Biomedical subjects

A Circolo

Publications and source records attributed to A Circolo.

At least 19 recordsLinked to original sources

PubMed2001

Complement component C3 is not required for full expression of immune complex glomerulonephritis in MRL/lpr mice.

Complement activation and tissue deposition of complement fragments occur during disease progression in lupus nephritis. Genetic deficiency of some complement components (e.g., Factor B) and infusion of complement inhibitors (e.g., Crry, anti-C5 Ab) protect against inflammatory renal disease. Paradoxically, genetic deficiencies of early components of the classical complement pathway (e.g., C1q, C4, and C2) are associated with an increased incidence of lupus in humans and lupus-like disease in murine knockout strains. Complement protein C3 is the converging point for activation of all three complement pathways and thus plays a critical role in biologic processes mediated by complement activation. To define the role of C3 in lupus nephritis, mice rendered C3 deficient by targeted deletion were backcrossed for eight generations to MRL/lpr mice, a mouse strain that spontaneously develops lupus-like disease. We derived homozygous knockout (C3(-/-)), heterozygous (C3(+/-)), and C3 wild-type (C3(+/+)) MRL/lpr mice. Serum levels of autoantibodies and circulating immune complexes were similar among the three groups. However, there was earlier and significantly greater albuminuria in the C3(-/-) mice compared with the other two groups. Glomerular IgG deposition was also significantly greater in the C3(-/-) mice than in the other two groups, although overall pathologic renal scores were similar. These results indicate that C3 and/or activation of C3 is not required for full expression of immune complex renal disease in MRL/lpr mice and may in fact play a beneficial role via clearance of immune complexes.

Albuminuria↗

PubMed2000

Modulation of renal disease in MRL/lpr mice genetically deficient in the alternative complement pathway factor B.

In systemic lupus erythematosus, the renal deposition of complement-containing immune complexes initiates an inflammatory cascade resulting in glomerulonephritis. Activation of the classical complement pathway with deposition of C3 is pathogenic in lupus nephritis. Although the alternative complement pathway is activated in lupus nephritis, its role in disease pathogenesis is unknown. To determine the role of the alternative pathway in lupus nephritis, complement factor B-deficient mice were backcrossed to MRL/lpr mice. MRL/lpr mice develop a spontaneous lupus-like disease characterized by immune complex glomerulonephritis. We derived complement factor B wild-type (B+/+), homozygous knockout (B-/-), and heterozygous (B+/-) MRL/lpr mice. Compared with B+/- or B+/+ mice, MRL/lpr B-/- mice developed significantly less proteinuria, less glomerular IgG deposition, and decreased renal scores as well as lower IgG3 cryoglobulin production and vasculitis. Serum C3 levels were normal in the B-/- mice compared with significantly decreased levels in the other two groups. These results suggest that: 1) factor B plays an important role in the pathogenesis of glomerulonephritis and vasculitis in MRL/lpr mice; and 2) activation of the alternative pathway, either by the amplification loop or by IgA immune complexes, has a prominent effect on serum C3 levels in this lupus model.

Animals↗

PubMed2000

Mutational analysis of the primary substrate specificity pocket of complement factor B. Asp(226) is a major structural determinant for p(1)-Arg binding.

Factor B is a serine protease, which despite its trypsin-like specificity has Asn instead of the typical Asp at the bottom of the S(1) pocket (position 189, chymotrypsinogen numbering). Asp residues are present at positions 187 and 226 and either one could conceivably provide the negative charge for binding the P(1)-Arg of the substrate. Determination of the crystal structure of the factor B serine protease domain has revealed that the side chain of Asp(226) is within the S(1) pocket, whereas Asp(187) is located outside the pocket. To investigate the possible role of these atypical structural features in substrate binding and catalysis, we constructed a panel of mutants of these residues. Replacement of Asp(187) caused moderate (50-60%) decrease in hemolytic activity, compared with wild type factor B, whereas replacement of Asn(189) resulted in more profound reductions (71-95%). Substitutions at these two positions did not significantly affect assembly of the alternative pathway C3 convertase. In contrast, elimination of the negative charge from Asp(226) completely abrogated hemolytic activity and also affected formation of the C3 convertase. Kinetic analyses of the hydrolysis of a P(1)-Arg containing thioester by selected mutants confirmed that residue Asp(226) is a primary structural determinant for P(1)-Arg binding and catalysis.

