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Results for “Complement Hemolytic Activity Assay”

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PubMed1990

Leukocyte depletion ameliorates free radical-mediated lung injury after cardiopulmonary bypass.

Activated leukocytes and oxygen free radicals have been implicated in the pathogenesis of lung injury associated with cardiopulmonary bypass. To determine whether leukocyte depletion could prevent this injury, we used a dog model simulating routine cardiac operations. Mongrel dogs (11 to 17 kg) were subjected to cardiopulmonary bypass with a bubble oxygenator and cooled to 27 degrees C. After aortic crossclamping and cardioplegic arrest for 90 minutes, control animals (n = 5) were rewarmed and weaned from bypass, and their condition was then stabilized for 90 minutes. Leukocyte-depleted animals (n = 5) had a leukocyte filter incorporated in the bypass circuit. During bypass, circulating leukocyte counts decreased by 60% in control dogs, and by 97% in leukocyte-depleted animals. Free radical generation (estimated by spectrophotometric assay of plasma conjugated dienes) was significantly reduced by leukocyte depletion during and after bypass. Total hemolytic complement activity and the titer of C5 decreased markedly immediately after the onset of bypass in both the control and leukocyte-depleted animals. Pulmonary function after bypass was better preserved in leukocyte-depleted animals. These data suggest that depletion of circulating leukocytes contributes to lung injury during cardiopulmonary bypass and is associated with increased oxygen radical activity, pulmonary edema, and vasoconstriction. Leukocyte depletion substantially reduced the pulmonary injury seen after cardiopulmonary bypass.

Animals↗

PubMed1991

The Fab/c fragment of IgG produced by cleavage at cyanocysteine residues.

The intra- and inter-heavy chain disulfides of rabbit IgG were cleaved by mild reduction with either dithiothreitol or sulfite and cyanocysteines generated by treatment with either 2-nitro-5-thiocyanobenzoic acid or KCN, respectively. When cleavage occurs at a cyanocysteine residue in the hinge region of one heavy chain alone the Fab/c fragment is produced. Fab/c was also produced by papain digestion of IgG. Fab/c made by papain digestion was able to active complement in haemolytic assays; this activity was lost after cleavage of its accessible disulfide bonds. Fab/c made by cyanylysis of sulfite-reduced IgG was also active in these assays, but Fab/c made by cyanylysis of dithiothreitol-reduced IgG was not. Treatment of the latter fragment with cysteine and cystine resulted in partial reformation of cleaved disulfide bonds. Fab/c was also made from human IgG and from murine IgG2a and IgG2b.

Animals↗

PubMed1992

Heparin and modified heparin inhibit complement activation in vivo.

Heparin regulates C activity in vitro, but has not been examined for this activity in vivo. The present study investigated the ability of commercial heparin and derivatized (N-desulfated, N-acetylated) heparin (Hep-NAc) with greatly diminished anticoagulant activity to inhibit C activation in guinea pigs. Catheters were placed in the right atrium of guinea pigs and kept patent with frequent saline flushes. The next day, heparin, Hep-NAc, or saline was given and 2.5 min later cobra venom factor or saline was given. Blood was drawn at intervals and assayed for total hemolytic C, C3 hemolytic activity, free hemoglobin, and activated partial thromboplastin time. Total hemolytic C and C3 activity decreased less rapidly in heparin- and Hep-NAc-pretreated animals than in non-pretreated animals, indicating that both heparins inhibited C activation. Heparin and Hep-NAc also inhibited cobra venom factor-induced hemolysis. This study demonstrates that commercial heparin and modified heparin inhibit C activation in vivo. This represents an important step in the development of an oligosaccharide drug to regulate C activation.

Animals↗

PubMed2006

Toxin(s), other than cholera toxin, produced by environmental non O1 non O139 Vibrio cholerae.

