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PubMed1985

Hemolytic complement activity assay in microtitration plates.

A new rapid technique is developed for the determination of complement activity in a large number of samples. Following serial dilution of complement, hemolysis is performed in the same microtiter plate. After the reaction, the degree of hemolysis in wells of the plate is determined spectrophotometrically by measurement of "absorbance" (light scattering) at 630 nm, without additional procedures. This method can find application in clinical and experimental biochemistry for the analysis of a large (up to thousands) number of samples.

Complement System Proteins↗
PubMedDate not supplied

Assessment of complement activation in vivo.

Hemolytic assays that measure the functional integrity of the complement system and the quantitation of individual components by immunochemical techniques have been widely used in the past for the assessment of in vivo complement activation. However, the complement system comprises a large number of interacting serum proteins which are subject to independent synthetic and catabolic processes. The fact that complement proteins are rapidly metabolized under in vivo conditions adds to the complexity of complement analysis. Assays that are based on monoclonal antibodies with specificities for activation-dependent neoepitopes now allow the direct determination of complement fragments in plasma. These methods are superior to the quantitation of native proteins. Several parameters that differentially affect the generation or the catabolism of individual complement activation products still have to be taken into account when elevated plasma levels of complement fragments suggest in vivo complement activation. These factors include the binding to complement fragment receptors, the degradation by serum proteases and renal or hepatic clearance. An accurate estimation of complement activation in vivo requires the simultaneous determination of both the native components and the activation products.

Animals↗
PubMed2004

Possible mechanism for medial smooth muscle cell injury in diabetic nephropathy: glycoxidation-mediated local complement activation.

BACKGROUND: Although recent studies have emphasized the pathogenic role of intrarenal muscular arteries in patients with diabetic nephropathy, notice has not been taken of their pathological characteristics. We focused on medial smooth muscle cell (SMC) injury and the involvement of glycoxidation and complement activation. METHODS: Renal samples were obtained at autopsy from patients with diabetes mellitus (DM), patients with hypertension without renal involvement (n = 9), patients with benign nephrosclerosis (n = 7), and age-matched control subjects (n = 12). Patients with DM had glomerulosclerosis classified as severe (n = 9; DM-sev), moderate (n = 11; DM-mod), and minimal (n = 7). Renal samples were immunohistochemically determined. Activation of plasma complement from healthy subjects using advanced glycation end products (AGEs) also was performed. RESULTS: A marked SMC loss was noted in the media of patients with DM-sev and DM-mod. A membrane attack complex (MAC) observed in the area with SMC loss correlated well with the loss. Considerable carboxymethyllysine (CML), an oxidatively modified AGE, was deposited in the area with SMC loss in patients with DM-mod and DM-sev. Degrees of MAC deposition, SMC loss, and CML deposition were greater in the DM-sev group than the non-DM groups. Another oxidative product, acrolein, colocalized with CML. Plasma complements were not activated by AGE-modified bovine serum albumin in our in vitro assays, which included a complement hemolytic activity assay and determination of complement fragments, including C4d, C3bB, iC3b, and MAC. CONCLUSION: It is strongly suggested that medial SMC injury in intrarenal arteries is caused by interaction between glycoxidation and complement activation and contributes to the progression of diabetic nephropathy.

Acrolein↗
PubMed1991

A simple radial immunohemolysis assay for the measurement of functional complement C2 activity.

1. This study describes a simple radial immunohemolysis method for determining the hemolytic activity of the second component of complement (C2) in human serum. The assay is based on the recovery of hemolytic activity of normal serum which has been pretreated to inactivate endogenous C2 and then mixed with test serum containing an unknown amount of C2. 2. The pretreated serum, designated R2 reagent, is obtained by heating normal human sera under carefully standardized conditions of temperature, time, volume and type of test tube. 3. R2 reagent is incorporated into agarose together with hemolysin-sensitized erythrocytes, and spread on a plate. The test serum is placed in wells cut in the agarose and, after appropriate incubation, the diameters of the hemolytic areas are measured. The area of hemolysis is directly proportional to the logarithm of the serum concentration. As a standard for C2 functional activity, dilutions of a pool of normal sera are tested on the same plate. 4. The method is specific for C2 and can detect as little as 20% of the C2 in normal serum (about 6 micrograms C2 protein/ml). The error in reproducibility is about 3% of the mean. In normal serum, the lower confidence limit of the distribution of the C2 values (based on a sample of 80 individuals) corresponded to 70% of undiluted serum. 5. This method is suitable for use in clinical laboratories since it is simple, rapid, quantitative and inexpensive, and does not require special equipment.

