C1q Assay Research Articles

14-OR Parallel C1q and IgG assays on single antigen beads reveal that the presence of IgM complement fixing antibodies can obscure clinically relevant IgG antibodies to the same allele

14-OR Parallel C1q and IgG assays on single antigen beads reveal that the presence of IgM complement fixing antibodies can obscure clinically relevant IgG antibodies to the same allele

53-P Which is the clinically relevant antibody in patients waiting for transplantation: Luminex SAB screens versus C1q assays?

53-P Which is the clinically relevant antibody in patients waiting for transplantation: Luminex SAB screens versus C1q assays?

Novel C1q assay reveals a clinically relevant subset of human leukocyte antigen antibodies independent of immunoglobulin G strength on single antigen beads

It has been known for 40 years that cytotoxic human leukocyte antigen (HLA) antibodies are associated with graft rejection. However, the complement-dependent cytotoxicity assay (CDC) used to define these clinically deleterious antibodies suffers from a lack of sensitivity and specificity. Recently, methods exploiting immunoglobulin G (IgG) antibody binding to HLA single antigen beads (SAB) have overcome sensitivity and specificity drawbacks but introduced a new dilemma: which of the much broader set of antibodies defined by these methods are clinically relevant. To address this, we developed a complement-fixing C1q assay on the HLA SAB that combines sensitivity, specificity, and functional potential into one assay. We compared the CDC, IgG, and C1q assays on 96 sera having 2,118 defined antibodies and determined that CDC detects only 19% of complement-fixing antibodies detected by C1q, whereas C1q detects only 47% of antibodies detected by IgG. In the same patient, there is no predictability by IgG mean fluorescence intensity (MFI) as to which of the antibodies will bind C1q because fixation is independent of MFI values. In 3 clinical studies, C1q + antibodies appear to be more highly correlated than those detected by IgG alone for antibody-mediated rejection in hearts as well as for kidney transplant glomerulopathy and graft failure.

C1q-Fixing Human Leukocyte Antigen Antibodies Are Specific for Predicting Transplant Glomerulopathy and Late Graft Failure After Kidney Transplantation

Human leukocyte antigen (HLA) antibodies, especially those that fix complement, are associated with antibody-mediated rejection and graft failure. The C1q assay on single antigen beads detects a subset of HLA antibodies that can fix complement and precede C4d deposition. The aim of this study was to determine whether C1q-fixing antibodies distinguish de novo donor-specific antibodies (DSA) that are clinically relevant and harmful. We retrospectively studied 31 of 274 kidney transplant recipients who had pretransplant and concurrent biopsy and serum specimens, 13 with C4d-positive and 18 with C4d-negative staining. We measured IgG and C1q DSA pretransplant and at the time of biopsy using single antigen bead assays. We identified 13 recipients who developed de novo DSA by IgG or C1q and examined associations with C4d deposition, transplant glomerulopathy, and graft failure. Testing for DSA by IgG is more sensitive for C4d deposition (IgG: 100%, 95% confidence interval [CI] 0.60-1; C1q: 75%, 95% CI 0.36-0.96). Testing for DSA by C1q is more specific for transplant glomerulopathy (C1q: 81%, 95% CI 0.57-0.94; IgG: 67%, 95% CI 0.43-0.85) and graft loss (C1q: 79%, 95% CI 0.54-0.93; IgG: 63%, 95% CI 0.39-0.83). Absence of de novo DSA by IgG and C1q has a high negative predictive value for the absence of C4d deposition (IgG: 100%, 95% CI 0.73-1; C1q: 88%, 95% CI 0.62-0.98), transplant glomerulopathy (IgG: 100%, 95% CI 0.73-1; C1q: 100%, 95% CI 0.77-1), and graft failure (IgG: 86%, 95% CI 0.56-0.97; C1q: 88%, 95% CI 0.62-0.98). Monitoring patients with the C1q assay, which detects antibodies that fix complement, offers a minimally invasive means of identifying patients at risk for transplant glomerulopathy and graft loss.

