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Results for “Complement Hemolytic Activity Assay”

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PubMed1991

A new quantitative assay for the determination of complement activity.

The suitability of the algal assay and the hemolysis method for the determination of complement activity was investigated with regard to reproducibility and quantification. It was shown that the day-to-day reproducibility is higher in the algal assay than in the hemolysis method in test tubes. The lysis of algal cells depends on serum concentrations. The extensive linear relationship between cytotoxic activity and serum concentration prevails at lysis rates between 20 and 80%. Therefore, it is often sufficient to measure the lysis rate at one serum concentration only. The algal assay enables the investigation of changes in complement activity, even if the changes are not expected to be drastic.

Animals↗

PubMed1993

Effect of low temperature storage on the haemolytic complement of cattle and sheep.

Sera from cattle and sheep stored at -70, -20 and -10 degrees C were examined for haemolytic complement at Days 0, 37, 54, 86, 133 and 245. The decline in complement followed a similar trend, both by classical pathway (CPW) and alternative pathway (APW) assay methods. Both cattle and sheep sera retained 80% of their complement activity till Day 245 at -70 degrees C whereas at -20 degrees C cattle sera had lost 67% and sheep sera 80% of the activity. At -10 degrees C the decline in complement levels was, in general, similar to -20 degrees C. The complement level of sheep sera declined more rapidly than that of cattle. For reliable results, it is suggested that sera of cattle and sheep should be assayed for complement within 4-5 months at -70 degrees C and within a month at -20 or -10 degrees C.

Animals↗

PubMed1993

Effects of a streptococcal lipoteichoic acid on complement activation in vitro.

This study describes activation of serum complement by lipoteichoic acid (LTA) from Streptococcus mutans OMZ 176, while in solution. Serum from 16 healthy students was taken. Test samples were incubated with increasing doses (1-5,000 micrograms/ml) of LTA or lipopolysaccharide (LPS) from Escherichia coli 0111:B4 for 1 h at 37 degrees C; then assayed for degradation of C3, C4 or factor B by crossed immunoelectrophoresis. Each preparation caused a significant (p < 0.05) dose-dependent conversion of C3. The response curves obtained were not statistically different. LPS was a stronger activator of the alternative pathway than LTA, as judged from analysis of C3 degradation in the presence of Mg2+/EGTA, and from their effects on factor B cleavage. LTA caused, however, pronounced alterations in the shape of C4 precipitation in the gels. Functional (hemolytic) assays showed that, when tested at 200 micrograms/ml, LTA and LPS triggered significant (p < 0.05) consumptions of both classical and alternative pathway proteins. LPS was a significantly (p < 0.05) stronger activator than LTA. Apparently, the C3 degradation found for this LTA involved the alternative pathway to a small extent; thus some other mechanism of fluid-phase C3 cleavage seemed also to be operative.

Complement Activation↗

PubMed2006

Which complement assays and typings are necessary for the diagnosis of complement deficiency in patients with lupus erythematosus? A study of 25 patients.

INTRODUCTION: Deficiencies in components of the classical pathway of complement activation are strong risk factors for lupus erythematosus (LE).Yet, it has not been addressed whether the conventional measurements of the serum hemolytic CH50 activity and antigenic concentrations of C3 and C4 are sufficient to asses a deficiency in C4A, C4B or C2 components, the most common deficiencies associated with LE. PATIENTS AND METHODS: In a retrospective series, we performed complement analyses in 35 patients with LE who were systematically screened for a complement deficiency. The majority of patients had cutaneous LE with mild systemic involvement and no complement consumption. Of 25 patients (72%) with complement deficiency we found 13 with a partial C4A deficiency, 2 with a complete C4A deficiency, 6 with a partial C4B deficiency, 2 with a complete C4B deficiency and 2 with a combined partial C2 and C4A deficiency. RESULTS: The total complement activity (CH50) was decreased in only one out of two patients with complete C4B deficiency. CH50 level was found to be low-normal (35-38 U/ml(-1)) in one patient with partial C4B deficiency, one patient with complete C4B deficiency and both patients with combined partial C4A and C2 deficiency. Total C4 levels were normal in 9 out of 13 the patients with a partial C4A deficiency and in 2 out of 6 patients with a complete C4B deficiency. The antigenic concentration of C3 was low in only 1 patients with a complete C4B deficiency and within the normal range in all the others patients. Overall, 50% of the patients had normal or elevated C3, C4, and CH50 levels. DISCUSSION: This study emphasizes that the usual measurements of CH50, C3 and C4 levels are not adequate to detect a C4 and/or C2 deficiency in patients with LE. In epidemiologic or investigative studies addressing the prevalence of complement deficiency, more elaborated diagnostic tests, such as C4 protein allotyping, C2 level measurement and genetic screening for type I C2 deficiency should also be performed.

Adult↗

PubMed2003

Validation of colorimetric assay to detect complement-mediated antibody-dependent bactericidal activity against serogroups B and C Neisseria meningitidis.

Colorimetric serum bactericidal assay (cSBA), based on the addition of glucose and a pH indicator to the culture medium after the bactericidal reaction, was validated. The precision measured as repeatability, intermediated precision, and reproducibility was determined as a percentage in titer coincidence between replicas >/=50. Moreover the use of the freeze-dried complement was evaluated in comparison to the traditionally stored by freezing. The results were the following: precision: titer +/-1 two-fold dilution (except for the highly positive serum against serogroup B, where there was titer +/-2 dilutions) percentage in titer coincidence: >/=50. The known titer or +/-1 two-fold dilution was found in the sera titrated with the complement either frozen or freeze-dried. Concluding, cSBA showed to be highly precise, allowing also the use of freeze-dried complement which is another important advantage for this kind of assay.

