Rat spleen leucocyte (RSL) radioimmunoassay for the detection and quantification of soluble immune complexes

A simple and sensitive method for the detection and quantification of soluble immune complexes in sera has been developed, by utilizing Fc receptors on rat spleen leucocyte (RSL). Aggregated human IgG (AHG) was used as an in vitro model of immune complexes and its uptake by RSL was quantified using ¹²⁵I-anti-human IgG.

RSL were found to bind AHG in medium as well as in ethylenediamine tetra-acetic acid (EDTA) inactivated and absorbed serum. The limit of detection of this method was 2 μg AHG/ml in medium and 4 μg AHG/ml in serum.

Sera samples from patients with thyroid disorders and from diabetics were incubated with RSL and the cells incubated with ¹²⁵I-anti-human IgG. The amount of label bound to the cells was determined and referred to a standard curve of radiolabelled antibody uptake by cells previously incubated with varying amounts of AHG in serum.

The results obtained were compared with those obtained using the Raji-cell radioimmunoassay.

Human anti-rat erythrocyte antibody was detected in normal human serum.

The amount of complexes formed in serum by heat treatment was quantified by this method.

     

Correlation between immune complexes and prognostic factors in Hodgkin''s disease

Serum or plasma samples from sixty-two patients with Hodgkin's disease (HD) were tested by four assays for circulating immune complexes or complement activation products: macromolecular C3 (MMC3), ¹²⁵I-labelled C1q binding (C1qBA), conglutinin binding (KgB) and plasma C3d levels. There was good agreement between the C1qBA, MMC3 and plasma C3d assays, and each gave significantly higher proportions of positive results in patients with symptoms of fever, night sweats and weight loss. The much lower proportion of positive assays by KgB, one out of thirty-three (3%) in asymptomatic and six out of twenty-nine (21%) in symptomatic patients, may reflect an inherent property of complexes or the presence of an inhibitor of conglutination in HD serum.

A positive result in one or more of these assays occurred in twenty-six out of twenty-nine (90%) of symptomatic patients compared with only ten out of thirty-three (30%) in asymptomatic patients. Most of the positive tests in the asymptomatic groups were by the C1qBA, and with one exception, these were from patients with histological types carrying a poor prognosis. In patients with poor prognostic histology, although the MMC3 and C3d assays detecting complement activation correlated with the symptoms, this was not found for immune complexes detected by the C1qBA. The close correlation between positive tests and two features known to adversely affect the prognosis in HD indicates that the detection of immune complexes may be of use in the staging and management of patients with this disease.

     

Mechanism of follicular antigen trapping: Evidence for a two cell mechanism using double isotope autoradiography

Normal mice were intravenously injected with ¹²⁵I-labelled HGG—anti-HGG complexes and 2 weeks later with ¹³¹I-labelled HGG—anti-HGG complexes. One week after the last injection the mice were killed and the localization of ¹²⁵I and ¹³¹I in the spleen was studied using a double isotope autoradiographic stripping technique. It appeared that ¹²⁵I-labelled HGG—anti-HGG and ¹³¹I-labelled HGG—anti-HGG complexes were both localized in exactly the same sites of the splenic follicle centres. It was concluded that both immune complexes were retained by the same dendritic cells.

¹³¹I-labelled HGG—anti-HGG complexes however were still localized in the periphery of the follicles shortly after injection at a time when ¹²⁵I-labelled HGG—anti-HGG complexes were already present in the follicle centres. If indeed cells carry immune complexes from the periphery of the follicles towards the follicle centres as suggested by previous studies, the present findings confirm that immune complexes are transferred from these cells to other cells (dendritic cells) in the follicle centres.

     

Ability of complement to release systemic lupus erythematosus immune complexes from cell receptors.

