C1q solid-phase radioimmunoassay: binding properties of solid-phase C1q and evidence that C1q-binding IgG complexes in systemic lupus erythematosus are not bound to endogenous C1q

The binding properties of C1q solid-phase radioimmunoassay (C1q SPRIA) were examined, using heat-aggregated IgG (HAG) as the model of immune complexes (IC). The free, liquid-phase C1q, which was added to the C1q-coated tubes prior to the addition of HAG, had little inhibitory effect on binding of HAG to the solid-phase C1q, suggesting that the solid-phase C1q has a higher affinity for HAG than the liquid-phase C1q. On the other hand, more than 60% inhibition was seen when HAG was preincubated with the liquid-phase C1q. These binding properties of HAG to the solid-phase C1q in the presence of the liquid-phase C1q were not essentially altered by the heat inactivation or the addition of EDTA, suggesting that these pretreatments are not essential in C1q SPRIA. Next, in similar kinds of experiments, the binding properties of C1q-binding IgG complexes in SLE sera were investigated. In contrast to HAG, the binding capacity of IgG complexes in SLE sera to the solid-phase C1q was not inhibited by the preincubation with excess liquid-phase C1q. These findings suggest that C1q-binding IgG complexes in SLE sera detected by C1q SPRIA may not be bound to endogenous C1q in the circulation.     

A microplate adaptation of the solid-phase C1q immune complex assay

A method has been developed for the detection of C1q binding immune complexes in serum in which microculture plates are used as the solid-phase matrix for adsorption of C1q. This micromethod used only one-tenth of the amount of both C1q and [¹²⁵I]anti-human immunoglobulin per test and enabled 7 times as many samples to be tested in triplicate in comparison with the number performed in duplicate by the standard tube assay.¹²⁵I-labelled C1q studies showed that adsorption varied with the brand of microplate used, some types of plate binding up to 66% of the labelled material. This is considerably more than that bound by the polystyrene tubes generally used for this assay.The increased capacity of the method allowed the binding to plates coated with the same batch of C1q to be assessed at various times after storage for 8–10 weeks at both 4°C and ?70°C. A marked decrease in binding to C1q of aggregates of IgG was observed on storage for longer periods. Results with different batches of aggregated IgG which had been ultracentrifuged suggested that this may be used as an effective standard, stable on storage at ?70°C.A comparison with the standard tube method of aggregated IgG, normal control sera and sera from patients with SLE showed that the micromethod adaptation of the C1q solid-phase binding radioimmunoassay is more economical and easier to perform and does not impair accuracy or standardization.     

Detection of immune complexes using a solid-phase C1q polystyrene ball assay

A polystyrene ball C1q solid-phase assay (PSB C1q SPA) has been developed for quantitating immune complexes in human serum. While similar to the previously reported solid-phase C1q assays in principle, the use of polystyrene balls with a specular finish has resulted in assay with significantly improved accuracy, sensitivity and reproducibility. The sensitivity of the assay based on the amount of AHGG bound per μg C1q added was approximately 12-fold higher in the PSB assay compared to the polystyrene tube solid-phase C1q method.

In order to correct for variable background contributions in different samples, values obtained with heat-inactivated C1q were subtracted from each experimetal result. Reproducibility studies yielded a coefficient of variation (CV) of 10 and 4% in day-to-day assays using 25 μg and 100 μg AHGG/ml normal serum, respectively compared to 11–15% for the tube technique. Within run measurements gave a CV of 37 and 20% at the low and high levels of AHGG.

Aggregated human gamma-globulin (AHGG) was used as a model immune complex and when chromatographed on Biogel A-15m yielded major fractions at 15,000,000 and 150,000 daltons. Maximum binding of AHGG to PSB C1q occurred with aggregates greater than 15,000,000 daltons.

Optimum binding of human albumin-anti-albumin complexes in the PSB C1q SPA occurred at a molar ratio of 1:1.5. The size distribution of this complex active in the assay determined by sucrose density gradients was 14–32 S with peaks at 21 and 27 S.

A normal range of immune complexes was determined as 15±8μg AHGG equivalents (±2 S.D., n = 65). Approximately 70% of rheumatoid arthritis, linear scleroderma, vasculitis, Sjögren's and glomerulonephritis and 40% of SLE patients were above 2 S.D. of normal. SLE patients demonstrated elevations in immune complexes only during periods of increased disease severity. The assay was a useful monitor of plasmapheresis in an SLE patient, showing decreases in immune complexes after each plasmapheresis. DNA did not interfere with AHGG binding whereas lipemia prevented detection of added AHGG.     

IgG subclass restriction of autoantibody to solid-phase C1q in membranoproliferative and lupus glomerulonephritis.