A total of 39 Vibrio cholerae non O1 non O139 strains were isolated from surface waters of different parts of Dhaka City, Bangladesh. All these strains showed lack of ctx or zot gene, as demonstrated by the PCR analysis. Eighteen representative strains were tested for enterotoxin production using a rabbit ileal loop model, of which live cells of 8 strains and culture filtrates of 6 strains produced fluid accumulation in ileal loops. However, none of them produced heat stable toxin (ST), as detected by suckling mouse assay. On the other hand, 15% of isolates produced cytotoxin as detected by the Chinese Hamster Ovary (CHO) cell assay. Fifty times concentrated culture filtrates of the representative strains did not give any precipitin band against the anti-cholera toxin, suggesting the strains produced an enterotoxin, which is antigenically different from known cholera toxin (CT). Eighty percent of the total isolates were found to be positive for heat labile haemolysin detected by tube method, whereas, 39% were found positive by the Christie-Atkins-Munch-Petersen (CAMP) method. However, 87% of the isolates were positive for haemagglutinin/protease and all of the strains were positive for mannose-sensitive-haemagglutinin assay.

Animals↗

PubMed1980

Activation of complement by plicatic acid, the chemical compound responsible for asthma due to western red cedar (Thuja plicata).

Plicatic acid, a low-molecular-weight compound responsible for western red cedar (Thuja plicata) asthma was tested for its ability to activate complement and to generate chemotactic activity from pooled normal human serum (NHS). Dose-dependent complement consumption was found as determined by hemolytic assay (CH50). Activation of complement by plicatic acid was also confirmed by the demonstration of conversion of C3 to C3b on immunoelectrophoresis. This activation was completely prevented by pretreating the serum with either edetate (EDTA) or ethylene glycol tetraacetic acid (EGTA), suggesting that complement was activated via the classical pathway. No conversion of factor B was seen in any of the samples. Leukocyte chemotactic activity was also generated when serum was incubated with plicatic acid. The consumption of C3 and CH50 was unimpaired in two samples of serum from patients with severe, untreated hypogammaglobulinemia and thus appears to be immunoglobulin independent. These observations suggest that plicatic acid could activate complement in vivo, thereby inducing an inflammatory response in the airways and contributing to the higher prevalence of industrial chronic bronchitis in exposed subjects. The pathogenetic role of complement activation in red cedar asthma has yet to elucidated.

Animals↗

PubMed2003

A simple and convenient microtiter plate assay for the detection of bactericidal antibodies to Vibrio cholerae O1 and Vibrio cholerae O139.

It is believed that the correlate of protection for cholera can be determined by the serum vibriocidal assay. The currently available vibriocidal assays, based on the conventional agar plating technique, are labor intensive. We developed a simple and convenient microtiter plate assay for the detection of vibriocidal antibodies that is equally as efficient for Vibrio cholerae O1 and for V. cholerae O139. The addition of succinate and neotetrazolium made it possible to measure the growth of surviving bacterial target cells by monitoring a color change. We evaluated assay parameters (target strains, growth of target cells, complement source and concentration) that may affect the reproducibility of the method for V. cholerae O139. The results obtained with the microtiter plate assay were uniformly similar to those obtained with the conventional agar plating assay, when testing both the Inaba and Ogawa serotypes of V. cholerae O1. The microtiter plate assay was also convenient for measuring the activity of animal sera and mouse monoclonal antibodies.

Animals↗

PubMed1991

[The complement activation system in acute poststreptococcal glomerulonephritis].

The activation of the complement system is very important part of the immunologic process. Measuring levels of products of complement activation is a useful diagnostic and prognostic procedure. This report presents the results of a follow-up of 54 children with acute poststreptococcal glomerulonephritis. At the beginning of the disease, all patients (i. e. 100%) had decreased levels of C3 component of the complement system, and 72.2% patients had decreased levels of the haemolytic complement. The mean value of the control measurements increased significantly from one measurement to another (with p < 0.01, and within 6 weeks after the beginning of the disease, it reached the normal range. According to our results, the activation of the complement system was induced by the classical pathway. We cannot explain why the component C4 did not take part in it. According to the results of the haemolytic assay, the alternative pathway of activation was used too. We found that there was a correlation between improvement of clinical features and decrease of metabolisation of complement components.

Acute Disease↗

PubMed2000

Susceptibility of Vibrio cholerae O139 to antibody-dependent, complement-mediated bacteriolysis.