Endogenous immune complexes present in sera from 10 different patients with systemic lupus erythematosus (SLE) in an active phase were allowed to bind to Raji cells; the ability of intact complement to release the cell-bound complexes from receptors was then examined. Fresh normal human serum, or, alternatively, zymosan-pretreated serum, was added to the complex-bearing Raji cells. Immune complexes remaining bound to Raji cell receptors after increasing times at 37 degrees C were quantitated by addition of 125I-labelled antiglobulin, after removal of serum by washing. In all 10 cases, complement-dependent release was observed. In parallel control studies performed under identical conditions, immune complexes prepared in vitro from bovine serum albumin (BSA) and guinea-pig anti-BSA antibody were used in place of the endogenous SLE complexes. The experimental complexes were released by fresh serum, but not by zymosan-treated serum, but not by zymosan-treated serum, when studied using either 125I-labelled anti-guinea-pig Ig or 125I-labelled complexes alone. The results suggest that intact complement can alter the immune complexes present in SLE sera and influence their interaction with receptors on lymphoid cells. The results further raise the possibility that hypocomplementaemia secondarily due to consumption of complement by immune complexes may contribute to the persistence of the complexes.     

Assay of anti-hapten antibody with the aid of hapten-coupled bacteriophage

Treatment of bacteriophage with 3-iodo-4-hydroxy-5-nitrophenyl acetic acid chloride (NIP) in aqueous medium killed a proportion of the phage but the survivors were made susceptible to inactivation by rabbit immune sera to NIP-chicken globulin conjugate. The serum factor inactivating NIP-phage (T2) was eluted from a Sephadex G-200 column as 7S γ-globulin and was neutralized by a sheep antiserum against electrophoretically purified rabbit γ-globulin. The inactivation was strongly inhibited by the hapten and its derivatives. As little as 0.001 micromole per millilitre of NIP—ε-amino caproic acid was inhibitory.

Inactivation of NIP-phage was ascribed to anti-NIP antibody.

Inactivation of NIP-phage (T2) in strong anti-NIP sera approximately followed first-order reaction kinetics until 99 per cent of the phage became inactivated. When incubated with phage for 6 hours concentrations of antiserum as low as 10^-7 (1.9×10^-5 μg/ml) or less caused measurable inactivation of the phage. The threshold quantity of antibody was estimated to be less than 10^-5 μg.

     

Radioimmunoassay for the measurement of mass and avidity of anti-nerve growth factor antibodies

A radioimmunoassay is described for measuring on a weight basis the total amount of humoral antibodies to the purified protein nerve growth factor (NGF). The technique is based on the primary interaction of the antibodies with ¹²⁵I-labelled NGF added in large molar excess. The resultant immune complexes are precipitated by ammonium sulfate at 37% saturation, while unbound ¹²⁵I-labelled NGF is removed with the supernatant. The assay permits measurement of antibodies in a range of sensitivity of 0.1–1.0 ng antibody protein per ml. It is specific and reproducible and permits estimates of the antibody avidity.     

Quantitative determination of anti-dsDNA antibodies and antibody/dsDNA stoichiometries in prepared,soluble complement-fixing antibody/dsDNA immune complexes

We have investigated quantitatively the complement-mediated binding of prepared, soluble ¹²⁵I-7S IgG antibody/³H-dsDNA immune complexes to human red blood cells (RBCs). We have performed these studies by using a detailed modification of the RBC-CF assay [Pedersen et al., J. Immun. Meth. 38, 2692–2280 (1980)] which now allows for the simultaneous measurement of both ³H-DNA and ¹²⁵I-binding to the cells. Our results indicate that, in the case of three SLE patients, their anti-dsDNA antibody titers are sufficiently high that a small fraction of their ¹²⁵I-7S IgG antibodies (ca 0.1–0.2%) can be identified as specifically anti-dsDNA. We have also used an indirect method (with ¹²⁵I-labelled rabbit anti-human IgG) for the determination of IgG anti-dsDNA antibodies in complement-fixing antibody/dsDNA immune complexes that bind to RBCs, and the results of these measurements are in reasonable agreement with the direct binding experiments. These studies have also allowed us to estimate the antibody/DNA stoichiometries in complement-fixing immune complexes. The results of these experiments may provide a useful standard for the analysis of monoclonal anti-dsDNA antibodies.     