Adolescent↗
PubMed1998

A small molecular weight factor in aqueous humor acts on C1q to prevent antibody-dependent complement activation.

PURPOSE: Aqueous humor inhibits activation of the classic complement pathway; however, the mechanism of this inhibition is unknown. We have examined at the molecular level potential factors responsible for the inhibition, and we have tried to determine where in the complement pathway the inhibition takes place. METHODS: Fresh rabbit aqueous humor was size fractionated by centrifuge concentrators and by size exclusion column chromatography, and each fraction was assayed for inhibition of the classic complement pathway in a standard CH50 hemolytic assay. Fractions with inhibitory activity were assayed for protein and the presence of ascorbic acid and were subjected to heat treatment. To identify where in the pathway the inhibitor(s) function, the expression of activated complement components bound to the surface of antibody-coated erythrocytes was analyzed by flow cytometry using fluorescein isothiocyanate-labeled antibodies to specific complement components. In addition, hemolytic assays were performed for the function of individual complement components. RESULTS: The most potent inhibition of the classic pathway was in a fraction of aqueous humor of less than 1.3 kDa. The inhibitory activity in the fraction was unassociated with detectable protein or ascorbic acid, and it remained present after heat treatment. The functional analysis through flow cytometry and hemolytic assays for individual complement components showed that the inhibitor in the less than 1.3-kDa fraction caused a blockade in the complement pathway at the level of C1q. CONCLUSIONS: The aqueous humor contains a unique potent anticomplementary factor that has a molecular weight less than 1.3 kDa. This heat-stable inhibitory factor inhibits the classic pathway at the level of C1q. These results imply that within the eye the complement pathway is inhibited at the earliest steps of its initiation. Such inhibition would prevent production of complement products that mediate inflammation and chemotaxis of inflammatory cells. Therefore, as part of the adaptation of immune privilege, the ocular microenvironment is protected from inflammation induced by antigen-antibody complexes.

Animals↗
PubMed1999

A novel assay for serum complement activity: C42 generation assay.

BACKGROUND: In most clinics, laboratory tests for serum complement are limited to immunochemical determinations of C3 and C4 and are occasionally extended to the hemolytic titration of total complement functional activity (CH50). However, these tests are often not sufficient for the analysis of low CH50 serum. METHODS: A novel assay for serum complement activity, the C42 generation assay, has been developed. The principle of this assay is based on the hemolysis of sensitized sheep erythrocytes (EA) by complement components in two sera: C42 (the classical pathway C3 convertase) is generated on EA by C1, C4 and C2 in the first serum, followed by a second reaction leading to hemolysis by C3-C9 supplied by the addition of the second serum in the presence of EDTA. RESULTS: This methodology permits the evaluation of two distinct serum complement activities of a test serum. The combined activity of C1, C4 and C2, as well as the combined activity of C3-C9, can be estimated from the observed degrees of hemolysis. Information obtained from this assay is helpful for the analysis of test serum determined to have decreased CH50. Several clinical cases are presented in which this assay was utilized. CONCLUSIONS: The C42 generation assay is another functional assay of serum complement which can provide information beyond that obtained from the typical serum CH50 assay. Since intermediate cells or isolated complement components are not necessary, this assay can be employed rapidly and economically in a clinical setting.

Animals↗
PubMed1999

Comparison of three different methods for measuring classical pathway complement activity.

The complement system plays an important role in host defense against infection and in most inflammatory processes. The standard 50% hemolytic complement (CH50) assay is the most commonly used method of screening patient sera for functional activity of the classical complement pathway. Our objective in this study was to compare two newer methods (the enzyme immunoassay and the liposome immunoassay) to a commercial CH50 assay for measuring total classical complement activity. We conclude that both newer methods compare well with a CH50 assay and are equally sensitive in screening routine clinical sera.

Adult↗
PubMed2001

[Anticomplement activity of Mahonia aquifolium bisbenzylisoquinoline alkaloids and berberine extract].

Crude extracts obtained from the stem bark of Mahonia aquifolium have been investigated as to the chemical composition and anticomplementary activity. The results show that their anticomplementary activity is mainly due to the alkaloid components. Especially the BBI alkaloid fraction and berberine showed a strong inhibitory effect on CH50 total hemolytic complement assay. The crude extract of M. aquifolium was less active than berberine or the fraction BBI alkaloids. The results indicate that the fraction of BBI alkaloids and berberine largely account for the immunomodulatory activity of the crude extract of M. aquifolium.

