A small proportion of cationic antibodies in immune complexes is sufficient to mediate their deposition in glomeruli

Positively charged antibodies mediate enhanced deposition of circulating immune complexes at the glomerular basement membrane. The presented experiments demonstrate that when soluble immune complexes were prepared with a mixture of antibodies containing 10 to 25% cationic antibodies, then noncationic antibodies in the complexes were deposited in mouse glomeruli. One or two cationic antibodies in each immune complex sufficed for deposition of the complexes. Proof for this was obtained by two kinds of experiments. First, the injected immune complexes were prepared in Ag excess from mixtures of radiolabeled noncationic rabbit antibodies to human serum albumin (HSA) and unlabeled cationized rabbit antibodies to HSA, thus permitting the specific quantitation of the deposition of noncationic antibodies in glomeruli because of the presence of cationized antibodies within the same complexes. As a control experiment, immune complexes prepared only with noncationic antibodies resulted in very little deposition in kidneys over the same time period. Second, detection of the localization of the noncationic antibody in deposits in glomeruli by immunofluorescence microscopy was accomplished using immune complexes prepared with mixtures of noncationic goat antibodies to HSA and cationized rabbit antibodies to HSA. Thus, the synthesis of a small population of cationic antibodies during the immune response may lead to the formation of circulating immune complexes with enhanced propensity for deposition in glomeruli in patients with SLE or other immune complex diseases.     

Use of larval, parasitic female and egg antigens from Strongyloides venezuelensis to detect parasite-specific IgG and immune complexes in immunodiagnosis of human strongyloidiasis

The aim of this study was to use larval, parasitic female and egg antigens from Strongyloides venezuelensis to detect parasite-specific IgG and immune complexes in human serum samples by enzyme-linked immunosorbent assay (ELISA). In total, 95 serum samples were analysed, consisting of 30 patients harbouring S. stercoralis larvae, 30 healthy subjects and 35 patients with other parasites. Sensitivity, specificity and diagnostic efficiency were calculated. A significant statistical difference was found in the detection of immune complexes and antibodies in patients harbouring S. stercoralis larvae from larval and eggs antigens, with higher positivity using larval antigen. The larval antigen showed the highest values for sensitivity, specificity and diagnostic efficiency in ELISA from detection of immune complexes. For the first time we used IgG anti-larvae, IgG anti-parasitic females or IgG anti-eggs for immune complex detection. We concluded that the association of antibody and immune complex detection could be used in the diagnosis of human strongyloidiasis.     

Evaluation of chemotherapy in experimental toxocarosis by determination of specific immune complexes

Parasitism by the larval phase of Toxocara canis is a chronic process in which the larvae survive in the tissues, resulting in the constant stimulation of the immune system. As a result, the detection of specific antibodies may not reflect the active state of the parasite. We have studied the dynamics of the production of specific immune complexes by ELISA with the monoclonal antibody TC-1 in rabbits inoculated with single and multiple doses of T. canis eggs. We also compared this with the production of specific antibodies and their possible modification after treatment with mebendazole. The specific antibodies against excretory-secretory antigen were detected with peaks at 10 and 12 weeks depending on the dose and remained positive during the entire experiment (62 weeks). Treatment caused an increase in the level of detectable antibodies dropping to similar levels to the controls. Specific immune complexes were detected only in multiple doses, and were then positive during the entire experiment. From the beginning of treatment the values of immune complexes fell quickly, remaining at undetectable levels during the rest of the experiment. For this reason the detection of specific immune complexes is a valid technique for monitoring the efficiency of treatment.     

The determination of antigen-specific immune complexes by an immunoenzyme method]

The methodological approach permitting the detection of immune complexes containing specific antibodies to a definite antigen in the enzyme-linked immunosorbent assay (ELISA) is described. The basic conditions of the assay were optimized. Immune complexes were precipitated from blood serum with 3.5% polyethylene glycol 6000 for 4 hours. The precipitate thus obtained was dissolved and incubated in polystyrene plates with immobilized antigen at 37 degrees C for a long time (at least 6 hours) in a humid chamber. The amount of bound antibodies, determined by ELISA techniques, was conjectured from the level of antigen-specific immune complexes. The proposed approach can be used in the immunodiagnosis of infectious diseases.     