Isolation of circulating immune complexes by conglutinin and separation of antigen from dissociated complexes by immobilized protein A.

A method for the isolation of complement-fixing immune complexes from human serum and the separation of antigen from antibody is described. In order to isolate the complexes, we used soluble bovine conglutinin in a three-step procedure: (1) serum containing immune complexes is reacted with conglutinin in the presence of 10 mM calcium; (2) the conglutinin-bound immune complexes are precipitated by anti-conglutinin rabbit serum; (3) the precipitate is washed and the complexes are eluted from the precipitate by EDTA (pH 7.5) which chelates calcium and releases C3-associated immune complexes from conglutinin. To separate the antigen from the antibody, the isolated complexes are acid-dissociated (pH 3.0), and the antibody is absorbed to staphylococcal protein A conjugated to Sepharose leaving the antigen in solution. The antibody bound to Sepharose-protein A is recovered by elution with 3.5 M magnesium chloride. This procedure permitted the isolation of immune complexes from sera of hepatitis B surface antigen (HBsAg) positive chronic active hepatitis. In addition, immune complexes were isolated from sera of patients with rheumatoid arthritis, systemic lupus erythematosus and primary biliary cirrhosis. The isolated immune complexes contained IgG, IgM, C3 and albumin. Specific antibodies such as rheumatoid factors, anti-nuclear antibodies and antimitochondrial antibodies in varying titres have been found to be present in the isolated immune complexes. The conglutinin method has proven to be a useful technique for the isolation of immune complexes and for the identification of antibody and could be applied to the identification of the antigen in immune complexes.     

Circulating antigen-antibody complexes in onchocerciasis

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 microplate adaptation of the solid-phase C1q immune complex assay

A method has been developed for the detection of C1q binding immune complexes in serum in which microculture plates are used as the solid-phase matrix for adsorption of C1q. This micromethod used only one-tenth of the amount of both C1q and [¹²⁵I]anti-human immunoglobulin per test and enabled 7 times as many samples to be tested in triplicate in comparison with the number performed in duplicate by the standard tube assay.¹²⁵I-labelled C1q studies showed that adsorption varied with the brand of microplate used, some types of plate binding up to 66% of the labelled material. This is considerably more than that bound by the polystyrene tubes generally used for this assay.The increased capacity of the method allowed the binding to plates coated with the same batch of C1q to be assessed at various times after storage for 8–10 weeks at both 4°C and ?70°C. A marked decrease in binding to C1q of aggregates of IgG was observed on storage for longer periods. Results with different batches of aggregated IgG which had been ultracentrifuged suggested that this may be used as an effective standard, stable on storage at ?70°C.A comparison with the standard tube method of aggregated IgG, normal control sera and sera from patients with SLE showed that the micromethod adaptation of the C1q solid-phase binding radioimmunoassay is more economical and easier to perform and does not impair accuracy or standardization.     

A solid-phase radioimmunoassay for anti-SNP antibodies.

A highly sensitive and discriminatory solid-phase radioimmunoassay has been developed to detect anti-SNP antibodies in sera. Polystyrene tubes are coated with SNP and incubated with the test sera. The fixed antibodies are detected by a double layer technique using rabbit anti-human γ-globulin antiserum followed by incubation with a ¹²⁵I-labelled sheep anti-rabbit γ-globulin antiserum. Results are expressed in ng of the ¹²⁵I reagent fixed by 30 ωl of serum. The mean binding of 47 normal human sera was 22 ng ± 12 ng: 22/50 SLE sera gave over 34 ng binding. The specificity of the assay was studied in 3 different types of experiment: inhibition of the binding of positive sera either by pre-incubation with NDNA, SNP or RNA, DNAse I digestion of the SNP coated tubes, and incubation of SNP coated tubes with sera of known reactivity. It was shown that the solid-phase assay detects mainly antibodies directed against NDNA; however antibodies directed against the DNA-protein complex or the protein alone can easily be detected. Our results obtained with the solid-phase assay correlated well with those of the Farr assay. However, this new assay presents major improvements: it is simple, highly reproducible, and avoids the need for labelled antigen. A single labelled antiglobulin reagent allows identification of the class or subclass of reactive antibodies in a given species. Quantitation is more precise, particularly for sera containing high amounts of antibodies.     

