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.     

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.     

Characterization of C1q-binding IgG complexes in systemic lupus erythematosus

The molecular size of C1q-binding immunoglobulin (Ig) G complexes in systemic lupus erythematosus (SLE) sera was studied by gel filtration using C1q solid-phase radioimmunoassay (C1q SPRIA). All 15 SLE sera tested contained predominantly small-sized IgG complexes, cofractionating with monomeric IgG. In contrast to heat-aggregated IgG, these small-sized IgG complexes retained C1q-binding activity even after pepsin digestion, exposure to low pH, or reduction and alkylation, suggesting that the F(ab')2 region is involved in C1q-binding activity of these complexes. To see whether anti-C1q antibodies or small antigen-IgG complexes, which bind to C1q via their antigens, are responsible for C1q-binding activity via the F(ab')2 region, the pepsin-digested Ig fractions of SLE sera were fractionated at high salt. C1q-binding activity in the fractions corresponding to the F(ab')2 region increased 2.5- to 3.9-fold at high salt. These results suggest that the C1q-binding, small-sized IgG complexes may be comprised mostly of anti-C1q antibodies and that some of the antibodies, which are dissociated with their antigens at high salt, might be cross-reactive with C1q.     

A solid-phase radioimmunoassay for the detection of nRNP immune complexes

We developed a solid-phase radioimmunoassay for complement (C)-fixing nuclear ribonucleoprotein (nRNP):anti-nRNP immune complexes (nRNP ICs). The assay was based on the ability of the C-fixing nRNP ICs to bind strongly to immobilized F(ab')2 anti-C3. The extent of binding was quantified by incubating the C-fixing nRNP ICs bound to anti-C3 with 125I-labeled anti-nRNP-specific IgG. The interaction between anti-C3 and C-fixing nRNP ICs was rapid, time- and concentration-dependent and sensitive over a broad range of nRNP IC concentrations in an antigen-antibody ratio of 8 : 1 (9.8-5000 ng of human aggregated IgG equivalent per ml). We found that the assay also detected an immunoreactive U1-RNP antigen in Sm : anti-Sm immune complexes but did not detect SSA : anti-SSA immune complexes. The assay was preliminary applied for serum samples obtained from patients with mixed connective tissue disease (MCTD), and elevated concentrations of nRNP immune complexes were found in 3 out of 5 patients with MCTD. This assay appears to be applicable to the detection and quantification of circulating nRNP ICs in patients with MCTD.     

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.     

A solid-phase radioimmunoassay for detection of human antibodies. I. Measurement of IgG antibody to bee venom antigens.

A solid-phase radioimmunoassay (SPRA) has been developed to measure IgG antibodies to bee venom (BV) and phospholipiase A2 (PLA) in human sera. The principle of the test is similar to that of the radioallergosorbent test (RAST) measuring IgE antibody. Cyanogen-bromide-activated paper discs coupled with BV or PLA followed by supplementary coupling with human serum albumin were incubated with standard or test sera, washed, and incubated with 125I-labeled anti IgG. The serum levels of the IgG antibody have been temporarily expressed in arbitrary units. the reaction between the antigen and antibody was specific and the results were reproducible. Sera from 19 beekeepers, 42 beesting-sensitive patients and 20 blood donors (controls) were assayed by the SPRA. IgG antibodies to BV and PLA could not be detected (less than 4 U/ml) in all control sera, in 25 of the 42 patients and in one beekeeper. The IgG antibodies in 17 patients ranged between 5 to 58 U/ml (mean 7.6 U/ml), and in the 18 beekeepers ranged between 8 to 160 U/ml (mean 59 U/ml).     

A solid-phase radioimmunoassay for IgG and IgM antigammaglobulin factor in rheumatoid arthritis sera.

Rheumatoid factors of IgG and IgM class were detected by means of a solid-phase radioimmunoassay using formalinized sheep erythrocytes sensitized with rabbit anti-sheep erythrocyte serum and 125I-labelled anti-human IgG or anti-human IgM antibody. 28 of 35 sera and all of 7 synovial fluids of patients with rheumatoid arthritis showed positive reaction for both IgG and IgM rheumatoid factor, though the activity of IgM factor was mostly higher than that of IgG factor. However, rheumatoid factor present in synovial fluids of 3 seronegative patients was mainly of IgG class. Of 14 sera of patient with other collagen diseases, 11 showed IgG and 6 showed IgM rheumatoid factor. The IgG rheumatoid factor seemed to be associated with autologous IgG to form macro-molecular complexes which could be dissociated into 7S molecules of IgG rheumatoid factor and IgG by acid treatment.     

The interference of fibrinogen and heparin with the determination of circulating immune complexes in the C1q-binding assay.

