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Results for “Complement Hemolytic Activity Assay”

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PubMed2001

Is hypocomplementemia useful for diagnosing or predicting extra-articular manifestations in patients with rheumatoid arthritis?

OBJECTIVE: Serum CH50 and C4 levels are usually normal or elevated in rheumatoid arthritis (RA) but are classically decreased in patients with serious extra-articular manifestations (SEAMs) of the disease. The objective of this study was to evaluate whether complement assays are useful in diagnosing or predicting SEAMs of RA. METHODS: First, a cross-sectional study of 405 patients admitted for RA compared patients with and without hypocomplementemia. Then, a retrospective longitudinal design was used to investigate within-patient complement level variations overtime. RESULTS: In the univariate analysis, patients with low CH50 and C4 levels were more likely to have vasculitis and/or cryoglobulinemia than those with normal CH50 and C4 levels, and nodules were more common in the patients with low than with normal C4 levels. In a multivariate model based on symptoms, low C4 was associated with vasculitis and pleurisy and low CH50 with vasculitis. However, these associations were too weak to make CH50 and C4 determination useful for detecting SEAMs, and the within-subject variations in patients with SEAMs limited the predictive value of these assays. CONCLUSION: Hypocomplementemia is of limited usefulness for detecting or predicting SEAMs.

Adult↗
PubMed1993

The association between hepatitis C virus infection and in vitro activation of the complement system.

The phenomenon of in vitro activation of the classical complement pathway at low temperatures (4-21 degrees C) is known as cold activation, and has been suggested to be associated with non-A, non-B hepatitis. We re-examined the association by using newly developed markers of hepatitis C virus (HCV) infection. Twenty-one cases randomly selected from those fulfilling the screening criteria of complement cold activation all showed evidence of HCV infection. More than half of 55 HCV antibody-positive cases selected randomly from our laboratory samples showed a tendency towards cold activation, whereas none of the HCV antibody-negative sera showed cold activation. All the HCV antibody-positive cases were negative in cold activation when CH50 was assayed with plasma. These results, taken together, indicate that cold activation of the complement system is strongly associated with HCV infection.

Cold Temperature↗
PubMed1990

Functional assay of C5-activating and nonactivating cobra venom factor preparations in the mouse system.

This paper deals with a new, functional assay of cobra venom factor (CVF) preparations with or without C5-activating property. Existing methods lack sensitivity and use diluted human complement as target of inactivation. An adapted assay using diluted mouse serum as complement source was hampered by underestimation of C3 depletion by bystander lysis and an overvaluation of C5 consumption resulting from C3 inactivation in the reagent used. These disadvantages prompted us to develop the new assay which is based on the incubation of CVF preparations with undiluted mouse serum. After incubation, residual total C activity, as well as functional C3 and C5 are estimated by titration. The procedure permits the assessment of CVF activities with minimal interference from undesired processes. The conditions in the new assay approach the in vivo situation in mice by the use of undiluted serum from the same animal species.

Animals↗
PubMed1999

One active C1r subunit is sufficient for the activity of the complement C1 complex: stabilization of C1r in the zymogen form by point mutations.

The binding of C1 (the first component of complement) to immune complexes leads to the autoactivation of C1r through the cleavage of the Arg463-Ile464 bond in the catalytic domain. Spontaneous activation of C1r (and C1) also occurs in the fluid phase, preventing the characterization of the zymogen form of C1r. To overcome this difficulty, the zymogen form of human C1r was stabilized by mutating the Arg in the Arg463-Ile464 bond to Gln. This mutant was designated as mutant QI. Recombinant C1r (wild type (wt) or mutant) was expressed in insect cells using serum-free medium in functionally pure form; therefore, the cell culture supernatant was suitable to reconstruct C1 for the hemolytic assay. Mutant QI was a stable, nonactivable zymogen and showed no hemolytic activity in reconstituted C1. However, this stable zymogen C1r mutant could form an active mixed dimer with the wt C1r, indicating that one active C1r subunit in the C1 complex is sufficient for the full activity of the entire complex. Our experiments also showed that the exchange of C1r monomers between the C1r dimers is completed in less than 16 h even at pH 7 and 4 degrees C. Two other mutants were also constructed by changing Arg463 to Lys, or Ile464 to Phe, and were designated as mutants KI and RF, respectively. Although these substitutions did increase the stability of the proenzyme in the cell culture supernatant, the mutant proteins retained their ability to autoactivate, and both had a wt-like hemolytic activity.