Volunteer studies with Vibrio cholerae O1 have shown that the best correlate of a vaccine's protective efficacy is its propensity to elicit serum bactericidal responses in its recipients. Attempts to detect such responses following infection with V. cholerae O139, however, have met with varying success. Using a tube-based assay which involves viable counting, we now report that strains of serogroup O139 can appear to be sensitive or resistant to a fixed concentration of complement in the presence of antibody, depending on assay conditions. Susceptibility to lysis is critically dependent on the availability of complement, but with O139 indicator strains this is not simply determined by the concentration of serum added to the reaction mix. The nature of the assay diluent and the concentration of indicator bacteria can also dramatically affect bactericidal end points, whereas such variables have minimal significance with O1 indicator bacteria. Although some laboratories use unencapsulated mutant strains to seek evidence of seroconversion following exposure to V. cholerae O139, this is not necessary, and our findings question the significance of capsule expression as a determinant of complement sensitivity when antibody is present. The medium used for growth of the indicator strain and the particular strain used appeared to be unimportant. Each of seven O139 isolates tested was found to be lysed by antibody and complement in our standard assay system, which allowed the detection of significant serum bactericidal responses in 9 of 11 cases of O139 disease.

Antibodies, Bacterial↗

PubMedDate not supplied

Assessment of the reproducibility of alginate encapsulation of pancreatic islets using the MTT colorimetric assay.

Radioisotope diffusion experiments demonstrate that alginate/polyaminoacid encapsulation can prevent antibody and cytotoxic cell contact in vitro. The unpredictable outcome of xenotransplantation of encapsulated islets may reflect incomplete encapsulation. We have assessed a cytotoxic/MTT (tetrazolium) assay to test antibody permeability of capsules. Samples of free porcine islet tissue, and islet tissue encapsulated in alginate/poly-L-lysine/alginate microspheres were incubated with fresh autologous pig serum or normal human serum overnight. Cell metabolism was assessed by the MTT assay. Data from eight experiments (10 replicates/experiment) were analyzed using the Mann-Whitney U-test. Values were deemed significant when p < 0.05. Free islets cultured in human serum showed a significant reduction in metabolism when compared with islets cultured in pig serum: percentage reduction 52 +/- 23% (mean +/- SD). The differences in formazan production were significant in all experiments (p < 0.05). Alginate encapsulation of islets or removal of xenoreactive antibodies in human serum by adsorption was shown to prevent the effects of complement-mediated lysis. However, in one of the eight experiments, there was a significant reduction in islet metabolism after exposure of encapsulated porcine islets to human serum. In conclusion, it has been shown that alginate encapsulation can prevent complement-mediated lysis. However, the encapsulation process employed was imperfect and did not prevent complement-mediated lysis of porcine islets in all experiments. The cytotoxicity/MTT assay allows investigation of the permeability of capsules to serum antibodies and could be performed to determine the viability of the islets and the integrity of microcapsules prior to transplantation.

Alginates↗

PubMed1979

Deficiency of the ninth component of complement in man.

The studies of serum from a case with C9 (the ninth component of complement) deficiency are described. A 29-year-old woman in good health was found to have low serum complement levels (CH50). C9 of her serum was undetectable by the hemolytic assay and by the immunochemical analysis but all other components were normal. It was demonstrated that low CH50 of her serum was due to the hemolysis of the sensitized sheep erythrocytes (EA) by the complement components from C1 to C8.

Adult↗

PubMed1998

[Studies on the conditions of blood sampling and storage for the liposome-based CH50 assay].

The CH50 values in the serum and plasma, especially those from chronic hepatitis caused by hepatitis C virus (HCV), are strongly affected and reduced through a process known as cold activation. We attempted to optimize the conditions of blood sampling and storage for the CH50 assay with a recently developed liposome-based assay kit. The bloods were obtained from HCV hepatitis patients as well as healthy donors. Regardless of the temperature (room temperature, 4 degrees C or 37 degrees C) at which samples were kept until the assay, higher values were always obtained in the serum than in the plasma. The plasma samples could either be heparinized or given any of the other anticoagulants, EDTA-2K and sodium citrate, at the time of sampling. We also attempted to optimize the temperature at which the fresh specimens were left during the period from sampling to assay and the temperatures to freeze them for storage and to thaw for assays. In the assays immediately after sampling, higher values were obtained when the specimens were left at 37 degrees C than at room temperature or 4 degrees C. To store at -80 degrees C rather than at -20 degrees C and to thaw rapidly at 37 degrees C rather than slowly at room temperature were found to be advantageous.