Clinical usefulness of a novel C1q assay to detect immunoglobulin G antibodies capable of fixing complement in sensitized pediatric heart transplant patients

Donor-specific antibodies (DSA) against human leukocyte antigens complicate transplantation with the potential for acute antibody-mediated rejection (AMR). Complement-fixing antibodies are required to initiate the complement cascade. Not all DSAs, however, can fix complement. A novel C1q assay was developed to detect the sub-set of immunoglobulin G (IgG) antibodies capable of fixing complement. Sera from 18 pediatric heart transplant patients were analyzed for DSAs using a Luminex platform (Luminex Inc, Austin, TX) and commercially available single-antigen bead assay kits. Biopsy specimens were assessed for AMR using histopathologic criteria and immunohistochemical staining. During the study period, 5 patients had AMR; of these, 2 were C1q virtual crossmatch positive (VXM+) and had persistent C1q DSAs after transplant, and 3 were C1q VXM- but antibody developed immediately after transplant. A positive C1q assay in the immediate post-transplant period had a positive predictive value (PPV) of 100% and a negative predictive value (NPV) of 100%, with 100% sensitivity and 100% specificity (Fisher exact p = 0.001). Of 11 patients who were IgG VXM+, 5 had AMR; the IgG VXM had a PPV of 45% and NPV of 100%, with 100% sensitivity and 54% specificity (Fisher exact p = 0.101). The C1q assay can detect a sub-set of antibodies capable of fixing complement and predicts AMR early after transplant. Avoiding only the donor antigens that would be recognized by the C1q assay may accelerate time to transplant by expansion of the donor pool and potentially allows transplantation of previously "incompatible" organs.

319: Antibody Mediated Rejection (AMR) Predicted by New C1q Assay

HLA antibodies (Ab) that fix complement are essential for AMR. Current single antigen bead assays (SABs) do not distinguish binding Ab from complement fixing Ab. We developed the C1q assay to detect the presence of complement fixing Ab and examined it's utility to predict early AMR after pediatric heart tx.

Autoimmune Pancreatitis and Complement Activation System

Autoimmune pancreatitis is characterized by increased serum level of IgG4, but its pathogenesis has not been fully elucidated. Because this disease is occasionally associated with decreased levels of complements, we sought to clarify which complement activation system was operating in its active state. We measured serum levels of complements, mannose-binding lectin, and circulating immune complex in patients with autoimmune pancreatitis, patients with chronic pancreatitis, and healthy controls. We found high serum circulating immune complex values, which decreased significantly after corticosteroid therapy. In patients with autoimmune pancreatitis, elevated levels of circulating immune complex, as determined by C1q assay, were significantly associated with increased serum levels of IgG1 and decreased levels of C4, as well as with a tendency toward decreased levels of C3. There were no significant differences in the serum levels of mannose-binding lectin or in the frequency of a mutant allele of mannose-binding lectin between patients with autoimmune pancreatitis and those with chronic calcifying pancreatitis. Furthermore, corticosteroid therapy had no effect on the level of mannose-binding lectin. Autoimmune pancreatitis exhibits a high serum circulating immune complex values in its active state, which links to a complement activation system with a classic pathway rather than the mannose-binding lectin pathway or alternative pathways.

Evaluation of Seven Commercial ELISA Kits Compared with the C1q Solid-Phase Binding RIA for Detection of Circulating Immune Complexes

Circulating immune complexes (CICs) are detectable in a variety of systemic disorders such as rheumatological and autoimmune diseases (1)(2)(3)(4); allergic diseases (5); viral, bacterial, and parasitic infections (1)(6)(7)(8); and malignancies (2)(9). Although detection of CICs is neither essential nor specific for any disease, it may provide useful clinical information regarding immunopathology, prognosis, and follow-up of rheumatic and autoimmune disorders. A variety of tests that can detect CICs have been described (10)(11)(12)(13)(14)(15)(16)(17)(18). The assays that are used routinely in clinical laboratories are based on (a) precipitation of CICs by polyethylene glycol, (b) interaction of CICs with complement (C1q assay), (c) detection of CICs bound to C3 (Raji cell assay, conglutinin assay), and (d) binding of CICs to Fc-recognizing molecules (mRF assay). In a comparative WHO study (10), only a few tests were described as sufficiently sensitive and reproducible. This study revealed that the C1q solid-phase RIA (C1q-SP RIA) discriminated well between sera from healthy persons and those from patients. Several studies have indicated that this assay correlated better with disease activity than do other immune complex tests (8). The clinical activity of systemic lupus erythematosus correlated well with the concentration of CICs in the solid-phase RIA (19)(20). Subsequently, this assay has been used frequently in clinical investigations. The working group therefore concluded that the C1q-SP test was one of the most reliable tests. For these reasons, we used this test as the reference method. Several ELISA kits have become commercially available …

Immunoglobulin- and Hepatitis B Surface Antigen-Specific Circulating Immune Complexes in Chronic Hepatitis B Virus Infection

