The Attachment of Serum-and Plasma-Derived C3 to Solid-Phase Immune Aggregates and its Relation to Complement-Mediated Solubilization of Immune Complexes

The interaction between immune aggregates and complement (C) was investigated. Solid-phase immune aggregates were prepared by coating microwells with heat-aggregated bovine serum albumin (BSA) followed by rabbit anti-BSA antibody. The immune aggregates were reacted with human serum or citrated plasma at 37 degrees C. The binding of C3 components was investigated with biotinylated F(ab')2 antibodies to C3c and C3d and avidin-coupled alkaline phosphatase. The form of the incorporated C3, whether C3b-iC3b or C3dg, can be deduced from the response with these two antibodies. The maximal binding of C3b-iC3b to the immune aggregates was observed within 5 min of incubation with serum or citrated plasma. The conversion to C3dg was evident by a decrease in bound anti-C3c concomitant with increasing anti-C3d reactivity within about 10 min of incubation. When the classical C pathway activation was inhibited, the binding of C3b-iC3b was delayed by 20-30 min, whereas stopping of the alternative pathway did not influence the initial kinetics of the reaction. The addition of human red blood cells had no measurable influence on the degradation of bound C3b-iC3b. 125I-labelled anti-BSA antibody bound to the solid-phase BSA was not released during the C3 incorporation. The incorporation of C3b into the immune aggregates was mediated equally well by serum and by citrated plasma. The incorporation of C3b-iC3b into immune complexes (IC) is thought to be responsible for the C-mediated solubilization (CMS) of IC. Citrated plasma, however, exerted no CMS capacity when measured by a radiometric assay. The CMS capacity of serum was inhibited by citrate, but could then be restored by adding Ca2+ and Mg2+, whereas no CMS could be demonstrated with citrated plasma to which divalent metal ions were added.     

Optimizing the solid-phase immunofiltration assay. A rapid alternative to immunoassays

The technical variables of the solid-phase immunofiltration assay (SPIA) for the detection of antibodies bound to antigens on a solid-phase filter have been investigated. The binding to solid-phase filters of 125I-labelled axial filament proteins derived from Treponema phagedenis and the optimal conditions for blocking non-specific protein binding were analysed. Axial filament was applied to nitrocellulose, Hybond Nylon and Zeta Probe. After extensive rinsing, the highest amount (68%) of axial filament was observed bound to Zeta Probe. However, blocking non-specific protein binding by pre-wetting the filter with rinsing buffer containing 0.5% Tween 20, prevented the binding of protein to the filter only when nitrocellulose was used as solid phase. Tween 20 (0.5%) in the rinsing and incubation solutions was found to be necessary for the reduction of non-specific binding of contaminants in turbid sera. However, the use of such solutions resulted in a substantial leakage of antigen (47%) during rinsing procedures. Binding of antigen-specific antibody was analysed using 125I-labelled protein A. The maximal possible binding of the antibody occurred within 5 min when the antibody solution was filtered. For optimal binding of 125I-labelled protein A an incubation time of 1 h was needed. It is suggested that solid-phase immunofiltration may provide a rapid alternative for radioimmunoassays or enzyme immunoassays for the detection of specific antibodies.     

A receptor for monomeric IgG2b on rat macrophages

The binding to rat splenic and peritoneal macrophages of affinity-purified monoclonal rat IgGs, representing all IgG subclasses, was measured by the direct binding of 125I-labelled proteins using an assay that did not require the removal of unbound Ig by washing. Only rat monomeric IgGs of the subclass IgG2b bound specifically and in large amounts to rat macrophages. The binding was temperature dependent and more IgG2b bound to the cells at 4 degrees than at 37 degrees. Spleen macrophages bound approximately 10 times more IgG2b than the same number of peritoneal macrophages, although the association constants (Kas) for the binding were similar for both types of macrophage. The calculated values for the Kas, which varied slightly with each experiment and increased with decrease in temperature, fell within the range 1.3-5.3 X 10(8) M-1; the number of binding sites was estimated as about 10(5)/splenic macrophage and 10(4)/peritoneal macrophage. The binding of 125I-IgG2b to splenic macrophages was inhibited only by unlabelled proteins of the IgG2b isotype and not by IgG1, IgG2a and IgG2c proteins. Soluble IgG2b-antigen complexes also bound to the FcR for monomer but a soluble IgG2a-antigen complex did not inhibit the binding of monomeric IgG2b.     

