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PubMed1990

[Current overview of diagnostic possibilities of complement analysis].

The complement system is composed of at least 21 serum proteins and 8 cell surface receptors sensitive to complement components or their fragments. Qualitative and quantitative analysis of complement components in clinical samples has become standard procedure in many laboratories thanks to the availability of easy-to-perform test kits. However, interpretation of results is still a task that requires great skill and the situation are rare, in which analysis of the complement system goes beyond diagnosis into therapeutic consequences. The present paper is an update of previous similar reviews now available in the international literature; in addition, the author attempts at bringing the often complicated world of complement and clinical practice together and thus contributes to answering the question: what should the clinician know about complement?

Antigen-Antibody Complex↗
PubMed1985

Anti-peripheral myelin antibody in patients with demyelinating neuropathy: quantitative and kinetic determination of serum antibody by complement component 1 fixation.

The role of anti-peripheral nerve myelin antibody (anti-PNM Ab) in the pathogenesis of acquired demyelination of peripheral nerve is unclear, in part, due to the poor correlation between antibody and disease activity. Previous studies show that only 27-50% of patients with acute demyelinating neuropathy or Guillain-Barré syndrome (GBS) had serum Abs to peripheral nerve or PNM as demonstrated by consumption of hemolytic activity of serum complement 1 (C1) fixation and transfer assay, quantitative determinations of anti-PNM Ab showed significantly high titers in the serum of patients with GBS, chronic and recurrent polyneuritis, and paraproteinemia associated with peripheral neuropathy. All 11 patients with acute-phase GBS had Ab titers 6-56 times higher than controls. In 6 GBS patients, serial Ab determinations showed that titers were highest on admission, fell rapidly the first week, and became undetectable or barely detectable by the third week. Declining Ab titers coincided with cessation of clinical progression. In 3 GBS patients, depletion of serum IgM lowered anti-PNM Ab titers significantly, whereas IgG depletion failed to produce a similar effect. This study shows that the C1 fixation and transfer assay is a sensitive method to detect anti-PNM Ab in the serum of patients with a variety of demyelinating neuropathies and provides good correlation between Ab level and the clinical course of GBS patients. It may provide important information about the pathogenesis of the demyelinating neuropathies.

Antibodies↗
PubMed1985

Double fluorescence technique for measurement of complement-fixing antibody to lymphocyte subsets.

A double fluorescent antibody method for quantitating human complement-fixing antibody to lymphocyte subclasses has been developed. The indicators in this system are a C6-deficient serum as a non-lytic source of complement, rhodamine-labeled anti-C3 and fluorescein-labeled murine monoclonal antibodies to human lymphocyte subsets. The basic procedure is to incubate lymphocytes with the unknown serum and then to add C6-deficient serum. The binding of C3 is indicated by staining with rhodamine-labeled anti-C3 and the subset class of the lymphocyte so stained is determined by binding of fluorescein-tagged anti-OKT4 or -OKT8 antibodies. The occurrence of both red and green cell surface fluorescence denotes the presence of a complement-binding antibody to the lymphocyte subset defined by the monoclonal antibody. In addition to defining the specificity of complement-fixing anti-lymphocyte antibodies, this technique is more sensitive than the microcytotoxicity assay.

Acquired Immunodeficiency Syndrome↗
PubMed1987

Quantitation of C3 nephritic factor of alternative complement pathway by an enzyme-linked immunosorbent assay.

We have developed an enzyme-linked immunosorbent assay (ELISA) for the quantitation of C3 nephritic factor of the alternative pathway of complement (NeFA). Incubation of the NeFA-positive serum (patient KS serum) with normal human serum (NHS) in Mg-EGTA resulted in the formation of C3-B-IgG complex. No complex was formed in EDTA. At first this was detected as three types of complexes: C3-IgG, B-IgG and B-C3, by the combination of antibodies. The reaction mixture in Mg-EGTA was filtered through an ACA 22 column, from which the complexes were eluted in the same part as the first protein peak. When IgG purified from KS serum was incubated with NHS in Mg-EGTA, B-C3 complex increased in proportion to the dose of IgG. These results indicated that only one kind of complex consisting of IgG, C3 and B (IgG-C3-B) was generated by the addition of NeFA to NHS. Serum NeFA could be quantified as the titer of B-C3 complex formed after its incubation with NHS in Mg-EGTA. Using the ELISA method, NeFA was positive in five out of six patients with membranoproliferative glomerulonephritis (MPGN) type II and in only one of 17 with MPGN type I. Titers obtained by the new method were in good accordance with those by C3 conversion and C3bBb stabilization assays for NeFA, and the new method was more exact and simple than the conventional methods.

