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Previous studies identified specific receptors for C1q on human blood platelets in purified systems using monomeric C1q. To assess the physiologic potential of platelet C1q receptors, C1q binding was evaluated in whole blood and in the presence of immune complexes or aggregated IgG. Blood was obtained from healthy volunteers and collected directly into EDTA (1 vol 100mM EDTA:9 vol whole blood) and purified, 125I-labeled C1q or 125I-C1q associated with albumin-anti-albumin immune complexes. Samples were incubated at 22 or 37 degrees C for 60 min, and total cell bound C1q and platelet associated C1q were quantified. Platelet-bound, monomeric C1q or immune complex-associated C1q represented 40-50% of total peripheral blood cell-associated C1q. C1q binding was unaffected by the incubation temperature, but the preincubation of 125I-C1q with immune complexes enhanced binding two- to threefold. This binding was partially inhibited by preincubating platelets with either the collagen-like amino-terminal fragments of C1q (c-C1q) or a monoclonal antibody Fab fragment recognizing platelet Fc receptors. A more complete inhibition was achieved if platelets were preincubated with both agents. Similar observations were made using washed platelets and 125I-C1q associated with aggregated IgG. The role of C1q and platelet C1q receptors in enhancing aggregated-IgG binding to platelets was further supported by experiments demonstrating increased 125I-aggregated IgG binding to platelets not only after preincubation of 125I-aggregated IgG with C1q but also following platelet preincubation with C1q. These data suggest that C1q receptors may participate in the localization and presentation of C1q-associated immune complexes on the platelet surface and demonstrate that platelets contribute significantly to the C1q binding activity of peripheral blood.

Circulating immune complexes (CICs) have been detected in the sera of patients with non-Hodgkin's lymphoma (NHL), Hodgkin's disease, chronic myeloid leukemia, and acute lymphoblastic leukemia by using C1q-binding and L1210-binding assays. Both assays gave broadly similar patterns of reactivity in terms of frequency and magnitude, though there are some differences. Significantly elevated CIC levels were observed in all pathologic groups. However, sera from NHL patients with an unfavorable prognosis consistently exhibited the highest frequency of positive values and mean CIC levels in both these assays. The two tests showed concordance in 66.6% of the NHL patients' sera and were significantly correlated. Of the sera from NHL patients 12.7% were positive in the C1q-binding assay only and 15.9% in the L1210-binding assay only. Both the assays gave positive results in some patients, and a degree of overlap indicates the presence of different types of CIC in cancer patients' sera. The combined use of two methods for detecting CICs may be useful for evaluation of the activity, the extent, and the prognosis of the malignant disease.

In order to study the relationship between the presence of circulating immune complexes (CIC) and the severity and duration of acute dysentery, we studied 11 adults and 45 children in Bangladesh with bacillary and amebic dysentery, using the Raji cell and solid-phase C1q (C1q-SPA) assays. CIC were found in 70% of patients with shigellosis and in all eight cases of amebic dysentery. Mild shigellosis was associated with positive samples in the first week of clinical illness, whereas severe cases, including those with the hemolytic-uremic syndrome, had negative admission assays but positive convalescent assays. Samples positive in the first two weeks of illness were more likely positive by the Raji cell assay alone whereas samples in the third and fourth weeks of illness were positive more often by the C1q-SPA assay. Only one shigellosis sample was positive by both assays. In amebiasis 11 of 13 samples were positive by the Raji assay alone. In dysenteric disease circulating immune complexes probably represents the failure of the inflamed mucosa to exclude microbial and dietary antigens, and suggests that the presence of CIC in any intestinal disease must be interpreted with caution.