Detection of immune complexes by their complement-dependent binding to bovine conglutinin: technical aspects of a solid-phase immunoenzyme microassay and its application to normal and pathological sera

A micromethod for the solid-phase conglutinin binding assay (Con BA) for the detection of circulating immune complexes (CIC) is described, in which the use of microplates, glucose oxidase-coupled anti-human immunoglobulins and automatic OD recorder contributed to the speed and low cost of this reproducible and sensitive test. The Con BA values of a large population of healthy blood donors had a wide distribution which in our series of 189 appeared to be trimodal. The Con BA values clearly reflected the levels of the main Ig classes (M, G, and A). This was confirmed by follow up of 6 individuals with high initial Con BA values. For 4 of them, a parallel decrease in Con BA value and IgG concentration was observed. In the 2 others, the sustained high level of Con BA remained unexplained. Complement components and activity of these sera were within normal limits. Because of fluctuation in the aggregated human gamma-globulins (AHG) reference curve, expression of results was based upon the standard deviation of 6 normal sera. In view of the above results, these sera were selected from those with the lowest Con BA values. On the basis that Con BA and C1q BA may detect CIC differing in their ag/ab ratio, sera from rheumatoid arthritis and chronic active B hepatitis patients with or without conventional rheumatoid factors (RF) were analyzed by both Con BA and C1q BA. RF-containing sera, likely to contain CIC in antigen excess, contributed exclusively to the highest C1q BA and lowest Con BA values. In contrast C1q BA-Con BA+ sera were preferentially RF negative. It is proposed that the complexes thus detected may be of idiotype-anti-idiotype nature.     

The intracellular localization of two antigens after uptake in vivo by peritoneal macrophages from normal mice

Normal mouse macrophages, which had ingested ferritin labelled with fluorescein isothiocyanate and human serum albumin labelled with tetramethylrhodamine isothiocyanate in vivo, were fixed in formalin and embedded for electron microscopy. The examination of sections 1–2 μ thick and adjacent ultrathin sections showed that the yellow-green fluorescent droplets (due to ferritin-FITC) seen by fluorescence microscopy were in the same position as the ferritin-containing phagolysosomes as seen by electron microscopy.

Normal mouse macrophages, which had ingested ¹²⁵I-labelled human serum albumin ([¹²⁵I]HSA) and unlabelled ferritin, were investigated by electron microscopic autoradiography. Both antigens were found to be situated within the same lysosomes.

     

Immune recognition of serum albumin—XIII. Autoreactivity with rabbit serum albumin of rabbit antibodies against bovine or human serum albumins and autoimmune recognition of rabbit serum albumin

Recently, this laboratory reported an autoreactivity of myoglobin (Mb)3 antisera with the Mb of the species in which the antisera are raised. Also, animals injected with autologous Mb mounted an autoimmune antibody and T-lymphocyte proliferative response against this protein. This posed the possibility that autoimmune recognition might be a general phenomenon not confined only to sequestered proteins such as Mb. Using RSA, we have demonstrated unambiguously that RSA cross-reacted with rabbit antisera to bovine (BSA) or to human (HSA) albumin. In exchange experiments, ¹²⁵I-labelled RSA was bound by the IgG in the immune complexes isolated from rabbit antisera to BSA or HSA. Also, ¹²⁵I-antibodies were bound by RSA-adsorbents. Both binding activities were inhibited specifically by RSA. RSA was isolated from three rabbits and each rabbit was immunized with its own RSA. In each rabbit autoantibodies were found by exchange between immune complexes and the rabbit's own [¹²⁵I]-RSA. Also, ¹²⁵I-antibodies from each rabbit were bound by adsorbents of the rabbit's own RSA. Inhibition studies, data on preimmune sera and on antisera against other proteins confirmed the specificity of the binding. The findings confirm the universality of autoimmune recognition and lend support to our previous suggestion that antigenic sites are ‘structurally-inherent’ in the protein.     

