C1 and human platelets. III. Role of C1 subcomponents in platelet aggregation induced by aggregated IgG.

Studies have been performed with platelets using C1 haemolytic assays and platelet aggregation induced by anti-C1q, anti-C1s and aggregated IgG in the presence of C1 subcomponents C1q, C1r and C1s. C1q was removed by EDTA or modified by collagenase from human platelets while after the same treatment C1s remained bound to the platelets. EDTA treated platelets were no longer aggregated by aggregated IgG. The addition of C1q restored the reactivity of the platelets to aggregated IgG while the addition of C1r or C1s was without effect. Furthermore, the addition of C1r or C1s to C1q inhibited the action of C1q in platelet aggregation induced by IgG.The possible association between the different C1 subcomponents and human platelets is discussed.     

Determination of circulating immune complexes by chicken anti-human C3 and anti-human C1q microELISA

The present study reports on an anti-human C1q and an anti-human C3 microELISA for measuring circulating immune complexes (CIC). Affinity-purified chicken anti-human C3 and anti-human C1q were used as capture antibodies and protein A-alkaline phosphatase conjugate for detection. Chicken antibodies do not activate mammalian complement, do not react with rheumatoid factor and are not bound to anti-mammalian IgG antibodies or protein A, which often are used for detection in ELISA. They are therefore suitable as capture antibodies in CIC assays. We have tested Bell's palsy patients and found an increase in both anti-C3- and anti-C1q-containing CIC in acute and convalescent sera compared with the normal.     

Anti-human C1q: Rapid and simple method for preparing monospecific antisera

A new simple, rapid and economical method is described for producing monospecific antisera to human C1q without using gel filtration or column chromatography. Moderately purified C1q is obtained by dialyzing fresh human serum in the presence of chelating agents at low ionic strength and then electrophoresing it in agarose. When injected into rabbits, the electrophoretically purified product induced potent antisera to three or four serum proteins including C1q, all with slow electrophoretic mobilities (γ to β) at pH 8.6. The antibodies to serum proteins other than C1q are easily removed by immunoadsorbents consisting of the insolubilized supernatants obtained from the dialysis used to make the original Clq-rich fraction.The monospecific antisera prepared by this technique form only one band of precipitate in the slow γ region in immunoelectrophoresis with whole human serum or C1q-rich solution, as well as in Ouchterlony double diffusion test. They agglutinate EAClq but not EA cells and detect the same antigen as standard monospecific antisera to human C1q obtained by another well-established method.     

Anti-C1q column: ligand specific purification of immune complexes from human serum or plasma. Analysis of the interaction between C1q and immune complexes

An efficient and reproducible procedure has been developed for the specific isolation of immune complexes. PEG precipitation of EDTA serum or plasma was an essential preliminary step to separate complex-bound from free C1q. PEG had no discernible effect on the molecular weight size of the extracted complexes. Redissolved complexes were incubated with a Sepharose-4B column coated with anti-human C1q antibodies and following removal of unbound material the bound complexes were sequentially eluted with 0.02 M EDTA, 0.5 M NaCl and 1 M propionic acid. Characteristics of the affinity column were established by the purification of 125I-labelled BSA-anti-BSA complexes and heat-aggregated IgG (HAGG) incubated in normal human serum (NHS). EDTA and NaCl eluted complexes were of similar molecular size and contained antigen, specific antibody, as well as human IgM, IgG, albumin, C3, C3c, C3d and C1q. Acid eluted complexes contained the highest yield of specific antigen and antibody and comprised in addition human C1q and C3d. Activation of complement components after C1q made the bond between C1q and immune complexes resistant to 0.5 M NaCl and interfered with the binding between solid phase anti-C1q and complex bound C1q. Using BSA-anti-BSA complexes and HAGG activated in NHS it was apparent that only a minority of the complexed material was isolated via the C1q ligand and this probably applies to the C1q binding assay. Most complexed material could be isolated using an anti-C3 affinity column.     