After a polyethylene glycol extraction which removes monomeric immunoglobulin G (IgG), circulating immune complexes were assayed by the FIAX fluorescence assay (Whittaker M. A. Bioproducts, Walkersville, Md.). The extraction portion of the procedure can be completed within a few hours after an overnight precipitation step, and the fluorescence assay takes about 0.5 h. The fluorescence assay is similar to the routine FIAX method for measuring IgG in cerebrospinal fluid except that a 10-microliter sample from sera is substituted for a 50-microliter sample. Good parallelism between endogenous immune complexes, monomeric IgG, and aggregated human globulin, along with good between-run precision (coefficients of variation, less than 10%), indicates that monomeric IgG calibrators from the kit could be used to standardize the assay. This standardization eliminates the need for aggregated human globulin, which is unstable and difficult to prepare. A reference range of 9 to 63 mg/liter followed a gaussian distribution. Correlation data indicate that the test provides information similar to the C1q-binding test (rho = 0.87; n = 30) but has better diagnostic sensitivity and better discrimination in the high range. Because of its simplicity, good reproducibility, and accessibility to equipment available in many laboratories, the method described here may be a preferred technique for measuring circulating immune complexes.

Circulating immune complexes were evaluated in 25 patients (age range 10 to 46 years) who had undergone splenectomy for non-malignant conditions by studying a polyethylene glycol insoluble serum fraction. Although the extent of binding to Clq was within normal limits, these patients had increased concentrations of factor B in the immune complex serum fraction. These findings indicate that an unusual type of circulating immune complex may be detected after splenectomy, suggesting a possible role for the spleen in the removal of circulating immune complexes.

The presence of circulating antigen-antibody complexes in the sera of patients with onchocerciasis was investigated using the Clq and conglutinin solid-phase binding assays. Only 50% of patients' sera had demonstrable complexes, levels of complexes were unrelated to microfilarial load and specific anti-onchocercal antibody titres and results with the two tests for complexes were not correlated. Both IgM- and IgG-containing complexes were commonly involved but there was no correlation between the levels of complexes containing these isotypes. Evidence for the presence of IgE in complexes of sera from a minority of individuals was also obtained.

A computer program is presented which enables iterative determination of the weighted standard curve and the quality control parameters calculation for the liquid phase radioimmunoassay which employs binding of labelled C1q as a measure of the presence of immunological complexes in serum samples. 22 independently performed laboratory tests were evaluated with respect to the presence of relationship between the dose (standardized human IgG aggregates) and response (fraction of radiolabelled C1q bound). On 14 occasions the plotted curve was sigmoidal in shape, the rest of the curves did not reach lower or upper asymptote. When the logistic transformation of this data was used the dose-response curve was linear and could be plotted with the use of the least square method. This allows calculation of the two other parameters of the sigmoidal curve (slope and midpoint). The slope of the weighted regression line, intercept point with the axis of ordinates and correlation coefficient were determined by means of the least square method for weighted linear regression with repetitions. When the iterative procedure was completed, content of immunological complexes in sera was calculated. The program is written in Fortran for the ODRA 1300 computer series. The printout consist of three parts. First part contains the original data, parameters and information about the form of input data. The second part of the printout consist of the results, obtained from non-weighted and weighted regression lines and the plotted regression line. The third part of the printout contains the final results of immune complexes determination.

Circulating immune complexes (CIC) with a molecular weight (M.W.) of 1.4-1.6 x 10(6) were observed in patients with liver disease. CIC containing abnormal albumin were found in patients with chronic active hepatitis. The abnormal albumin from CIC displayed an apparent M.W. of 105-110,000 on Sephadex G-100 and Sepharose 6B column chromatograph before reduction, and only 50,000 on reduced sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). It is postulated that abnormal albumin contained within CIC may be a dimeric-S-S-linked small albumin (sAlb-S-)2. Immunoglobulins in the CIC containing (sAlb-S-)2 were identified as IgM and IgG. Based on the constituents of CIC and their M.W., the assumed molecular formula of CIC is (IgM)1 (sAlb-S-)2 (IgG)3. Cause and effect of these CIC in liver disease, especially a possible role of altered host components in the initiation of autoimmune reactions, are discussed.

