A sensitivie and simple procedure for the detection and quantitation of soluble complement (C)- fixing immune complexes in sera of patients with various disease states has been developed by utilizing C receptors on Raji cells. These cells lack membrane-bound immunoglobulin but have receptors for IgG Fc, C3b, C3d, and possibly with other C proteins. Uptake experiments showed that both aggregated human gamma globulin (AHG) and 7S IgG bound to receptors for IgG Fc; however, AHG reacted with C bound to cells only via receptors for C and this binding was much more efficient than via IgG Fc receptors. AHG was used as an in vitro model of human immune complexes and its uptake by Raji cells was quantitated by 125I-radiolabeled antihuman IgG. The limit of sensitivity of this test was 6 mug AHG/ml serum. The ability of Raji cells to detect AHG in serum depended on the amount of radioactive antibody used and the size of aggregates. The presence of an excess of C somewhat inhibited binding of AHG containing C to Raji cells. The efficient binding of AHG by receptors for C on Raji cells was used for the detection and quantitation of immune complexes in human sera. Raji cells were incubated with sera to be tested and then reacted with excess radiolabeled antihuman IgG; the amount of radioactivity bound to the washed cells was determined and referred to a standard curve of radioactive antibody uptake by cells previously incubated with increasing amounts of AHG in serum. Thereby immune complexes were detected and quantitated in serum hepatitis, systemic lupus erythematosus, vasculitis, subacute sclerosing panencephalitis, dengue hemorrhagic fever, and malignancies.

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The Raji cell radioimmune assay for detecting immune complexes in human sera.

Previous in vitro studies have shown that immune complexes (IC) that fix complement can bind to the C3b receptor on primate erythrocytes. The in vivo function of this erythrocyte receptor, however, is unknown. This study was undertaken to determine whether the binding of IC to erythrocytes in vivo might play a role in the removal of IC from the circulation. Baboons and rhesus monkeys were prepared with a catheter in the ascending aorta to infuse IC and in the abdominal aorta, renal, hepatic, and portal veins to monitor changes in binding and clearance of IC across kidney, liver, and spleen + gut, respectively. Autologous 51Cr-labeled erythrocytes were infused intravenously and allowed to equilibrate. Preformed IC (125I-labeled bovine serum albumin [BSA] rabbit anti-BSA) were then infused into the ascending aorta at a constant rate for 120 s. Blood samples were drawn at frequent intervals for 30 min from all catheters below the IC injection site. Each blood sample was then centrifuged on percoll to separate IC bound to erythrocytes from IC in plasma or bound to buffy coat cells. This resulted in an "erythrocyte fraction" beneath the percoll that contained the IC bound to erythrocytes, and a "plasma/buffy coat fraction" above the percoll that contained the IC in plasma and IC bound to buffy coat cells. Analysis of these data showed that the majority of the IC infused into the circulation rapidly became bound to erythrocytes. However, by 5 min after beginning the IC infusion, most of this IC load had been removed from the erythrocytes as they traversed the liver. In contrast, IC on erythrocytes did not deposit in kidney. The IC-bearing erythrocytes themselves were not trapped or detained by any organ. IC in the plasma/buffy coat fraction of blood were removed from the circulation but at a relatively low rate and almost entirely by the liver. These findings suggest that primate erythrocytes intercept large complement-fixing IC in the circulation causing the IC to adhere to the erythrocyte until th e IC-bearing erythrocyte traverses liver where the IC is deposited, and the erythrocyte is returned to the circulation. This primate erythrocyte-IC-clearing mechanism may be important in the protection against diseases mediated by deposition of circulating IC.

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Primate erythrocyte-immune complex-clearing mechanism.

Publisher Summary This chapter describes the interaction of immune complexes (ICs) with complement and with the cells of the immune system. The effect of immune complexes depends to a great extent on their antigen–antibody ratio so that their influence, either stimulatory or suppressive, is itself modulated by quantitative aspects of the immune response. The development of numerous techniques for the detection and quantitation of immune complexes has stimulated clinically related research and expanded the list of diseases in which immune complexes appear to play an important role. An extension of this diagnostic technology is the ability to isolate immune complexes and, in turn, their antigenic component, thereby making it possible to identify the antigens involved in immune processes of a great many diseases, including those of unknown etiology. In vivo and in vitro experiments have clarified many factors involved in IC formation, removal, and localization as well as the mechanisms of IC-induced inflammatory reactions. An individual can make an immune response to a large number of exogenous and a smaller number of endogenous antigens. Depending upon the availability of antigen, the antibodies so produced form ICs, which for the most part serve the purpose of aiding the host in eliminating potential pathogens.