For assessing the role of circulating immune complexes (CIC) in chronic hepatitis B virus (HBV) infection, CICs containing IgM, IgG, and HBsAg were determined by C1q and conglutinin (K) assays in 216 patients with chronic HBV infection and 54 healthy controls. The concentration of each type of CIC in patients is higher than in controls ( P= 0.0001). CIC is a common feature of chronic HBV infection with 95.8% of cases having at least one abnormal test result. At least one type of HBsAg-CIC is positive in 54.2% of patients. HBsAg-CIC positivity is associated with HBeAg positivity ( P= 0.0001), higher aminotransferase levels ( P< 0.002), and younger age ( P= 0.001). IgG-CIC or IgM-HBsAg-CIC correlates with higher aminotransferase activity ( P= 0.001). In conclusion, HBsAg-CIC correlates with HBV replication. IgG-CIC and/or IgM-HBsAg-CIC correlate with disease activity. Immune-mediated injury may play a role in the pathogenesis of chronic HBV infection.

Circulating Immune Complexes in Chronic Hepatitis Related to Hepatitis C and B Viruses Infection

Circulating immune complexes (CIC) may be involved in tissue damage and/or viral clearance in viral hepatitis. To assess the frequency of raised CIC in chronic hepatitis related to hepatitis B and C, IgM, IgG, and hepatitis B surface antigen (HBsAg) containing CIC were determined, by conglutinin (K) and C1q assays, in 101 patients with chronic hepatitis B alone, 24 patients with chronic hepatitis B and C, 48 patients with chronic hepatitis C alone, and 54 healthy controls. Compared to patients with hepatitis B alone, patients with dual infection had higher frequency of raised IgM-C1q CIC ( P < 0.001) and IgM-K CIC ( P < 0.01). There is no difference in the prevalence of HBsAg-CIC between patients with hepatitis B alone and those with dual infection. Among patients with chronic hepatitis C alone, conglutinin-binding CIC is the predominant type of raised CIC and correlated with more severe liver damage. In conclusion, CIC may play a role in the pathogenesis of chronic hepatitis C virus infection.

Circulating immune complexes involving the ABO system after platelet transfusion.

It has been proposed that when ABO unmatched platelets are transfused circulating immune complexes (CIC) may be formed between the patient's soluble ABH antigens and the transfused antibodies. Platelets might then be destroyed by bystander mechanisms or by the binding of CIC to the Fc receptor or to C3 binding membrane proteins on the platelet. An ELISA C1q assay was used to detect CIC in 40 patients with haematological diseases who had received multiple platelet transfusions. A significantly larger number of refractory patients were positive (41%) in the assay than non-refractory patients (13%) or normal blood donors (0%). The presence of circulating IgG anti-A was sought in six group A refractory patients who had been transfused with ABO unmatched platelets. To determine whether the IgG anti-A was monomeric or in high molecular weight complexes, the serum was fractionated by gel exclusion chromatography and fractions were tested for the presence of IgG anti-A. In all six patients the peak of IgG anti-A binding occurred in fractions of high molecular weight (200-900 kD). Five out of six patients also demonstrated anti-A activity in fractions corresponding to monomeric IgG (about 150-180 kD). Fractions containing high molecular weight anti-A were purified using a protein G column and the eluates were tested for the presence of group A antigen using dot immunoblotting. Group A antigen was associated with the purified IgG anti-A in 4/5 patients tested. Appropriate transfused and non-transfused controls had no anti-A in any fractions. Although not unexpected, these studies demonstrate for the first time that refractory patients receiving ABO unmatched platelets have CIC composed of ABO antigens and their corresponding antibodies present in their serum that circulate for at least several days. It also confirms the hypothesis that some CIC in haematological patients are induced by transfusion.