A quantitative analysis of complex formation between IgM and immobilized ligand using atomic force microscopy

Specific interaction between human IgM and polyclonal antibodies immobilized on support was studied by atomic force microscopy. Human IgMs are responsible for a number of side effects arising during the xenotransplantation of mammalian organs to man. On the basis of atomic force microscopy, a quantitative analysis of complexes with IgM was performed. The data of the analysis agree well with the results of enzyme immunoassay. It was shown that the method of detection of immune complexes based on atomic force microscopy is able to detect specific antibodies/antigens in serum.     

Circulating immune complexes in the diagnosis of Mycoplasma pneumoniae infection

The possibility of detecting M. pneumoniae antigen and antibodies to it, incorporated into immune complexes, in the sera of patients with acute pneumonia by means of erythrocyte diagnosticums was studied, and the immunological characterization of these complexes was made. In patients with mycoplasmal pneumonia M. pneumoniae antigen and specific antibodies, both free and incorporated into immune complexes, were found to circulate in the blood. In children, antigenemia was detected twice as frequently as in adults. Dissociated M. pneumoniae antigens had different molecular weight, their location on the gel chromatogram of the serum being in fractions 7S and 19S. The dissociation of immune complexes permits the detection of M. pneumoniae antigen and antibodies to it in a bound state by means of the passive hemagglutination test, thus increasing the frequency of positive results in the diagnosis of M. pneumoniae infection.     

G-quadruplex DNAzyme-based microcystin-LR (toxin) determination by a novel immunosensor

In this paper, we demonstrate the application of versatile G-quadruplex-hemin DNAzymes in an immunoassay for detecting Microcystin-LR (MC-LR). Taking advantage of the high peroxidase activity of G-quadruplex-hemin complexes and the enhancement effect of gold nanoparticles (AuNPs), the method showed simple, high sensitive and selectivity detection of target toxin residues in water samples. The coated antigen, MC-LR-ovalbumin (OVA) coated on a plate, competed for MC-LR antibody with added target analyte to form antibody-antigen immune complexes. Subsequently, the immune complex reacted with G-quadruplex-labeled secondary antibodies for colorimetric detection of MC-LR. This assay specifically determined MC-LR in the linear range of 0.1-10 ng/ml, with a limit of detection (LOD) of 0.05 ng/mL for MC-LR. The results indicated that the novel immunoassay was an alternative to traditional plate-based immunoassay for MC-LR residue screening due to this method met the standard of World Health Organization (WHO) requirements for MC-LR content in drinking water (1 ng/mL).     

Immunosensor for Mycobacterium tuberculosis on screen-printed carbon electrodes

In this work, two methods have been compared to produce enzymatic voltammetric immunosensors for the determination of Mycobacterium tuberculosis antigens (Ag360 and Ag231), using a pre-oxidised screen-printed carbon electrode (SPCE) as a signal transduction element. The enzyme alkaline phosphatase (AP) was used in combination with the substrate 3-indoxyl phosphate (3-IP). In one design, the immune complexes between M. tuberculosis antigens and monoclonal antibodies against M. tuberculosis were formed out of the electrode surface. Then, the immune complexes were captured by biotinylated rabbit anti-M. tuberculosis antibodies, immobilised on the streptavidin modified SPCEs through the streptavidin:biotin reaction. Finally, an alkaline phosphatase (AP) labelled rabbit IgG anti-mouse immunoglobulin G was used as a detector antibody. In the other design, the M. tuberculosis antigens were captured by monoclonal antibodies against M. tuberculosis, which were immobilised on the electrode surface through the reaction with rabbit IgG passively adsorbed on the SPCEs. The biotinylated rabbit anti-M. tuberculosis antibodies were used with an alkaline phosphatase labelled streptavidin as detector antibodies. The best results for M. tuberculosis antigen determination were obtained using the immunosensor on the streptavidin modified SPCEs and the immune complexes between antigen Ag231 and monoclonal antibodies MabF184-3, with a detection limit of 1.0 ng/ml. The immunosensor was also applied to Ag231 spiked proteic matrices.     