Animals↗

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↗

PubMed2004

Investigation of the activation of a human serum complement protein, C3, by orthopedic prosthetic particulates.

Myriad molecular, cellular, and physiological processes underlie the inflammatory and osteolytic processes induced by particles of biomaterials resulting from the wear of implants such as total joint replacement prostheses. The objective this study was to investigate the role that the complement system may be playing in these phenomena. The aim was to evaluate the degree to which particles of selected orthopaedic materials--high density and ultrahigh molecular weight polyethylene, polymethylmethacrylate, and commercially pure titanium--cause the elevation of a key complement molecule, C3a, in an in vitro assay that directly measured the concentration of C3a. The results demonstrated that HDPE particles, at high concentration, are capable of causing the elevation of C3a in the in vitro assay. This finding is discussed in the context of other work and the mechanics of the complement system as it may affect the osteolytic process.

Biocompatible Materials↗

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↗

PubMed2004

The complement system is involved in acute inflammation but not in the hemorrhage produced by a Bothrops atrox snake venom low molecular mass proteinase.

Low molecular weight hemorrhagins were purified from crude Bothrops atrox snake venom by gel filtration followed by ionic strength chromatography. The protein fractions obtained, designated HI-1 to HI-8, contained proteins with molecular masses lower than 30 kDa. HI-5, the most representative among of these fractions, exhibited, in vitro, proteolytic and C inactivating properties, as analyzed by proteolysis of a protein substrate, and C system consumptive activities as assayed by reduction of the hemolytic C activity in normal human serum and by cleavage of partially purified component C3. HI-5 hemorrhagin injected i.m. into C-sufficient BALB/c mice induced a local inflammation characterized by edema, accumulation of polymorphonuclear leucocytes (PMN) and hemorrhage. In contrast, when injected into BALB/c mice previously C-depleted, the number of PMN per tissue section, but not hemorrhage, was significantly reduced (129.668 +/- 31.341 cells per microscopic field) as compared with the control C-sufficient mice (812.168 +/- 111.194 cells per microscopic field). The observations were confirmed by using C5-deficient mice instead of C-depleted mice. The average number of PMN per tissue section in C5-defficient A/J mice was 72.666 +/- 19.416 cells per microscopic field. These data indicate that the C system is involved in PMN accumulation, but not in the hemorrhage, at the local induced lesions by low molecular mass B. atrox hemorrhagins. HI-5 apparently is not contaminated with other direct or indirect inflammation mediators, PMN accumulation and hemorrhage, however, an independent phenomenon, could be mediated by the same hemorrhagin proteinase domain.

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↗

PubMed2004

Prevention of complement-mediated immune hemolysis by a small molecule compound.

Complement sensitization of red blood cells (RBCs) can result in transfusion reactions and hemolytic anemias. We hypothesized that manipulating the complement system using small organic molecules might prevent RBC destruction, thereby prolonging RBC survival in patients. Using a simple, rapid, large-scale hemolytic assay, we screened a 10,000 compound library, enriched in anti-inflammatory compounds at a final concentration of 25 microM, and identified a 549Da compound (C(34)H(24)N(6)O(2)) with a symmetrical structure containing two benzimidazole rings that, as compared to a known anti-complement molecule FUT-175, was more effective in reducing hemolysis by the classical pathway and had comparable anti-hemolytic activity against the alternative pathway. Furthermore, in a xenotransfusion mouse model, treatment of mice with 1.2mg/kg of the compound significantly prolonged the survival of transfused RBCs, reducing C3 deposition, but not the deposition of control IgG or IgM, for the first hour post-transfusion. These data suggest that further studies are warranted to determine if this compound has usefulness in a transfusion setting.

Anemia, Hemolytic↗

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↗

PubMed2004

The role of complement in the early phase of Leishmania (Leishmania) amazonensis infection in BALB/c mice.

Complement-depleted and -non-depleted BALB/c mice were inoculated with Leishmania (Leishmania) amazonensis promastigotes into the hind footpad to study the role of the complement system in cutaneous leishmaniasis. Total serum complement activity was measured by hemolytic assay and C3 fragment deposit at the inoculation site was determined by direct immunofluorescence in the early period of infection, i.e., at 3, 24, 48 h and 7 days post-infection. The inflammatory reaction and the parasite burden were evaluated in the skin lesion at 7 and 30 days post-infection. Total serum complement activity decreased in the early phase of infection, from 3 to 24 h, in non-depleted mice compared to non-infected and non-depleted mice. C3 fragment deposit at the site of parasite inoculation was present throughout the period of infection in non-depleted mice. In contrast, no C3 fragment deposit was observed at the inoculation site in complement-depleted mice. Complement-depleted mice showed a significant decrease in the inflammatory response and a significant increase in the number of parasites (70.0 +/- 5.3 vs 5.3 +/- 1.5) at 7 days of infection (P<0.05). A higher number of parasites were also present at 30 days of infection at the inoculation site of complement-depleted mice (78.5 +/- 24.9 vs 6.3 +/- 5.7). These experiments indicate that complement has an important role at the beginning of experimental cutaneous leishmaniasis caused by L. (L.) amazonensis by controlling the number of parasites in the lesion.

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↗