Rheumatoid factors and circulating immune complexes in HIV-infected individuals

We studied serological aspects of autoimmunity in patients with AIDS, AIDS-related complex (ARC) and in individuals at risk for AIDS. Immunoglobulin (Ig) M, IgG and IgA rheumatoid factors (RF) were quantified by enzyme-linked immunosorbent assay (ELISA), Ig by radial immunodiffusion, and circulating immune complexes (CIC) by the CIC-conglutinin and CIC-complement 1q (C1q) assays. Mean IgM RF levels were normal in AIDS patients, but those of ARC patients were higher and more frequent than the levels defined by agglutination methods. Similar observations were made for intravenous drug users (IVDU) and for both HIV-seropositive and HIV-seronegative homosexual men. Mean IgG RF levels were normal in AIDS and ARC patients but high in homosexual men and, to a lesser degree, in IVDU. IgA RF levels were high in many AIDS and ARC patients, in homosexual men, and in haemophiliac and control groups. The selective increase of the IgA isotype in AIDS was confirmed by the Ig results, which also showed an IgG increase in all groups. IgM were mainly high in people with ARC. CIC were detected in 68% of ARC patients by both methods, and in 55% of AIDS patients by CIC-Clq. A high incidence of positive samples in all at-risk populations, but particularly in seronegative individuals, was observed using CIC-conglutinin. CIC-C1q also revealed larger amounts of CIC in HIV-seronegative individuals, mainly in homosexual men. The study of these humoral aspects of autoimmunity provides useful information on the impairment of B-cells in patients with AIDS and ARC.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of immune complexes containing cytomegalovirus-specific IgM antibodies following a kidney graft.

In order to demonstrate the viral specificity of IgM-containing immune complexes (IgM-CIC) detected by a C1q assay in renal allograft recipients developing a CMV infection, a technique is described allowing: 1) the dissociation of IgM-CIC by action of an acid buffer, and 2) the characterization of the viral specificity of IgM antibodies released by this treatment. This step was performed by ELISA and Western Blot. When technique was applied to the follow-up of a renal allograft recipient developing a recurrent CMV infection within 2 months post-graft, it was found that the IgM-CIC detected on the day of the graft were not CMV-specific, whereas the IgM-CIC detected during the second month after transplantation contained CMV-specific IgM antibodies. These CMV-specific IgM-CIC were detected as early as the urinary viral excretion. It was shown by Western Blot analysis that these IgM antibodies reacted with a 45-47 kDa viral polypeptide which is a viral target for specific humoral response at the early phase of CMV infection.

Idiopathic Membranous Nephropathy, Associated with HLA-DRw3 and Not Related to Monocyte-Phagocyte System Fc Receptor Dysfunction, in Father and Son

Familial idiopathic membranous nephropathy, an immune-complex-associated glomerulopathy, has not been previously reported in father and son, despite its striking immunogenetic correlation, especially with HLA-DR3. As a dysfunction of the monocyte-phagocyte system (MPS), it has been observed linked to DR3 antigen, so we studied the MPS Fc receptor function in a father and his son with a histologically proven membranous nephropathy, associated with the haplotype A9-B35-DR3-DQw2. The Fc receptor function of the MPS was examined by measuring the clearance of IgG-sensitized, 51Cr-labeled erythrocytes and by measuring the ability of isolated monocytes to ingest autologous red blood cells coated with IgG anti-Rh (D) antibody. Immune clearance and in vitro phagocytosis was normal in both patients and not related to their levels of immune complexes (as measured by ELISA C1q and Conglutinin solid-phase binding assay). This report suggest that genetic factors may play an important role in the development of membranous nephropathy, and it seems not to be related to a dysfunction of MPS as measured by these tests.

Immune Complexes in Pediatric Human Immunodeficiency Virus Infection

Circulating immune complexes (CIC) were analyzed in a cohort of 30 children infected with the human immunodeficiency virus. Elevated CIC were detected by the C1q assay in 70% (21/30) of all patients and by the Raji cell assay in 93% (28/30) of all patients. While only less than one third of patients with elevated CIC had free serum antibodies to Epstein-Barr virus, 80% (16/20) of them had detectable antibodies to Epstein-Barr virus associated with CIC. Enriched CIC in human immunodeficiency virus-infected children contained low levels of complement. These findings document that, as an expression of the humoral immunodeficiency, CIC in human immunodeficiency virus-infected children are deficient in complement and can thus be underestimated if complement-precipitating methods are used for their detection.

Immunoglobulin and hepatitis B surface antigen‐specific circulating immune complexes in chronic hepatitis with hepatitis delta virus infection