The relationship between immune complexes and insulin antibodies was evaluated in 237 insulin treated subjects with a duration of diabetes of more than 1 year. Ninety-seven diabetics were selected at random (group 1) whereas 140 according to the presence of diabetic microangiopathy (group 2). Immune complexes were evaluated by the solid phase C1q binding test in all patients and by conglutinin radioimmune assay in most of them. Insulin antibodies were determined by Christiansen's and Anderson's methods. Immune complexes as detected by the solid phase C1q method were found increased in group 1 and there was an inverse correlation between these complexes and insulin antibody levels. In group 2 patients with microangiopathy the amount of this kind of complex was significantly greater than in those without microvascular lesions and there was no correlation with insulin antibodies. Immune complexes as detected by conglutinin were found increased in group 2 patients and these were significantly correlated with the level of insulin antibodies. No increase in these immune complexes was found in patients with microangiopathy when compared with patients without microangiopathy. Insulin antibodies were not correlated with the presence of complications. Overall, immune complexes detected by C1q binding were significantly correlated with the presence of microangiopathy. In patients with high insulin antibody levels the complexes formed were not detected by C1q binding. The immune complexes detected by conglutinin are correlated with insulin antibodies, but not with the presence of microangiopathy.

A microscale ELISA system, designated as the 'C1q-micro ELISA,' has been developed for the measurement of circulating immune complexes. The sensitivity of the method ranged between 0.1 and 3 microgram equivalents of aggregated human gamma-globulins. The procedure requires small quantities of serum and reagents, and it can be performed with rapidity without the loss of accuracy and reproducibility.

Immune complexes (IC) were assessed in serum and CSF from 11 patients with acute inflammatory polyneuritis (AIP). An 125 I-labeled Clq binding assay (Clq BA) was used. IC were present in the sera of four patients and in the CSF of six. CSF-Clq BA of AIP (Group 1) were compared with 12 patients with other inflammatory neurological diseases (Group 2) and 22 patients with non-inflammatory neurological diseases (Group 3). There was only a significant difference between Group 1 compared to Group 3 (p less than 0.05). Serum and CSF IC did not correlate either with blood-brain barrier lesions or with immunoglobulin deposits in sural nerve biopsy.

ZnSO4 promotes the binding of C1q to immune complexes over the same concentration range (10(-5)-10(-4) M) that it inhibits binding of C1 to cell-bound immunoglobulin [Biochem. Biophys. Res. Commun. (1981) 103, 856-862]. At higher concentrations (10(-3)-2 X 10(-2) M) ZnSO4 inhibited the binding of C1q to immune complexes, [Ki = (6 +/- 2) X 10(-3) M]. This inhibition could be correlated with a ZnSO4-induced change in the tryptophan fluorescence of C1q [delta F 25%, Kd = (9.9 +/- 1.0) X 10(-3) M].

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.

Soluble immune complexes containing IgM antibodies (IgM.IC) were generated in vivo utilizing a passive induction model, whereby purified antibodies were injected into rabbits with circulating radiolabelled bovine serum albumin (BSA) as antigen. A triphasic response was obtained consisting of an initial rapid elimination of TCA-precipitable antigen in the first 30 min, followed by a progressive diminution in the clearance velocity as antigen from the tissues moved back into the circulation to re-equilibrate, and subsequent elimination of the antigen at a rate close to that of free BSA. The dynamics of IC formation and disappearance were studied by a combination of Farr assay and solid-phase C1q binding. The results show that the rate of clearance decreased as the complexes progressively moved into antigen excess, and that the decrease in the proportion of complexed antigen was mirrored by a similar decrease in the ability of the complexes to bind C1q. Depletion of complement by treatment with cobra venom factor did not inhibit the clearance of the antigen, but may have inhibited solubilization of the complexes in vivo. Tissue localization experiments indicated that the liver is the organ predominantly involved in the uptake and catabolism of in vivo-generated IgM.IC. These results show that the clearance velocity of soluble IgM.IC is critically dependent on the antigen/antibody ratio, and that clearance is mediated via a C3b-independent mechanism in the RES.