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.     

Functional affinity constants of the reaction between 125I-labelled C1q and C1q binders and their use in the measurement of plasma C1q concentrations.

Purified 125I-labelled C1q has been found to react with glutaraldehyde-treated red cells (glut-RBC), with a value for the functional affinity constant, K, of 1-3 X 10(8) M-1, based on measurement of concentrations of bound and free reactants at equilibrium. Values of K obtained for other C1q-binders were as follows: diaminopropane, 2 X 10(2) M-1; monomer IgG, 5 X 10(4) M-1; heat-aggregated IgG, 0-5-2-5 X 10(8) M-1; IgG-anti-IgG complexes, 0-31 X 10(8) M-1. The functional rate constant for association (ka) between 125I-labelled C1q and glut-RBC was 5 X 10(5) M-1 S-1 at 37 degrees in 0-17 M NaC1. The rate of dissociation of the C1q-glut-RBC complex was biphasic with rate constants (kd) of 2 X 10(2) S-1 and 2 X 10(5) S-1. Calculated values of K from the ration ka/kd gave values of 2-5 X 10(7) M-1 and 2-5 X 10(10) M-1. It is suggested that the range of values of K reflects the involvement of 1,2 or more binding sites on the C1q molecule. Reduction of the ionic strength of the medium from 0-17 M to 0-14 M increases the rate of association of C1q and glut-RBC eleven-fold, indicating involvement of ionized groups at the binding site. A method is described for measuring plasma C1q concentrations by saturation assay, using 125I-labelled C1q and glut-RBC. Plasma C1q concentrations fell in the range 170-250 microng/ml.     

Binding of native alpha 2-macroglobulin to human group G streptococci.

Binding of human alpha 2-macroglobulin (alpha 2M) to group G streptococci and to their immunoglobulin G (IgG)-binding proteins (protein G) was investigated. Native alpha 2M bound specifically to strain G-148 with an apparent dissociation constant of (2.2 +/- 1.5) x 10(-9) M. Proteinase-complexed alpha 2M did not compete for the binding sites, and 125I-labelled proteinase-complexed alpha 2M did not bind to the bacteria. Binding of native alpha 2M to the cells was not affected by IgG or protein G consisting of only IgG-binding domains. 125I-labelled recombinant protein G did not bind to native or proteinase-complexed alpha 2M. However, a lysate of G-148 cells inhibited binding of alpha 2M to the bacteria, and immobilized wild-type protein G bound alpha 2M directly from fresh human plasma. In 13 group G streptococcal isolates, IgG-binding proteins were immunologically identified as protein G. In 11 isolates, these molecules reacted also with alpha 2M and human serum albumin (HSA). Western blots (immunoblots) of two wild-type protein G variants revealed identical bands reactive with goat IgG, HSA, and native alpha 2M. Digestion of wild-type protein G with clostripain destroyed in both variants the binding sites for alpha 2M but not for albumin and IgG. N-terminal fragments of protein G (lacking the IgG-binding region) bound both alpha 2M and HSA, whereas a similar HSA-binding peptide lacking the first 80 amino acids did not react with alpha 2M. Our findings are consistent with a specific binding site for native alpha 2M in the N-terminal region of protein G and suggest that binding of alpha 2M via IgG-binding proteins may be a general feature of human group G streptococci.     

