Antibody production in mice: II. The mechanism of antigenic stimulation in the secondary immune response

To study the mechanism of antigenic stimulation in the secondary immune response, primed lymphoid cells were stimulated with antigen in various ways in vitro. The effect of antigenic stimulation was assessed by the antibody titres obtained after in vivo culture of primed cells in X-irradiated recipients.

Primed cells were much more effectively stimulated by antigen—antibody (Ag—Ab) complex than by free antigen. Primed cells could also be stimulated by spleen or lymph node cells from normal mice which had been exposed to free antigen or Ag—Ab complex in vitro or in vivo and thoroughly washed. Under these conditions, Ag—Ab complex was again much more effective than free antigen. When the cells were incubated with Ag—Ab complex, the dose of antigen bound to the cells was somewhat increased. But this increased binding of antigen could not solely account for the increase in immunogenicity.

It is suggested that the ingestion of antigen by macrophages is facilitated by the presence of antibody and that the macrophages mediate the effective immune stimulus to memory cells.

The effect of antibody in increasing the immunogenicity of antigen was lost completely when antibody was digested with pepsin. Thus, the Fc portion of antibody seemed to be important for this effect. However, it was demonstrated that antibody does not operate by becoming attached to macrophages as cytophilic antibody, and that complement is not involved in this process. The augmenting mechanism of antibody on the antigenic stimulation mediated by macrophages was discussed.

     

Antigen—antibody complexes in the immune response: I. A analysis of the effectiveness of complexes on the primary antibody response

Soluble crystalline bacterial α-amylase (BαA)-mouse anti-BαA antibody complexes (Ag—Ab complexes) elicited a primary antibody response in mice with a single intravenous injection, while free BαA could not. The response was dose dependent. Ag—Ab complexes were not only phagocytosed but also degraded more rapidly than free BαA in vivo and in vitro but these characteristics themselves were not important for immunogencity of the complexes.

The Ag—Ab complexes phagocytosed by cells in normal spleen and lymph node elicited a primary antibody response when injected into non-irradiated mice but the response was suppressed when anti-BαA antibody was simultaneously injected. On the other hand, free BαA phagocytosed by cells could not elicit the response.

The degraded products of complexes phagocytosed by normal spleen and lymph node cells were highly immunogenic and probably retain antigenic fragments. They elicited an even higher primary antibody response than the original complexes and were also more effective in eliciting a secondary response from primed cells than the original complexes or free BαA. The degraded products of free BαA, however, were ineffective not only for the primary response but also for primed cells.

Ag—Ab complexes prepared with heterologous rabbit antibody were ineffective for the primary antibody response.

     

Kinetics of interaction of immune complexes with complement receptors on human blood cells: modification of complexes during interaction with red cells.

Antigen-antibody complexes, composed of 125I-BSA and guinea-pig or rabbit antibody, were incubated at 37 degrees C with human blood cells suspended in autologous serum and kinetics of binding analysed. When purified polymorphonuclear (PMN) or mononuclear cells (MNC) were studied, maximum binding was observed within 8 min, and immune complexes (IC) remained associated with cells even after 1 hr. When cells were studied unseparated (in the same amount of serum), maximum binding was observed slightly earlier (within 4 min), but within 15 min most of the IC were found in the serum. Separation of cell types at the time of maximal binding and studies with cell preparations depleted of different elements revealed that binding was principally to red blood cells (RBC). IC recovered in the serum 16 min after addition to unseparated cells bound very slowly to purified PMN or MNC; binding after 30 min was 10-15% of that observed with fresh IC at 8 min. Ultracentrifugal analysis revealed that reduction in binding efficiency correlated with decrease in the size of IC. RBC isolated after binding and release of IC bound newly-formed IC was identical rapidity and capacity as fresh RBC, indicating that receptors were not altered by IC. Kinetics studies with serum in the absence of cells suggested that interaction with RBC accelerated the rate of change in binding properties of IC. Rates of binding and release were independent of antigen/antibody ratio but were slowed and binding to RBC sustained when diluted or hypocomplementaemic (SLE) serum was substituted for neat serum. Our results suggest that competition for IC by RBC is associated with loss of ability of IC to bind to other blood cell types and reduction in size of IC, and that abnormalities of complement can lead to prolonged association of IC with RBC.     

