Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “quantitative complementation”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 91 records · Page 5Linked to original sources
PubMed1979

Antigenic similarity of morphologic type CIII1 bacteriophages.

The aim of the study was to demonstrate antigenic differentiation among bacteriophages belonging to the same morphologic type--CIII1 according to Krzywy and Slopek or A2 according to Ackermann. Twenty-six bacteriophages which multiplied on various strains of bacteria of the genera Escherichia, Shigella and Klebsiella, were studied. Serologic tests were done by the quantitative complement fixation test. Immune sera against 5 phi E. coli. D8 Sh. flexneri, G35 Sh. sonnei and Kl7 Klebsiella bacteriophages were obtained from rabbits. It was shown that bacteriophages of CIII1 morphologic type, with morphologically identical virion had different antigenic specificity. On the basis of results obtained the phages could be divided into eight serologic groups. The bacteriophages for which antisera were obtained belonged to four different serologic groups. All bacteriophages had some common antigens with 5 phi E. coli phage. The similarity points to a phylogenetic relationship among phages of CIII1 morphologic type.

Antibodies, Viral↗
PubMed1984

Quantitative determination of complement components produced by purified hepatocytes.

In this report we describe, on a quantitative basis, the secretion of complement components by hepatocytes. Primary cultures were established after isolation of the cells from guinea-pig liver and the synthesis of C3, C5, C4 and C2 was measured. The cells were isolated by collagenase perfusion of the liver followed by differential centrifugation. The contamination of the hepatocyte suspension with non-parenchymal cells was less than 1%. At 24 h after plating the cells the kinetics of complement production were measured. C3 and C5 content in the culture medium harvested at different time intervals was determined by a sensitive ELISA. Secretion of C2 and C4 was measured haemolytically using C2 or C4 deficient guinea-pig serum. Under the conditions used hepatocytes secreted C3 at a rate of about 100 ng/10(6) cells/h with a plateau of secretion after 24 h of culture corresponding to about 350,000 molecules/cell/h. C5 secretion was detectable after 3-6 h of culture. The C5 secretion rate was about 15 ng/10(6) cells/24 h. The functional activity of C4 and C2 in the supernatants amounted to about 80 SFU/cell/h if the culture medium was changed every 3 h but dropped significantly if the medium was changed every 12 h. The decrease of the haemolytic activity became stronger if the medium was changed every 24 h. Cycloheximide reversibly inhibited the complement production. Our results show that guinea-pig hepatocytes synthesize considerably more C3 and C5 compared to peritoneal macrophages supporting the hypothesis that hepatocytes provide the major source of plasma complement.

Animals↗
PubMed1992

Assessment of complement activation in clinical samples. Comparison of immunochemical and functional measurements of complement components with quantitation of activation fragments.

We have compared functional and immunochemical measurements of complement components with assays measuring the generation of activation fragments, for the assessment of classical pathway activation in vitro and in vivo. The generation of the C3a, C3b/C3bi cleavage fragments of C3, and of the C4d cleavage fragment of C4 measured by ELISA and RIA, was correlated with the decrease in total complement hemolytic activity (CH50) and in functional activity of C3 and C4 in normal human serum in which the classical pathway had been activated with aggregated IgG. The decrease in CH50 in in vitro activated serum was also correlated with the generation of C5a and soluble SC5b-9 complexes. In contrast, no or little increase in the concentration of C3a, C3b/C3bi and C4d was observed in plasma samples from patients with low CH50 and with low levels of immunochemical C3 and C4, indicating that fragment quantitation assays provide no information on the presence and extent of complement activation in vivo. Analysis of samples from patients expressing the four C4 genes and patients having one or two C4 null alleles indicated that a ratio of hemolytic C4 to C2 > or = 1 was indicative of complement activation without C4 deficiency, whereas a ratio below 1 was indicative of C4 deficiency with or without classical pathway consumption. Classical pathway activation and C4 deficiency in clinical samples are best predicted by the concomitant assessment of immunochemical levels of C3 and C4 and hemolytic levels of C4, C2 and C3.

Complement Activation↗
PubMed1984

Development and application of an enzyme-linked immunosorbent assay for the quantitation of alternative complement pathway activation in human serum.