Pathological (190) and normal (33) sera were tested for their content of circulating immune complexes (CIC) by a battery of 13 assays performed in 11 laboratories. Statistical processing was done both by pooling all pathological samples and by extracting those falling into well-defined disease groups, i.e., rheumatoid arthritis, diabetes, lupus, melanoma, and glomerulonephritis. Highly significant correlations between methods--taken two at a time--for each disease differed in proportion (ranging from 6 to 30%) and in the pattern displayed on a checkerboard. Disease-linked patterns were also found when a function maximizing discrimination between pathological and normal samples was derived by combining the information from all methods. Here the order and the weight attributed by the computer to the methods differed for each of the disease groups. Taken together these results are interpreted as an indication that all assays may not determine the same classes of CIC, and thus vary in sensitivity depending on the prevailing properties of the complexes present in the serum, which in turn may depend on the etiology, pathogenesis, and stage of the disease.

The low titer and incidence of autologous antibody to melanoma has hampered its evaluation. Through acid dissociation and ultrafiltration of serum, we have been able to augment the autologous immune response in 9 of 10 patients studied. This result suggests that autologous antibody is present in most patients with melanoma, but is obscured by circulating antigen and the formation of immune complexes. Because native antibody and antibody derived from circulating immune complexes are produced by the host against physiologically relevant antigens, correlations can be made to clinical course. Serological studies of three patients with melanoma were performed with serum samples obtained over many months; these studies demonstrated correlations with tumor progression and clinical course. Serial serologic studies may yet provide one of the better ways to evaluate these relationships. They have the advantage of detecting transient events that may occur with the inception of metastatic disease or autoimmune phenomena, and of avoiding the difficulties encountered in comparing antibody responses between different individuals.

We developed a simplified, relatively rapid, inexpensive, antigen-nonspecific, enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG)- and C3-containing circulating immune complexes (CICs), adapted from a solid-phase anti-C3 radioimmunoassay (RIA). Standards (containing purified, heat-aggregated IgG and fresh human serum) or samples were allowed to react with goat F(ab')2 antihuman C3 bound to the matrix of microtiter plates. Then alkaline phosphatase conjugated to goat IgG fraction antihuman IgG was added, followed by p-nitrophenylphosphate, optical densities determined, and concentrations of CICs calculated. We found excellent correlations between serum and plasma CIC levels by either ELISA (r = 0.95, p less than 0.01) or RIA (r = 0.89, p less than 0.01). Furthermore, ELISA quantitation of CICs correlated well with RIA (serum, n = 75, r = 0.64, p less than 0.01; plasma, n = 101, r = 0.56, p less than 0.01). By ELISA we found 32 normal subjects had 38 +/- 12 micrograms CIC/ml in serum and 34 +/- 10 micrograms CIC/ml in plasma. Patients with systemic lupus erythematosus (39% of 27 patients, p less than 0.05) had significantly elevated CIC levels compared with normal (serum, 157 +/- 50 micrograms/ml, p less than 0.01; plasma, 89 +/- 23 micrograms/ml, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

The present studies were initiated to investigate whether soluble immune complexes, upon interaction with complement, can bind to endothelial cells. Human umbilical vein endothelial cells (HUVE) were incubated with purified human 125I-labeled C1q at 4 degrees C in RPMI-0.5% bovine serum albumin and assayed for binding. Optimal binding of 125I-labeled C1q to HUVE was reached within 2 h, and saturation of binding was found at concentrations of 5 micrograms/well input. The binding of 125I-labeled C1q was inhibitable with unlabeled C1q and by the collagenous region of pepsin-cleaved C1q. No inhibition was observed with the globular heads of C1q, suggesting that C1q binds to HUVE via the collagenous region of C1q. When HUVE were first reacted with various concentrations of C1q, washed and subsequently incubated with 125I-labeled aggregated human IgM (AIgM), binding of 125I-labeled AIgM to HUVE occurred depending on the dose of C1q. Only those aggregates of IgM which react with C1q in a solid-phase C1q binding assay were able to bind to HUVE presensitized with C1q. In addition it was shown that C1q mediated binding of aggregated IgG to HUVE. Furthermore, immune complexes (IC), that were prepared with bovine thyroglobulin (BTg) and rabbit anti-BTg, bound to C1q-preincubated HUVE. These studies suggest that localization of IC on endothelium can be enhanced following interaction of the IC with complement.