Complement fixation by small, DNase-resistant DNA-anti-DNA immune complexes

125I-ds DNA-anti-DNA immune complexes (IC) formed at antibody excess and containing DNA of 300-350 base pairs (bp) fixed complement, incorporated C3b and bound to the C3b receptors (CR1) on human red blood cells (RBC). When the IC were treated with DNase to generate small, DNase-resistant IC, some of the IC incorporated C3b, but did not bind to RBC. In order to examine C3b incorporation and RBC binding by IC of specific sizes, the DNase treated IC were fractionated by sucrose density gradient (SDG) ultracentrifugation. Small IC containing one, two, three or four IgG molecules per fragment of 125I-ds DNA were identified by autoradiography after electrophoresis of the SDG fractions on 3-12% linear polyacrylamide gradient gels. The SDG fractions were tested for C3b incorporation and RBC binding ability. There was neither C3b incorporation nor RBC binding activity in fractions which corresponded to 9-11S (containing IC with one IgG/DNA). Fractions which corresponded to 12-22S (containing IC with up to four IgG/DNA fragment) demonstrated increased C3b incorporation with increased size, but did not show significant RBC binding activity. Fractions with IC containing four or more IgGs (22-24S) incorporated C3b and bound to RBC at approximately the same level. It is concluded that DNase digested IC which contain three-four IgG/DNA fragment are large enough to activate complement and incorporate C3b, but are too small to bind to RBC CR1. These IC could therefore escape rapid clearance from the circulation via the erythrocyte CR1 clearance mechanism. Such IC could persist in the circulation and potentially elicit pathogenic effects in patients with systemic lupus erythematosus.     

Release of Immune Complexes Bound to CR1 on Erythrocytes; Suramin Inhibits Factor I in the Presence of EDTA

An experimental model was established in order to study the release of immune complexes (IC) bound by complement C3b receptors (CR1) on human erythrocytes (RBC). Soluble tetanus toxoid anti-tetanus toxoid complexes were incubated with RBC in the presence of autologous serum at optimal conditions for binding. The RBC carrying complement-opsonized complexes were incubated with appropriate serum reagents, and it was shown that factor I was required for release of the complexes, which occurred without loss of CR1. Suramin was, irrespective of factor I, found to induce release of CR1-bound IC in the absence of EDTA, whereas factor I-mediated release was inhibited by suramin in the presence of EDTA. EDTA probably interfered through a charge-dependent interaction. These observations are decisive for the interpretation of in vitro experiments involving these reagents. The combination of EDTA and suramin was found inappropriate for use in quantitative determination of in vivo CR1-bound IC.     

Immune complex alterations occur on the human red blood cell membrane.

Experimental antigen-antibody complexes (Ag-Ab) were incubated at 37 degrees with human red blood cells (RBC) suspended in autologous normal serum and the reaction stopped after progressively increasing times. Bound antigen-antibody-complement complexes (Ag-Ab-C) were eluted from C3b receptors and the eluted Ag-Ab-C re-incubated with different blood cell types suspended in serum, or centrifuged (along with unbound Ag-Ab-C found in the serum) through 20-50% sucrose gradients. Ag-Ab-C recovered from C3b receptors shortly after initial binding to RBC bound efficiently to other RBC, polymorphonuclear and mononuclear cells, and sedimented rapidly. Ag-Ab-C simultaneously present in the serum sedimented with a similar velocity. Ag-Ab-C recovered at a subsequent time during RBC interaction bound less well to each blood cell type, and sedimented less rapidly. Decreased amounts of rapidly sedimenting Ag-Ab-C were present in the serum. Ag-Ab-C recovered from C3b receptors at a still later time in the course of RBC interaction bound poorly to each cell type, and sedimented slowly. Increased amounts of slowly sedimenting Ag-Ab-C were found in the serum. These findings indicate that alterations in properties of immune complexes can occur while they are associated with C3b receptors on RBC membrane in solid phase.     