Immune deposition of C1q and anti-C1q antibodies in the kidney is dependent on the presence of glomerular IgG

Anti-C1q autoantibodies are found frequently in patients with Systemic Lupus Erythematosus (SLE) and several studies indicate that these autoantibodies are associated with renal involvement. We have shown earlier that administration of anti-C1q antibodies to normal BALB/c mice results in the deposition of these antibodies and C1q in the kidney. In the present study we have investigated which factors are essential for this C1q-anti-C1q deposition. Injection of anti-C1q antibodies in C57BL/6 mice results in deposition of both C1q and anti-C1q in glomeruli, while administration of equal concentrations of anti-C1q to immunoglobulin deficient Rag2-/- mice did not result in deposition of anti-C1q antibodies. Analysis of renal sections of naive Rag2-/- mice revealed absence of mouse IgG and C1q in the glomeruli, while circulating C1q was within normal levels. Reconstitution of Rag2-/- mice with IgG, either by injection with purified mouse IgG or by splenocyte transfer, resulted in restored localization of mouse IgG together with C1q in the kidney. Subsequent injection of anti-C1q antibodies in these IgG reconstituted mice resulted in clear deposition of C1q together with anti-C1q in the kidneys comparable to that found in C57BL/6 mice receiving anti-C1q. We propose that the continuous presence of serum derived non-immune IgG in the glomerulus serves as a target for low affinity interactions with C1q, which then can serve as antigen for anti-C1q antibodies. Therefore we hypothesize that high and fluctuating levels of IgG as observed in patients with SLE may contribute to flares of renal inflammation in those patients with anti-C1q autoantibodies.     

Anti-C1q autoantibodies from active lupus nephritis patients could inhibit the clearance of apoptotic cells and complement classical pathway activation mediated by C1q in vitro

Anti-C1q antibodies are prevalent in patients with active lupus nephritis and were found to be closely associated with renal involvement and predictive for a flare of nephritis. However, the pathogenesis of anti-C1q antibodies involved in human lupus nephritis remains unclear. C1q, which plays a key role in apoptotic cell and immune complex removal, is a very important functional molecule in the pathogenesis of SLE. The aim of this study was to investigate the influence of anti-C1q autoantibodies from active lupus nephritis patients on the bio-functions of C1q in vitro. We purified IgG autoantibodies against C1q from lupus nephritis patients, and found that they could recognize C1q bound on early apoptotic cells at 30 μg/ml, and could significantly decrease the phagocytosis by macrophages of early apoptotic cells opsonized by 50 μg/ml C1q in comparison with normal IgG. Levels of circulating immune complexes of the ten patients were measured by a circulating immune complexes (CIC)-C1q Enzyme Immunoassay Kit. Anti-C1q autoantibodies affinity purified by microtiter plates could significantly inhibit the deposition of C3c on CIC-C1q in a dose dependent manner in comparison with IgG from 10 healthy blood donors. The binding of opsonized immune complexes to RBCs was significantly inhibited by anti-C1q autoantibodies purified by microtiter plates in a dose dependent manner. Our observations suggest that serum anti-C1q autoantibodies from active lupus nephritis patients could interfere with some biological function of C1q in vitro.     

Isolation and characterization of mouse C1q

C1q was isolated from mouse serum and ascites fluid by absorption onto human IgG-coated latex beads followed by seperation on 3–10% exponential gradient sodium dodecyl sulfate (SDS) polyacrylamide gels. Mouse C1q was also purified by low ionic strength precipitation of mouse serum. The purified C1q was heat-labile (56°C, 30 min) both structurally and functionally, contained 4.3% hydroxyproline, 1.38% hydroxylsine, and 18.5% glycine, had an apparent molecular weight of 380,000 daltons, and reconstituted the hemolytic complement activity of C1q-depleted mouse serum. The negatively stained ultrastructural appearance of this purified material consisted of 6 globular units connected by strands. These data demonstrate that mouse C1q structurally and functionally is similar to human and rabbit C1q. A portion of polyacrylamide gel containing mouse C1q was injected into rabbits resulting in the production of monospecific antisera against mouse C1q. Thus, this procedure is a new, rapid and simple method to obtain monospecific antisera against mouse C1q.     

Anti-C1q autoantibodies specific against the globular domain of the C1qB-chain from patient with lupus nephritis inhibit C1q binding to IgG and CRP

Lupus nephritis is one of the most severe manifestations of systemic lupus erythematosus. Higher titers of serum anti-C1q autoantibodies correlate with disease activity in patients with lupus nephritis. Anti-C1q autoantibodies have been shown to bind neo-epitopes within the collagen region of human C1q. In a preliminary study, we recently reported that the anti-C1q autoantibodies could also recognize epitopes within the globular domain (gC1q) of the C1q molecule. Here, 38 sera from patients with renal biopsy-proven lupus nephritis were screened for the presence of anti-gC1q autoantibodies, using recombinant globular head regions of individual A (ghA), B (ghB) and C (ghC) chains of human C1q. We isolated anti-gC1q autoantibodies from three selected patients. Human C1q was pre-incubated with increasing concentrations of the isolated anti-ghA, anti-ghB or anti-ghC autoantibodies and its binding to different C1q target molecules such as IgG and CRP was then evaluated. Anti-ghB, but not anti-ghA and anti-ghC autoantibodies, markedly inhibited C1q interaction with IgG as well as CRP. These results appear to suggest that the anti-ghB autoantibodies may partially induce acquired functional C1q deficiency and thus may interfere with the biological function of C1q.     