Amino Acid Sequence↗

PubMed1999

Deficiency of human complement protein C4 due to identical frameshift mutations in the C4A and C4B genes.

The complement protein C4, encoded by two genes (C4A and C4B) on chromosome 6p, is the most polymorphic among the MHC III gene products. We investigated the molecular basis of C4 deficiency in a Finnish woman with systemic lupus erythematosus. C4-specific mRNA was present at low concentrations in C4-deficient (C4D) patient fibroblasts, but no pro-C4 protein was detected. This defect in C4 expression was specific in that synthesis of two other complement proteins was normal. Analysis of genomic DNA showed that the proposita had both deleted and nonexpressed C4 genes. Each of her nonexpressed genes, a C4A null gene inherited from the mother, a C4A null gene, and a C4B null gene inherited from the father, all contained an identical 2-bp insertion (TC) after nucleotide 5880 in exon 29, providing the first confirmatory proof of the C4B pseudogene. This mutation has been previously found only in C4A null genes. Although the exon 29/30 junction is spliced accurately, this frameshift mutation generates a premature stop at codon 3 in exon 30. These truncated C4A and C4B gene products were confirmed through RT-PCR and sequence analysis. Among the possible genetic mechanisms that produce identical mutations is both genes, the most likely is a mutation in C4A followed by a gene conversion to generate the mutated C4B allele.

Adult↗

PubMed1999

Genetic disruption of the murine complement C3 promoter region generates deficient mice with extrahepatic expression of C3 mRNA.

Genetic deficiencies of the complement protein C3 occur naturally in humans and animal models and have been induced in mice by targeted deletion of the C3 gene. The study of these deficiencies has provided evidence for C3 functions in immune responses. C3 deficient mice were generated by replacing the 5'-flanking region of the C3 gene with the neomycin-resistance (neo) gene. Serum from these mice had no detectable C3 protein or complement activity. Challenge with Streptococcus pneumoniae revealed approximately 2000-fold increase in bacteremia as compared to littermate controls. C3 mRNA was absent in the liver, but it was detected in the lung, kidney, fat tissue, heart and spleen. Metabolic labeling of the lung tissue and peritoneal macrophages showed synthesis of pro-C3, but no post-synthetic intracellular processing of the protein and no secretion of mature C3. cDNA analysis at the cap site indicated that extrahepatic transcription of the targeted gene was initiated in the neo cassette, close to the C3/neo junction and predicted a primary translation product lacking the leader peptide. The data indicate that these mice provide a good animal model for the study of complete C3 deficiencies and a potential probe for tissue-specific C3 gene regulatory elements.

Animals↗

PubMed1998

Molecular heterogeneity in deficiency of complement protein C2 type I.

Deficiency of the complement protein C2 (C2D), one of the most common genetic deficiencies of the complement system, is associated with rheumatological disorders and increased susceptibility to infection. Two types of C2D have been recognized, each in the context of specific major histocompatibility complex (MHC) haplotypes; type I, a deletion, frameshift and premature stop codon resulting in absence of detectable C2 protein synthesis, and type II, missense mutations resulting in a block in secretion of C2 proteins. Analysis of C2 expression in a child with C2 deficiency, a MHC haplotype different from those associated with type I or II C2D, and recurrent infections revealed additional molecular heterogeneity among C2 deficient patients. No detectable C2 protein was synthesized in the child's fibroblasts under conditions supporting C2 synthesis and secretion in normals and the child's C2 mRNA was reduced to 42% of normal. Nucleotide sequencing of RT-PCR fibroblast mRNA and genomic DNA revealed a type I C2 deficiency (28 base-pair deletion) on one allele and a previously unrecognized two base-pair deletion in exon 2 on the other. Expression of the closely linked factor B gene was markedly decreased (Bf mRNA 25% of normal), though Bf was up-regulated appropriately by interferon-gamma and the flanking sequence containing the Bf promoter was normal in this C2-deficient patient. Moreover, the concentration of Bf protein was normal in the patient's plasma.

Bacteremia↗

PubMed1997

Abrogation of the alternative complement pathway by targeted deletion of murine factor B.

