Clq Binding Assay Research Articles

A case of polymyalgia rheumatica with the elevation of circulating immune complexes

A 72-year-old man was admitted to the hospital because of muscle pain, stiffness involving the neck, shoulders and limbs, and joint pain. He was well until one month earlier, when high fever and myalgia appeared. The pain was worse with active motion and it was difficult for him to get around. Fatigue and malaise were prominent. He lost 5kg in one month.On examination, general phisical findings were unremarkable except for high fever, myalgia and joint pain. His right wrist was slightly swollen. The temporal arteries were normal.Laboratory data proved no muscle damage (GOT 27 IU/1, CPK 45 IU/1, aldolase 1.9mU/ml). ESR was 70mm in 1hour. WBC was 11, 800/cumm. Total protein was 7.5g/dl. Serum protein electrophoresis revealed an increase of α2-globulin to 14.1%. Circulating immune complexes were detected by means of Clq binding assay, while CH50 was slightly elevated. RAtest, HBsAg, HBsAb and HB immune complex were all negative. Biopsy of the left quadoriceps femoris muscle revealed slightly inflammatory cells surrounding arterioles. Biopsy of the right temporal artery revealed atherosclerotic change, but no evidence for giant cell arteritis was found.The above findings are compatible with polymyalgia rheumatica. He was started on predonine (30 mg/day). His response to the therapy was dramatic and predonine was gradually decreased in dosage Two weeks of predonine therapy resulted in a marked improvement of his myalgia, and other symptoms disappeared. Circulating immune complexes dropped gradually after treatment.

Felty syndrome: autoimmune neutropenia or immune-complex-mediated disease?

Immunofluorescence on polymorphonuclear cells (PMN) of patients with Felty syndrome (FS) revealed increased amounts of IgG, IgA, and IgM bound to the PMN surface compared with PMN of patients with rheumatoid arthritis alone. A positive correlation was found between the score for surface-bound immunoglobulins on FS-PMN and the results of the Clq binding assay in FS sera. After preincubation with sera from 20 patients with FS, immunofluorescence on PMN from healthy controls (HC) showed that these cells had bound IgG, IgA, and IgM. However F(ab')2 fragments of IgG from FS sera did not bind to PMN, although the antigen-binding reactivity of the F(ab')2 fragments was maintained as shown by control experiments. Immunoglobulins eluted from FS-PMN failed to bind to HC-PMN, whereas the corresponding IgG of patients with autoimmune neutropenia was bound. Gel filtration of FS sera on Sepharose 4B showed that the binding of IgG in FS sera to PMN did not coincide with the 7S peak but occurred mainly in fractions containing larger material. No binding of IgA and IgM to HC-PMN was found after incubation with FS sera pretreated with polyethylene glycol (PEG) to precipitate immune complexes. These results indicate that in sera of patients with FS the PMN-binding reactivity of IgG, IgA, and IgM is due to the binding of immune complexes containing these immunoglobulins and not to presence of autoantibodies directed to antigens on the neutrophil surface.

Immune complexes and antinuclear, antinucleolar, and anticentromere antibodies in scleroderma

Forty-one patients with various forms of systemic sclerosis (scleroderma) and positive antinuclear antibodies of nucleolar (ten patients), speckled (eleven patients), or centromere pattern (twenty patients) were selected for study of immune complexes by the radioisotope labeled Clq binding and the radioisotope labeled protein A binding methods. The presence of immune complexes was found by the Clq binding assay in sixteen patients (39%) and by a protein A binding assay in eight patients (20%). Overall, 46% of patients (19/41) had immune complexes. A lower incidence of organ involvement and fewer positive results in the screening of serum immune complexes were observed in patients with centromere antibody (35%) than in patients with nucleolar (60%) or speckled pattern (55%). Patients with immune complexes had higher frequencies of kidney, heart, and muscle involvement and digital ulceration than did patients with no detectable immune complexes, but the differences were not statistically significant. Diffuse skin involvement was not related to the presence of immune complexes.

Binding of complement subcomponent Clq to mouse IgGl, IgG2a AND IgG2b: A novel Clq binding assay

A Clq binding assay is presented which is suitable for use in comparison of the binding ability of different antibodies, and which allows the quantitative determination of their binding constants. The assay system uses IgG bound to a hapten-derivatized Affigel support. No non-specific binding is observed to a DNP-derivatized support, allowing the use of anti-DNP antibodies. With mouse anti-DNP hybridoma IgGs it was found that Clq binding followed the series IgG2a > IgG2b > IgGI, in accordance with the complement fixing ability of these subclasses. Since it is relatively simple to couple any antigen to Affigel, this assay system should be generally applicable to any antibody-antigen system.