The IgG subclass distribution for autoantibodies to solid-phase C1q (anti-spC1q) in sera from 14 patients with membranoproliferative glomerulonephritis (MPGN) and 10 patients with systemic lupus erythematosus (SLE) nephritis was determined by an enzyme-linked immunosorbant assay employing C1q as the immunosorbant in the presence of 2 M NaCl to prevent Fc binding and monoclonal anti-human IgG subclass reagents. The autoantibody to spC1q in MPGN, especially in types I (7 patients) and II (3 patients), was almost entirely restricted to IgG3. In contrast, in SLE anti-spC1q was completely restricted to IgG2 in 3 patients while predominantly IgG2 in the other 7 patients. The different subclass restriction of anti-spC1q in these two disorders suggests that antibody formation is either in response to different epitopes on the collagen-like region of C1q or that patients with SLE and MPGN mount different immunologic responses to the same antigenic stimulus.     

Rapid three-step purification procedure for isolation of the C1q component of human complement and its use in a solid-phase C1q binding assay

A new procedure for the isolation of the C1q subcomponent of complement from human sera has been devised. The 3-step protocol employs DEAE Sephadex A-50, hydroxyapatite and Sephacryl S-200 chromatographies and can be performed within 9 h. It yields immunoglobulin-free homogeneous C1q protein with about 80% recovery. The isolated C1q protein is biologically active and may be used for the detection of circulating immune complexes in sera by the solid-phase C1q binding assay.     

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.     

Porcine C1q and the solid-phase immunoassay of human immune complexes

Experiments were undertaken to determine if porcine C1q could replace human C1q in the solid-phase immunoassay of human immune complexes (ICs). Porcine C1q was obtained by a two-cycle precipitation method involving dialysis against chelating agents in low ionic strength buffer. C1q was adsorbed to polystyrene beads and in vivo- or in vitro-formed ICs binding to the solid-phase C1q were detected with ¹²⁵I-labeled or horseradish peroxidase-conjugated anti-human gamma antibodies. Unfractioned, heat-aggregated human gamma globulin (ΔIgG) could be detected at 20 ng/ml when diluted in buffer only. The detection threshold changed to 40–80 ng ΔIgG/ml when the assay was run with buffer containing normal human serum diluted 1 : 1000 (the serum dilution used for detecting natural ICs). Analysis of systemic lupus erythematosus sera revealed that 60% contained highly significant levels of ICs (binding ?3 S.D. above the mean of controls). Comparison with platelet aggregation test results revealed a highly significant correlation between the two methods (P < 0.0001), even though each assay detected ICs in several serum specimens negative in the other test. These results demonstrate that porcine C1q can functionally replace human C1q in the solid-phase immunoassay of human ICs. Since porcine blood is normally a waste product of the meat-processing industry, it is an obvious source of easily isolated C1q for use in such an assay.     

Carbodiimide crosslinking of human C1q and rabbit IgG

The water soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was used to covalently link carboxyl groups on rabbit IgG to lysyl groups on complement protein C1q. The interaction between C1q and IgG was disrupted by varying the pH, modifying essential residues in the IgG binding site of C1q and by reducing the interchain disulfides of IgG. Under each of these conditions the correlation found between binding and crosslinking indicated a strong requirement for the proteins to bind normally in order for crosslinking to occur. SDS-PAGE analysis of the crosslinked material showed a 210 kDa band consistent with one IgG crosslinked to two disulfide linked C1q chains. Blotting and autoradiography showed the crosslinking involved the A and/or B and C chains of C1q. The lysines flanking the intrachain half cystines are proposed as the likely candidates for crosslinking to IgG, thus delineating the immunoglobulin binding site of C1q.     

Disulfide bridge formation between C1q and IgG in vitro

The globular heads of C1q are known to possess free-SH groups. Here we show that these groups, which are concealed in the native molecule, are exposed by interaction of C1q with dialysis membrane. During iodination, I+ and I2 oxidize these sulfhydryls to produce disulfide-linked C1q aggregates. Approximately 15% of C1q bound to immunoglobulin aggregates is resistant to high conductivity elution and reducing agent is required to release it. These data show that dialysis, adsorption to Ig and iodination of C1q result in structural and functional changes in the molecule, and suggest a mechanism by which these changes occur. Disulfide bridging between C1q and IgG in vitro suggests that this may be a normal physiological function of C1q for which the free cysteines of human, mouse and guinea pig C1q have been conserved.     

A simple immunofluorescence assay for C1q binding

Antibody-independent binding of C1q to Crithidia luciliae kinetoplast DNA was demonstrated by immunofluorescence microscopy. The method allows detection of C1q in human sera diluted 1/20–1/40 and in 5–10 μg/ml concentrations of isolated C1q. Various substances known to interfere with the early events of complement activation inhibited binding of C1q to kinetoplasts. The method may be used as a functional assay for serum C1q.     