To investigate the role of complement protein factor B (Bf) and alternative pathway activity in vivo, and to test the hypothesized potential genetic lethal effect of Bf deficiency, the murine Bf gene was interrupted by exchange of exon 3 through exon 7 (including the factor D cleaving site) with the neor gene. Mice heterozygous for the targeted Bf allele were interbred, yielding Bf-deficient offspring after the F1 generation at a frequency suggesting that Bf deficiency alone has no major effect on fertility or fetal development. However, in the context of one or more genes derived from the 129 mouse strain, offspring homozygous for Bf deficiency were generated at less than expected numbers (P = 0.012). Bf-deficient mice showed no gross phenotypic difference from wild-type littermates. Sera from Bf-deficient mice lacked detectable alternative complement pathway activity; purified mouse Bf overcame the deficit. Classical pathway-dependent total hemolytic activity was lower in Bf-deficient than wild-type mice, possibly reflecting loss of the alternative pathway amplification loop. Lymphoid organ structure and IgG1 antibody response to a T-dependent antigen appeared normal in Bf-deficient mice. Sensitivity to lethal endotoxic shock was not significantly altered in Bf-deficient mice. Thus, deficiency of Bf and alternative complement activation pathway led to a less dramatic phenotype than expected. Nevertheless, these mice provide an excellent model for the assessment of the role of Bf and the alternative pathway in host defense and other functions in vivo.

Animals↗

PubMed1996

Constitutive expression of murine complement factor B gene is regulated by the interaction of its upstream promoter with hepatocyte nuclear factor 4.

Factor B (Bf) is a constituent of the alternative pathway of complement activation encoded within the major histocompatibility complex. Transcription of the murine gene from two initiation sites generates two Bf mRNA species differing in size and tissue distribution. Striking genetic, tissue-specific differences in Bf mRNA levels at extrahepatic sites (kidney and intestine) among mouse strains correlate with a DNA sequence polymorphism in the 5'-flanking region of the gene and differential nuclear protein binding at the Bf upstream transcriptional initiation site (UIS). To ascertain the functional consequences of this polymorphism in the Bf promoter, we analyzed the effects of strain-specific sequences in the Bf 5' region on the expression of a chloramphenicol acetyltransferase (CAT) reporter gene transfected in human and mouse hepatoma cells. The CAT activity and mRNA level produced when transcription was driven by the sequence of strains with high extrahepatic expression were reduced to background levels when the sequence specific to the low expressor strains was used. Eighty percent of this difference was accounted for by a point substitution that affects DNA-protein interaction at the UIS, the sequence of higher affinity conferring higher expression. Hepatocyte nuclear factor 4 (HNF-4), derived from HepG2, mouse liver and kidney or cell-free translation of HNF-4 RNA, is the nuclear protein that preferentially binds to the high expressor UIS. Bf-CAT is not expressed in cells that lack HNF-4 (CV-1). However, co-transfection of HNF-4 into CV-1 cells drives Bf-CAT expression and reproduces the differences derived from the substitution that affect HNF-4 binding in vitro. These data show that interaction of HNF-4 with polymorphic variants of the upstream Bf promoter is the major determinant of strain-specific extrahepatic factor B expression.

Animals↗

PubMed1996

Impaired production of both normal and mutant C1 inhibitor proteins in type I hereditary angioedema with a duplication in exon 8.

In the autosomal dominant disorder type I hereditary angioedema, reduced levels of C1 inhibitor may be due in part to increased turnover and decreased synthesis of normal C1 inhibitor protein. A type I hereditary angioedema patient was recently described in whom the C1 inhibitor mutation consisted of a 20-bp duplication of nucleotides 1414 to 1433 in exon 8 that introduced a frame shift predicting the loss of a normal stop codon and the translation of a protein 52 amino acids longer than normal. In this study, we analyzed the expression of C1 inhibitor in fibroblasts obtained from a skin biopsy of this patient. Two proteins of approximately 78 and 94 kDa were found intracellularly, corresponding to the products of normal and mutated alleles, respectively. Pulse-chase analysis showed a complete lack of secretion of the mutated form. In addition, there was decreased extracellular production of the normal C1 inhibitor, suggesting either decreased secretion or increased intracellular catabolism of the normal protein because of the presence of the mutant allele. The production of other complement proteins was normal. This study provides a model for further analysis of autosomal dominant genetic disorders in which production of the functional protein may be affected by the product of the mutated allele.

Alleles↗

PubMed1995

Translational regulation of murine complement factor B alternative transcripts by upstream AUG codons.