The alternative complement pathway control protein H binds to immune complexes and serves their detection

During solubilization of immune complexes C3b becomes fixed to the immunoglobulin part and serves as a receptor for the alternative complement pathway control protein H. The H-C3b immune complex interaction can be made detectable using 4% polyethyleneglycol to separate free from bound 125I-H. Tetanus toxoid (Te)/anti-Te complexes kept soluble with fresh serum and containing 125 IU of specific antibody bound 18% of 125I-H; when fresh serum was chelated with 10 mM EDTA, 125I-H binding was only 5%. On sucrose density gradients, the H-binding material sedimented in the range of 12 to 30 S. In 36 serum samples from rheumatoid arthritis (RA) patients and in 12 serum samples from patients with systemic lupus erythematosus (SLE), 125I-H binding was significantly elevated to 9.5 +/- 4.7% (mean +/- 1 SD) and 13.3 +/- 5.6%, respectively, while 125I-H binding by 36 normal human sera was 4 +/- 2%. RA samples (17/36, 47%) and SLE samples (9/12, 75%) had H-binding values increased by more than 2 SD above the normal mean. The serum samples were also assessed for conglutinin- and C1q-binding activities; a significant correlation between H and C1q binding was observed (P less than 0.001); there was no correlation between H and conglutinin binding. Although binding to immune complexes through its interaction with C3b, H clearly detects a population of complexes other than conglutinin, thus expanding the possibilities of further characterizing pathological complexes.     

Immunological unresponsiveness to protein antigens in rabbits: II. The nature of the subsequent antibody response

Rabbits made immunologically unresponsive by neonatal administration of HSA, HGG or BSA were given a course of intravenous injections of the respective antigens, adsorbed on alum, after a lapse of 13–27 months since the last administration of antigen. 8/12 responded to HSA, 4/5 to HGG, 9/10 to BSA, as judged by immune elimination of antigen, but this was delayed in onset and slow compared with that in previously untreated rabbits. The antibody formed was small in quantity and usually failed to precipitate with antigen.

The sedimentation coefficients of ¹³¹I-labelled antigens, in the presence of excess antibody, were measured by ultracentrifugation through a sucrose density gradient. These showed that only small complexes were formed in some of the non-precipitating antisera. In one instance the diffusion coefficient of the complex was also measured, by a technique based on diffusion through agar gel. The calculated molecular weight of the complex, 330,000 indicated the presence of only two combining sites on the antigen.

Combination of the anti-HSA sera with an HSA fragment was also measured. Whereas the amount of the fragment bound by ordinary hyperimmune anti-HSA sera was about one-fifth the HSA bound, the amounts bound by the test sera were relatively much less. Some non-precipitating sera failed to bind the fragment, although they bound HSA.

These findings indicate that following neonatally induced immunological unresponsiveness the capacity to respond to antigen returns piecemeal in respect of different parts of the antigenic mosaic, and that it may be severely restricted. The theoretical implications are discussed.

     

Purification of soluble immune complexes from serum using polymethylmetacrylate beads coated with conglutinin or C1q. Application to the analysis of the components of in vitro formed immune complexes and of immune complexes occurring in vivo during leishmaniasis.

A procedure for the isolation of immune complexes from human sera has been developed. Two steps are involved: (1) lipid-free serum is precipitated by polyethylene glycol; (2) the solubilized precipitate is absorbed on a column of polymethylmethacrylate beads coated with conglutinin (K) or C1q; the column is washed, the complexes are then eluted, using 0.02 M EDTA (for K column) or 0.5 M NaCl (for C1q column). This procedure permitted the purification and the characterization of soluble 125I-BSA-anti-BSA, 125 I-tetanus toxoid-anti-tetanus toxoid, and 125-I-HBsAg-anti-HBsAg complexes made in vitro in the presence of fresh human serum. The isolated complexes were shown to contain antigen, antibody, C1q, C1r, C1s and C3. When normal human serum was submitted to such a procedure, no detectable amount of protein was present in the final eluted fraction. Immune complexes formed in vivo were also purified by conglutinin column from the serum of a patient with disseminated leishmaniasis. The isolated material was found to contain IgM, IgG, C1q, C1r, C1s, C3c and C3d. The purified complexes dissociated at acid pH were found to contain anti-IgG and anti-leishmania antibodies.     