HLA Alloantibodies and the Mechanism of the Antiglobulin-Augmented Lymphocytotoxicity Procedure

ABSTRACT: HLA Class I alloantigens express multiple epitopes which can be defined serologically using human HLA alloantibodies (aAb). We have shown that the vast majority of HLA antisera exhibit the CYNAP phenomenon (complement-dependent cytotoxicity (CDC) negative, adsorption positive) which can be identified by conversion to direct CDC positive reactivity with the addition of an antihuman immunoglobulin (Ig) light chain (AHG) reagent.

In this study, the immunochemical mechanisms responsible for the CYNAP phenomena and how AHG overrides CYNAP have been further characterized using affinity-purified HLA aAb, class-specific anti-IgH reagents and human C1q binding assays quantified by flow cytometry. We have found that CYNAP reactions are not the result of low affinity aAb or generally caused by non-complement fixing HLA aAb. Our experiments illustrate that only anti-human IgL AHG reagents can consistently augment CDC and override CYNAP; anti-IgH have not effective. Two noncompeting HLA aAb of different epitopic specificity or one aAb in conjunction with the AHG-augmenting reagent results in striking synergy with a 200 to 400% increase in binding of C1q.

We conclude from these and other experiments detailed in this article that an IgM aAb or either two adjacent, noncompeting IgG HLA aAb bound to spatially distinct epitopes on a single HLA molecule or a monospecific IgG HLA aAb in concert with the AHG binding to this HLA aAb, is required for efficient (bivalent) C1q binding and initiation of C-mediated lympholysis. In contrast, the CYNAP phenomenon usually occurs because monospecific HLA aAb directed against a single epitope cannot effect high affinity, bivalent interaction with C1q and activate complement that would ultimately lead to cytolysis.     

Xenoantiserum to human C3 receptors: its preparation and effect on the C3b and C3d receptors of tonsil cells and the C3b receptors of erythrocytes and neutrophils.

Antisera directed against complement (C3) receptors on human tonsil cells were prepared and tested for their capacity to block specifically C3 receptors on various types of human cells. The antisera were capable of blocking both membrane-bound and solubilized C3 receptors of human tonsil cells. The C3b receptors of human erythrocytes and granulocytes were also blocked by the anti-C3 receptor sera. Sheep erythrocyte rosette formation was not affected. IgG-EoxA rosette formation was only slightly reduced by the anti-C3 receptor sera. Immunofluorescent staining with anti-C3 receptor sera revealed only a faint or negative staining of T cells and a distinct staining of EAC-reactive tonsil cells, lymphocytic leukaemia cells, and granulocytes. Absorption of the antisera with human serum proteins, brain, thymus, liver, EU-1 cell line cells, or trypsinized tonsil cells did not influence the capacity of the anti-C3 receptor sera to inhibit C3 receptors, whereas absorption with splenic tissue or tonsil cells completely removed the blocking activity of the anti-C3 receptor sera. Absorption with human erythrocytes or kidney removed only the inhibitory effect of the antisera on C3b receptors of tonsil cells, human erythrocytes, and granulocytes, but not on C3d receptors of tonsil cells. The results indicate that (a) the antisera prepared with the described procedure contained significant amounts of antibody against C3 receptors, (b) the receptors for C3b and C3d differe in antigenicity, and (c) the C3b receptors of tonsil cells, human erythrocytes, granulocytes, and probably glomerular cells have common antigenic sites.     