Interaction of Complement-Solubilized Immune Complexes with CR1 Receptors on Human Erythrocytes. The Binding Reaction

The binding of complement (C)-solubilized ¹²⁵I bovine serum albumin (BSA) anti-BSA immune complexes (IC) to CR1 receptors on human red blond cells (RBC-CR1) was studied. The binding of IC to CRI was strongly dependent on the molar antigen lo antibody ratio, and IC formed in moderate antigen excess showed no binding. IC solubilized, in 50% human serum in the presence of autologous RBC bound rapidly lo RBC-CRI, with maximal binding within less than 1 min at 37°C. Release of CRI-hound IC under these conditions occurred slowly, requiring more than.30 min. Only binding of 'partially' solubilized, e.g., anti C3c (C4c) and conglutinin-reactive 1C occurred, whereas fully solubilized complexes (IC-C3dg. C4d) showed virtually no binding. Solubilization of IC in the presence of Mg-EGTA or in C2-deficient serum resulted in a markedly delayed binding of IC ti RBC, indicating the importance of an intact classical pathway in preparing the IC for binding to RBC-CR1. C-solobilized IC could be absorbed to solid-phase conglutinin or antibody to C3c abd C4c, and tgese kugabds were able to inhibit the binding of solubilized IC to RBC. Heparin also exerted a marked, dose-dependent inhibitory effect on the binding of presolubilized IC to RBC-CR1, whereas the binding was unaffected by the addition of monosaccharides or by the concentration of Ca²⁻ or Mg²⁻ ions.     

The covalent interaction of C3 with IgG immune complexes

Antigens (Ags) are converted into immune complexes (antigen-antibody complexes, IC) as soon as they encounter their specific antibodies (Abs). In fluids containing complement, the process of IC formation and fixation of complement components occur simultaneously. Hence, the formation of Ag-Ab-complement complexes is the normal way of eliminating Ags from a host. C3b-C3b-IgG covalent complexes are immediately formed on interaction of serum C3 with IgG-IC. These C3b-C3b dimers constitute the core for the assembly of C3/C5-convertase on the IC, which are subsequently converted into iC3b-iC3b-IgG by the complement regulators. These complexes are detected on SDS-PAGE by two bands of molecular composition, C3alpha65-C3alpha43 (band A) and C3alpha65-heavy chain of the Ab (band B), which correspond to C3b-C3b and C3b-IgG covalent interaction respectively, and that identify opsonized IC (C3b-IC). C3b can attach to Fab and Fc regions of the Ab molecule with similar efficiency. The presence of multiple C3b binding regions on IgG is considered an advantageous characteristic that facilitates the elimination of Ags in the form of C3b(n)-IC. Ab molecules on the IC recognize the Ag, and also serve as a very good acceptor for C3b binding. In this way, Ags, even if they have no acceptor sites for C3b, can be efficiently processed and removed. When C3 is activated in serum by IC or other activators, secondary C3b-IgG covalent complexes are generated, with bystander monomeric circulating IgG, and thus constitute, physiological products of complement activation. These complexes gain importance when IgG concentration is extremely high as in cases of infusion of intravenous IgG (IVIG) in several pathologies. The covalent attachment of activated complement C3 (C3b, iC3b, C3 d,g) to Ags or IC links innate and adaptative immunity by targeting Ags to different cells of the immune system (follicular dendritic cells, phagocytes, B cells). Hence C3b marks Ags definitively, from the earliest contact with the innate immune system until their complete elimination from the host.     

Non-complement-dependent adsorption of soluble immune complexes to human red cells

Heat aggregated IgG and soluble immune complexes (IC) prepared by combining human serum albumin with rabbit anti-serum albumin and tetanus toxoid with rabbit antiserum to tetanus toxoid were shown to bind to human O+ RBC. The binding was greater for soluble IC prepared at antigen excess, and although it was usually maximal when IC were pre-opsonized, it could also be demonstrated using non-opsonized heat aggregated IgG or soluble IC prepared in the absence of complement. These observations suggest that two types of receptors may be involved in binding of soluble IC to human RBC: the classical C3b receptor, and a non-complement-dependent receptor, perhaps recognizing the Fc region of the immunoglobulin molecule exposed after heat aggregation or antigen-antibody reaction.     

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.     