When citrate plasma and serum of the same individual were tested simultaneously in the C1q-binding assay (C1qBA), binding levels in plasma were found to be 90-400% higher than in serum. The difference in 125I-Clq binding was due to the presence of fibrinogen in plasma. It was shown that complex formation between fibrinogen and 125I-Clq occurs and that this complex precipitates in the presence of polyethylene glycol, leading to the false positive results in the ClqBA. When heparin plasma was used to the assay, heparin itself also induced an increase in 125I-C1q binding that was not based on the presence of immune complexes. The effect of both fibrinogen and heparin could be inhibited by addition of protamine sulphate. Therefore, pretreatment of plasma with protamine sulphate makes it possible to use plasma samples for a reliable determination of C1q-binding levels. However, serum that is well clotted should be used preferentially.     

A solid-phase radioimmunoassay for bacterial lipopolysaccharide.

A radioimmunoassay for E. coli 055:B5 lipopolysaccharide (LPS) is described. The LPS was derivatised by two new methods and subsequently radiolabeled with 125I to a specific activity of 2-4 mCi/mg without apparent loss in its biophysical, immunological or biological activities. Using antibody-coated polystyrene tubes, a solid-phase radioimmunoassay was developed with a sensitivity of 10-500 ng/ml of LPS.     

A solid-phase radioimmunoassay for anti-SNP antibodies.

A highly sensitive and discriminatory solid-phase radioimmunoassay has been developed to detect anti-SNP antibodies in sera. Polystyrene tubes are coated with SNP and incubated with the test sera. The fixed antibodies are detected by a double layer technique using rabbit anti-human γ-globulin antiserum followed by incubation with a ¹²⁵I-labelled sheep anti-rabbit γ-globulin antiserum. Results are expressed in ng of the ¹²⁵I reagent fixed by 30 ωl of serum. The mean binding of 47 normal human sera was 22 ng ± 12 ng: 22/50 SLE sera gave over 34 ng binding. The specificity of the assay was studied in 3 different types of experiment: inhibition of the binding of positive sera either by pre-incubation with NDNA, SNP or RNA, DNAse I digestion of the SNP coated tubes, and incubation of SNP coated tubes with sera of known reactivity. It was shown that the solid-phase assay detects mainly antibodies directed against NDNA; however antibodies directed against the DNA-protein complex or the protein alone can easily be detected. Our results obtained with the solid-phase assay correlated well with those of the Farr assay. However, this new assay presents major improvements: it is simple, highly reproducible, and avoids the need for labelled antigen. A single labelled antiglobulin reagent allows identification of the class or subclass of reactive antibodies in a given species. Quantitation is more precise, particularly for sera containing high amounts of antibodies.     

A solid-phase radioimmunoassay for IgG gliadin antibodies using 125I-labelled staphylococcal protein A

A sensitive radioimmunoassay for IgG gliadin antibodies is described. Serum specimens were added to wells of plastic microtitre plates coated with gliadin. After removal of the unbound material, gliadin antibodies were detected by adding 125I-labelled staphylococcal protein A (125I-SpA). Serum specimens from coeliac patients on a normal diet or on a gluten-free diet were tested, as well as sera from an age-matched control group. Measurements to obtain precise quantitative values were made with gliadin antibody-rich serum as reference standard. High titres of gliadin antibodies were found in 18 out of 19 coeliac patients on a normal diet (95%); in patients on a strict gluten-free diet serum values did not exceed 2 S.D. of the control mean. Due to the high sensitivity of the method a low but detectable amount of gliadin antibody was present in the sera of all controls.     

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.     

A simple solid-phase radioimmunoassay for the measurement of IgG secreted in vitro by human lymphocytes.

A radioimmunoassay is described for measuring IgG, based on the ability of immunoglobulins of this class to inhibit the binding of radioiodinated staphylococcal protein A to IgG linked to a solid phase. The solid phase is represented by ox erythrocytes coated with anti-ox erythrocyte rabbit IgG, a reagent used for detecting cells equipped with receptors for the Fc fragment of IgG. By this assay the IgG secreted in vitro by human peripheral blood lymphocytes stimulated with PWM and those present in samples of very diluted human sera were measured. It was found that the assay is a very rapid, simple and reproducible procedure for the detection of IgG immunoglobulin at the nanogram level.     

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.     

A simplified solid-phase radioimmunoassay for carcinoembryonic antigen.

A simple and rapid solid-phase radioimmunoassay for the measurement of carcinoembryonic antigen (CEA) levels in sera or plasma is described. The procedure involves perchloric acid (PCA) extraction of the samples, followed by a rapid neutralization of excess acid with alkaline buffer solution. The PCA extracts are assayed uing antibody-coated plastic tubes and radio-labeled anti-CEA antibody as a marker. The assay may be completed in one day. The solid-phase direct-binding assay demonstrated a sensitivity of 0.5 ng CEA/ml plasmin and a marked tolerance to variations in pH and ionic strength of the system. A fair correlation between the CEA levels determined by the solid-phase radioimmunoassay and zirconium phosphate gel method was observed.     

IgA and IgG immune complexes increase human macrophage C3 biosynthesis.