Antigen-Antibody Complex↗
PubMed1990

Effect of amnionitis on the complement system of preterm infants.

The development of the complement system was studied by quantitation of total hemolytic complement activity (CH50), C1q, C3, C4, and C3 split product (C3d) in cord plasma of nine human fetuses (17-22 weeks of gestation), 110 preterm (24-36 weeks of gestation) and 30 term neonates. The complement levels were analyzed in relation to various illnesses of preterm infants. Histological examination of the placenta revealed a higher incidence of amnionitis in the placenta of less than 34 gestational weeks. In cases without amnionitis, there were significant correlations between complement levels and gestational age. In cases with amnionitis, the complement system was activated even in infants of less than 28 weeks gestation. The complement levels correlated with the extent of the inflammation in the placentas and umbilical cords except for C1q. In infants with Wilson-Mikity syndrome, complement levels other than C1q were significantly elevated in comparison with those of infants with respiratory distress syndrome. In the group of preterm infants without amnionitis, no differences were found between infants with intrauterine growth retardation and those with growth appropriate for gestational age.

Chorioamnionitis↗
PubMed1976

Serum immunoglobulin and complement levels in patients with psoriasis vulgaris.

In 102 patients with psoriasis vulgaris, divided into group with slight psoriatic form and the group with severe psoriatic form, the serum immunoglobulin and complement levels were determined. The quantitation of immunglobulin and complement was carried out by single radial immundiffusion. It was found that the IgM levels were significantly decreased in both investigated groups. The IgA and IgG levels were significantly increased only in patients with severe psoriatic form. While the beta1A (C'3) levels were significantly increased in both investigated groups the beta1E (C') levels were not significantly changed compared to control group. It is concluded that the immunoglobulin imbalance in patients with psoriasis, opposite to complement, is depended on the severity of clinical form.

Adolescent↗
PubMed1976

Critical role of the conversion of the third complement component C3 (beta 1C/beta 1A) for its immunochemical quantitation.

For the immunochemical quantitation of the third component of complement C3 the state of conversion of this protein in the sample and the reference serum has to be guarded. Antisera raised against C3 may show different antibody specificity for the antigenic pattern of the C3 proteins which change during proteolysis by activation and also by ageing. It is shown that anti-C3-sera gave differences from -17% to + 91% comparing fresh and aged aliquots of the same sera directly on the same immunodiffusion plate corresponding to a particular anti-C3-serum. The use of three different monospecific antisera directed against the A determinant of C3 seemed to give results with good correlation in respect to C3 conversion. Glass and polystyrene plastic tubes did not have a significantly different effect on the C3 values in respect to C3 conversion. The in vitro acceleration of C3 conversion by thrombin or an inulin-like polysaccharide did not give consistent results in this context.

Complement C3↗
PubMed1993

Characterization of kinetics and target proteins for binding of human complement component C3 to the surface-exposed outer membrane of Chlamydia trachomatis serovar L2.

In order to characterize the interaction of human complement with Chlamydia trachomatis, flow cytometry was used to quantitate binding of complement component C3 to elementary bodies of C. trachomatis serovar L2 preincubated in fresh serum in the presence or absence of human polyclonal chlamydial antibody. Isolation of each of the complement activation pathways revealed that C3 was activated most effectively by the alternative pathway. The degree of binding by the classical pathway was proportional to the concentration of antibody, but dual-pathway-mediated binding was not greater than antibody-independent alternative pathway binding. Electrophoresis and immunoblotting of detergent-extracted outer membrane protein-C3b complexes indicated that the chlamydial major outer membrane protein was the primary cell surface moiety binding C3b in both the presence and absence of specific antibody. Hydroxylamine cleavage of outer membrane protein-C3b complexes provided evidence that the majority of C3b is bound to the major outer membrane protein by hydroxyl ester bonds. This result was also unchanged by the presence of specific antibody. An unexpected finding was the apparent binding of anti-C3 antibody to a 40-kDa protein of the chlamydial outer membrane complex, perhaps indicating C3 mimicry on the part of the chlamydial major outer membrane protein.