Animals↗
PubMed1994

Hereditary deficiency of the seventh component of complement and recurrent meningococcal infection: investigations of an Irish family using a novel haemolytic screening assay for complement activity and C7 M/N allotyping.

Terminal complement component deficiency predisposes to meningococcal infection and is inherited in an autosomal co-dominant manner. An Irish family is described, in which 2 of 3 brothers had recurrent meningococcal infection. A novel screening assay was used to investigate for terminal complement deficiency and the 2 affected brothers were found to be completely deficient in the seventh component of complement (C7). Enzyme-linked immunosorbent assay for C7 revealed lower than normal levels in the remaining brother and parents. C7 M/N protein polymorphism allotyping, used to investigate the segregation of the C7 deficiency genes, showed that the apparently complement sufficient brother was heterozygous C7 deficient and a carrier of one of the deficiency genes. Complement screening should be carried out in any individual suffering recurrent meningococcal infection or infection with an uncommon meningococcal serogroup. Identification of complement deficient patients allows the implementation of strategies to prevent recurrent infection.

Adolescent↗
PubMed1992

Murine ascitic fluids contain varying amounts of an inhibitor that interferes with complement-mediated effector functions of monoclonal antibodies.

The ability of murine monoclonal antibodies (mAbs), directed to the inner core of Gram-negative bacterial lipopolysaccharide (LPS, endotoxin), to enhance complement-mediated killing of bacteria, was investigated. The mAbs were tested as present in ascitic fluid. It was found that ascites contains an factor that inhibited the activity of complement. This effect was evident in assays for complement-mediated lysis of antibody-coated Gram-negative bacteria (bacterial killing) or of opsonised red blood cells. Moreover, the amount of inhibitor was found to vary from one ascites to another and spanned a 60-fold range. Thus, in vitro or in vivo experiments where complement is known to play a determining role may yield incorrect results when ascites is used as a source of antibody; the use of ascites prepared from irrelevant antibody as a negative control does not eliminate this problem.

Animals↗
PubMed1995

Molecular structure alteration of IgG increased anticomplementary activity of intravenous immunoglobulin.

AIM: To study the cause of anticomplementary activities (ACA) of intravenous immunoglobulins (IVIG). METHODS: ACA were determined by two assays (limit test and 100CH50 test), distributions of IgG molecular size (polymer, dimer, monomer and fragment) by HPLC, IgG subclasses and IgA contents by radial immunodiffusion, prekallikrein activator (PKA) and kallikrein (KK) activities by chromogenic assay, potency of antibody against hepatitis B surface antigen (Anti-HBs) by enzyme-linked immunosorbent assay (ELISA). RESULTS: The two ACA assays showed good correlation. However, ACA levels were not associated with distributions of IgG molecular size, IgG subclasses and IgA contents, PKA and KK activities. After heating incubation, ACA levels increased markedly and Anti-HBs decreased notably, distributions of IgG molecular size remained relatively constant. CONCLUSION: Molecular structure alteration of IgG increased spontaneous complement activation of IVIG.

Complement Activation↗
PubMed2002

Differentiation between the complement modulating effects of an arabinogalactan-protein from Echinacea purpurea and heparin.