The relation of clinical disease to antibody titre, proliferative response and neurophysiology in murine experimental autoimmune myasthenia gravis

The influence of the lattice of immune complexes on the C1q solid phase assay was examined using covalently cross-linked 125I-labelled immune complexes, separated into pools of varying and stable lattice. The C1q binding of antibodies alone, of Ag1Ab1, and of Ag2Ab2 could not be distinguished from each other statistically; but with increasing, higher lattices, immune complexes bound more efficiently to C1q. The binding of these immune complexes to C1q was also measured with 131I-labelled antibodies to IgG in the immune complexes. The detection of bound immune complexes by this indirect method showed the same order of binding efficiency as that observed by the direct measurement of immune complex binding. Up to a critical level, the binding of 131I-antibodies to IgG was proportional to the 125I-IgG in the bound complexes, and was independent of the lattice of complexes. This proportionality, however, was lost at higher levels of binding. The presence of serum diminished the binding of both large latticed and small latticed immune complexes, but serum did not alter the order of binding efficiency and the order of detection of binding using 131I-antibodies to IgG. The conclusion was reached that no single ideal standard for this assay can be currently designed to permit accurate quantitation of the concentration of immune complexes of varying lattice.     

Surface receptors for human serum albumin in Peptococcus magnus strains

Eighty-one bacterial strains representing 16 anaerobic species were tested in a sensitive binding assay for uptake of 125I-labelled human serum proteins. Fifteen of 36 Peptococcus magnus strains (42%) bound significant amounts of human serum albumin (HSA). None of the other bacterial species showed any affinity for HSA. All strains studied were incapable of uptake of human fibrinogen, fibronectin, haptoglobin or aggregated beta 2-microglobulin. P. magnus strain Ra 4 was tested for binding of purified serum albumin from 11 animal species, and showed a binding profile similar to human group-C and -G streptococci, but different from Streptococcus pyogenes, Strep. zooepidemicus and Strep. dysgalactiae. Kinetic experiments showed that albumin binding was a rapid displaceable, time-dependent process, that could take place over a wide range of pH or salt concentrations. The albumin-binding component of P. magnus strain Ra 4 was resistant to heat and to periodate treatment, but sensitive to proteolytic enzymes.     

Quantification of antibodies to the C3d subcomponent of human C3.

Purified soluble C3d has been employed to measure the concentration of anti-C3d antibodies in immune rabbit sera. Multiple batches of C3d, prepared from C3-C3b substrate by treatment with C3b-inactivator (KAF), after labelling with 125I, retained 80% immunoreactivity, and were stable on storage at -50 degrees and +4 degrees. Concentrations of anti-C3d were determined by Scatchard analysis of equilibrium concentrations of bound and free C3d in a mixture of 125I-labelled C3d and anti-C3d. Separation of bound from free C3d was by G-75 Sephadex filtration. Assuming a 1:1 molar ratio in the antibody-C3d complex, anti-C3d antibody concentrations for four rabbit whole antisera and four IgG preparations fell in the range 288-2433 microgram/ml, with Ko values of 6-2 X 10(8)-2-9 X 10(9) litres/mol. One commercial antiglobulin-serum contained 3-6 microgram anti C3d/ml and had a Ko value of 1-7 X 10(8) litres/mol. Values for anti-C3d concentrations measured independently by an indirect method employing 125I-labelled sheep anti-rabbit IgG averaged 20% lower than those obtained with 125I-labelled C3d. Antibody concentrations were correlated with antiglobulin agglutination titres against C3d-coated red cells; a titre of 1 was given by an anti-C3d concentration of 0-5 microgram/ml.     

Studies in vivo of cobra factor and murine C3.

The effect of the isolated C3-cleaving factor (CoF) of cobra venom on murine C3 in vivo and in vitro was studied. Optimal quantities of 100-200 units (0.5 minus 1.0 mg) of CoF per kg administered to mice by intraperitoneal injection in divided doses caused plasma C3 levels to fall to less than 5 per cent of normal from 1 to at least 4 days afterwards. Passive anti-CoF serum promptly abrogated the in vivo plasma C3 depletion, and under optimal conditions C3 levels reached 50 per cent of normal after approximately 15 hours. Injection of as little as 20 mug per mouse of CoF in saline induced a precipitating anti-CoF antibody response which prevented subsequent depletion of plasma C3 by CoF. The in vivo half-life of 125I-labelled CoF in normal mice estimated by whole body elimination and clearance from the blood was 24 hours. The presence in vivo of antibodies to CoF caused rapid clearance from the blood and elimination of 125I-labelled CoF, and also localization of some CoF in the spleen, liver and kidneys.     