Circulating immune complexes containing IgG, IgM and hepatitis B surface antigen (HBsAg) in sera from groups of patients with various liver diseases were detected by both the C1q and conglutinin solid phase assays. Elevated levels of antigen non-specific immune complexes were observed in sera from all groups and complexes containing IgG were present to a greater extent than were IgM-containing complexes. Higher levels of complexes were generally obtained using the conglutinin assay than the C1q assay and the two assays were shown to preferentially bind complexes of different size ranges and antigen-antibody ratios. Only sera from HBsAg-positive patients had complexes containing HBsAg, and although serum HBsAg titres and levels of HBsAg-containing complexes were correlated, the correlation coefficient was low. The mean levels of immune complexes and the frequency of positive sera varied between different disease categories, but there was little correlation between levels of the three types of complexes detected by the two tests. Assay of immune complexes in sequential serum samples from an individual patient revealed considerable variation in the levels of the three complex types, demonstrating that the measurement of complexes in single serum samples is of limited value in assessing the potential significance of circulating immune complexes in hepatitis B.

In order to characterize the immune complexes detected in patients with Sjogren's syndrome (SS) and with rheumatoid arthritis (RA), the sera of 19 patients with SS alone and 11 with SS plus RA were examined. Elevated quantities of circulating immune complexes (CIC) were detected in 67% by the C1q-binding assay (C1q-BA), 73% by the C1q-solid phase (C1q-SP) assay, 43% by the monoclonal rheumatoid factor solid phase assay (mRF-SP) and 33% by the mRF-inhibition assay (MRF-Inh). Elevated concentrations of IgM RF were detected in 83% and of IgG RF in 73% of the sera by radioimmunoassay. Strong correlations existed between RF of the IgM and IgG classes and both the C1q-BA and the C1q-SP. Three lines of evidence indicated that RF were important components of the immune complexes detected by these radioimmunoassays. These results indicated that in those patients with RA plus SS, as well as those with SS alone, both IgM and IgG RF made substantial contributions to immune complexes detected both by C1q-BA and C1q-SP.

In this report we have investigated the association of fibronectin with antigen-antibody-C1q complexes incubated in fibronectin-depleted and C1q-depleted plasma. When BSA--anti-BSA immune aggregates are incubated in plasma depleted of both fibronectin and C1q to which 125I-fibronectin has been reconstituted, little radio-activity is bound to the immune complexes. However, pre-incubation of immune complexes with purified C1q prior to incubation in the plasma causes an approximately 10-fold increase in the amount of radioactivity bound. The binding of 125I-fibronectin to preformed antigen-antibody-C1q complexes is specific, since the reaction is inhibited by the addition of unlabelled fibronectin but not by ovalbumin. When antigen-antibody-C1q complexes are incubated in C1q-depleted plasma containing physiological concentrations of fibronectin, and analysed by immunoblotting, fibronectin antigens are detected on the immune complexes. Identical results are obtained using immune complexes composed of sheep erythrocyte rabbit anti-sheep erythrocyte C1q (EAC1q) cells. There is no specific requirement for preformed antigen-antibody-C1q complexes, since fibronectin can be detected on antigen-antibody complexes after incubation in normal human serum or in C1q-depleted ethylenediamineteraacetic acid (EDTA) serum reconstituted with purified C1q prior to incubation with the complexes. Finally, we also demonstrate that in the presence of C1q, 125I-fibronectin will associate with soluble antigen-antibody complexes.