Biosynthesis of the subcomponents C1q, C1r and C1s of the first component of complement (C1) by guinea pig hepatocyte primary cultures

Thus far, the synthesis of C1q by liver cells has not been demonstrated. To investigate this possibility, viable hepatocytes were isolated from the liver of guinea pigs and primary cultures were established. The cells (10(6) cells/ml) were cultured under serum-free conditions for 8 days and the culture medium was changed every 24 h. The few contaminating Kupffer cells were lysed by preincubating the cell cultures with a monoclonal (22C4-8) antibody directed against a nonpolymorphic Ia determinant and preabsorbed rabbit serum. The hemolytic activity of C1 and its subcomponents C1q and C1r/C1s was tested in the supernatants. Guinea pig hepatocyte primary cultures synthesize and secrete up to 3 X 10(3) effective C1q molecules/cell/24 h and 34 X 10(3) effective C1r/C1s molecules/cell/24 h. The synthesis of C1q and C1r/C1s could be reversibly inhibited by cycloheximide (50 micrograms/ml). Furthermore, to demonstrate de novo synthesis of the C1q subcomponent, endogeneous labeling with 3H-proline (or 14C-proline) was performed. The immunoprecipitated C1q from cellular lysates and culture medium was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Compared to biosynthetically labeled guinea pig C1q from peritoneal macrophages, three corresponding bands (30, 28 and 24 kDa, respectively) were detectable in the fluorograph. The data show that guinea pig hepatocytes are able to synthesize C1 subcomponents, whereby the synthesis of C1q and C1r/C1s occurs independently.     

Glomerular deposition of C1q and anti-C1q antibodies in mice following injection of antimouse C1q antibodies

Anti-C1q autoantibodies are present in the serum of patients with different autoimmune diseases such as systemic lupus erythematosus (SLE). The occurrence of these autoantibodies correlates with renal involvement. In the present study we examined whether injection of rabbit antimouse C1q antibodies in mice leads to deposition in kidneys. Injection of healthy mice with a single dose of rabbit IgG antimouse C1q antibodies resulted in deposition of both C1q and IgG anti-C1q in glomeruli. The pattern of deposition observed in the glomeruli of mice injected with antimouse C1q antibodies both at 24 h and 2 weeks was both glomerular basement membrane (GBM)-associated and mesangial. Injection of control IgG did not have a detectable effect on circulating C1q levels, and no deposition of either C1q or rabbit IgG was seen at 24 h. The deposition of rabbit antimouse C1q and C1q in glomeruli resulted in complement activation, as assessed by C3 deposition, and influx of leucocytes associated with albuminuria in some, but not all mice. In none of the control mice was albuminuria observed. This report is the first to show that anti-C1q antibodies deposit in the healthy glomerulus together with autologous C1q. This deposition is stable for at least 2 weeks, causes complement activation, leucocyte influx and can lead to mild albuminuria.     

Detection of C1q-bearing immune complexes by a monoclonal anti-C1q ELISA system

A monoclonal antibody directed against the collagenous portion of human C1q was used to detect C1q-bearing immune complexes in patients with rheumatic disorders. Sera of patients with rheumatoid arthritis, systemic lupus erythematosus (SLE), osteoarthritis, as well as normal human sera (NHS) used as controls were tested in an ELISA system. C1q-bearing immune complexes were bound to a solid-phase monoclonal anti-C1q antibody, and detected with F(ab')2 antibodies to human IgG. Heat-aggregated human IgG was adjusted to the same concentration as the WHO standard for immune complexes and used for the standard curve in NHS. The mean value in NHS was 19.5 micrograms/ml equivalents of aggregated IgG. Using 2 SD over the mean as the upper limit for normal values, samples greater than 43 micrograms/ml were considered positive. Patients with osteoarthritis were negative; high levels of C1q-bearing immune complexes were detected in patients with rheumatoid arthritis (up to 800 micrograms/ml equivalents of aggregated IgG). With our assay C1q-bearing immune complexes were detected with high frequency (81%) in the sera of patients with rheumatoid arthritis, while a C1q solid-phase binding assay (C1q SPBA) revealed positive results only in 67% of rheumatoid arthritis sera. Compared to NHS, CH50 titers and C1q values of sera from patients with rheumatoid arthritis were frequently high. In contrast, the sera of SLE patients with low CH50 titers and low C1q levels had IgG immune complexes which could be detected only in the C1q-SPBA. C1q-bearing immune complexes were not detectable in the sera of patients with SLE. Since C1q triggers activation of the classical C pathway, this assay with monoclonal anti-C1q antibody appears to be useful for detecting immune complexes in rheumatoid arthritis patients with normal or elevated CH50 and C1q values, especially in the early stage of the disease.     