Serum and Plasma Fibronectin Binds to Complement Reacted Immune Complexes Primarily via C1q

The binding of fibronectin to human Clq, C3b, and complement-reacted immune complexes (IC) was investigated by enzyme-linked immunosorbent assays. Microplates were coated with BSA followed by incubation with rabbit-anti-BSA IgG or F(ab')2 fragments of rabbit anti-BSA. Incubation of the solid phase with serum at 37 degrees C caused attachment of Clq and C3b. Addition of EDTA to the serum inhibited the binding of C3b, but not Clq, whereas substitution of the anti-BSA IgG on the solid phase with the F(ab')2 fragments abrogated the Clq, but not the C3b binding. Fibronectin binding was observed after incubation of the solid-phase IC with serum or plasma at conditions where Clq was also bound, whereas only minor amounts of fibronectin bound to the solid phase IC via C3b. Purified fibronectin showed a dose-dependent binding to solid-phase IC pretreated with Clq or fibronectin-depleted serum, confirming that the binding of fibronectin to IC largely occurred via Clq. Significantly smaller amounts of fibronectin were bound to solid-phase IC incubated with plasma instead of serum, despite the higher fibronectin concentration in plasma. This difference was found not to be due to a fibrinogen-fibronectin interaction in plasma. Binding of fibronectin to preformed fluid phase IC incubated with serum was demonstrated by SDS-PAGE analysis of PEG-precipitated IC.     

Increased non-specific binding of heat-treated proteins to plastic surfaces analyzed by ELISA and HPLC-fractionation

Heat-treatment of proteins from human sera and bovine serum albumin caused an increase in their binding to microplates in a temperature-dependent manner. Quantitative data were obtained for 125I-labelled human IgG, with binding of up to 400 ng of 56 degrees-treated IgG per untreated well. However, only a minor increase in available antibody activity of adsorbed rabbit antibody was found upon pretreatment at 56 degrees. The increased binding to microplates as a result of pre-incubation at raised temperatures was by high pressure liquid gel permeation chromatography demonstrated to be accompanied by polymerization. Inhibition of the direct binding of the proteins to plastic surface was achieved most efficiently by coating with non-interfering proteins followed by non-ionic detergent (Tween 20), which should also be present in the wash buffer.     

Selective acquisition of plasma proteins by Trichomonas vaginalis and human lipoproteins as a growth requirement for this species

Trichomonas vaginalis avidly bound numerous host macromolecules which were not removed by repeated washing in phosphate buffered saline. The use of radioiodinated Cohn plasma fractions in binding studies allowed the identification of plasminogen, fibrinogen, immunoglobulin G, lipoproteins A and B, transferrin, α₁-antitrypsin, and albumin on intact organisms. The binding of immunoglobulin G, albumin, transferrin, and lipoproteins to intact, motile trichomonads was further demonstrated using ¹²⁵I-labeled plasma that was chromatographically depleted of these proteins. Kinetic studies indicated that ¹²⁵I-labeled lipoproteins bind to T. vaginalis in a receptor-ligand-like manner. The surface localization and uptake of bound lipoproteins was shown by treatment of intact organisms with pronase at various times after incubation with lipoproteins. Purified lipoproteins could be substituted for plasma or serum as a growth supplement in a complex medium of trypticase/yeast extract/maltose and supported growth and multiplication rates equal to those in the same medium with plasma.     