Immune complexes with cationic antibodies deposit in glomeruli more effectively than cationic antibodies alone

In previously published studies, highly cationized antibodies alone and in immune complexes bound to glomeruli by charge-charge interaction, but only immune complexes persisted in glomeruli. Because normal IgG does not deposit in glomeruli, studies were conducted to determine whether cationized antibodies can be prepared which deposit in glomeruli when bound to antigen but not when free in circulation. A series of cationized rabbit antiHSA was prepared with the number of added amino groups ranging from 13.3 to 60.2 per antibody molecule. Antibodies alone or in preformed soluble immune complexes, prepared at fivefold or 50-fold antigen excess, were administered to mice. With the injection of a fixed dose of 100 micrograms per mouse, antibodies alone did not deposit in glomeruli with less than 29.6 added amino groups by immunofluorescence microscopy. In contrast, 100 micrograms of antibodies with 23.5 added amino groups in immune complexes, made at fivefold antigen excess, formed immune deposits in glomeruli. With selected preparations of cationized, radiolabeled antibodies, deposition in glomeruli was quantified by isolation of mouse glomeruli. These quantitative data were in good agreement with the results of immunofluorescence microscopy. Immune complexes made at 50-fold antigen excess, containing only small-latticed immune complexes with no more than two antibody molecules per complex, deposited in glomeruli similar to antibodies alone. Selected cationized antibodies alone or in immune complexes were administered to mice in varying doses. In these experiments, glomerular deposition of immune complexes, made at fivefold antigen excess, was detected with five- to 10-fold smaller doses than the deposition of the same antibodies alone. These studies demonstrate that antibody molecules in immune complexes are more likely to deposit in glomeruli by charge-charge interactions than antibodies alone.     

Immunoenzyme analysis for determining class G and M antibodies to HBsAg

A scheme of the purification of hepatitis B virus surface antigen (HBsAg) as applied to the enzyme immunoassay (EIA) for the detection of antibodies to HBsAg is described. An indirect EIA technique for the detection of IgG and IgM antibodies to HBsAg has been developed and the diagnostic assay system based on the use of immunoreagents and solid-phase carriers produced in the USSR has been obtained. The sensitivity of the indirect EIA technique in the detection of IgG antibodies to HBsAg exceeds that of double immunodiffusion in gel used for this purpose 2,500- to 5,000-fold. The study has shown the possibility of using the indirect EIA technique for the detection of antibodies to HBsAg, both free and bound in immune complexes, of detecting antibodies to HBsAg in patients with acute and chronic viral hepatitis B, as well as of simultaneous detection of IgG and IgM antibodies to HBsAg without pseudonegative results.     

Relationships between the content of circulating immune complexes, the concentration of serum immunoglobulins and the titers of specific antibodies in patients with tick-borne encephalitis

In the blood serum of tick-borne encephalitis (TBE) patients the detection rate and concentration of circulating immune complexes, as well as the content of serum IgA, IgM and IgG, were evaluated. The formation of immune complexes was found to depend on IgM and IgG specific antibodies to TBE virus, the period of the disease and the clinical form of virus infection.     

Application of antigen retrieval by heating for double-label fluorescent immunohistochemistry with identical species-derived primary antibodies.

Double-label fluorescent immunohistochemistry (IHC) is frequently used to identify cellular and subcellular co-localization of independent antigens. In general, primary antibodies for double labeling should be derived from independent species. However, such convenient pairs of antibodies are not always available. To overcome this problem, several methods for double labeling with primary antibodies from identical species have been proposed. Among them are methods using monovalent secondary antibodies, such as Fab fragments. Soluble immune complexes consisting of primary and monovalent secondary antibodies are first formed. After absorption of the excess secondary antibody with nonspecific immunoglobulin, the immune complexes are applied to sections. By this procedure, unwanted cross-reaction between false pairs of antibodies is avoidable. However, soluble immune complexes often show reduced or no immunoreactivity to antigens on sections. I noted that antigen retrieval (AR) of tissues by heating often but not always showed improved immunoreactivity for soluble immune complexes. Here I demonstrate the examination of conditions for this soluble immune complex method using AR-treated sections and show examples of double-label fluorescent IHC with identical species-derived primary antibodies.     

Recent advances in the immunological aspects of renal disease.