Factor B (Bf), a constituent of the alternative pathway of complement activation, is encoded by a single gene that is located within the MHC. In murine kidney and intestine, two Bf transcripts (Bf short and Bf long), generated from distinct transcriptional initiation sites, are expressed in approximately equal amounts. In the liver, > 95% of Bf mRNA is the short transcript. To ascertain the biologic consequences of this tissue-specific mRNA polymorphism, we quantitated the effect of structural differences between the two transcripts on net Bf protein synthesis. Cell-free translation of Bf mRNA in vitro revealed that the rate of translation of Bf short is about twice that of Bf long. The 5' extension of Bf long includes four short open reading frames upstream of the authentic translational initiation codons. Mutation of all four upstream AUGs generates a Bf long transcript with a translational rate about equal to that of Bf short. This effect was primarily accounted for by mutation of the second AUG from the 5' end. Similar studies of Bf expression in vivo showed an approximately twofold difference in translational rate between Bf long and the transcript mutated at the upstream AUGs. Because systemic and local inflammation in kidney alters the ratio of Bf long and short, net production of complement protein Bf in extrahepatic tissues is regulated by both transcriptional and translational control mechanisms.

Alternative Splicing↗

PubMed1994

Regulation of synthesis of complement protein C4 in human fibroblasts: cell- and gene-specific effects of cytokines and lipopolysaccharide.

Synthesis and secretion of the class III major histocompatibility complex (MHC) gene product, C4, were detected in human skin fibroblasts by metabolic labelling, immunoprecipitation and SDS-PAGE analysis. Pro-C4 (approximately 185,000 MW) was present in intracellular lysates, and the mature protein was present in extracellular media, with three bands of approximately 93,000, 75,000 and 33,000 MW, corresponding to the alpha, beta and gamma chains, respectively. C4 expression was increased in a dose-dependent manner by interferon-gamma (IFN-gamma), but was unaffected by interleukin-1 beta (IL-1 beta), IL-6 and tumor necrosis factor-alpha (TNF-alpha) alone, each of which augmented the expression of factor B, C3 and other complement proteins synthesized in fibroblasts. Simultaneous incubation of fibroblasts with IFN-gamma and TNF resulted in a synergistic increase in C4 synthesis. RNA blot analyses indicated that regulation of C4 synthesis by IFN-gamma and the combination of IFN-gamma and TNF was mediated primarily at a pretranslational level. Lipopolysaccharide (LPS) had no effect on C4 or HLA-DR synthesis in fibroblasts, either constitutive or IFN-gamma-regulated. These results are in contrast to the effects of LPS in monocytes, where LPS decreased constitutive synthesis and counter-regulated the IFN-gamma-enhanced expression of both C4 and HLA-DR. C2 expression in fibroblasts was also increased primarily by IFN-gamma. However, C2 synthesis was increased by LPS, 1L-1 and TNF, although to a lesser extent than the increase in synthesis of factor B stimulated by these mediators. These results show that up-regulation by IFN-gamma is a common feature of C2 and C4 expression in human cells that constitutively synthesize these proteins. In contrast, regulation of MHC class III and class II genes by LPS, TNF, IL-1, and IL-6 is cell- and gene-specific.

Blotting, Northern↗

PubMed1991

Differential regulation of the expression of proteinases/antiproteinases in fibroblasts. Effects of interleukin-1 and platelet-derived growth factor.

We have previously reported that polypeptide growth factors had an anti-inflammatory effect by decreasing the cytokine-enhanced expression of factor B (FB), an activator of the alternative complement pathway, in human fibroblasts. To further characterize the role of cytokines and growth factors in the inflammatory/repair continuum, we have studied the effects of interleukin-1 (IL-1) and platelet-derived growth factor (PDGF) on the expression of metalloproteinases/antiproteinases of the extracellular matrix in cultured human fibroblasts. Co-incubation of IL-1 and PDGF synergistically increased the expression of stromelysin and interstitial collagenase to 23-fold (for both proteins) over background, while PDGF decreased the IL-1-enhanced expression of FB by 82%. PDGF, but not IL-1, alone or in combination, increased the synthesis of tissue inhibitor of metalloproteinases. RNA blot analysis indicated that the changes in protein synthesis were regulated at a pretranslational level. Cycloheximide treatment indicated that the effects of PDGF on the metalloproteinases/antiproteinases were not protein-dependent, in contrast to results obtained for FB. The effect of the three dimeric forms of PDGF (AA, AB, and BB) on the synthesis of metalloproteinases and FB was also analyzed. The effects were qualitatively similar for each of the dimeric forms; however, the BB and AB isoforms had considerably greater effects than PDGF-AA. It has been reported that the PDGF receptors found in human fibroblasts have higher binding affinity for the BB and AB isoforms of the growth factor. The results presented in this paper are in accord with the possibility that differences in the biological activity of the three isoforms of PDGF are due to differences in the number or affinity of the binding sites of the target cells, rather than to different activation pathways of the receptor. Thus, PDGF increased cytokine effects on metalloproteinases, while decreasing cytokine effects or complement activator FB. The net effect of these changes may be to decrease inflammation and enhance remodeling early in repair and to enhance matrix stability later in the repair process.