Alkaloids↗
PubMed1990

A new hemolytic assay for bovine serum complement and its application during experimental bovine anaplasmosis.

A new hemolytic assay for bovine complement is presented. Using this assay we found a significant reduction in bovine serum complement activity during the acute phase of anaplasmosis, and an increase in the sensitivity of the red blood cells (RBC) to bovine complement lysis in vitro. The new hemolytic test is performed with bovine RBC, rabbit anti-bovine RBC serum and bovine serum complement. An isotonic sucrose Tris-buffered saline solution of ionic strength 0.094 and pH 7.2 was found to be adequate for this test. The titres obtained with this new assay, which uses autologous RBC, are comparable with those obtained using the guinea pig RBC assay. The finding of a reduction in bovine serum complement during anaplasmosis may be suggestive of a mechanism responsible for the pathology of this disease.

Anaplasmosis↗
PubMed2002

[Study on CH50 levels in factor D-depleted serum].

It is generally accepted that levels of serum whole complement activity (CH50) reflect the activities of complement (C) components of the classical C pathway (CP), since CH50 is assayed by use of sensitized sheep erythrocytes (EA). However, the alternative C pathway (AP) is considered to be also activated simultaneously in the process of activation of serum CP by EA. Thus, serum CH50 levels may possibly reflect not only CP but also AP activation in CH50 assay. We studied on the influence of AP activation during CH50 assay on CH50 levels, by comparison of CH50 levels in serum samples before and after treatment of factor D depletion. Polystyrene beads carrying polyanion, poly (2-acrylamide 2-methylpropane sulfonate) (PAMPS-beads), on the surface were prepared and used for preparation for factor D-depleted serum. After treatment of pooled normal human serum (NHS) with PAMPS-beads (2.5 mg/ml of serum), serum ACH50 level decreased to be undetectable, indicating that AP activation is prohibited in PAMPS-beads-treated serum. When isolated factor D was added to this PAMPS-beads-treated serum, ACH50 level recovered to that of before treatment. Immunoblot analysis revealed that factor D band observed in NHS disappeared completely after PAMPS-beads treatment. From these results, it is clear that factor-D deficient serum is prepared by PAMPS-beads treatment. Besides, since serum CH50 level was not decreased by PAMPS-beads treatment, it may be concluded that CH50 level is not affected by AP activation during CH50 assay.

Adult↗
PubMed1997

Molecular, genetic, and functional analysis of homozygous C8 beta-chain deficiency in two siblings.

UNLABELLED: C8 deficiency is associated with an increased susceptibility to neisserial infections. We present a case of an 11 year old boy who suffered from infection with Neisseria meningitidis. Medical history of the patient and his family (n = 5) did not indicate any previous immunodeficiency symptoms. Results from the analysis of phagocyte and lymphocyte functions were within the normal range. No hemolytic activities of the classical (CH50) and the alternative (APH50) pathways of complement were measurable, and SC5b-9 protein complexes could not be detected in the patient's plasma. Further analysis by highly sensitive ELISA and functional assays revealed a complete deficiency of C8. Upon the reconstitution with purified C8 total hemolytic activity could be restored. SDS-PAGE and Western blot analysis established a deficiency of the C8 beta chain. Genetic analysis at the genomic DNA level demonstrated the common C-T mutation in exon 9 of the C8B gene. Family analysis presented the older sister with non-detectable function of C8 in serum, both parents with about half-normal C8 titres, and the younger sister with normal C8 function. The parents and both sisters were asymptomatic, although the older of the sisters presented with the same complete C8 beta-chain deficiency as the patient described. IN CONCLUSION: the common C-T mutation in the C8B genes is the genetic basis of C8 beta-chain deficiency in two members of this Bosnian family.

Alleles↗
PubMed2005

Activation of the complement system and leukocyte recruitment by Tityus serrulatus scorpion venom.

The scorpion Tityus serrulatus is considered one of the most dangerous species in Brazil. Its venom evokes an inflammatory response, although the exact mechanism of this effect is still unknown. The aim of the present study was to investigate the effect of Tityus serrulatus venom (TsV) on the complement system (CS) and on leukocyte recruitment. Complement consumption by TsV was evaluated using in vitro hemolytic assays, immunoelectrophoresis and two-dimensional immunoelectrophoresis of complement components (factor B and C3). In order to evaluate neutrophil migration induced in normal human serum (NHS) in the presence of TsV, in vitro chemotaxis assays were performed using the Boyden chamber model. In vitro TsV induced a concentration- and time-dependent reduction in hemolytic activity of the classical/lectin and alternative complement pathways, with samples of 43.0 microg and 43.4 microg, respectively, inhibiting 50% of the lytic activity. Alterations in C3 and factor B electrophoretic mobility after incubation of NHS with TsV, were identical to those obtained with zymosan (positive control). Incubation of NHS with TsV induced neutrophil chemotaxis similar to that observed with zymosan-activated serum. Our results show that TsV activates the CS, leading to factor B and C3 cleavage, to reduction of serum lytic activity and generation of complement chemotactic factors. Therefore, CS may play an important role in the inflammatory response observed upon scorpion envenomation.