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

A new procedure for the isolation of the Clq 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 Clq protein with about 80% recovery. The isolated Clq protein is biologically active and may be used for the detection of circulating immune complexes in sera by the solid-phase Clq binding assay.

Immune complex-like material in the serum and plasma exchange in patients with metastatic cancer.

Clinical significance of immune complex-like material in the serum was investigated in tumour patients undergoing plasma exchange with albumin-saline solution and subsequent chemotherapy. Immune complexes were detected by the Clq binding assay or the Raji cell radioimmunoassay in nine out of forty-five patients before this therapy. Levels of immune complexes were decreased to 10-30% of the initial value by plasma exchange depending on exchanged plasma volume. In contrast to other serum proteins like alpha 1-antitrypsin and alpha 2-macroglobulin, which showed protein specific increase during follow up after plasma exchange in all patients, recovery rates of immune complexes and IgG were highly individual but parallel in each patient. Clinical response to this protocol did not correlate with immune complex status, suggesting that removal of the measured immune complex like material had little clinical significance or was not longlasting enough to provide therapeutic benefit.

Behçet's syndrome: Immune regulation, circulating immune complexes, neutrophil migration, and colchicine therapy

Immune regulatory dysfunction, circulating immune complexes (CIC), and polymorphonuclear (PMN) cell migration were investigated in patients with Behçet's syndrome. Six patients meeting rigorous clinical criteria were evaluated. Only one patient showed evidence of immune regulatory dysfunction (increased T4/T8 ratio). Although Clq binding and Raji cell assays for CIC yielded positive results in only one of five patients, all five patients had in vivo “histamine trap test” evidence of CIC (all controls had normal results). Sera from all Behçet's syndrome patients increased migration of neutrophils to zymosan-activated serum. Colchicine therapy abolished the enhancing effect of the patient's sera on movement of PMN cells from patients and controls. An immune complex-mediated injury that is followed by an excessive accumulation of PMN cells may lead to the cutaneous lesions and other lesions in Behçet's syndrome. Further evaluation of colchicine therapy is warranted on the basis of these studies.

Circulating immune complexes and hypertension in pregnancy.

Eighteen patients with pre-eclampsia, 10 patients with essential and 9 with transient hypertension during pregnancy, were investigated regarding circulating immune complexes by a Clq-binding assay and a PEG precipitation assay. The women were studied during pregnancy, 2 and 5 days after childbirth, and also 3 and 6 months afterwards. The frequency of circulating immune complexes was not significantly increased in any of the groups when compared with that in 18 normotensive pregnant control subjects and 19 non-pregnant controls. Thus Clq-binding and PEG-precipitable immune complexes are a feature neither of normal pregnancy, nor of pregnancy complicated by hypertension or pre-eclampsia.

Circulating immune complexes in childhood malignancies: a Pediatric Oncology Group study.

Pretreatment serum samples obtained at diagnosis from 89 children with various pediatric malignancies were examined for circulating immune complexes (CIC) using the [125I]Clq binding assay. The study population consisted of 35 children with acute lymphocytic leukemia (ALL), 22 children with acute non-lymphocytic leukemia (ALL), 24 with neuroblastoma (NB), and eight with osteosarcoma (OS). Concomitant quantitation of immunoglobulins was performed in 55 patients, revealing normal values for age. Increased levels of CIC at diagnosis were found in 9%, 22%, 42%, and 50% of children with ALL, AML, NB, and OS, respectively. Except for a higher proportion of CIC-positive patients observed in stage IV NB (nine of 17) compared to stage I-III NB (one of seven), no correlation was observed between initial CIC level and presenting clinical features, response to treatment, prognosis, or presence of infection. Longitudinal sampling of six NB and two OS patients did not reveal a clear relationship between disease activity and quantity of CIC. For the pediatric malignancies studied, these data demonstrate minimal value in quantitating CIC as a means of assessing disease activity or predicting response to treatment and are in contrast to the apparently adverse effect of elevated pretreatment CIC on response to therapy and survival observed in adults with ALL, AML, and OS.

Circulating immune complexes in haemophilia and von Willebrand's disease.