Blood Preservation↗

PubMed1989

[Enterobacterial antigen in human peripheral blood lymphocytes].

The following study has as prior history the research reports which have shown the existence of an antigenic tissue deposit in gram-negative enterobacteria. The antigens of the enterobacteria have also been found in the lymphocytic membranes and cytoplasm. Since intestinal lymphoid tissue cells can recirculate by means of the thoracic duct to the peripheral venous system, it was proposed that the circulating lymphocytes in healthy people could also contain small amounts of a common enterobacterial antigen. The study was carried out in 15 human venous blood samples, of which the lymphocytic population was separated to later be used in the preparation of 15 alcohol soluble extracts. This material was used for inhibiting the immuno-hemolysis assay in three occasions in order to show the presence of antigens shared by different enterobacterias, using as reference a fraction separated from the LPS of Escherichia coli 08. The results showed that the human lymphocytes also had antigenic determinants common to gram-negative bacteria.

Antigens, Bacterial↗

PubMed1978

A microassay for the determination of hemolytic complement activity in mouse serum.

A 51Cr release microhemolytic complement assay is described to detect hemolytic complement activity in mouse serum, 51Cr-labeled sheep erythrocytes (SRBC) which have been sensitized with a subagglutinating amount of the 7S IgG fraction of rabbit anti-SRBC serum are placed in microtiter plates and further antibody added to each well prior to the addition of the complement (C) source. The IgG antibody was found to be more efficient in the lytic assay than the 19S IgM antibody. The assay is simple to perform, reproducible, and requires small volumes of mouse serum. Comparative hemolytic values were established for serum from a variety of murine strains using pooled BALB/c serum as the C reference. No apparent relationship was noted between the H-2 type of inbred mouse strains and the presence or absence of hemolytic complement.

Animals↗

PubMed1991

Haemolysin occurrence among Aeromonas hydrophila, Aeromonas caviae and Aeromonas sobria strains isolated from different aquatic ecosystems.

A total of 909 Aeromonas spp. isolates from different aquatic ecosystems were tested for haemolysin production by both sheep and horse blood agar-plate assays and by rabbit erythrocytes in broth assay. A comparison of these different methods was undertaken in order to appreciate their capacity to evaluate the haemolytic activity of Aeromonas spp. isolated from aquatic ecosystems. The haemolytic activity was associated particularly with A. hydrophila and A. sobria (about 95% of strains), whereas A. caviae did not produce haemolysin (about 95% of strains). A method suitable for use in routine diagnostic microbiology laboratories is proposed for quantifying both groups of A. hydrophila/A. sobria and A. caviae in environmental water.

Aeromonas↗

PubMed1994

Liposome-complement interactions in rat serum: implications for liposome survival studies.

Serum complement opsonizes particles such as bacteria for clearance by the reticuloendothelial system. Complement has been reported to interact with liposomes and therefore may mediate the reticuloendothelial system clearance of liposomes. This study has used a rat serum model to define some of the characteristics of liposomes which modulate their ability to activate complement. Using functional hemolytic assays and C3/C3b crossed immunoelectrophoresis, we have demonstrated that liposomes activated rat complement in a dose-dependent manner with higher concentrations of liposomes activating higher levels of complement. The detection of complement activation required the inclusion of phospholipids bearing a net charge. Complement activation occurred via the classical pathway; no alternative pathway activation was detected. The presence of cholesterol contributed to complement activation in a dose-dependent manner. Phospholipid fatty acyl chain length did not influence complement activation while the introduction of unsaturated acyl chains markedly decreased levels of complement activation. Liposome size also influenced complement activation with 400 nm unilamellar vesicles more effectively activating complement than 50 nm vesicles for equivalent amounts of exposed lipid. These studies demonstrate that the composition of the liposome greatly affects the in vitro activation of rat serum complement and suggest that the biological half-life of liposomes in the circulation of rats may be altered by changing the liposome composition to reduce complement activation.