Blotting, Northern↗

PubMed1990

Antiinflammatory effects of polypeptide growth factors. Platelet-derived growth factor, epidermal growth factor, and fibroblast growth factor inhibit the cytokine-induced expression of the alternative complement pathway activator factor B in human fibroblasts.

The synthesis of complement components in human fibroblasts is modulated by mediators of inflammation such as cytokines. In particular, interleukin-1 (IL-1) and tumor necrosis factor (TNF) induce time- and dose-dependent increases in the synthesis of complement proteins factor B (FB), C3, and factor H (FH). Polypeptide growth factors are also soluble mediators released during inflammation and able to modulate many fibroblast functions. We have studied the effects of polypeptide growth factors platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF) on the synthesis of complement proteins in cultured human fibroblasts. PDGF, EGF, and FGF alone did not affect the level of synthesis of any of the complement proteins analyzed, but simultaneous incubation of PDGF, EGF, or FGF with IL-1 and TNF resulted in a dose-dependent inhibition of the cytokine-enhanced expression of FB. Inhibition of FB synthesis was observed between 4 and 8 h of exposure to PDGF and persisted for 4 h after the removal of the growth factor. Analysis of steady-state levels of specific FB mRNA suggested that PDGF-induced inhibition of FB synthesis is mediated at a pretranslational level and that it requires new protein synthesis. The effect of the growth factors was limited to FB, with marginal or no inhibition on the cytokine-enhanced synthesis of C3 and FH, excluding the possibility that the inhibitory effects of PDGF, EGF, and FGF on FB synthesis were due to a negative modulation of the growth factors on cytokine cell membrane receptors. Specific inhibition of cytokine-induced increases in FB synthesis by the growth factors may represent down regulation of the acute inflammatory process, further permitting progression to processes of tissue repair and remodeling. Study of the interactions between cytokines and growth factors in the regulation of synthesis of complement proteins may also provide a system for investigating mechanisms of signal transduction of both polypeptide growth factors and cytokines.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1987

Activation of human C1: analysis with Western blotting reveals slow self-activation.

The first component of human complement was separated from C1-INH by sucrose linear gradient ultracentrifugation. Activation of C1 was studied in the absence and presence of immune complexes; activation was monitored by SDS-PAGE and Western blot. When the partially purified native C1 preparation was incubated at 37 degrees C without immune complexes, activated C1s appeared after 30 min in the case of eightfold dilution with respect to the original serum, and after 45 min with 32-fold dilution. Kinetics of appearance of activated C1r was the same as that of activated C1s. From the following results, we concluded that spontaneous activation may be partially due to proteolytic enzymes contaminating the preparation: 1) a nonspecific protease inhibitor, PMSF, completely inhibited spontaneous activation but did not inhibit the activation of C1 by immune complexes; 2) alpha 2-macroglobulin partially inhibited spontaneous activation, and 3) although spontaneous activation in the absence of PMSF was relatively slow, activated C1 accelerated spontaneous activation that was completely blocked by C1-INH. In contrast to spontaneous activation, the partially purified native C1 was rapidly activated by immune complexes: within 5 min almost all C1 was activated by rabbit IgG anti-human IgM-human IgM complexes. These results support conclusions derived from activation studies when using native C1 and hemolytic assays, and do not support those derived from the activation studies with reconstituted C1 and SDS-PAGE analysis. We suggest that the contradictions can be resolved if one assumes that C1 activation can be both an intra- and intermolecular process; which process dominates is determined by the state of C1 and by experimental conditions.

Antigen-Antibody Complex↗

PubMed1987

Anti-IgM antibody decreases dissociability of cell bound IgM anti-hapten antibodies as measured with fluid phase hapten.