Sera from 63 patients with haemophilia A, 21 with haemophilia B and 29 with von Willebrand's disease were screened for the presence of circulating immune complexes (CICs), serological markers of hepatitis A and B virus, autoantibodies and factor VIII or factor IX inhibitors. CICs were detected by the 125J Clq binding assay (ClqBA), the solid phase conglutinin assay (KgBSP) and the solid phase Clq assay (ClqSP). The incidence of CICs detected by the ClqBA and the ClqSP methods in haemophiliacs and in von Willebrand patients was higher than that observed in normal subjects, while the prevalence of CICs detected by the KgBSP method was not. The presence of CICs was not correlated with patient age, severity of disease, presence of hepatitis B virus serological markers, abnormal liver function tests or factor VIII inhibitors. A significant connection was demonstrated between CICs detected by the ClqBA method and replacement therapy when the dose administered over 1 year was over 20 000 U of factor VIII or IX concentrates. The high proportion of CICs in von Willebrand's disease, not connected with the replacement therapy or the presence of serological markers of hepatitis virus, is in agreement with the possibility that immune complexes may be related to the disease itself and independent, at least in part, of exogenous agents.

The interaction of immune serum globulin and immune globulin intravenous with complement

The in vitro anticomplementary activity of untreated and heat-aggregated (63°C, 10 min) immune serum globulin (ISG) and immune globulin intravenous (IGIV) prepared by partial reduction and alkylation have been evaluated by three assays, C3 activation, binding to Clq and enhancement of alternative pathway lysis of rabbit erythrocytes. Crossed immunoelectrophoresis was used to quantitatively measure the ability of ISG and IGIV to activate endogenous C3 in normal serum. Binding to Clq was determined according to the ability to inhibit binding of 125I-Clq to solid phase IgG. ISG and IGIV enhancement of lysis of rabbit erythrocytes by normal human serum adsorbed with rabbit erythrocytes in the presence of MgEGTA was used to determine activity in the alternative complement pathway. Unheated IGIV at 10mg/ml only marginally activated endogenous C3 in normal serum, had about a 5-fold lower affinity for 125I-Clq ( K i = 138 to 356 μM vs K i = 62.5 μM for ISG), but was very similar in ability to ISG on a weight basis in enhancing complement alternative pathway activity ( RCH50 = 0.23 to 0.40 mg for IGIV vs 0.17mg for ISG). Heat-aggregated IGIV at 5 mg/ml in normal human serum was about 2-fold less effective than heat-aggregated ISG in the activation of C3 in normal serum and had approximately 2- to 3-fold lower affinity in the Clq binding assay ( K i = 45 to 83 n M for heat-aggregated IGIV vs K i = 14.6 n M for heat-aggregated ISG). These data suggest that IGIV prepared by chemical modification retains sufficient specific receptor activity to allow in vivo efficacy in complement-mediated amplification of host defense reactions, but is safe for intravenous use due to a lower capacity to initiate nonspecific complement activation.

Recovery of immune complexes from serum-based samples is dependent on the concentration of reactants: heat-inactivated and unheated samples compared by nephelometric assay

We examined heat-treated and untreated samples for immune complexes (IC) with a nephelometric type Clq binding assay. Curves of absorbance vs. concentration derived from aggregated γ-globulin in serum samples showed curvilinear behavior, while curves obtained from aqueous samples were highly linear. Curves from heat-treated sera bent upward, while those from unheated sera tended to level off, causing the curves to cross. Heated sera showed greater recovery of IC in the high range and unheated sera greater recovery of IC in the low. Recovery from serum-based samples varied as the concentrations of both Clq and IC in the reaction mixture changed when aqueous curves were used as calibrators. This behavior in serum-based samples seems to be caused by endogenous binders of IC, and may be an important reason for inter-laboratory variation in results from similar samples.

Effect of pregnancy on immune complexes and rheumatoid factors in patients with rheumatoid arthritis

Rheumatoid arthritis is associated with circulating and intra-articular immune complexes and rheumatoid factors. The clinical activity of rheumatoid arthritis improves during pregnancy in the majority of women, with exacerbation following delivery. Concentrations of immune complexes, as detected by the Clq-binding assay, the Clq-solid phase assay, and the monoclonal rheumatoid factor-solid phase assay, decreased during gestation, with elevations following delivery. Concentrations of IgM-rheumatoid factor and IgG-rheumatoid factor, analyzed by radioimmunoassay, changed variably during pregnancy, increasing in some patients and decreasing in others. When examined serially before, during, and following pregnancy, changes in the concentration of circulating immune complexes and/or rheumatoid factors corresponded with the clinical changes in three patients. These observations document the significant effect of gestation on the concentration of circulating immune complexes in patients with rheumatoid arthritis. They also support the role of these laboratory tests in monitoring the clinical course of rheumatoid arthritis.