We have developed a new, specific, and highly sensitive enzyme-linked immunosorbent assay (ELISA) which quantitates activation of the alternative pathway in human serum, plasma, or on the surface of activators. The ELISA detects the third component of complement (C3b), proteolytic fragment of complement Factor B (Bb), and properdin (P) complex or its derivative product, C3b,P. In the method, activator-plasma mixtures, plasma containing an activated alternative pathway, or other samples are added to the wells of microtitration plates precoated with antibody to P. C3b, Bb,P or C3b,P complexes which become bound are quantitated by subsequently added, enzyme-labeled, anti-C3. The resulting hydrolysis of the chromogenic substrate is expressed as nanograms of C3b by reference to a C3 standard curve. In addition to absolute specificity for activation of the pathway because of the nature of the complex detected by the assay, the ELISA is highly sensitive and able to reproducibly detect 10-20 ng/ml of C3b,P complexes in serum. This value corresponds to 0.0015% of the C3 in serum. In a series of studies to validate the parameters of the ELISA, reactivity was found to be dependent on the presence of alternative pathway proteins, the functional integrity of the pathway, and on the presence of magnesium. Sheep erythrocytes were converted to activators by treatment with neuraminidase. By using a variety of activators, the kinetics of activation and the numbers of bound C3b molecules quantitated by the ELISA were very similar to those measured by C3b deposition. The ELISA also detected identical activation kinetics when MgEGTA-serum and a mixture of the purified alternative pathway proteins were used as sources of the pathway. ELISA reaction kinetics also correlated with the restriction index, a measure of alternative pathway-activating ability. These studies cumulatively validate the ELISA as a direct and quantitative assay for alternative pathway activation. The sensitivity of the ELISA has permitted its use to detect direct alternative pathway activation by several viruses. The ELISA has also shown that certain classical pathway activators trigger the amplification loop of the alternative pathway while others do not. In addition, stable ELISA reactive complexes appeared in the supernatant of mixtures of serum with certain, but not other activators. The ability of the ELISA to detect activation which has already occurred and the stability of the reactive complexes permits studies of clinical sera. Normal human sera (20) contained low levels (5-20 ng/ml) of ELISA-reactive complexes. A proportion of sera from individuals with the adult respiratory distress syndrome (9-10), typhoid fever (8-10), malaria (3-5), gram-negative sepsis (9 of 47), acute trauma and shock (6 f 25), and systemic lupus erythematosus (3 of 29) showed elevated levels of complexes reactive in the alternative pathway ELISA. In contrast, nine sera from patients with circulating C3 nephritic factor were not reactive in the ELISA.

Animals↗
PubMed1988

Radioassays for quantitation of intact complement proteins C2 and B in human serum.

Availability of polyclonal and monoclonal antibodies recognizing determinants on the major cleavage fragments of complement proteins C2 and B enabled development of sensitive radioassays which can be used to quantitate the intact proteins in human sera. Changes in C2 and B concentrations indicative of classical or alternative pathway activation, or both, were seen in normal serum after incubation with complement activators. We determined the normal range (mean +/- 2 SD) of C2 concentration to be 11-35 micrograms/ml in 32 healthy individuals, and that of protein B to be 74-286 micrograms/ml. Sera from patients with systemic lupus erythematosus (SLE), septic shock, infections, and following orthopedic surgery were then assayed. Mean protein B concentration was significantly higher in SLE sera (P = 0.002) and in the infected and post-operative (acute-phase) sera (P less than 0.001), and the mean C2 concentration in the septic shock group (P less than 0.001) was significantly lower than the mean of healthy individuals. Intact C2 was not detected in known C2-deficient individuals. These assays allow parallel quantitation of the structurally and functionally homologous proteins of the classical (C2) and alternative (B) pathways, which is of interest in patients with genetic and acquired hypocomplementemia.

Adult↗
PubMedDate not supplied

Comparison of chicken plasma and guinea pig serum in a quantitative microtiter method of determining microbial complement resistance.

A quantitative microtiter method using chicken plasma is described for determining the degree of complement resistance or sensitivity of avian Escherichia coli isolates. Results obtained with the microtiter method using chicken plasma were compred with results obtained using commercially available standardized guinea pig serum as the source of complement. The test organisms consisted of five isolates of E. coli isolated from chickens. Three isolates were from flocks with colisepticemia; one was from a flock with omphalitis; and one isolate was a non-pathogenic control. Data were accumulated from the five avian E. coli isolates incubated at 35 C with either chicken plasma or guinea pig serum and with heat-inactivated chicken plasma or guinea pig serum. The microtiter results of the chicken plasma and guinea pig serum had a statistically positive correlation. The use of commercially available guinea pig serum in the test system will allow for standardization of this method.

Animals↗
PubMed1985

A new and rapid quantitative assay for complement activation: an ELISA for C3d.

Complement activation occurs in many pathological conditions. Assays to evaluate the presence and extent of this activation may be limited by being qualitative, time-consuming, or radioactive. We have recently devised an enzyme-linked immunoassay that quantitatively measures the presence of the complement activation product C3d in plasma. The assay is rapid: Results can be available within 8 hours of submission. Intra-assay variation was low (4.9%) as was interassay variability (8.7%). This assay was then used to demonstrate that patients with systemic lupus erythematosus (SLE) have increased levels of circulating C3d as compared to those of normals (p less than 0.001). This assay may be useful to demonstrate continuing complement activation and inflammation in patients, even those without clinical symptoms.