We have studied the effect of IgA- and IgG-containing immune complexes on the production of complement proteins C3, factor B and C2 by human monocyte-derived macrophages, using biosynthetic labelling, immunoprecipitation, sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE) and autoradiography. There was a consistent increase in C3 production and secretion with both IgA and IgG immune complexes. This increase appeared after a 24-hr incubation period of the macrophages in the presence of immune complexes. No change in the biosynthesis of factor B and C2 proteins was observed in these experiments. Concomitant with the enhanced C3 biosynthesis, the immune complexes caused an increase in macrophage tumour necrosis factor (TNF) production; 310 + 24 U/ml/5 x 10(5) cells and 430 + 51 U/ml/5 x 10(5) cells for IgA and IgG immune complexes, respectively, versus 12 + 8 U/ml/5 x 10(5) cells in the control cells. The presence of prednisolone (2 x 10(-5) M) or dexamethasone (1 x 10(-7) M) inhibited the immune complex-induced TNF production, but had no effect on C3-increased synthesis, suggesting that the effect of immune complexes was not mediated by endogenous TNF production. These findings may be relevant to the local inflammatory response in IgA immune complex-mediated diseases, including IgA nephropathy.     

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.     

The covalent interaction of C3 with IgG immune complexes

Antigens (Ags) are converted into immune complexes (antigen-antibody complexes, IC) as soon as they encounter their specific antibodies (Abs). In fluids containing complement, the process of IC formation and fixation of complement components occur simultaneously. Hence, the formation of Ag-Ab-complement complexes is the normal way of eliminating Ags from a host. C3b-C3b-IgG covalent complexes are immediately formed on interaction of serum C3 with IgG-IC. These C3b-C3b dimers constitute the core for the assembly of C3/C5-convertase on the IC, which are subsequently converted into iC3b-iC3b-IgG by the complement regulators. These complexes are detected on SDS-PAGE by two bands of molecular composition, C3alpha65-C3alpha43 (band A) and C3alpha65-heavy chain of the Ab (band B), which correspond to C3b-C3b and C3b-IgG covalent interaction respectively, and that identify opsonized IC (C3b-IC). C3b can attach to Fab and Fc regions of the Ab molecule with similar efficiency. The presence of multiple C3b binding regions on IgG is considered an advantageous characteristic that facilitates the elimination of Ags in the form of C3b(n)-IC. Ab molecules on the IC recognize the Ag, and also serve as a very good acceptor for C3b binding. In this way, Ags, even if they have no acceptor sites for C3b, can be efficiently processed and removed. When C3 is activated in serum by IC or other activators, secondary C3b-IgG covalent complexes are generated, with bystander monomeric circulating IgG, and thus constitute, physiological products of complement activation. These complexes gain importance when IgG concentration is extremely high as in cases of infusion of intravenous IgG (IVIG) in several pathologies. The covalent attachment of activated complement C3 (C3b, iC3b, C3 d,g) to Ags or IC links innate and adaptative immunity by targeting Ags to different cells of the immune system (follicular dendritic cells, phagocytes, B cells). Hence C3b marks Ags definitively, from the earliest contact with the innate immune system until their complete elimination from the host.     

Precipitated immune complexes of IgM as well as of IgG can bind to rabbit polymorphonuclear leucocytes but only the immune complexes of IgG are readily phagocytosed.

We have shown by in vitro experiments, using immunofluorescence techniques, that precipitated immune complexes of IgM antibodies and ovalbumin (ICIgM) are able to bind to rabbit blood polymorphonuclear leucocytes (PMN), as well as immune complexes of IgG antibodies (ICIgG). This binding capacity for both classes of immune complexes is exhibited by more than 80% of the PMN cell population and is independent of Ca2+ in the medium. For ICIgG the binding to PMN can be completely inhibited by preincubation of the cells with soluble IgG used at physiological concentrations (competition for the Fc gamma receptors) while for ICIgM there is no such inhibition by fluid-phase IgM. After binding to the leucocytes there was a striking difference in the fate of ICIgM and ICIgG: whereas the ICIgG was readily phagocytosed (endocytosed), the ICIgM remained mostly on the cell surface, being only poorly endocytosed after 1 hr incubation at 37 degrees. This was demonstrated by a quantitative fluorimetric method developed to assay phagocytosis of immune complexes, and was confirmed by a qualitative fluorescence quenching technique. These results may have implications for understanding the fate of these classes of immune complexes formed in circulation or deposited in tissues, and the participation of PMN in inflammatory reactions and tissue injury in immune complex diseases.     

Hepatic uptake of circulating IgG immune complexes.

IgG antibodies were found to increase the uptake of circulating dinintrophenylated human serum albumin (DNP-HSA) preparations by the nonparenchymal liver cells in rats. Highly DNP-conjugated HSA was taken up by the Kupffer cells both when given alone and when complexed by IgG. More lightly DNP-conjugated HSA was taken up mainly by the liver endothelial cells. Here, IgG promoted the antigen uptake both by the Kupffer cells and by the endothelial cells. Uptake of IgG immune complexes (IgG-ICs) by the sinusoidal endothelial cells of the liver is a new aspect on the function of these cells. Whether or not this phenomenon is Fc receptor-mediated is discussed. A heat-labile serum factor was found to direct the ICs to the Kupffer cells. This implies that serum complement and hepatic C3 receptors are essential for the physiological clearance of circulating immune complexes.