Animals↗

PubMed1998

Sphingomyelinases in the venom of the spider Loxosceles intermedia are responsible for both dermonecrosis and complement-dependent hemolysis.

The bite of spiders of the genus Loxosceles can induce a variety of biological effects, including dermonecrosis and complement (C) dependent haemolysis. The aim of this study was to characterise the toxins in the venom responsible for the different biological effects. We have previously shown that a 35 kDa protein, named F35, purified from Loxosceles intermedia venom, incorporates into the membranes of human erythrocytes and renders them susceptible to the alternative pathway of autologous C. Here we have further purified the F35 protein which was resolved by reversed phase chromatography into three tightly contiguous peaks termed P1, P2, and P3. P1 and P2 were shown to be homogeneous by SDS-PAGE and N-terminal aminoacid analysis, while P3 consisted of two highly homologous proteins. N-terminal sequencing of all four proteins showed a high degree of homology, which was confirmed by cross-reactivity of antisera raised against the individual purified proteins. Functional characterisation of P1 and P2 indicated the presence of sphingomyelinase activity and either protein in isolation was capable of inducing all the in vivo effects seen with whole spider venom, including C-dependent haemolysis and dermonecrosis. In all assays, P2 was more active than P1, while P3 was completely inactive. These data show that different biological effects of L. intermedia venom can be assigned to the sphingomyelinase activity of two highly homologous proteins, P1 and P2. Identification of these proteins as inducers of the principal pathological effects induced by whole venom will aid studies of the mechanism of action of the venom and the development of a effective therapy.

Amino Acid Sequence↗

PubMed2004

Adjuvant synergy: the effects of nasal coadministration of adjuvants.

Modern peptide and protein subunit vaccines suffer from poor immunogenicity and require the use of adjuvants. However, none of the currently licensed adjuvants can elicit cell-mediated immunity or are suitable for mucosal immunization. In this study we explored the immunological effect of nasal co-administration of adjuvants with distinct functions: cholera toxin subunit B, a potent mucosal adjuvant that induces strong humoral responses, muramy di-peptide (MDP), an adjuvant known to elicit cell mediated immunity but rarely used nasally, and chitosan, an adjuvant that achieves specific physiological effects on mucosal membranes that improve antigen uptake. Groups of five female BALB/c mice received on days 1 and 56 nasal instillations of the recombinant Helicobacter pylori antigen urease admixed to single or multiple adjuvant combinations. Serum IgG kinetics were followed over 24 weeks. At the conclusion of the experiment, local antibody responses were determined and antigen-specific recall responses in splenocyte cultures were assayed for proliferation and cytokine production. The combination of adjuvants was shown to further contribute to the increased antigenicity of recombinant H. pylori urease. The data presented here outline and support facilitation of increased immunomodulation by an adjuvant previously defined as an effective mucosal adjuvant (chitosan) for another adjuvant (MDP) that is not normally effective via this route.

Adjuvants, Immunologic↗

PubMed1991

Functionally active complement proteins C6 and C7 detected in C6- and C7-deficient individuals.

Two sensitive sandwich ELISAs based on monoclonal antibodies directed to native C6 and C7 allowed the detection and quantitation of these complement proteins in 20 out of 37 serum samples from individuals who had previously been classified as deficient in these proteins as assessed by immunochemical and/or functional assays. Furthermore, serum from four C6-deficient and one combined C6-/C7-deficient individual showed an increase in the terminal complement complex (TCC) and a decrease in native C6 and C7 after complement activation as assayed by specific ELISAs. Despite their (incomplete) deficiencies, these individuals therefore possess functionally active terminal complement proteins with respect to their ability to generate the TCC. As these individuals have no history of a susceptibility to neisserial infections, even low concentrations of functionally active C6 and C7 may provide sufficient protection against those micro-organisms whose destruction requires TCC formation.

Antibodies, Monoclonal↗