起重要作用，已被越来越多地用于研究大量累积的序列数据，获得许多重要发现。对抗人Ｃ１ｑ轻链可变基因（Ｃ１ｑＶ_Ｌ）的分析结果表明：Ｃ１ｑＶＬ属鼠ＩｇＶＫ基因家族，但与其它种属的Ｉｇ基因也显示较高同源性。它的另─个显著特点是与自身抗体编码基因高度同源。研究结果对我们进─步认识抗－Ｃ１ｑ抗体生理、病理作用具有─定价值。     

Antibodies against C1q in patients with systemic lupus erythematosus

The first component of the classical pathway of complement (C1q) is considered to be involved in the pathogenesis of systemic lupus erythematosus (SLE). This view is based on the observation that a substantial number of patients with SLE develop hypocomplementemia with depletion of the classical pathway components, and C1q has been shown to play an important role in the clearance of immune complexes and apoptotic bodies. In addition, homozygous C1q deficiency is the strongest disease susceptibility gene for the development of SLE that has been characterised in humans. However, most SLE patients have no primary complement deficiency. Hypocomplementemia in SLE patients is a secondary event and often associated with antibodies against C1q (anti-C1q). Although anti-C1q have been found in a number of distinct autoimmune disorders, they are best described in patients with SLE where they strongly correlate with renal flares. Current data suggest that the occurrence of anti-C1q in SLE patients is necessary but not sufficient for the development of proliferative lupus nephritis, suggesting an interference with the normal function of the complement system.     

New insight into the autoimmunogenicity of the complement protein C1q

C1q along with its physiological role in maintenance of homeostasis and normal function of the immune system is involved in pathological conditions associated with repetitive generation of anti-C1q autoantibodies. The time and events that cause their first appearance are still unknown. We addressed this issue by analyzing the immunogenicity of C1q in two target groups—one of non-diseased humans and the other of lupus nephritis (LN) patients whose autoimmune disorder is associated with high titers of anti-C1q autoantibodies. The non-diseased humans were represented by pregnant women because the sex hormones are thought to be involved in triggering autoimmune pathologies by their ability to tip the balance of female adaptive immune response to production of antibodies.We screened, using ELISA, 31 sera from healthy pregnant women for the presence of IgM and IgG classes of autoantibodies, recognizing epitopes within the native C1q molecule, its collagen-like region (CLR) and globular head fragment (gC1q). The latter was represented by recombinant analogs of the three globular fragments of A, B and C chains, comprising C1q-ghA, ghB and ghC. We did not find IgM antibodies for all test-antigens which suggest that the natural IgM antibodies are not involved in triggering autoimmunity to C1q. Still more, we did not detect anti-CLR antibodies which have been proved pathogenic in already manifested LN. We completed the analysis with comparative epitope mapping of gC1q and we found similar immunogenic behavior in both target groups—ghA and ghC contained the immunodominant epitopes. This implies that the initial immune response to C1q might occur when the molecule has interacted with its ligands via ghB as part of gC1q. The presence of anti-gC1q in both healthy and diseased humans also implies that these antibodies, unlike anti-CLR, may have a contribution to an onset of autoimmunity.     

Human Alveolar Macrophages Synthesize Active Complement Components C6, C7, and C8 in Vitro

We investigated whether serum-free human alveolar macrophage cultures synthesize active C6, C7, and C8. There was a significant binding of polyclonal anti-human C6 antibodies to agarose beads incubated with unstimulated macrophages for 24 or 48 h. Endotoxin stimulation of the macrophages was necessary for significant binding of polyclonal anti-C7 and anti-C8 antibodies to agarose beads co-cultured for 48 or 96 h. Two monoclonal antibodies (poly C9-MA and MCaE11) specific for a neoantigen of polymerized C9 in the terminal complement complex (TCC), bound to beads mainly incubated with endotoxin stimulated macrophages. The MCaE11 was more sensitive than the poly C9-MA in detecting the C9 neoantigen on beads incubated with the macrophages or human serum diluted 1:16. We thus conclude that human alveolar macrophages synthesize active C6, C7, and C9 that together with C5 and C9, assemble as the TCC on co-cultured agarose beads. Activation of the alternative pathway on the agarose with generation of fixed C3 and C5 convertases is a prerequisite for the subsequent generation of the TCC.     

Characterization of immune complexes in acute lymphoblastic leukaemia.

Sera from 120 children and young adults with acute leukaemia (59), various other tumours (53) and histiocytosis X (eight) were studied for the presence and characteristics of circulating immune complexes (CIC). Serial and parallel testing was performed using: C1q binding (solid phase), Raji cell radioimmunoassay and anti-C3 (solid phase). CIC were detected in 36 of 56 (64%) patients with acute lymphoblastic leukaemia (ALL) and in 62% of other tumour subjects. In the ALL sera, the mean positive C1q binding was 5.4 s.d., Raji cell 4.2 s.d. and anti-C3 4.4 s.d. In 12 ALL sera CIC were characterized for molecular size by sucrose gradient centrifugation. Most samples showed high molecular weight (19S) complexes but intermediate (11-14S) and smaller (8-9S) complexes were also detected. There was no apparent relationship between the presence, amount or physical size of the detectable CICs and clinical course of the patients studied; 12 patients with ALL in long term remission showed presence of CIC at some time during their course. Immune complexes precipitated from leukaemic sera were also examined for the presence of common ALL antigen (cALL) and Ia(DR) antigens utilizing rabbit antisera and mouse monoclonal antibodies. Experiments with isolated immune complexes from ALL sera provided no positive evidence for the presence of cALL antigen or Ia antigen within immune complex materials from ALL patients.