Modulation of receptors for the colony-stimulating factor, CSF-1, by bacterial lipopolysaccharide and CSF-1

The ability of the mononuclear phagocyte-specific colony-stimulating factor, CSF-1, to down-regulate its receptor on peritoneal exudate macrophages (PEM) was examined. Because of the essentially irreversible binding of CSF-1 to its receptor at 2 degrees C, unoccupied cell surface receptors could be measured by rapidly cooling PEM to 2 degrees C and determining the amount of 125I-CSF-1 bound at this temperature. On incubation with 125I-CSF-1 at 37 degrees C more receptors were lost than could be accounted for by 125I-CSF-1 binding. This receptor loss, apparently caused by CSF-1 itself, was shown to be due in large part to the presence of contaminating lipopolysaccharide (LPS), which at 10 ng/ml was by itself able to cause complete loss of the CSF-1 receptors. LPS also induced loss of the insulin receptor by PEM. LPS did not cause apparent CSF-1 receptor loss by binding to the receptor or by stimulating the release of CSF-1 or substances which compete for the binding of 125I-CSF-1 to the receptor. However, LPS did stimulate release of factors by LPS responsive (C3H/HeN) PEM which caused CSF-1 receptor loss by LPS non-responsive (C3H/HeJ) PEM. In the absence of LPS induced effects, incubation of 125I-CSF-1 with PEM at 37 degrees C resulted in down-regulation of the CSF-1 receptors. The number of CSF-1 receptor sites down-regulated corresponded to the number of CSF-1 molecules that were cell-associated plus the number that were intracellularly degraded and released.     

Specific binding of human plasma high density lipoprotein (cruzin) to Trypanosoma cruzi

Binding of high density lipoprotein (HDL) to Trypanosoma cruzi was examined because of its ability to specifically inhibit the parasite's neuraminidase. 125I-Labeled HDL bound to live and glutaraldehyde-fixed parasites equally well either at 37 degrees C or at 4 degrees C. Binding was saturable and inhibited by unlabeled HDL but not by unrelated plasma proteins. Specificity of the T. cruzi-HDL interaction was confirmed using fluorescein labeled HDL which bound to T. cruzi but not to T. rangeli, a species whose neuraminidase is not inhibited by HDL. Binding of HDL to T. cruzi paralleled the neuraminidase activity exhibited by the parasite's different stages and strains. In agreement with this finding, Steck and Wallach analysis of the binding data showed that the number of HDL binding sites was greater in infective trypomastigotes and on strains with high neuraminidase activity. However, the association constant of the binding did not change within the various developmental forms and strains of T. cruzi, suggesting that HDL bound to the same receptor, presumably having neuraminidase activity.     

Rapid down-regulation of substance P binding to guinea-pig pancreatic acinar cells during homologous desensitization.

Binding of 125I-labelled peptides, cytoplasmic Ca2+ concentration ([Ca2+]i) and amylase release were studied in guinea-pig pancreatic acinar cells during exposure to substance P (SP), and cholecystokinin octapeptide (CCK-8). Pre-incubation of cells at 22 degrees C with 0.03 nM to 1 microM SP for 10 min or at 37 degrees C for 5 min followed by acid or neutral washes reduced subsequent binding of 125I-Bolton-Hunter reagent-labelled SP (125I-BH-SP) in a biphasic manner by up to 95%. Incubation at 4 degrees C eliminated high-affinity binding of 125I-BH-SP and concentrations of SP above 1 nM were required for inhibition of subsequent tracer binding. Pre-incubation of cells at 37 degrees C with 1 nM to 1 microM CCK-8 for 10 min followed by neutral washes reduced subsequent binding of 125I-BH-CCK-8 by up to 65%. In cell suspensions, the [Ca2+]i response to SP was gradually reduced by pre-exposure to increasing agonist concentrations from 0.2 to 20 nM. Pre-incubation with high SP concentrations for 10 min caused profound reduction of subsequent amylase responses to SP, whereas secretion was little affected in corresponding experiments with CCK-8. Down-regulation of receptor binding is not important during short exposure to CCK-8, but it is a pronounced and rapid phenomenon during SP exposure, which explains tachyphylaxis of [Ca2+]i and amylase responses.     