Amino Acid Sequence↗
PubMed1984

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

We have investigated quantitatively the complement-mediated binding of prepared, soluble 125I-7S IgG antibody/3H-dsDNA immune complexes to human red blood cells (RBCs). We have performed these studies by using a detailed modification of the RBC-CF assay [Pedersen et al., J. Immun. Meth. 38, 269-280 (1980)] which now allows for the simultaneous measurement of both 3H-DNA and 125I-binding to the cells. Our results indicate that, in the case of three SLE patients, their anti-dsDNA antibody titers are sufficiently high that a small fraction of their 125I-7S IgG antibodies (ca 0.1-0.2%) can be identified as specifically anti-dsDNA. We have also used an indirect method (with 125I-labelled rabbit anti-human IgG) for the determination of IgG anti-dsDNA antibodies in complement-fixing antibody/dsDNA immune complexes that bind to RBCs, and the results of these measurements are in reasonable agreement with the direct binding experiments. These studies have also allowed us to estimate the antibody/DNA stoichiometries in complement-fixing immune complexes. The results of these experiments may provide a useful standard for the analysis of monoclonal anti-dsDNA antibodies.

Antibodies, Antinuclear↗
PubMed1994

Antibody-mediated complement activation on nucleated cells. A quantitative analysis of the individual reaction steps.

The sequential molecular events of the initiation, amplification, and membrane attack phases of classical C pathway activation on nucleated cells were investigated. As a model system, C-susceptible human melanoma cells (SK-MEL-93-2) expressing the disialoganglioside Ag GD3 were studied. Activation of the classical C pathway was initiated by the anti-GD3 mAb R24 (murine IgG3). The initiation phase is characterized by a very inefficient molar ratio of deposited C1q per Ab molecule. At an Ab density of 5.86 x 10(6) molecules/cell, only 3% of cell-bound R24 molecules form suitable pairs for C1q binding. During the amplification phase maximally 2.44 x 10(6) molecules of C4 and 0.67 x 10(6) molecules of C2/cell are being bound to form the C3 convertase. Despite the rather inefficient binding of C2, the C3 convertase is highly active in depositing high numbers of C3b molecules on the cell surface. Maximum binding of C3b occurred within 5 min of incubation with a total number of 2.1 x 10(7) molecules/cell. This indicates amplification factors at the level of C4 and C3 of 28 (C4/C1q) and 241 (C3/C1q), respectively. C3b was found to be rapidly cleaved into iC3b. As a result of this rapid C3b degradation, the membrane attack phase is initiated with a relatively inefficient C5 activation. The maximal number of 9.5 x 10(5) molecules C5b/cell corresponds to a molar ratio of C5:C3 of only 1:22. The deposition of C5b led to the subsequent maximum binding of the following numbers of molecules of terminal C components per cell: C6, 0.8 x 10(6); C7, 0.89 x 10(6); C8, 0.82 x 10(6); C9, 1.8 x 10(6). These numbers correspond to average molar ratios (calculated per C5b molecule) of C5b/C6/C7/C8/C9 of 1/0.85/0.94/0.86/1.88. In addition to the monomeric C9, dimeric and polymeric (12- to 16-mer) forms of the molecule could be demonstrated. Collectively, our data represent a first comprehensive quantitative analysis of classical pathway activation on a nucleated cell.

Antibodies, Monoclonal↗
PubMed1980

Artificially transformed schistosomula of Schistosoma mansoni: mechanism of acquisition of protection against antibody-mediated killing.

Incorporation of labelled amino acid into tegumental proteins and acquisition of protection by schistosomula against antibody-mediated killing in vitro were simultaneously stimulated by serum factors and inhibited by puromycin. Comparison of polyacrylamide gel electrophoresis patterns with fluorographic autoradiography indicates that the majority of proteins in the parasite tegument were labelled with the isotope after incubation for 3 h. No new, clearly defined band was observed in the autoradiography pattern. During this period a decreasing susceptibility of the schistosomula to antibody plus complement was observed. Quantitative fluorescence assay shows that schistosomula insensitive to antibody plus complement were still able to bind the same amount of antibody as the unprotected parasites. Pre-culture of schistosomula in the presence of inactivated normal rabbit serum also decreased the susceptibility of the parasites to the in vivo killing mechanism.

Animals↗
PubMed1979

Analysis of genetic complementation by whole-cell microtechniques in fibroblast heterokaryons.