We studied the effect of an IgG anti-IgM on the dissociation of cell bound IgM anti-hapten antibody, as measured with fluid phase hapten. Methotrexate (MTX) covalently bound to the red cells surface was used as hapten; rabbit anti-MTX IgM and rabbit anti-allotype IgG reactive with the IgM were used as antibodies. The amount of cell bound antibodies was measured with 125I-labelled protein A. In the absence of the anti-allotype antibody, most of the anti-hapten IgM was prevented from reassociation by fluid phase hapten. In the presence of the anti-allotype IgG most of the anti-hapten IgM was non-dissociable, with about a 1000-fold increase in the apparent binding constant. We found that (1) dissociability of the anti-hapten IgM depended on the density of the cell bound hapten, (2) decrease in dissociability was a function of the anti-allotype antibody concn and (3) even in the presence of excess anti-allotype antibody dissociability was an inverse function of anti-hapten IgM concn.

Animals↗

PubMed1986

Anti-hapten IgG antibodies bound to cell surface hapten: anti-IgG antibody prevents dissociation as measured with fluid phase hapten.

We investigated the dissociation by fluid phase hapten of IgG antibodies bound to cell surface hapten in the presence and absence of anti-IgG antibodies. Dissociation was quantitated with fluid phase hapten, preventing reassociation of the anti-hapten antibodies. More than 90% of the anti-hapten IgG alone was prevented from reassociation by low concentrations of fluid phase hapten (nanogram to microgram range). In contrast, no dissociation of some IgG-anti-IgG complexes could be measured even at 24 hr incubation in the presence of very large excess of fluid phase hapten (100 mg/ml). We excluded aggregate formation between anti-hapten antibodies due to cross-linking by anti-antibodies as a cause for decreased dissociability by 1) performing the experiments in large excess of anti-antibody, 2) showing that the phenomenon was independent of anti-hapten antibody density, 3) showing that decreased dissociation also occurred at 4 degrees C, and 4) showing that aggregation by protein A did induce decreased dissociability, albeit three orders of magnitude lower than the anti-antibody. It was concluded that anti-antibody directly affected the "avidity" of cell hapten bound anti-hapten IgG in an unknown manner.

Animals↗

PubMed1985

Efficiency of activation of complement by anti-hapten antibodies at the red cell surface: effect of patchy vs random distribution of hapten.

Binding and activation of complement (C) by anti-hapten IgG and IgM antibodies (Abs) bound to a cell surface are dependent on the density and presumably on the distribution of cell-bound hapten. The purpose of this study was to find out if altering the distribution of the hapten on a red cell surface could modify the ability of anti-hapten IgG or IgM Ab to activate C. To test this we devised methods for comparing the C binding and activating efficiency of Abs bound to a hapten distributed randomly or in patches on cells. Random distribution was achieved by the covalent binding of methotrexate (MTX) directly to sheep red blood cells (E), while patchy distribution was achieved by the convalent binding to E of bovine serum albumin-MTX complexes. We considered bound albumin molecules as patches of MTX molecules. The results showed that, for IgG Ab, the number of hapten/E and the number of anti-hapten Ab molecules/cell required to generate one C-activating IgG complex were about an order of magnitude lower for hapten bound in patches than for randomly bound hapten. In contrast, IgM Ab bound to a hapten distributed in patches on an E surface lacked the ability to activate the lytic sequence of C, although maintaining a full ability to binding C1.

Animals↗

PubMed1985

Activation of hemolytic complement by mouse monoclonal IgG1 antibody: comparison with rabbit IgG.

We investigated the ability of a mouse anti-hapten monoclonal IgG1 antibody (Ab) to bind to cell-bound specific hapten and to fix and activate C1 and thus the lytic sequence of complement (C). In a comparative study with polyclonal rabbit anti-hapten IgG Ab, we found that about 6 times more monoclonal Ab molecules than polyclonal were necessary for the generation of 1 hemolytic site/cell: the data were interpreted to mean that a cluster of four cell-bound monoclonal Ab molecules was necessary to bind C1 and activate C-mediated hemolysis. Experiments performed under conditions of low density of cell-bound hapten and excess of antibody showed that both monoclonal and polyclonal IgG Abs were able to react only with 20-30% of the cell-bound hapten and that both Abs recognized the same hapten specificity. We also found that even though monoclonal IgG1 Ab was able to bind strongly to a protein A-Sepharose column and could be eluted only by a low-pH buffer, the purified Ab, when bound to cell surface hapten, showed a weak ability to react with free protein A.

Animals↗