Animals↗
PubMed1990

[A simplified method for the assessment of C1 esterase inhibitor function].

From the result that the activated form of C1-s(C1-s) prolonged the kinetics of hemolysis via complement, this assay was applied to assess C1 esterase inhibitor (C1INH) function. In the kinetic assay, the complement hemolytic activity was evaluated by the time which required to cause 50% reduction of the initial turbidity of sensitized sheep erythrocytes, and was expressed as T1/2. (1) T1/2 of pooled normal human sera (p-NHS) showed dose-dependent prolongation by the addition of various amounts of C1-s. (2) Preincubation of various amounts of functionally pure C1INH with the constant amounts of C1-s inhibited dose-dependently the prolongation of T1/2 by C1-s. (3) The C1INH activity of NHS was 840 +/- 80 units/ml (n = 6) and that of the C1INH deficient serum was 80 units/ml, which were calculated from the standard curve established by the addition of various amounts of purified C1INH. This test requiring only C1-s and sensitized sheep erythrocytes is simple technically and high in sensitivity, and seems to be useful for the routine assay for C1INH function of human sera.

Animals↗
PubMed2003

Possible immunomodulatory actions of Carica papaya seed extract.

Carica papaya seed extract is currently being marketed as a nutritional supplement with purported ability "to rejuvenate the body condition and to increase energy". The product claims to improve immunity against common infection and body functioning. The present study was initiated to analyze the chemical constituents of the Carica Seed Extract and determine the potential immunomodulatory properties of the different bioactive fractions. These immunomodulatory activities of crude Carica Seed Extract and its bioactive fractions were examined in vitro using lymphocyte proliferation assays and complement-mediated hemolytic assay. Three major observations were made in this study: (1) the crude Carica Seed Extract and two other bioactive fractions significantly enhanced the phytohemagglutinin responsiveness of lymphocytes; (2) none of the Carica Seed Extract (at the concentrations used in this study) was able to protect the lymphocytes from the toxic effects of chromium; and (3) some of the bioactive fractions of Carica Seed Extract were able to significantly inhibit the classical complement-mediated hemolytic pathway. These findings provide evidence for immunostimulatory and anti-inflammatory actions of Carica Seed Extract. No single compound is likely responsible for these activities. Further purification, isolation and characterization of the active components are needed.

Adjuvants, Immunologic↗
PubMed1993

Activation of serum complement by polysaccharide-containing antigens of Porphyromonas gingivalis.

We previously reported that hot aqueous phenol extraction of Porphyromonas gingivalis yields a preparation containing both lipopolysaccharide (LPS) and an antigenically distinct capsular polysaccharide (PS). In the present study, we examined the capacity of phenol-water extracts from a number of strains of P. gingivalis to activate human serum complement. Anticomplementary activity of extracts from two invasive and two noninvasive strains of P. gingivalis was assessed in a sheep erythrocyte hemolytic assay and in an alternative pathway-selective rabbit erythrocyte hemolytic assay. In the sheep erythrocyte assay, extracts from noninvasive strains were found to exhibit greater anticomplementary activity than extracts derived from invasive strains. A phenol-water extract from invasive strain ATCC 53977 was further resolved into its LPS and PS fractions. Whereas isolated LPS from this strain exhibited strong anticomplementary activity, the PS fraction was only weakly active. Phenol-water extracts from three of four strains were found to be potent activators of the alternative pathway, with extracts from the two noninvasive strains being most active. The extract from the remaining strain (ATCC 53977) was a poor activator of the alternative pathway. Further analysis of this extract revealed, however, that the LPS fraction was a potent activator of the alternative pathway, although the PS fraction exhibited negligible activity. The results of this study indicate that phenol-water extracts of invasive and noninvasive strains of P. gingivalis differ in their respective anticomplementary activities, with invasive strains being less active. Although extracts from both invasive and noninvasive strains activated the alternative pathway, this activity appears to be attributable to the LPS, rather than the PS, component.

Antigens, Bacterial↗
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↗