A Comparative Immunologic Study of IgA Nephropathy

A conglutinin binding assay has been used to detect circulating immune complexes (CIC) containing IgA, IgG, or IgM in sera from patients with IgA nephropathy. IgA class CIC were detected in 40.7% of patient. IgG class CIC were detected only in patients with glomercular IgG deposits. IgM class CIC were detected more often in patients with glomerular IgM deposits than in patients without glomerular IgM deposits. These results demonstrate an association between the immunoglobulin in CIC and those in glomerular deposits. CIC were not detected in sera from most patients with IgA nephropathy by a Clq binding assay, however, since this assay does not detect IgA class CIC. Immunoelectronmicroscopic studies of IgA nephropathy have shown that C3 deposits are localized to the same areas as IgA deposits. In conclusion, we suggest that mesangial IgA deposits are composed of immune complexes and may be derived from CIC.

Circulating immune complexes in chronic myeloid leukemia patients at various stages of the disease

Circulating immune complexes (CICs) in sera from patients suffering from chronic myeloid leukemia (CML) at initial diagnosis, in ‘remission’, at relapse and in blastic crisis have been quantitated using fluid [ 125I]Clq binding assay in terms of per cent binding activity and μg/ml aggregated human globulin (AHG) equivalents. The Clq binding activity (Clq-BA) has been compared within the groups of CML patients in different phases of the disease as well as with sera obtained from normal healthy donors. The results showed that the mean Clq-BA was significantly increased in CML patients at initial diagnosis (25.74 ± 3.48, p < 0.001), in relapse (53.36 ± 6.9, p < 0.001) and in blastic crisis (60.5 ± 8.7, p < 0.001) when compared to control sera. Sera of ‘remission’ patients showed significant decrease in Clq-BA when compared to sera collected in active phases of the disease, however, the values were still significantly higher (12.87 ± 1.58, p < 0.02) than those of normal healthy donors. When the levels of CICs as assessed by Clq-BA were compared with the WBC/blast counts of CML patients in chronic as well as blastic phase, it was noted that the variations in numbers of circulating leukemic cells do not correlate with the CIC levels. The significance of assessment of CIC levels in monitoring the disease in CML patients is discussed.

The role of circulating immune complexes in the pathogenesis of Graves' disease

It has recently been reported that many immunological abnormalities including the presence of TSH-receptor antibody (TRAb) were found in Graves' disease (GD). Circulating immune complexes (CIC) have also been detected in the serum of patients with GD as observed in systemic lupus erythematosus, which is thought to be a typical model of immune complex disease. The role of CIC in pathogenesis of hyperthyroidism, however, remains to be elucidated. Therefore, to clarify pathophysiological functions of CIC in GD, the levels of it in those patients were compared with their symptoms, those of TRAb, and lymphoblastogenesis (LBG) induced by phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM). The subjects were forty patients with GD without any medication, one hundred and nine patients with GD on medication with methimazole (MMI), and fifteen healthy volunteers. CIC was measured by three different methods; polyethyleneglycol precipitation method (PEG), Clq binding assay (Clq), and Protein A binding assay (PA). The normal range was estimated with the mean plus or minus two times the standard deviation of normal controls. In untreated GD, CIC determined by PEG, Clq and PA widely distributed from normal range to high levels. The positive rates of CIC determined by PEG, Clq, PA, and any one method of these three were 17.5%, 22.5%, 30.0% and 52.5%, respectively. LBG using incorporation of tritiated thymidine showed the decreases in PHA and Con A, and the increases in PWM in patients with GD. The positive rates of CIC determined by PEG and PA were significantly higher in patients without goiter or with small one than those with large one (p less than 0.05). CIC measured by all three of PEG, Clq and PA showed negative correlation with TRAb significantly (p less than 0.05, p less than 0.01, p less than 0.01, respectively). On the other hand, CIC measured by Clq showed significant negative correlation with serum thyroxine concentration (p less than 0.01). The levels of CIC, TRAb and PWM-induced LBG decreased following the tapering dose of MMI sufficient to keep patients in euthyroid state. In consequence, there were no longer any correlations between CIC and TRAb after thyroid function was normalized. These observations suggest that CIC's which have huge molecular weight or have ability to bind Fc receptor on K cell, macrophage, neutrophil, and other immune cells may be one of the factors to inhibit the goitrogenic action of TRAb, and that CIC's which have ability to activate the complement system may be one of the factors to inhibit the stimulation of secretion of thyroid hormone by TRAb.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunopathological Studies of Polyarteritis Nodosa and Wegener's Granulomatosis: A Report of 43 Patients with 51 Renal Biopsies