Anti-DNA antibody derived from a systemic lupus erythematosus (SLE) patient forms histone–DNA–anti-DNA complexes that bind to rat glomeruli in vivo

Histone can mediate the binding of both free DNA and DNA complexed to anti-DNA antibody to the glomerular capillary wall. We tested whether preformed histone–DNA–anti-DNA immune complexes (IC) could bind to the glomerular capillary wall. The immune complex, generated with anti-DNA antibody derived from an SLE patient and excess of ¹²⁵I-DNA followed by digestion with DNase, was mixed with histones. The complex containing 4 μg DNA was injected via the aorta into the left kidney of rats. At 15 min, 1.3% of the histone–DNA–anti-DNA antibody complex bound (measured as ¹²⁵I-DNA), when histone was omitted less than 0.1% of the DNA–anti-DNA antibody complex bound. By immunofluorescence human immunoglobulins and histones, representing the IC, could be observed in a capillary pattern; but no complement deposition was detected. Electron microscopy revealed discrete, electron dense deposits in a subendothelial, subepithelial and mesangial localization at 15 min. These results provide direct evidence that antibodies from serum of SLE patients can form soluble histone–DNA–anti-DNA immune complexes that bind to the glomerular capillary wall in vivo     

Analysis of Human Serum Immunoglobulin G against O-Acetyl-Positive and O-Acetyl-Negative Serogroup W135 Meningococcal Capsular Polysaccharide

The capsular polysaccharide of Neisseria meningitidis serogroup W135 is expressed in both O-acetyl-positive (OA⁺) and O-acetyl-negative (OA⁻) forms. This study investigates the impact of OA status (OA⁺ versus OA⁻) on serological measurements of anti-W135 immunoglobulin G (IgG) antibodies in immunized adults. W135-specific serum antibody assignments were made for 28 postimmunization sera from adults by enzyme-linked immunosorbent assay using the meningococcal standard reference serum CDC1992. The established IgG concentration in micrograms per milliliter ([IgG]μg/ml) for CDC1992 against OA⁺ antigen (16.2 μg/ml) was used as a reference to assign a concentration of 10.13 μg/ml IgG against OA⁻ antigen by cross-standardization. Overall, the IgG assignments for these sera were higher against OA⁺ antigen (geometric mean concentration [GMC] = 7.16 μg/ml) than against OA⁻ antigen (GMC = 2.84 μg/ml). However, seven sera showed higher specific [IgG]μg/ml values against the OA⁺ antigen than against the OA⁻ antigen. These sera were also distinguished by the inability of fluid-phase OA⁻ antigen to compete for antibody binding to OA⁺ solid-phase antigen. Although there was no overall difference in functional activity measured by complement-mediated serum bactericidal assay (SBA) against OA⁺ and OA⁻ target bacteria (geometric mean titers of 9,642 and 9,045, respectively), three serum specimens showed a large difference in SBA antibody titers against OA⁺ versus OA⁻ W135 target bacteria, which may reflect different epitope specificities for these sera. Our data indicate that, for some sera, the agreement in anti-OA⁺ versus anti-OA⁻ W135 IgG assignments is serum specific and does not reflect the functional (killing) activity in vitro.     

The quantitation of C6 in rabbit and human sera

C6 quantitation was carried out in rabbit and human sera by the single radial immunodiffusion technique. The C6 content of the rabbit and human sera used as standards was estimated by precipitin analysis, using an anti-C6 antiserum labelled with ¹²⁵I. The mean C6 level in normal human serum was 11 μg/ml, whereas in normal rabbit serum it was 35 μg/ml. Sera from forty rabbits heterozygous for C6 deficiency were found to have a mean concentration of C6 of 14 μg/ml.