The Biology and Detection of Immune Complexes

A sensitive and reproducible procedure for the detection of soluble immune complexes in sera from patients with various immunopathological disorders is reported. Radiolabeled C1q is reacted with sera containing immune complexes. Separation of free from complex bound [(125)I]C1q is achieved by selective precipitation with polyethylene glycol (PEG). The method is based on both the large molecular size and the C1q-binding property characterizing immune complexes. The minimal amount of aggregated immunoglobulins thus detected is about 10 mug and that of soluble human IgG-anti-IgG complexes is about 3 mug of complexed antibody. Some immune complexes formed in large antigen excess (Ag(2)Ab) can still be detected by this radiolabeled C1q binding assay. The specificity of the radiolabeled C1q binding test was documented by the inability of antigen-F(ab')(2) antibody complexes to lead to a precipitation of [(125)I]C1q in PEG. In a second step, this radiolabeled C1q binding assay was applied to an experimental model of immune complex disease and was shown to be efficient for the detection of in vivo formed immune complexes.Finally, the technique could be applied to the study of sera from patients with systemic lupus erythematosus (SLE) or to carriers of the hepatitis B antigen (HB-Ag). Significantly increased [(125)I]-C1q binding values were observed in 52 sera from SLE patients when compared to values obtained with healthy blood donors (P<0.001). Particularly high values were seen in active disease, a finding which was confirmed by follow-up studies performed with four SLE patients. No increased [(125)I]C1q binding was seen in 18 healthy carriers of the HB-Ag; whereas, sera from carriers with hepatitis appear to precipitate increased [(125)I]C1q percentages: 7/24 cases with acute transient and 4/7 cases with chronic persistent hepatitis were found to increasingly bind [(125)I]C1q. The results were also used for a correlative study of [(125)I]C1q binding to IgG levels in the sera but increased [(125)I]C1q binding could not be attributed to high serum IgG levels which are likely to account for gammaglobulin aggregates. These examples suggest the utility of the radiolabeled C1q binding assay for the evaluation of immune complex diseases in human pathology.

Circulating Immune Complexes in the Serum in Systemic Lupus Erythematosus and in Carriers of Hepatitis B Antigen QUANTITATION BY BINDING TO RADIOLABELED Clq

In vitro, collagen and collagen-like material in GBM, were demonstrated to have a particular high affinity for any DNA tested (mammalian, bacterial, viral, and plant). GBM fixed DNA 40-80 times more than HGG and BSA and 10-40 times more than bacterial LPS. GBM has a higher affinity for SSDNA than for DSDNA. This binding was inhibited at low pH, low ionic strength, and in the presence of anionic detergents, indicating that the highly negatively charged DNA may interact with the basic site on collagen or GBM by electrostatic forces. This interaction was competitively interfered with by DNA-binding proteins such as Clq. Complexes formed of DNA and anti-DNA antibodies did not exhibit the same binding property as free DNA. However, DNA which was already bound to GBM or to collagen could very efficiently bind anti-DNA antibodies and form immune complexes which would remain on these structures. The biological significance of the binding of DNA to GBM or to collagen should be particularly considered in relation to the pathogenesis of SLE. It is possible that DNA released from disrupted or degenerating cells would bind to surrounding collagen fibers or to basement membranes and then act as an immunoabsorbant for circulating anti-DNA antibodies. Some evidence for an in vivo binding of SSDNA to renal structures was obtained in mice treated with bacterial LPS 2 days before the injection of SSDNA.

https://rupress.org/jem/article-pdf/144/2/428/1087948/428.pdf

In vitro demonstration of a particular affinity of glomerular basement membrane and collagen for DNA. A possible basis for a local formation of DNA-anti-DNA complexes in systemic lupus erythematosus.

Furthermore, PEG has been used at lower concentrations in order to precipitate soluble antigen-antibody complexes in conditions at which free antigen or free antibody would be soluble. This second method could be applied to larger antigens such as IgG or thyroglobulin.

P. A. Miescher

Papers

Detection of Antibodies and Soluble Antigen-Antibody Complexes by Precipitation with Polyethylene Glycol