Interaction of C1q with its receptor on cultured cell lines induces an anti-proliferative response

The receptor for human C1q (C1qR) is expressed on a wide variety of somatic cells, including cultured cell lines of different lineages such as Raji, Daudi, Wil2WT, U937, and Molt4. In this report, we present evidence which shows that culturing of C1qR-expressing cell lines with C1q inhibits their growth. When each of the different cell lines were cultured for 5 days with or without various concentrations (5-50 micrograms/ml) of micro-filtered (0.22 micron) C1q, cell proliferation was inhibited in a dose-dependent manner with maximal inhibition (90%) occurring at a concentration of 50 micrograms/ml at Day 4 of culture. This anti-proliferative effect of C1q was inhibited when 30 micrograms/ml of F(ab')2 anti-C1q was included in the culture with C1q while the antibody alone did not have any effect. The specificity of this interaction was further substantiated by the finding that neither macromolecular C1, or subcomponents C1r and C1s, nor human or murine IgG nor IgM had any inhibitory activity when cultured with these cell lines. That this C1q-induced inhibition of cell growth is mediated by C1qR was deduced from experiments in which (i) culturing of cells in the presence of two IgM monoclonal antibodies II1/D1 and II1/B5, directed against the C1q-binding site of C1qR resulted in the inhibition of cell growth while nonimmune murine IgM did not, and (ii) the collagenous portion of C1q (c-C1q) which contains the intact, C1qR-binding domain was also capable of inhibiting cell proliferation in a manner similar to intact C1q. The effect of C1q was not cytotoxic but cytostatic since the number of dead cells in the C1q-treated cultures was not significantly different than that in the untreated cells (5% vs 4%), a figure which represents the normal wear and tear of tissue culture conditions. On the basis of these findings we propose that the C1qR alone or in conjunction with other cellular factors may function as a molecule which supports cell growth. Upon ligand binding, however, the ligand-receptor interaction may suppress postreceptor events which are necessary for cell proliferation.     

In vivo reduction of circulating C1q binding immune complexes by intravenous gammaglobulin administration

Six patients with systemic lupus erythematosus were treated with high-dose intravenous gammaglobulin. Immunological parameters were studied and included solid-phase immune complex determinations, quantitative immunoglobulins G, A, and M, as well as C3 and C4 concentrations. Pretreatment values of circulating immune complex concentrations as measured by either C1q binding or anti-C3 binding assays were elevated in all patients. Posttreatment values showed reductions in all C1q binding immune complexes (p less than 0.01) and anti-C3 binding immune complexes also decreased in 5 out of 6 patients. These assays are described in detail and were also used to define in vitro interactions between the intravenous gammaglobulin preparation and heat-aggregated IgG or sera containing elevated circulating immune complexes. No reduction of immune complex levels were observed when IgG was incubated in vitro with either heat-aggregated IgG or sera with elevated immune complex concentrations. The duration of the in vivo effect and the patients' clinical responses are described. These findings show that high-dose intravenous gammaglobulin administration can reduce certain types of immune complexes in patients with elevated levels of these substances.     

Specific antisera to C1r. Quantitation of C1r in normal and pathological human sera

Monospecific neutralizing and precipitating antisera to C1r, a subunit of the first complement component, were obtained. These antisera neutralized C1r activity in purified preparations and in macromolecular C1 and did not react with C1q or C1s. They formed one line of precipitation in the β-globulin region with normal human serum, C1^hu and C1r at various stages of purification. Using anti-C1r antiserum and a radial immunodiffusion technique, the concentration of C1r was determined in normal, SLE and RA sera. It was 101 μg/ml in normal sera and lower in sera of active SLE patients (69·7 μg/ml). No significant variations from normal were found in sera of SLE patients in periods of remission or in RA patients.     

Porcine C1q and the solid-phase immunoassay of human immune complexes

Experiments were undertaken to determine if porcine C1q could replace human C1q in the solid-phase immunoassay of human immune complexes (ICs). Porcine C1q was obtained by a two-cycle precipitation method involving dialysis against chelating agents in low ionic strength buffer. C1q was adsorbed to polystyrene beads and in vivo- or in vitro-formed ICs binding to the solid-phase C1q were detected with ¹²⁵I-labeled or horseradish peroxidase-conjugated anti-human gamma antibodies. Unfractioned, heat-aggregated human gamma globulin (ΔIgG) could be detected at 20 ng/ml when diluted in buffer only. The detection threshold changed to 40–80 ng ΔIgG/ml when the assay was run with buffer containing normal human serum diluted 1 : 1000 (the serum dilution used for detecting natural ICs). Analysis of systemic lupus erythematosus sera revealed that 60% contained highly significant levels of ICs (binding ?3 S.D. above the mean of controls). Comparison with platelet aggregation test results revealed a highly significant correlation between the two methods (P < 0.0001), even though each assay detected ICs in several serum specimens negative in the other test. These results demonstrate that porcine C1q can functionally replace human C1q in the solid-phase immunoassay of human ICs. Since porcine blood is normally a waste product of the meat-processing industry, it is an obvious source of easily isolated C1q for use in such an assay.     