Lymphocyte cytotoxicity induced by preincubation with serum from patients with Hashimoto thyroiditis

Lymphocytes from healthy donors were incubated with serum samples from nine patients with Hashimoto thyroiditis and subsequently shown to be cytotoxic to chicken red blood cells (Ch. RBC) coated with thyroglobulin. Target cell death was estimated using a standard ⁵¹Cr release assay system.

Lymphocytes pre-incubated with Hashimoto serum caused a mean% ⁵¹Cr release of 13·11±2·83 (SEM) from thyroglobulin-coated Ch. RBC and a mean% ⁵¹Cr release of 1·22±0·65 from uncoated Ch. RBC.

Untreated lymphocytes caused no significant isotope release from either uncoated or thyroglobulin coated target cells.

     

Enhanced binding of immune complexes processed by erythrocyte CR1 (CD 35) receptors to purified CR2 (CD 21) receptors from tonsillar mononuclear cells

The binding of immune complexes (IC) opsonized by serum complement (C) and IC processed by CR1 (CD 35) receptors on human erythrocytes (E) to purified CR2 (CD 21) receptors was compared. Soluble CR2 was prepared from tonsillar mononuclear cells and purified by antibody affinity chromatography. Solid phase CR2 as well as CR2 subjected to PAGE and blotted onto nitro-cellulose membranes bound 125I-labelled BSA anti-BSA IC which had been opsonized by C and processed by CR1 up to ten times more efficiently than IC reacted with serum only. Radiolabelled monomeric C3d also bound to solid phase CR2. The binding of IC to purified and solid phase bound CR2 could be inhibited by anti-CR2 antibodies or by preincubation of the IC with polyclonal antibodies reacting with C3d or C3b/iC3b. Thus, both C3dg and iC3b appeared to mediate binding of IC to CR2. Preincubation of solid phase CR2 with purified monomeric C3d did not inhibit the subsequent binding of E-CR1 processed IC. The data indicate that E-CR1 have an important role in generating IC which bind effectively to CR2 receptors on B lymphocytes.     

Alternative pathway-mediated rebinding of immune complexes to human red blood cells.

Antigen-antibody complexes (Ag-Ab) prepared from 125I-bovine serum albumin (BSA) and guinea-pig anti-BSA were (i) incubated at 37 degrees for 4 min with undiluted normal human serum and autologous red blood cells (RBC) together, or (ii) incubated first at 37 degrees for 30 min with serum diluted optimally for binding and then with RBC. Reactions were stopped by dilution and cooling, and RBC bearing antigen-antibody-complement complexes (Ag-Ab-C) were washed and resuspended in either untreated normal human serum (undiluted or diluted), serum treated with zymosan (SZYM), ethylenediamine tetracetic acid (SEDTA) or ethyleneglycol tetracetic acid-Mg++ (SEGTA), serum heated at 56 degrees for 30 or 120 min (S delta 30 or S delta 120), or buffer alone. The mixtures were placed at 37 degrees and the percentage of Ag-Ab-C dissociated from RBC after progressively increasing times determined. (i) Ag:Ab:C bound to RBC with undiluted serum dissociated more rapidly following resuspension in SZYM, SEDTA, or S delta 30 than following resuspension in untreated serum. Rate of dissociation in SEGTA paralleled that in untreated serum. (ii) Ag-Ab-C bound to RBC with diluted serum dissociated following resuspension in SZYM, SEDTA, or S delta 30, but rebound and dissociated a second time following resuspension in untreated serum or SEGTA. Initial dissociation occurred in less than 1 min in undiluted serum, took place at 0 degrees as well as 37 degrees, and was diminished but detectable in greater than 8- and greater than 64-fold-diluted serum, respectively. Rebinding required 37 degrees, factors B and D and C3, and was maximal at 4-8 min. Subsequent dissociation had similar complement requirements to initial dissociation, but occurred only at 37 degrees and was 90% complete at 15 min. No dissociation of Ag-Ab-C bound to RBC in (i) or in (ii) occurred following resuspension in S delta 120 or in buffer. These findings suggest that after initial binding, release of experimental immune complexes from RBC in whole serum involves concurrent dissociation and alternative pathway-dependent rebinding.     

The binding of immune complexes to human red cells: complement requirements and fate of the RBC-bound IC after interaction with human phagocytic cells.