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 IgG aggregates or immune complexes using solid-phase C1q and protein A-rich Staphylococcus aureus as an indicator system.

A radioimmunoassay for detection of C1q-binding IgG aggregates and antigen-IgG antibody complexes is described. The assay makes use of solid-phase C1q and 32p-labelled protein A-rich Staphylococcus aureus as an indicator system. Both 19S and heavier IgG aggregates that fixed C1q were detected. The sensitivity of the assay permitted detection of heavy (19-25S) IgG aggregates at a concentration of 8 mug/ml or less. The results indicated that detection of IgG in this assay is dependent on the degree of IgG polymerization and the molar ratio between the solid-phase C1q and the IgG polymers. Albumin-anti-albumin complexes, preformed at equilibrium with antibody to antigen molar ratios of 2:1 to 3:1 and at antigen concentrations of 25 to 40 mug/ml, were also detectable using the described radioimmunoassay.     

The reaction between the complement subcomponent C1q, IgG complexes and polyionic molecules.

The strength of the bond between 125I-labelled C1q and immune complexes, Fc piece, dextran sulphate, polyglutamic acid and polylysine has been investigated. The binding of C1q to Fc piece, small molecular weight (less than 10,000) dextran sulphate, polyglutamic acid and polylysine have value; for the functional affinity constant (Ko) in the range of 0.2-1.5 X 10(4) M-1. In contrast the binding of C1q to immune complexes and large molecular weight polyions (greater than 100,000 is much greater and lies in the range 3 X 10(7)--4 X 10(8) M-1. The differences in the binding constants between the two groups can be explained if the Fc piece and small molecular weight compounds bind to only 1 head of the C1q molecule but the immune complexes and large molecules bind to 2 heads. There are probably 6 binding sites on the C1q molecule for dextran sulphate. The enhancement of the binding affinity of C1q by reduction in ionic strength and the reaction with polyions, indicate that ionic groups are present near or within the binding sites.     

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.     

IgG autoantibodies to C1q do not detectably influence complement activation in vivo and in vitro in systemic lupus erythematosus

The influence of IgG antibodies to C1q (C1qAb) on activation of the classical pathway of the complement system was investigated in patients with systemic lupus erythematosus (SLE). In in vivo experiments, a prototype for immune complexes was administered intravenously to 14 patients and 9 healthy controls. Eight SLE patients had increased C1qAb titers. The increase of C3a levels, which was measured as a parameter of C1 activation, was significantly lower in SLE patients than in the healthy controls (p = 0.01). No correlation was found between C3a increases and C1qAb titers. In in vitro experiments the influence on C1 activation of monomeric IgG isolated from serum of 11 SLE patients, 7 of whom had increased C1qAb titers, was measured in a C4 consumption assay. The presence of C1qAb did not influence C4 consumption. The results demonstrate that C1qAb do not influence C1 activation by immune complexes in SLE patients.     

IgG4 antibodies from patients with asymptomatic bancroftian filariasis inhibit the binding of IgG1 and IgG2 to C1q in a Fc-Fc-dependent mechanism

A striking feature of lymphatic filariasis (LF) is the clinical heterogeneity among exposed individuals. While endemic normals (EN) remain free of infection despite constant exposure to the infective larvae, a small group of patients, generally microfilaria free (Mf-) develops severe pathology (CP) such as lymphedema or hydrocele. Another group of infected individuals remains asymptomatic while expressing large amounts of microfilariae (Mf+). This Mf+ group is characterized by an immune-suppressed profile with high levels of anti-inflammatory cytokines and elevated IgG4. This particular immunoglobulin is unable to activate the complement. The complement system plays a critical role in both innate and adaptive immunity. However, its importance and regulation during LF is not fully understood. Using affinity chromatography and solid-phase-enzyme-immunoassays, we investigated the ability of antibody isotypes from LF clinical groups to bind C1q, the first element of the complement’s classical pathway. The results indicate that while C1q is similarly expressed in all LF clinical groups, IgG1–2 in the plasma from Mf+ individuals presented significantly lower affinity to C1q compared to EN, Mf−, and CP. In addition, selective depletion of IgG4 significantly enhanced the affinity of IgG1–2 to C1q in Mf+ individuals. Strikingly, no effect was seen on the ability of IgG3 to bind C1q in the same conditions. More interestingly, papain-generated IgG4-Fc-portions interacted with Fc portions of IgG1–2 as revealed by far-western blot analysis. These data suggest that while being unable to bind C1q, IgG4 inhibits the first steps of the complement classical pathway by IgG1 or IgG2 via Fc-Fc interactions.