Anti-basement membrane antibodies and tissue deposition of immune complexes appear to be responsible for most glomerulonephritides and for some tubulo-interstitium injury accompanying glomerulonephritis or occuring primarily. Anti-tubular basement membrane antibodies complicate immunologic and toxic renal injury, including transplantation, and widespread tubulo-intersitial immune complex deposits are present in most patients with systemic immune complex disease, such as lupus erythematosus. Radioimmunoassay is now available for detecting and monitoring circulating anti-glomerular basement membrane antibodies. The effect of aggressive therapy with immunosuppression and plasma exchange is being studied to determine is value in minimizing tissue damage produced by the usual transient production of anti-glomerular basement membrane antibodies. Techniques are being explored to detect circulating immune complexes. Vigorous efforts are under way to identify antigen-antibody systems involved in the production of nephritogenic immune complexes, which may lead to antigen irradiation or specific manipulation of the immune response or its products.     

Circulating immune complexes in the serum of diabetes mellitus in childhood by a modified 125I-C1q binding test.

The 125I-C1q binding test for the detection of soluble immune complexes in native unheated human serum was applied to the study of sera from 52 patients with diabetes mellitus in childhood. This radiolabeled C1q binding test is more sensitive and reproducible among the various methods proposed for the detection of immune complexes. The 125I-C1q binding activity in 52 sera from diabetes mellitus in childhood was 9.47 +/- 0.36% compared to 6.94 +/- 0.74% in normal controls. 125I-C1q binding values in diabetes mellitus in childhood were significantly higher than normal controls. Slight high values were seen in 3 patients with positive anti-DNA-antibodies in diabetes mellitus in childhood. 125I-C1q binding was not significantly increased in patients with positive antithyroid antibodies and insulin antibodies. There was no significant correlation between the duration of diabetes and 125I-C1q binding activity.     

Application of magnetite nanoparticles for the development of highly sensitive immunochromatographic test systems for mycotoxin detection

Highly sensitive immunochromatographic test systems were developed for the detection of zearalenone (ZEA) and T-2 toxin (T2T) using magnetite nanoparticles (MNPs) for the labeling. In order to detect an analyte with high sensitivity, the competitive reaction was performed with free specific antibodies, while immune complexes were detected by the reaction with label-conjugated anti-species antibodies. The conditions for the synthesis of magnetite nanoparticles conjugated to anti-species antibodies were optimized. The concentrations of specific reagents that provided the highly sensitive detection of T-2 toxin and zearalenone were found. The instrumental detection limit for the determination of T-2 toxin and zearalenone in baby food samples (oat flakes) was 0.1 and 0.05 ng/mL (2.0 and 1.0 ng/g), respectively. The assay time was 15 min. The results of the present study confirm the possibility of the practical use of magnetite nanoparticles for immunochromatographic assay labeling.     

A comparative analysis of the efficiency of bird and mammalian antibodies in HBsAg sandwich assay

The antigen-binding capacities of chicken and goat polyclonal antibodies and mouse monoclonal antibodies specific for the hepatitis B virus surface antigen were compared via enzyme immunoassay. It was shown that the use of both adsorbed and detecting chicken antibodies reduces significantly the analytical sensitivity of the sandwich method for the detection of this antigen. It was assumed that the reduced flexibility of IgY molecules hinders bivalent binding to antigen, which leads to the formation of more stable immune complexes.     

A radiometric immunosorbent assay for the detection of anti-hormone-binding protein antibodies

A radiometric immunosorbent assay (RISA) for the detection of monoclonal antibodies to hormone-binding proteins has been developed. The assay involves incubating hybridoma supernatants in microtiter wells that have been coated with goat anti-mouse IgG antibodies. Any mouse IgG in the test supernatant is thus specifically retained in the wells. Radioactive ligand-binding protein complexes are then incubated in the wells. The presence of anti-binding protein antibodies in the supernatant is indicated by specific retention of radioactive ligand-binding protein complexes in the wells. Crude antigen preparations, such as tissue homogenates, can be used to detect antibodies. The assay is capable of detecting antibody at concentrations 20 ng/ml (approximately 100 pM IgG). The RISA has been used successfully to screen for monoclonal antibodies to the intracellular receptor for 1,25-dihydroxyvitamin D3 and should be useful for the detection of antibodies to ligand-binding proteins in general.     