Soluble immune complexes (IC) are known to bind to human red blood cells (HRBC). Most authors have attributed this binding to the interaction between IC-bound C3b and a red cell CR1 receptor, but contradictory data has been published concerning the ability of IC to bind to HRBC in the absence of complement. Using soluble tetanus toxoid-rabbit anti-tetanus toxoid (TT-ATT) IC, we have shown that binding through the CR1 receptor takes place when IC are formed at antibody excess, while IC formed at antigen excess do not require complement for erythrocyte binding. Once absorbed to HRBC, IC are recognized by CR1 and/or Fc receptors on phagocytic cells. This interaction is not associated with red cell engulfment, but using radiolabelled S. aureus protein A as a probe, we have demonstrated the transfer of IC from HRBC to phagocytic cells. Such transfer without red blood cell (RBC) damage agrees with the postulated role of RBC in the elimination of soluble IC from circulation. However, we have also demonstrated that the interaction between HRBC-IC and phagocytic cells is associated with the release of mediators of inflammation. It is, therefore, not absolutely clear whether the interaction of RBC-adsorbed IC and phagocytic cells will always have beneficial consequences.     

The elution of 35S-labelled C′ from antigen—antibody-35S complement precipitates

C′₁ haemolytic activity and radioactivity were detected in EDTA eluates of immune precipitates formed in the presence of either whole guinea-pig serum or euglobulin fraction labelled with ³⁵S in vivo. Eluted C′₁ and ³⁵S were fixed by sensitized sheep cells which subsequently lysed on exposure to guinea-pig C′ reagents lacking C′₁. The dissociated material contributed only a minute fraction of the protein N taken up by Ag—Ab precipitates from fresh C′.     

Biological studies with an iodine-containing synthetic immunological determinant 4-hydroxy-3-iodo-5-nitrophenylacetic acid (NIP) and related compounds

Immunization with 4-hydroxy-3-iodo-5-nitrophenylacetic acid (NIP)—protein conjugates, and assay of antisera by binding of N¹³¹IP—ε-amino-n-caproic acid (N¹³¹IP—aminocap), is used to investigate the response of CBA mice. Primary immunization is obtained more efficiently with alum-precipitated conjugates mixed with pertussis, than with conjugates alone or in Freund's complete adjuvant, and is most efficient with conjugates of chicken serum globulin (NIP—CG) or edestin. The mice invariably respond to efficient immunization. Secondary responses have been obtained by stimulation in vitro or in vivo of spleen cells transferred from primed donors. Cells taken from donors primed with NIP—ovalbumin respond detectably to as little as ~0.001 μg NIP—ovalbumin/mouse, but are less sensitive to stimulation with NIP—bovine serum albumin (NIP—BSA). Secondary stimulation by NIP-conjugate is subject to competitive inhibition by NIP—aminocap. Prolonged administration of NIP—BSA or NIP—BGG renders mice partially unresponsive to immunization with NIP—CG, but the degree of immunological paralysis obtained is less than that obtained towards the carrier protein.     

Activation of complement by soluble IgG aggregates and their subsequent interaction with macrophages

The mononuclear phagocyte system (MPS) plays an important role in the removal of both particulate and soluble immunogenic material from the circulation. E.IgG adhere to mononuclear phagocytes if the Fc portion of the IgG can interact with the phagocytes' Fc receptors. Simultaneous sensitization with IgG and C3b enhances the effectiveness of binding and ingestion. If soluble material cannot adhere to the surface of macrophages, it will be endocytosed in vitro via fluid-phase pinocytosis at the concentration that is present in the medium. If the material adheres to the cell's surface via its chemical properties or via specific receptors, it will be selectively concentrated at the cell's surface and endocytosed by an adsorptive pinocytosis. Ingestion of IC via Fc gamma R and C3b depends on the ability of the antibodies to interact with Fc gamma R and their capacity to activate the complement system. IC-bound C3b enhances the adsorptive pinocytosis of IC. Soluble AIgG are also pinocytosed more efficiently when C3b is bound to AIgG. The degree of endocytosis varies with the level of C3b sensitization. The highly effective C3b-mediated pinocytosis can be abolished by treating the macrophages with trypsin to inactivate C3bR. This observation illustrates that C3-mediated pinocytosis can replace Fc-mediated pinocytosis in unstimulated macrophages. Soluble IC and AIgG are removed from the circulation mainly by hepatic Kupffer cells. It seems that the size, the Ag/Ab ratio, the capacity of the IC to bind C1, activate C1, and allow deposition of C3b together with the degree of phagocyte activation determine the degree of binding and subsequent degradation of soluble IC.(ABSTRACT TRUNCATED AT 250 WORDS)     