Forty-one patients with progressive systemic sclerosis were studied for the presence of immune complexes by the fluid- and solid-phase C1q binding, C1 activation, and the fluid-phase conglutinin assays. Complement activation and autoantibodies were also studied. Immune complexes were detected in only 6 patients (15%); activation of complement was found in 5 others. The clinical and serologic features of patients with complexes were compared with those in whom complexes were not identified. No significant difference was found with respect to serology. Organ involvement was generally more frequent in the group with immune complexes, but the difference was statistically significant only with respect to lung involvement. The present data suggest that, although complement-fixing immune complexes are infrequently detected in progressive systemic sclerosis, they may play a role in the pathogenesis of lung lesions associated with the disease.

Circulating immune complex (CIC) levels were serially studied in healthy subjects and in normal rats. In human 3 methods (Clq solubility, complement consumption, and granulocyte phagocytosis tests) were used for the measurement of CIC; in rats, CIC levels were determined by the modified complement consumption test. A marked fluctuation in the CIC level was observed both in healthy human subjects and in normal rats. No correlation between results of the 3 assays used for CIC detection was found indicating that not only the level but the composition of CIC changes continuously in health.

Circulating immune complexes have been described in most liver diseases, including alcoholic liver disease, although their pathogenic significance remains unclear. Currently available immune complex assays do not distinguish immunoglobulin aggregates from antigen-antibody complexes. Immunoglobulin aggregate formation occurs in vitro at 37 degrees C in the presence of hypergammaglobulinemia and/or hypoalbuminemia, conditions common in liver disease. To determine if hypergammaglobulinemia and/or hypoalbuminemia could predispose to immunoglobulin aggregate formation in vivo, 25 patients with alcoholic liver disease were studied. Using sucrose density gradient fractionation followed by quantitation of IgG by radioimmunoassay, high molecular weight IgG complexes (greater than 11S) were frequently present in alcoholic liver disease sera, and correlated with the degree of hypergammaglobulinemia and/or hypoalbuminemia, and with 125I-C1q binding activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of these complexes revealed only IgG and nonspecific trapping of several normal serum proteins. A specific complex-associated antigen could not be identified. While small, undetectable quantities of true antigen-antibody complexes might also be present, our data suggest that IgG complexes in alcoholic liver disease may represent immunoglobulin aggregate formation in vivo.

Immune complexes (IC) were isolated from the serum of streptozotocin (Sz)-induced diabetic hamsters by coprecipitation with an equine rheumatoid-C1q factor (RhC). The IC, comprised of an unknown antigen(s) and IgG, occurred at 2 distinct time intervals during the 12-week chronological study. Although all diabetic hamsters with hyperglycemia of 300 mg/dl had IC, there was no correlation between the occurrence or concentration of IC and the degree of hyperglycemia. The cycling nature of IC in the serum of diabetic hamsters suggests that the antigen component either fluctuates chronologically or that the IC contain different antigens that do not occur simultaneously.

A rapid and simple method for detecting circulating immune complexes based on turbidity measurements following polyethylene glycol precipitation was studied with regard to its suitability as a routine assay in clinical laboratories. This method was found to have an acceptable degree of precision provided the temperature was carefully controlled. The mean value obtained with a group for 70 blood donors was 0.09 (SD 0.05) and the 90th percentile value was 0.16. There was no significant difference between values obtained from groups divided on the basis of age or sex. Of 70 diabetic sera assayed by the polyethylene glycol turbidity method, 20% gave positive values although only 10% were strongly positive. The corresponding figures for the solid phase Clq binding method were 15.7% and 14.3%, respectively. Correlation between the two methods was poor. It was concluded that although both methods have a similar likelihood of detecting immune complexes in randomly selected diabetics, it is probable that different immune complexes were being detected.

Brown-Norway rats maintained under conventional housing conditions showed a significant increase in the C1q binding activity of serum and to a lesser extent of the Raji cell assay whereas no change was observed, in BN rats maintained under specific pathogen free (SPF) conditions. Glomerular IgG deposits were encountered among rats with circulating immune complexes (CIC). This suggests that microbiological environment is a major factor in the spontaneous appearance of CIC which could be of pathogenic significance.