A whole-cell microtechnique for the determination of complementation of human metabolic disorders is presented. This procedure permits the isolation of individual multinucleate cells produced by cell fusion for the quantitative evaluation of complementation. Mutant fibroblasts with a deficiency of propionyl-CoA carboxylase activity (EC 6.4.1.3) that had been mapped to complementation groups pcc and bio were used to evaluate the microtechnique. Complementation was monitored by the determination of [14C]propionate incorporation into cellular macromolecules. Single cells or a small number of cells were isolated from plastic film dishes after radioactive incubation by cutting out the portion of the plastic film holding the desired cells. Isotope incorporation was linear in 10-50 unfused cells and in 10-50 fused normal cells containing five or more nuclei. There was also a direct correlation between the nuclear content of cells and the amount of isotope incorporated. Three pcc and two bio mutants were fused in pairwise combinations by means of polyethylene glycol and complementation was determined by isotope incorporation in sets of 50 multinucleate cells, each cell isolated individually. The results agreed with autoradiographic data for both complementing and noncomplementing strains. The method is quantitative and gives severalfold higher sensitivity than current procedures. The method can be applied to the complementation analysis of a wide variety of inherited disorders of intermediary metabolism.

Acyl Coenzyme A↗
PubMed1995

Serum concentrations of C4 isotypes and factor B in type I C2 deficiency suggest haplotype-dependent quantitative expression of MHC class III complement genes.

The complement protein C4 exists as two isotypes, C4A and C4B, encoded by genes in the major histocompatibility complex (MHC) class III region. The serum concentrations of C4A4 were lower than those of C4B2 in serum from 19 individuals homozygous for type I C2 deficiency (p < 0.0002). These individuals all had the S042 complotype and most of them were homozygous for the haplotype HLA-B18,S042,DR2. In 14 individuals heterozygous for the C2Q0 gene and with the C4A4, C4B2 phenotype and in 51 individuals with the C4A3, C4B1 phenotype, the isotype concentrations were equal. Factor B concentrations in the C2-deficient individuals were lower than those in individuals with the C4A3, C4B1 phenotype (p < 0.0001). The findings strongly suggest that the quantitative expression of C4 isotypes and factor B is MHC haplotype dependent. C4 null alleles cannot be accurately determined by measuring relative C4 isotype serum concentrations.

Complement C2↗
PubMed1979

Complement in cystic fibrosis.

Quantitative and functional assessments were made of both the classical and alternative pathways of complement activation in sera from 23 patients with cystic fibrosis. The classical pathway functioned similarly in patients and controls as measured by CH50 titre. Alternative pathway function, initiated in patient sera by incubation with inulin, was equal to that of controls as determined by cleavage of Factor B and C3, and by the consumption of terminal components. Factor B, however, was more readily activated in patient than in control sera. This rapid alteration of Factor B did not lead to accelerated or more extensive activation of the terminal complement components via the alternative pathway when assessed by C3 cleavage and the consumption of terminal components. Thus, a complement deficiency was not found. The importance of the easily activated Factor B is undefined.

Adolescent↗
PubMedDate not supplied

Epitope specificities and quantitative and serologic aspects of monoclonal complement (C3c and C3d) antibodies.

Twenty-two monoclonal antibodies to human C3c and ten to C3d were obtained by hybridization after the immunization of mice with complement-coated human red cells and/or purified human complement components. C3c antibodies were variable in their agglutination reactions with cells coated with C3 by antibody in vitro; more consistent and potent reactions with these cells were observed with anti-C3d, and all anti-C3d reacted with red cells coated with C3 in vivo. Immunoradiometric assays were used to estimate antibody concentration, affinity, and epitope specificity. The antibody content in ascitic fluids varied from less than 0.1 mg per ml to 5.6 mg per ml. The estimated values of antibody affinities for Sepharose-coupled C3 ranged from 2.8 X 10(6) l per M to 5.0 X 10(8) l per M; on average, IgM antibodies had higher affinities than IgG antibodies. Competitive binding assays showed that the monoclonal antibodies recognized at least seven different epitopes, four on the C3c and three on the C3d fragment of C3. When the results of serologic and quantitative assays were compared, no convincing relationship was found between serologic performance and epitope specificity, antibody concentration, or affinity. IgM antibodies generally gave higher agglutination scores than IgG antibodies, and Ig class was the only useful predictor of serologic efficacy.

Animals↗