Although it is generally considered that vasculitis of the polyarteritis nodosa (PAN) group and Wegener's granulomatosis (WG) is immune complex (IC) mediated, there are no simultaneous data on circulating IC, complement levels and deposits of Ig and complement in the kidney. Therefore we have performed a retrospective study of 43 patients suffering from PAN and WG. Ig glomerular deposits were uncommon and scanty, except in two patients with WG; C3 deposits were detected in 12 patients, whereas fibrinogen was constantly found when lesions were recent and active. Similar data were obtained for the renal vessel walls. Contrasting with these results, rheumatoid factors and cryoglobulins, suggestive of the presence of circulating IC, were detected respectively in nine of 39 and seven of 37 patients, and IC 'activity' assessed by the Raji cell assay and the Clq binding assay was found respectively in six of 17 and nine of 10 patients before treatment, and in none of 10 and five of seven patients in remission. Haemolytic complement activity and complement components were never decreased, but the C3d breakdown product of C3 was elevated in all the eight patients studied before treatment. Signs of persistent hepatitis B virus (HBV) infection were detected in five of 25 patients of the PAN group, whereas three of eight patients with WG had only anti-HBV antibodies. Furthermore, cytomegalovirus (CMV) could be isolated from the blood in a case of WG before the treatment was started. Persistent interferonaemia was detected in one of five patients. These results suggest either that renal deposition of CIC is transient, the paucity of Ig deposits being due to rapid clearance of IC by phagocytic cells; or alternatively that vascular and glomerular lesions are not caused by CIC, as in some cases of experimental vasculitis induced by infectious agents.

Serum Immune Complex Levels in Multiple Sclerosis Cases and Their Unaffected Siblings

To determine whether the multiple sclerosis susceptibility (MSS) gene has an effect on serum immune complex (IC) levels, we used the solid-phase Clq binding assay to test for the presence of IC in sera of MS patients and their unaffected siblings. Using HLA markers, the siblings were classified on the probability of sharing the MSS gene with the proband. Although we found that familial membership exerts a strong effect on circulating IC levels, no other significant differences were seen. Investigation of serum and spinal fluid during exacerbations should be done before concluding that ICs have no role in the pathopoiesis of MS.

Complexes immunoglobulines-lipoproteines dans l'infection par Plasmodium chabaudi

Immunoglobulin-lipoprotein complexes have been investigated in the course of P. chabaudi acute infection of Swiss mice. Serum ultracentrifugation in a sucrose density gradient was used to demonstrate the presence of immunoglobulins in the enriched lipoprotein fraction of density lower than 1100. The levels of lipoprotein-bound immunoglobulins (Ig-Lp) were measured using the rate-nephelometric Immuno-Chemistry-System. IgM-Lp and IgG-Lp were significantly increased at day 9 and reached a peak at day 13 post-infection. From day 13 to day 16 they dramatically decreased. This kinetic effect was similar to that of non-specific circulating immune complexes detected by the [ 125I]Clq binding assay. A radioimmuno-precipitation-PEG-assay (RIPEGAssay) with [ 125I]Lp revealed the highest Lp precipitation with day 13 post-infection mouse sera compared to controls. Similar results were recorded when control sera were 8 times as concentrated in order to obtain Ig levels identical in both control and experimental sera. Part of the lipoprotein-bound immunoglobulins were demonstrated to be anti-Lp antibodies by the way of the RIPEGAssay using F(ab′) 2 fragments from infected mouse sera. Moreover, the [ 125I]Clq bound to the Ig-Lp complexes isolated from day 13 mouse sera. The present data suggest that the alteration of lipid metabolism which characterizes malaria infection could lead to the complexation of lipoproteins to immunoglobulins.

Clq-binding substances in pemphigus.

ABSTRACTSerial serum samples from sixteen patients with pemphigus were assayed for Clq binding activity (ClqBA) using a modified solid phase Clq binding assay. The modification included use of horseradish peroxidase instead of 125I or 131I. ClqBA was detected in six of the sixteen patients. Sequential samples from those positive patients demonstrated a decrease in ClqBA in response to steroid therapy.