Due to the important physiological role of the complement system, complement modulation, either inhibition or stimulation, is an interesting target for drug development. Several plant polysaccharides are known to exhibit complement modulating activities. Sometimes these effects are described as complement inhibition, although the basic mechanism is a stimulation of the complement activation. This misinterpretation is due to the observed reduced haemolysis in the widely used haemolytic complement assay, which does not allow to differentiate between complement activators and inhibitors, when it is performed in the classical manner. The aim of the presented study was to demonstrate that by simple modifications of the classical procedure this assay becomes an efficient tool to distinguish between real complement inhibitors and complement activating compounds without performing expensive, molecular mechanistic investigations. As practical examples heparin with proven complement inhibiting activity and AGP, a new arabinogalacatan-protein type II isolated from pressed juice of the aerial parts of Echinacea purpurea, as a potential complement activating compound were included in the study. By means of varying the preincubation time of the test compound with complement, AGP was clearly identified as a stimulator of both the classical and alternative pathway of complement activation. These findings correspond to the results of molecular mechanistic investigations. Selective removal of the arabinose side chains of AGP resulted in considerably reduced activity. Therefore, the three-dimensional structure of the polysaccharide, i. e., a backbone branched by side chains, is supposed to be important for the interactions with the complement system. The complement activating effects of AGP may contribute to the well-established immunostimulating effects of the pressed juice from Echinacea purpurea. Abbreviations. AGP:arabinogalactan-protein AGP-hydr.:hydrolysed arabinogalactan-protein AP-CA:haemolytic complement assay for the alternative pathway CP-CA:haemolytic complement assay for the classical pathway EGTA-VB:veronal buffered saline containing EGTA and Mg 2+HPS:human pooled serum RT:room temperature LPS:lipopolysaccharide RaE:rabbit erythrocytes RT:room temperature ShE(A):(sensitised) sheep erythrocytes VB:veronal buffered saline containing Ca 2+ and Mg 2+

Adjuvants, Immunologic↗
PubMed1992

Determination of optimal in vitro conditions for caprine alternative complement pathway assay.

Optimal in vitro testing conditions for caprine alternative complement pathway assay were determined. Effects of the following variables were tested: heterologous erythrocytes; pH, ionic strength and Mg2+ ion concentration of the complement diluent; incubation time and temperature. Rabbit erythrocytes were the optimal target cells. The optimal buffer conditions were: pH 8.0, ionic strength 0.06 mmol NaCl and 5mmol Mg2+ ion. Optimal incubation time and temperature were 75 min and 30 degrees C, respectively.

Animals↗
PubMed1993

Estimation of the genetic variation in complement activity of common carp (Cyprinus carpio L.).

The complement status of hybrid, laboratory raised carp was determined by an in vitro approach of the alternate complement activity (ACH50) and total haemolytic activity (CH50), and by measurement of serum C3 levels. The lysis of target sheep red blood cells (RBC) in the haemolytic assay for CH50 activity depended, amongst others, on the haemolysin concentration in the assay. Rocket electrophoresis showed a mean serum C3 concentration of 0.95 mg ml-1. The variation for both ACH50 and CH50 haemolytic activity was approximately 30%. The degree of genetic determination of the parameters was investigated by estimation of their repeatabilities, which were relatively high for CH50 (0.71) and ACH50 activity (0.72), but lower for C3 levels (0.54). Correlations between ACH50 values and C3 levels were significant, but moderate (0.54-0.58).

Animals↗
PubMed1993

Human complement factor B: functional properties of a recombinant zymogen of the alternative activation pathway convertase.

The human complement factor B is a centrally important component of the alternative pathway activation of the complement system. Here we report the isolation, characterization and eukaryotic expression of the first full length cDNA transcript for human factor B. In a factor B dependent haemolysis assay, the recombinant human factor B generated by transient COS cell transfection was shown to reconstitute haemolytic activity of factor B depleted human serum. To study the biological activities assigned to factor B, the availability of recombinant polypeptides representing definite portions of the human factor B molecule is desirable.