Immune complex-induced C1 activation and fluid phase C1 autoactivation have been compared in order to elucidate the immune complex role in the C1 activation process. Kinetic analyses revealed that immune complex-bound C1 activates seven times faster than fluid phase C1 spontaneously activates. The rate of spontaneous C1 activation increased after decreasing the solution ionic strength. In fact at one-half physiologic ionic strength (i.e., 0.08 M), the kinetics of spontaneous C1 activation were indistinguishable from the kinetics of activation of immune complex-bound C1 at physiologic ionic strength. The enhanced fluid phase C1 activation at low ionic strength resulted neither from C1 nor C1q aggregation, nor from selective effects on the C1r2S2 subunit; however, at the reduced ionic strength, the C1 association constant (defined for C1q + C1r2S2 in equilibrium C1qr2S2) did increase to 2.3 X 10(8) M-1, which is equal to that for C1 bound to an immune complex at physiologic ionic strength. Therefore, C1 can spontaneously activate in the fluid phase as rapidly as C1 on an immune complex when the strength of interaction between C1q and C1r2S2 is the same in both systems. In conclusion, under physiologic conditions, C1q and C1r2S2 are two weakly interacting proteins. Immune complexes provide a site for the assembly of a stable C1 complex, in which C1q and C1r2S2 remain associated long enough for C1q to activate C1r2S2. Thus, immune complexes enhance the intrinsic C1 autoactivation process by strengthening the association of C1q with C1r2S2.

Ovalbumin-antiovalbumin IgG immune aggregates were incubated with normal human serum in the presence of iodo[1-14C]acetamide, in conditions in which only the alternative pathway of complement was activated. The [14C]C3b-IgG covalent complexes formed were digested with pepsin, and analysed by SDS/polyacrylamide-gel electrophoresis and fluorography. Covalent complexes of [14C]C3-Fd and [14C]C3-pFc' were visualized, demonstrating that, during complement activation by the alternative pathway, C3 is covalently incorporated into the C gamma 3 domain of IgG, as well as into the Fd region. The C gamma 2 domain becomes protected from pepsin action by the bound C3b. All the covalent linkages between C3 and the IgG were sensitive to hydroxylamine. When [14C]C3-pFc' covalent complexes were treated with 1 M-NH2OH and loaded onto a Bio-Gel P-4 column, a radioactive peak of 3 kDa was obtained. The material released from [14C]C3-pFc' and [14C]C3-F(ab')2 complexes after treatment with 1 M-NH2OH was mixed and analysed in the Bio-Gel P-4 column. A similar radioactive peak of 3 kDa was obtained. When this peak, either from [14C]C3-pFc' alone or from the mixture of [14C]C3-F(ab')2 and [14C]C3-pFc', was fractionated by h.p.l.c., virtually the same radioactive peptide profile was obtained, indicating that very similar C3 peptides remained covalently bound to both regions (Fab and C gamma 3) of the antibody molecule. It is suggested that C3 bound to the C gamma 3 domain of IgG may interfere with the Fc-Fc interactions of immune aggregates and thus may be involved in several biological properties displayed by these complement-activating aggregates.

The fluid phase C1q binding, the solid phase C1q binding and the Raji cell assay are the most widely employed methods for the detection of complement fixing immune complexes in human disease. However, their binding characteristics for complexes formed in native serum have not been compared and studied in detail. For this purpose, Tetanustoxoid (TT): anti-TT complexes were formed closely to in vivo conditions by incubating sera of immunized people with TT. Then the molecular characteristics of those complexes, which could be detected by the 3 immune complex assays, were defined. The methods used are precipitation curve, gel chromatography and measuring the complexed antibody with a sensitive Elisa method. All 3 assays detected only complexes near the equivalence zone; the Raji cell assay being the most sensitive detecting 1.5 micrograms complexed antibody/ml serum followed by the solid phase (6 micrograms/ml) and the fluid phase C1q binding assay (50 micrograms/ml). As estimated by gel chromatography, both the C1q binding assays measured most sensitively complexes with a molecular weight distinctly over 2,000,000 daltons, whereas the Raji cell assay detected preferentially complexes in the range of 2,000,000 daltons. Smaller TT: anti-TT complexes with a molecular weight less than 600,000 daltons were not detectable by the 3 assays. In conclusion, the 3 assays, while differing in sensitivity, showed similar binding patterns with preference for high molecular complexes near equivalence. Subsequently, they might be insensitive to small complexes persisting in the circulation.

