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.     

Solid phase C1q microassay for the rapid detection of circulating immune complexes

A C1q solid phase microassay was designed for the rapid detection of circulating immune complexes. Its level of sensitivity is comparable to that of the Raji cell and greater than the C1q binding assay; furthermore, it is faster and low in cost. These conditions make it more practical and applicable in the clinical setting.     

Detection of immune complex by C1q solid phase enzyme immunoassay]

We have developed a new and improved method for detecting immune complexes (IC) by C1q solid-phase enzyme immunoassay (CSP-EIA). The sensitivity of this method was between 0.62 micrograms/ml and 1.25 micrograms/ml, and values in normal individuals were 1.8 micrograms/ml and less. The positive rate of IC of sera in which abnormal values were detected by autoimmune disease associated laboratory examinations was 50.0% in RA (2+), 20.4% in ANA (+), 60.0% in CH50 (less than 10), 41.7% in LE latex (+). In sera of RA (2+), the higher the value of CRP detected by a semi-quantitative analysis was, the higher the frequency having abnormal high IC values was. The number of IC positive sera, in which enzyme-linked immunoglobulins were detected, was 18 of 68 (positive rate 26.5%). The number of IC positive samples in asymptomatic carriers of sera, whose titer of anti-HTLV-I antibody was positive by gelatin particle agglutination assay (PA), was 14 of 67 (pos. rate 18.4%). All of these 14 samples were high positivity of anti-HTLV-I antibody (titer greater than or equal to 256 times). In urine of some patients with urogenital diseases, IC-like substances to show positive reaction by our CSP-EIA were detected. However, any positive reaction was not detected either by an anti-C1q- or an anti-C3d method. Studies are in progress to elucidate detailed characterization of the IC-like substances.     

Preparation of a defined immune complex for use in C1q binding assays

Soluble immune complexes of human β₂ microglobulin with its IgG antibodies from Macacca irus antisera have been tested for suitability as a reference preparation for immune complex assays in human disease. The defined complex described is stable on storage, behaves reproducibly in a C1q solid phase binding assay and fulfills the criteria required for an international reference standard.     

Measurement of immune complexes with the liquid phase C1q binding assay: ten years experience in a routine diagnostic laboratory

We describe our 10 years experience in assaying over 15,000 clinical specimens for immune complexes (IC) using the C1q binding assay. Normal ranges were initially established using a large panel of blood donor sera and precision of the assay was optimized by inclusion of heat aggregated IgG (HAGG) as standards. Nevertheless some variability was observed due to variation in C1q binding from batch to batch and with aging of this reagent. In an empirically selected 2 year period involving over 3,000 clinical specimens, 25% had elevated concentrations of IC. Of these the majority were from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), other connective tissue disorders, infective endocarditis (IE), diffuse interstitial lung disease (DILD) and vasculitis (VASC). In RA, IE and VASC, significant correlations were observed between concentrations of IC and rheumatoid factor (RF) and the addition of a purified monoclonal RF to normal serum caused increased C1q binding. Longitudinal studies in RA and IE demonstrated a striking decline in IC in response to effective treatment. We conclude that the measurement of IC provides little additional useful diagnostic information in those diseases associated with high levels of RF but appears more useful in disorders such as SLE, IE and DILD in which RF is absent or present in low concentration. Sequential monitoring of IC in RA and IE reflects response to treatment.     

C1q binding substances in pemphigus and bullous pemphigoid. Detection with a [131I] C1q binding assay.

A modification of the [125I]C1q binding assay was developed to allow the estimation of C1q binding activity (C1q BA) in pemphigus and bullous pemphigoid sera. The modifications include lower final concentration of PEG 6000 (1-5%) which permitted the use of sera that had been stored at -20 degrees C for extended periods of time; use of 131I instead of 125I and an [131I] C1q concentration of 5 microng/ml rather than 1 microng/ml. EDTA was used at a final concentration of 0-13 M to obviate the need for heat inactivation of sera. Sera from seventy-one patients with pemphigus and from 142 patients with bullous pemphigoid were tested for C1q BA. Of these 40% of the pemphigus and 20% of the bullous pemphigoid patients showed elevated C1q BA. A relationship between elevated C1q BA in serum and active disease was noted. Sequential samples from forty patients with pemphigus and thirty-seven patients with bullous pemphigoid demonstrated two different types of relationship between serum antibody titres to cutaneous antigens and C1a BA. In some patients serum antibody titres and C1q BA increased and decreased simultaneously; in others, increase of C1q BA followed increase of antibody titre and coincided with its decrease. The latter relationship supports the hypothesis that C1q BA may represent at least in part antigen-antibody complexes containing cutaneous antigens.     

C1q binding and Raji immune complex assays: a comparison using defined immunoglobulin aggregates

Aggregated IgG is frequently employed as a standard in systems for the measurement of immune complexes in man and animals. In this paper aggregates prepared by heat or alkali denaturation of human IgG were fractionated by column chromatography through LKB AcA 22 Ultrogel. Heat aggregation yields preparations containing considerably more monomer than alkali treatment (47% and 6.3% respectively). The bulk of aggregated material prepared by both methods was of size 19 S or greater. Smaller aggregates were present in assayable quantities only in the alkali aggregated material. The sized fractions of aggregates IgG were tested in the presence of a human complement source for their efficiency in the C1q binding and Raji radioimmunoassay for immune complexes. Both techniques efficiently measured large aggregates (greater than or equal to 19 S) but the C1q binding assay measured smaller material with greater efficiency than did the Raji cell assay. Neither technique detected monomeric IgG. The date presented is relevant to the binding characteristics of the 2 assay systems studied and suggests that when used together they are capable of measuring immune complexes present over a wide range of sizes.     

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

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

C1q binding to human vascular smooth muscle cells mediates immune complex deposition and superoxide generation

Evidence was obtained for the binding of Clq to the membrane of cultured vascular smooth muscle cells derived from human umbilical cord veins. Clq was fixed to the cell membrane at 4°C, whereas it was ingested into the cytoplasm, as a cytoplasmic inclusion, when tested at 37°C. The addition of Clq in advance inhibited the subsequent binding of Clq. Neither fibronectin nor laminin was detected on the cell membrane. Aggregated IgG bound to vascular smooth muscle cells in the case of preincubation with Clq at 4°C, whereas aggregated IgG did not bind to the cells in the absence of Clq. The addition of Clq molecules to the cells in suspension enhanced Superoxide generation by vascular smooth muscle cells. There was no effect of Clq on Superoxide generation by the cells in monolayer. These results suggest that Clq binds on the membrane of vascular smooth muscle cells via its specific receptor that mediates immune complex binding to the cells and Superoxide generation. These properties elucidate the mechanisms by which circulating immune complexes deposit in the vascular wall, and subsequent degradation of tissue components surrounding vascular smooth muscle cells occurs through oxidative burst of the cells.     

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.     

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.     

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.     

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.     

Enhancement of the binding of C1q to immune complexes by polyethylene glycol

The binding of 125I-labelled human C1q to insoluble rabbit IgG:ovalbumin immune complexes was enhanced by polyethylene glycol (PEG, Mr 8 x 10(3)) in the concn range 0-2.5% (w/v). C1q with native immunoglobulin bindings sites rendered inactive by diethylpyrocarbonate treatment did not bind to immune complexes in the presence of PEG. The ionic strength dependence of the binding was independent of the presence of PEG. There was a linear relationship between the logarithm of the apparent affinity constant of the C1q:immune complex interaction and PEG concn.