Animals↗
PubMed1991

Transfection of human CD59 complementary DNA into rat cells confers resistance to human complement.

We have examined the role of the human CD59 antigen in inhibiting complement-mediated lysis by transfer and expression of a CD59 cDNA in rat cells. A cDNA encoding CD59 was subcloned into the expression vector pSFSVneo and stably transfected into the rat T cell line NB2-6TG. Indirect immunofluorescence staining using the anti-CD59 monoclonal antibody YTH53.1 demonstrated the presence of human CD59 antigen on transfected cells and its attachment to the cell surface by a rat glycolipid anchor. Transfected cells were found to contain a single 3.3-kb species of CD59 mRNA by Northern blot hybridization. Immunoblotting revealed that this encoded a protein band of the same size as that observed in human erythrocytes. To determine the biological effect of expression of human CD59 in rat cells, an assay was devised which measured the relative lysis of transfected cells compared to untransfected cells in the presence of human complement and a lytic monoclonal antibody. It was observed that CD59-transfected rat cells are less susceptible to lysis by human complement and that this effect was blocked by a F(ab')2 fragment of YTH53.1. These experiments provide a direct demonstration that CD59 can function as an homologous complement restriction factor for nucleated cells.

Animals↗
PubMed1997

Neutralization of cytomegalovirus virions: the role of complement.

Complement provides a key immunologic defense against invading pathogens; thus, a clear understanding of the interactions between cytomegalovirus (CMV) and complement may permit the development of strategies to enhance CMV neutralization. In the presence of specific anti-CMV antibodies, complement enhanced the neutralizing ability of serum by 2- to 3-fold. However, in the absence of specific anti-CMV antibodies, complement was ineffective in neutralizing CMV virions by plaque assay. Although complement alone did not mediate any neutralizing effect, CMV consumed complement activity from seronegative serum, resulting in the deposition of C3 on the virion. However, only in the presence of specific anti-CMV antibody did complement activation continue to the deposition of C9 on the virions. These results strongly suggest complement regulation by CMV virions that is modulated by anti-CMV antibody; this regulation may be attributed to three host complement regulators on the virions: CD55, CD46, and CD59.

Antibodies, Viral↗
PubMed1999

[Effects on the amount of total hemolytic complement levels(CH50) and immunoglobulin in serum induced by the implantation of biomaterials into rats].

Three kinds of biomaterials, PES, YP and SR, were implanted into the subcutaneous tissue of rats and stayed there for 7, 14, 30, 60 and 90 days. Then using the immunity assaying system (Beckman, ICS) and the standard method of measuring the value of total hemolytic complement levels (CH50), we determined the values of immunoglobulin and CH50 in serum of rats. The results showed the amounts of IgG and IgA of rats with the implanted YP and SR materials were significantly higher than those of the control groups, and their value of CH50 was significantly lower than that of the control group. There was no significant difference between the PES group and the control group.

Animals↗
PubMed1993

Vitronectin-mediated inhibition of complement: evidence for different binding sites for C5b-7 and C9.

In the activated complement system, vitronectin (complement S-protein) occupies the metastable membrane binding site of the nascent precursor complex C5b-7, so that the newly formed SC5b-7 is unable to insert into cell membranes. Some evidence also indicates that vitronectin limits on-going membrane-associated pore formation by inhibiting C9 polymerization. It has been assumed that these two stages of terminal complement complex (TCC) inhibition take place through charge interactions between the heparin-binding region of vitronectin and homologous cysteine-rich sequences of the late complement proteins C6, C7, C8 and C9. We examined SC5b-7 formation and inhibition of C9 binding in the TCC using separate haemolytic assays. The mode of action of vitronectin in these assays was compared with two 15mer peptides which span residues 348-379 of the heparin-binding region, and a heparin-affinity polypeptide, protamine sulphate. The results showed that vitronectin acts predominantly through SC5b-7 production with a lesser effect on the inhibition of C9 lytic pore formation. In contrast, protamine sulphate did not prevent C5b-7 membrane attachment, but was a potent inhibitor of C9-mediated lysis. The peptides did not inhibit C5b-7 membrane insertion and only one affected C9 binding. These data suggest that the two stages of TCC inhibition involve separate binding sites on the vitronectin molecule. The site for association with nascent C5b-7 is unknown, whereas inhibition of C9 binding and pore formation takes place through the heparin-binding region.