Serum immune complexes were measured in 92 patients with progressive systemic sclerosis, and elevated levels were found as follows: Raji cell assay 72% (59% after pronase treatment of Raji cell), agarose gel electrophoresis 52%, and C1q binding 24%. Forty-three (47%) had abnormal results on two or more of these tests, but only 17 (18%) had normal results by all three assays. Computer-assisted analysis of immune complex results and extensive clinical and laboratory data compiled on these patients revealed that the patients with abnormal Raji cell assays more often had diffuse scleroderma, tendon friction rubs, and positive serum antinuclear antibody tests than did patients with negative results on Raji cell assays. Individuals with immune complexes detected by C1q binding had evidence of pulmonary involvement and positive serum rheumatoid factor more frequently than did patients whose C1q tests were negative.

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.

Seventeen patients with peripheral corneal thinning and ulcers (four with Mooren's corneal ulcer, five with Terrien's marginal degeneration, and eight with collagen vascular disease) were tested for immune complexes in their serum. Circulating immune complexes, measured by Raji cell and C1q binding assays, were compared with levels in serum samples from normal controls and seven patients with staphylococcal marginal corneal ulcers. Comparison with normal controls showed significantly higher levels of circulating immune complexes in patients with collagen disease by the C1q binding assay and in patients with Mooren's ulcer by the Raji cell assay. Circulating immune complexes may play a role in the pathogenesis of Mooren's ulcer and marginal ulceration in the presence of collagen vascular disease. Their presence, however, may represent an epiphenomenon nonspecifically associated with corneal ulceration.

Inhibition assay of 125I-C1q binding to IgG-p-azobenzamidoethyl Sepharose 6B (IgG-Sepharose) by immune complexes was developed for the detection of circulating soluble immune complexes in the liver disease and was compared with polyclonal rheumatoid factor (pRF) binding inhibition assay and with C1q binding assay. The C1q inhibition assay was proved to be very sensitive, reproducible and rapid. Sucrose density gradient ultracentrifugal analysis showed that the assay could detect aggregates of human IgG (AHGG) larger than 19s. C1q inhibition activity (C1qIA) correlated with severity of the liver disease, defined by histological criteria. The highest C1qIA was observed in sera of patients with primary biliary cirrhosis, followed by liver cirrhosis, fulminant hepatitis, chronic aggressive hepatitis (2B), lupoid hepatitis and hepatocellular carcinoma in the order. There were correlations of C1qIA with serum gamma-globulin levels, sero-positivity for rheumatoid factor and hepatitis B surface antigen, and significant correlations existed also among pRFIA, C1qIA and C1qBA. Ultracentrifugal analysis of sera from patients with the liver disease showed that ClqIA demonstrated two sizes of immune complexes, 7s and larger than 19s, while complexes larger than 8s were seen in pRFIA.

The immune complex binding activity of human Clq was lost following treatment of the protein with the arginine-selective reagents cyclohexane 1,2-dione and phenylglyoxal. Both inactivations followed pseudo-first-order kinetics. The affinity of Clq for immune complexes was reduced 7-fold following cyclohexane-1,2-dione treatment, and could be substantially restored by treatment of the modified protein with hydroxylamine. Heat-aggregated IgG protected Clq against inactivation by both reagents. Incorporation of 25 molecules of [7-14C]phenylglyoxal per Clq molecule completely inactivated the protein. These data are consistent with the presence of arginyl residues in the immunoglobulin recognition sites of human Clq.