IgM, IgG, and HBsAg containing circulating immune complexes (CIC) were determined, by conglutinin (K) and C1q assays, for assessing the role of CIC in hepatitis delta virus (HDV) infection in 54 HBsAg-negative controls and 85 HBsAg-positive patients with chronic hepatitis. The prevalence of HDV markers (HDV antigen and anti-HD) was 24.70% (21/85). CIC were a common feature of HDV infection with 95.24% of patients having at least one abnormal test result. The prevalence of elevated IgM-K, IgG-K, IgM-C1q, and IgG-C1q CIC were 85.71, 85.71, 57.14, and 85.71%, respectively. The prevalence of IgM class CIC were statistically higher in patients with HDV infection than in those without (P = .001 for the K assay and P = .023 for the C1q assay). There was no difference in the prevalence of IgG class CIC. Patients with HDV infection also have significantly higher median levels of IgM K-CIC (P = .002), IgG K-CIC (P = .049), and IgG C1q-CIC (P = .008). In patients with HDV infection, there was positive correlation between IgM C1q-CIC and transaminase levels (r = .519, P = .016 for AST; r = .500, P = .021 for ALT). There was no difference in the prevalence of HBsAg containing CIC between patients with HDV infection (76.19%) and those without (74.60%). In conclusion, IgM class CIC are the major CIC and correlate with disease activity in HDV infection. CIC may play a role in the pathogenesis of HDV infection.

Cellular and Humoral Immune Responses in Experimental Aspergillosis in Mice

Invasive aspergillosis was established in a naive murine model. Both humoral and cellular aspects of the immunological mechanism responded to invasion. Circulating immune complexes containing IgG in the infected group of animals were detected by both the C1q and conglutinin solid phase assays. Attempts to identify these complexes as Aspergillus antigen-specific complexes were unsuccessful. Cellular responses, as measured by blastogenic activity and inhibition of migration of sensitized cells were significantly elevated in the infected group.

Effects of penicillamine on serum immunoglobulins and immune complex-reactive material in primary biliary cirrhosis

Although penicillamine is used in the treatment of primary biliary cirrhosis, its mechanism of action in this disease is unknown. As an immunologic action had been attributed to the drug, we investigated whether penicillamine might alter serum immunoglobulin levels or immune complex-reactive material in patients with primary biliary cirrhosis. Immunoglobulin levels and immune complex reactivity were measured and clinical tests were performed in 53 consecutive patients entering a double-blind randomized trial of 750 mg vs. 250 mg of penicillamine. Measurement of immune complex reactivity was determined by laser nephelometry, 125I-C1q binding, and Raji cell assays. Immune complex reactivity was detected by at least one assay in 75% of patients tested before treatment. Sixty-two percent were positive in the C1q assay, 28% in the Raji cell assay, and 39% by nephelometry. After therapy with either dose, we found no change in immune complex-reactive material by any assay. Concentrations of immunoglobulins G and M fell (p < 0.05) after 12 mo of therapy. Concentrations of immunoglobulin A decreased (p < 0.05) only in the high-dose group. Correlation was not consistent between results of immune complex assays and clinical liver tests. Although immunoglobulin levels fell during penicillamine therapy, no decrease in immune complex-reactive material was detected. The effect of penicillamine in primary biliary cirrhosis is not mediated through alteration of immune complex-reactive material.

Circulating IgA immune complexes in AIDS.

Circulating immune complexes were isolated from the serum of patients with AIDS, as well as patients with other acute and chronic viral diseases. Analysis of these immune complexes by methods of flow cytometry and by radioimmune (Raji cell) assay revealed a prevalence of IgA complexes in the serum of AIDS patients and a prevalence of IgG complexes in the serum of patients with other viral diseases. Raji cells bind immune complexes via Fc and complement (C3) receptors and may detect IgA immune complexes more efficiently than a C1q assay since IgA has no affinity for C1q.

Sequential studies of complement activation in systemic lupus erythematosus.

C1 and C3 activation, measured as C1r-C1s-C1 inactivator C1s-C1r-C1IA complexes in serum and circulating C3d were studied in serial samples from 33 patients with SLE. All patients demonstrated exacerbations during observation periods of 10-30 months and were divided into groups according to principal clincal features (mild SLE, severe extra-renal SLE, and lupus glomerulonephritis). Increased C1 activation was consistently found during exacerbation. C3d in plasma was a feature associated with severe disease flares. Activation of C1, but not of C3, was documented before flare-ups of disease activity, but such predictive information was mostly restricted to patients with extra-renal disease. C2 cleavage in plasma, studied serially in a few patients, appeared to be closely associated with C1 activation. Circulating immune complexes, measured with solid-phase C1q assay, did not always increase before development of clinical manifestations. Remission of symptoms was paralleled by decreasing concentrations of C1r-C1s-C1IA and of, when present, C3d. Similar findings were made for immune complexes but only in severe disease. Persisting C3d was observed in 3 patients, who subsequently developed renal failure. C1q levels were transiently low during flare-ups of lupus glomerulonephritis, but otherwise the concentrations of C1q, C4 and C3 did not show consistent patterns of variation in relation to disease activity.