L-ficolin binding and lectin pathway activation by acetylated low-density lipoprotein

L-ficolin, like mannan-binding lectin (MBL), is a lectin pathway activator present in normal human plasma. Upon binding ligand, l-ficolin similarly initiates C4 cleavage via the serine protease MBL-associated serine protease-2 (MASP-2). We sought further insight into l-ficolin binding reactions and MASP-2 activation by passing plasma through GlcNAc-derivatized Sepharose. l-Ficolin bound in 1.0 M NaCl-ethylenediamine tetraacetic acid (EDTA), and remained bound in NaCl-free EDTA, while MASP-2 eluted in proenzyme form ( approximately 20% yield, > 40 000-fold purification). L-Ficolin was eluted with GlcNAc in 1.0 M NaCl ( approximately 10% yield, > 3000-fold purification), with trace amounts of C3, alpha(2)-macroglobulin and both native and activated MASP-2. These preparations were utilized to investigate l-ficolin reactivities with acetylated low-density lipoprotein (A-LDL) as a model ligand in albumin-free systems. L-Ficolin bound strongly to A-LDL in the absence as well as presence of calcium, including saline-EDTA, and was optimal in 1.0 M NaCl-EDTA, but binding failed to occur in EDTA in the absence of NaCl. The addition of l-ficolin to immobilized A-LDL resulted in activation of MASP-2 in unmodified but not ficolin-depleted plasma unless l-ficolin was restored. We conclude that A-LDL is a useful ligand for investigation of l-ficolin function; both binding and activation are optimally examined in systems free of albumin; and ligand binding in 1.0 M NaCl in EDTA can be useful in the isolation of l-ficolin and native MASP-2.     

Defective Immune Complex Degradation by Monocytes in Patients with Systemic Lupus Erythematosus.

The binding of 125I-labelled anti-bovine serum albumin (BSA)-BSA immune complexes (IC), giving a final molar antibody to antigen ratio of 1:1, to monocytes isolated from 18 patients with systemic lupus erythematosus (SLE) and from 10 normal healthy donors was quantitatively investigated. The degradation of the bound IC by the same monocytes was kinetically determined at the same time. The assays were performed on monocyte monolayers. Scatchard plots at 4 degrees C demonstrated that monocytes from patients with active SLE expressed a mean Fc gamma receptor (FcR) number that was 22% higher than that of the controls, although this did not reach statistical significance. The FcR number of normal monocytes and the degradation rate of anti-BSA-BSA complexes by the same cells showed a positive correlation. At the same time, the digestion of anti-BSA-BSA complexes by monocytes of SLE patients with active disease was prolonged, despite their enhanced FcR-ligand binding. The dissociation of FcR-ligand binding and FcR-mediated degradation suggests that the IC degradation is controlled by altered biochemical mechanisms in the monocytes of SLE patients.     

Detection of immune complexes in mice infected with Mycobacterium lepraemurium.

A specific binding test was used to detect immune complexes containing antigens of Mycobacterium lepraemurium in the serum and tissues of infected mice. Complexes were precipitated by antiserum against immunoglogulin, free antigen removed by washing and the presence of bound antigen demonstrated by measurement of uptake of radioactively labelled specific antibody by the precipitate. Tests were done both with 125I-labelled FAB prepared from an immune from rabbit antiserum against M. Lepraemurium and with 125I-labelled IgG precipitate. Out of seventy-nine serum samples taken monthly up to the 5th month after infection, only there were positive (one at 2 months and two at 3 months). Kidneys taken from infected mice were also examined for immune complexes. Although deposits of IgM and sometimes of IgG were observed by immunoflourescence in glomeruli of normal mice, deposits of IgG were more frequent later on in infected mice. Nevertheless, binding tests done on acid eluates were positive in only one out of fifty-three infected mice.     

Characterization of the IgA receptor from human polymorphonuclear leucocytes.

Human polymorphonuclear leucocytes (PMNs) will phagocytose yeasts opsonized with specific affinity-purified human serum IgA. PMNs also bind to Sepharose beads coated with IgA or IgG, but not to beads coated with bovine serum albumin (BSA) or horseradish peroxidase (HRP). Binding to IgA-Sepharose stimulates the cells to release lysozyme. Affinity chromatography of 125I-labelled PMN membrane proteins on IgA-Sepharose results in isolation of a polypeptide of apparent 60,000 MW. The protein, which is not bound to IgG-Sepharose under the same conditions, appears as a diffuse band on SDS-PAGE, suggesting it is heavily glycosylated.