Echinostoma caproni in mice: ultrastructural studies on the formation of immune complexes on the surface of an intestinal trematode

The binding of mouse antibodies to the surface antigens of juvenile and 7 and 28 day old Echinostoma caproni was examined by transmission electron microscopy of thin sections of parasites, which were treated with antibodies in a double sandwich technique with ferritin-conjugated antibody. The surface of freshly recovered mature adult parasites was covered with an irregular but often rather intensive mouse antibody containing matrix, which probably represents a layer of mouse antibody/parasite antigen complexes. The complexes were lost after in vitro culturing of the parasites for 24 h, but incubation of the in vitro-maintained antibody-negative adult parasites with immune mouse serum led to reformation of a similar but less intensive cover with immune complexes. Juvenile and young stages of E. caproni, which had never been exposed to host antibodies, obtained a layer of immune complexes on their surface after incubation with immune mouse serum in vitro. In both young and mature parasites, the antibody-antigen complexes were observed to be rather loosely attached to the outer surface of the parasites, where the antigens probably constitute a part of the irregular glycocalyx of the organisms. It may also be that the antigens are present as isolated excretion along the surface of the parasites. Several sections indicated that the parasite surface antigens may be present in the tegument in vesicles which fuse with the outer membrane of the parasite whereby their contents are released to the exterior.     

Analysis of vascular lesions in murine SLE. I. Association with serologic abnormalities

In murine SLE, two different vascular lesions can develop. A necrotizing polyarteritis (NPA), exclusively found in MRL/I mice, is characterized by a dense infiltration of PMN and fibrinoid necrosis of the arterial wall. The second, a degenerative vascular lesion, occurs in a low incidence in all SLE mice, except the (NZW X BXSB)F1 (WBF1) male, in which its incidence is 100%. This lesion shows subendothelial deposits of immunoglobulins with minimal or no inflammatory or proliferative reaction. This degenerative vascular disease (DVD) is predominantly localized in the coronary arteries and is highly correlated with myocardial infarction. Serologic analysis revealed that NPA in MRL/I mice was associated with relatively late development of high levels of autoantibodies and circulating immune complexes; DVD in WBF1 mice was associated with an early onset of autoantibody production of a low magnitude that gave rise to a persistent low level of circulating immune complexes. Characterization of circulating immune complexes in MRL/I mice showed these complexes were mainly of intermediate size (7S-19S) and contained predominantly anti-DNA antibodies. In WBF1 mice, complexes were barely detectable and contained mostly anti-gp70 antibodies. Elution of kidneys showed that the major antibody deposited in MRL/I mice has an anti-DNA specificity, whereas in WBF1 animals, the major antibody was anti-gp70. Furthermore, a 10 times greater amount of immunoglobulins could be eluted from WBF1 hearts with DVD than from MRL/I and BXSB hearts. Additionally, we found that the lack of an inflammatory reaction in DVD was not because of a preferential deposition of noncomplement-fixing IgG1 antibodies nor could it be related to a defective inflammatory response, because WBF1 mice had an undiminished reverse passive Arthus reaction throughout their lives. It is concluded that NPA develops secondary to high levels of autoantibodies with a concomitant rise in immune complexes, whereas DVD is associated with sustained low levels of circulating immune complexes.     

Complement-mediated solubilization of immune precipitates prepared with antibodies of different avidity.

Complement-mediated solubilization of immune precipitates prepared with HSA and rat IgG anti-HSA has been quantitatively analyzed. Early and late IgG anti-HSA antibodies were obtained 27 and 49 days after immunization, respectively. Immune precipitates prepared with early IgG anti-HSA were solubilized by rat serum to a larger extent than complexes prepared with late IgG anti-HSA. The affinities for HSA of the early and late antibodies were not significantly different. The quantitative differences in solubilization were neither due to differences in the Ab/Ag ratios of the immune precipitates, nor appeared to be brought about by changes in the distribution of the antibodies over the IgG sub-classes. The avidity of the late IgG anti-HSA antibodies was higher than the avidity of the early IgG antibodies. Presumably, the avidity of the antibodies greatly affected the complement-mediated solubilization of the immune precipitates. In addition, the solubilization was found to be dependent on the conditions employed to prepare the immune precipitates.