Amino Acid Sequence↗
PubMed1991

Complement-dependent antibody-mediated cytotoxicity (C'AMC) to pancreatic islet cells in the spontaneously diabetic BB/OK rat: interference from cell-bound and soluble inhibitors.

Sera from 30 newly diagnosed diabetic BB/OK rats were analyzed cross-sectionally for complement-dependent antibody-mediated cytotoxicity (C'AMC) to pancreatic islet cells using different 51Cr release test systems. The sera contained enough active complement to lyse either sheep erythrocytes or 51Cr-labelled rat islet cells that had been sensitized with specific rabbit antibodies, but, in the presence of BB/OK rat islet cell antibody they released significant amounts of islet cell-bound 51Cr only after addition of rabbit complement. In a one-step assay, 19 out of the 30 sera produced lysis significantly above that by sera from the non-diabetes-prone WOK strain. This was increased 2.5-fold (p less than 0.01) by briefly washing the serum-treated cells before adding complement (two-step assay), indicating that C'AMC inhibitory activity was present in the diabetic sera. Some inhibitory activity could still be detected in heated sera but only when they were added to the cells immediately before the rabbit complement. Islet cell lysis was still substantial after preferential inactivation of factor B of the alternative complement activation pathway (by heating at 50 degrees C), and thus mainly depended on the classical pathway. From these findings it is concluded that (a) islet cell surface antibodies in diabetic rats activate heterologous complement via the classical pathway, (b) anti-islet C'AMC is sensitive both to species-restricted interference in interactions between homologous antibody and homologous complement in the target cell membrane, and to a distinct serum inhibitor that impairs the ability of membrane-fixed BB/OK rat antibody to interact with rabbit complement.

Animals↗
PubMed1989

A hemolytic assay for the measurement of equine complement.

A hemolytic assay was developed for the measurement of functional equine complement activity. The assay utilizes antibody sensitized chicken erythrocytes as the target cell and was specific for classical pathway (antibody dependent) complement activity. The assay was found to be reproducible and more sensitive than previous reports using other species of target cells. Total serum complement (CH50) values were determined for five mares and their foals and followed over a period of 3 months.

Animals↗
PubMed1992

The effects of complement depletion on corneal inflammation in rats.

There are indications that complement activation may be involved in inflammatory processes of the cornea. To investigate the role of the complement system in experimental keratitis, rats were depleted of their plasma complement by treatment with cobra venom factor (CVF). intraperitoneal injection of CVF resulted in undetectable complement serum activity for 6 days when measured by a hemolytic assay. The corneal inflammatory response, induced by a single intracorneal injection of heterologous serum into nonsensitized CVF-treated rats, was suppressed significantly. The onset of the clinical symptoms of keratitis was delayed, and the severity was reduced. In addition, analysis of the serum antibody titers showed impaired antibody synthesis in the CVF-treated group. When keratitis was induced by an intracorneal antigen challenge in sensitized rats, no difference was observed when comparing the clinical signs of keratitis of CVF-treated animals with sham-treated animals. In addition, CVF treatment did not alter the course of lipopolysaccharide (LPS)-induced corneal inflammation. These findings suggest that the role of plasma complement activation in antigen- and LPS-induced keratitis appears to be limited. The implications for the immunopathologic mechanisms underlying keratitis are discussed.

Animals↗