Lack of binding of C3 to IgG antibodies during the activation of the classical complement pathway on the red cell

We have studied the interaction between C3 and natural human and rabbit anti-MTX IgM and hyperimmune IgG antibody bound to red cells to which MTX was covalently coupled. IgM Ab molecules bound to the cell surface were measured by their interaction with rabbit anti-human mu-chain IgG Ab: the bound anti-mu-chain or anti-MTX IgG was quantitated with 125I-labelled PA. C3 uptake by EMTX AC142 complexes from purified preparation of C3 was detected by the interaction of bound C3 with rabbit anti-C3 IgG Ab; the bound IgG was then measured with radiolabelled PA. In some experiments the uptake of C3 by the EMTX AC142 complexes was measured by using 125I-labelled C3. To find out if C3 was bound to Ab molecules, anti-MTX Abs were eluted from the cells by excess fluid-phase MTX. After dialysis the eluted anti-MTX IgM or IgG Ab was than reattached to fresh EMTX. All cells were then analyzed for Ab and C3 content by a radioimmunoassay. It was found that when anti-MTX IgM or IgG was eluted from EMTX AC423 complexes no C3 was removed from the cells and that eluted IgM or IgG did not carry with them either C3 antigen or 125I-labelled C3. It was concluded that, during the activation of the classical C-pathway at the red cell surface, no C3 was bound to IgM (rabbit or human) or IgG (rabbit) antibody molecules.     

Antibodies against C1q in anti-glomerular basement membrane nephritis.

The prevalence of antibodies against the collagen-like region of the subcomponent of the first component of complement, C1q, was investigated in 11 patients with anti-glomerular basement membrane (GBM) nephritis. Anti-C1q antibodies (anti-C1qAb) were detected in seven patients. IgG anti-C1qAb were found in four and IgA anti-C1qAb in five patients. During follow up of the patients a relationship was observed between the levels of IgG anti-C1qAb and the levels of anti-GBM antibodies (anti-GBMAb). Gelfiltration experiments indicated that both IgG anti-C1qAb as well as IgG anti-GBMAb were monomeric and that binding also occurred with the F(ab')2 fragments of the antibodies. Although anti-C1qAb and anti-GBMAb are both directed against a collagen-like structure, it was demonstrated by means of inhibition experiments that anti-C1qAb and anti-GBMAb are directed against different antigenic sites. Comparison of patients with anti-GBM nephritis with and without anti-C1qAb revealed that there were no differences in disease activity or disease severity. Therefore, the results of this study suggest that anti-C1qAb do not play a direct pathogenetic role in anti-GBM nephritis.     

Binding of the human complement subcomponent C1q to hybrid mouse monoclonal antibodies

Activation of the classical pathway of the complement system is initiated by the binding of C1q to antibody complexes. Here we evaluated the C1q binding capacity of series of monospecific and bispecific hybrid mouse monoclonal antibodies (mAb) and compared them with parental (conventional) mAb. The hierarchy in C1q binding capacity of the bispecific anti-HuIgA1/HRP mAb with homologous H-H chain combinations (IgG2a-2a, IgG2b-2b and IgG1-1) and the parental anti-HuIgA1 or anti-HRP mAb was identical; IgG2a greater than IgG2b much greater than IgG1. Hybrid IgG1-2a mAb bind intermediate amounts of C1q when compared with the IgG1 and IgG2a parental antibodies. IgG1-2b and IgG1-1 hybrid mAb did not bind any C1q, like the IgG1 mAb. We could not observe any difference in C1q binding efficiency between monovalently bound IgG1-2a, IgG2a-2a and IgG2b-2b anti-HuIgA1 HRP mAb and the bivalently bound IgG1-2a, IgG2a-2a and IgG2b-2b anti-HuIgA1 mAb, respectively. Furthermore, these hybrid ms anti-HuIgA1 and bs anti-HRP/HuIgA1 mAb were able to lyse HuIgA1-coated erythrocytes, in the presence of 50% human serum, as efficiently as their parental counterparts. These data indicate that a simultaneous binding of both F(ab') fragment to antigen is not a necessary prerequisite for binding and activation of C1q.