B cells and antibodies in MS

When the B-cell response was examined by enumeration of immunoglobulin (Ig)-secreting cells, normal cerebrospinal fluid (CSF)--in contrast to previous beliefs--contained IgG-secreting cells, indeed at an 8-fold higher proportion per 10(4) mononuclear cells (MNC) than blood. As expected, the proportion of IgG-producing cells was greatly increased in MS CSF. Evaluation of antibody (Ab) responses at the cellular level, thereby bypassing draw-backs inherent in determinations of circulating Ab levels, such as Ab binding to target, revealed that in one MS patient group, 57% had, in CSF, cells secreting IgG Ab against myelin basic protein (MBP) and, in another MS group, 55% had, in CSF, cells producing IgG Ab against myelin-associated glycoprotein (MAG); both MBP and MAG are possible targets for immune attack in MS. Anti-MBP and anti-MAG IgG antibody-secreting cells could occur in parallel or independently. They were rarely detected in blood, reflecting strong sequestration in CNS CSF. Their possible role in MS pathogenesis is envisaged in light of recently suggested coupling between polyclonal B-cell hyperresponsiveness and antigen-driven specific responses in autoimmune-prone individuals.     

A study of cells present in lymph draining from a contact allergic reaction in pigs sensitized to DNFB.

The binding of the third component of the complement system (C3) to Con-A-stimulated murine spleen cells (MSPC) and the role of C3 for the formation of cell aggregates was examined. C3 became bound up to 15% of the T blasts (Thy-1.2 positive cells) while only 2.5% of the non-activated T cells bound C3 when they were incubated with autologous serum. Application of purified human C3 or heat-inactivated autologous serum did not result in a detectable C3 binding. The highest percentage of C3-carrying T blasts (15% of Thy-1.2 positive cells) was observed concomitantly with maximum [³H]-thymidine incorporation when cells were harvested between 24 and 38 hours of culturing. Treatment of the Con-A-stimulated MSPC with phenylmethylsulphonylfluoride (PMSF) and soybean trypsin inhibitor (SBTI) induced a decrease of the percentage of C3-binding T blasts. However, at small concentrations the protease inhibitors (PI) effected increased C3 binding depending on the time of harvesting the cells. The PI when applied in concentrations causing an enhancement of the percentage of C3-positive T cells also increased the aggregation of Con-A-stimulated MSPC. In contrast Con-A-stimulated MSPC aggregated less when treated with autologous mouse serum containing anti-mouse C3. These observations suggest that the cell contact among Con-A-stimulated MSPC in the presence of serum is at least in part mediated by C3 bound to T-cell blasts. Based on these and previous findings that certain concentrations of DFP enhance rosette formation of blast-transformed B cells with EAC1423b, we hypothesize that PI are involved in regulation of B- and T-blast membrane metabolism, thereby influencing C3-deposition on T-cell blasts and C3-dependent cell—cell interaction.     

Isolation of Immune Complexes by an Equine CIQ-Like Factor

A method for the isolation of circulating immune complexes by co-precipitation with an equine Clq-like factor is described. The Clq-like factor co-precipitated 80% of the ¹²⁵I-labeled aggregated human IgG (AHG) following dialysis against 0.05 M Tris-HCl, pH 8.1 compared to only 2% of the unaggregated human IgG (HG). When incubated with bovine serum albumin (BSA) anti-BSA soluble immune complexes (IC) prepared at antigen (Ag)-antibody (Ab) equivalence or in Ab excess, the Clq-like factor co-precipitated 100Z of the available complexes. Under conditions of 2x Ag excess, Clq-like factor was less efficient as demonstrated by co-precipitation of 40% of the available IC. Although the nature and specificity of binding of the Clq-like factor is unknown, its isolation and partial characterization are reported.