Complement Activation↗
PubMed1983

Comparison of immunodiffusion techniques with standard complement fixation assay for quantitation of coccidioidal antibodies.

Quantitative immunodiffusion (QID) and complement fixation (CF) methods were compared for their agreement in detecting coccidioidal antibodies. For these studies, we assayed 719 sera from 181 patients with coccidioidomycosis. Over 60% of the specimens had CF results of 1:2 to 1:256. A total of 43 patients had five or more specimens obtained over periods of between 1 and 8 years. The QID method, as originally performed, agreed within a twofold dilution of the CF titer in 191 of 267 sera (71.5%). Modification of QID by repeated filling of the antigen and serum wells improved agreement to 84.7% (383 of 452 sera). The degree of CF titer change in patients over time periods was more closely matched by the modified than by the original QID method. Discrepancies between the CF and QID methods appeared not to be due to a subpopulation of patients. QID measurement of coccidioidal antibodies may be a useful substitute for the CF assay in certain clinical laboratories.

Antibodies, Fungal↗
PubMed1977

Quantitation of antibody-complement mediated lysis of tumor cells.

An assay for quantitating antibody-complement mediated killing based on the release of 125I from 125IUdR labelled target cell is described. The temporal delay between antibody--complement damage and the release of nuclear material was shortened by treatment of the cells with a combination of trypsin and DNase. This treatment increased the rate of release of the labelled nuclear material from damaged cells without causing labelled nuclear material to be released from undamaged cells. The low level of spontaneous release of 125I from the target cells allows this assay to be used for experiments carried out over long time periods or in experiments involving extensive manipulations of the cells.

Animals↗
PubMed1975

Immunologic studies in hypersensitivity pneumonitis--quantitative precipitins and complement-fixing antibodies in symptomatic and asymptomatic pigeon breeders.

Quantitative precipitation and complement fixation tests were performed on sera from a group of patients with hypersensitivity pneumonitis (pigeon breeders' disease). Their antibody levels were compared to a selected group of similarly exposed but asymptomatic subjects. The quantity of precipitating antibodies was generally greater in serum from symptomatic breeders, although quantities in the serum from some of the asymptomatic subjects were greater than that seen in ill breeders. Complement-fixing antibodies were detected in the serum of both groups. Here again, complement-fixing activity was generally greater in the serum of ill breeders. However, one asymptomatic breeder showed complement-fixing activity greater than most of the symptomatic breeder group. These findings indicate that the presence of complement-fixing antibodies per se in serum to pigeon gamma-globulin is not sufficient to cause the symptoms of pigeon breeders' disease. We suggest that other factor(s) may be involved, such as lgE- or cell-mediated hypersensitivity.

Animals↗
PubMed1985

Rapid rate-kinetic turbidometric assay for quantitation of viral complement-fixing antibodies.

A rapid rate-kinetic turbidometric assay for the quantitation of viral complement fixing antibodies has been developed, using adenovirus as a model. The procedure is based on the turbidometric quantitation of intact sheep erythrocytes and measures the rate of hemolysis (change in absorbance at 640 nm/min/), at maximum velocity, occurring in the presence of residual complement not fixed by the antigen-antibody reaction. Reagents were standardized and assays performed using a microprocessor-controlled spectrophotometer with kinetic assay capability and a thermoregulated cell compartment. Eleven sera were assayed for complement fixing antibodies both by the conventional microtiter technique and by the rapid turbidometric method described here. Good correlation (r = 0.89) was obtained between the two procedures. Unlike the conventional complement fixation test, the rate-kinetic turbidometric complement fixation assay was found to be tolerant of variation in complement and antigen concentration, endpoint titers were objectively quantitated and, once reagents had been standardized, results could be obtained within 45-60 min. The technique is potentially adaptable to large-scale automation.

Adenovirus Infections, Human↗
PubMed1986

Clinical utility of complement anaphylatoxin assays.

Meaningful quantitation of complement activation is difficult by conventional techniques, because of the dynamic equilibrium status of complement components. Assays for complement-derived split products have been useful adjuncts, but until recently have been cumbersome and only semiquantitative. The recent introduction of superior assay methods, especially immunoassays for the complement derived anaphylatoxins, has enhanced such study by allowing more sensitivity, specificity and reproducibility. Such assays are already being applied to the study of diseases in which complement activation may play a role, as well as to the evaluation of biocompatibility of various prosthetic materials and drugs. 'Beside' clinical utility of the tests is still limited, but may grow rapidly if assays are devised which are capable of more rapid and less expensive performance.

Anaphylatoxins↗
PubMed1979

Comparison of several in vitro assay methods for the quantitative determination of complement consumption ('binding') by gammaglobulin preparations.

Unspecifically induced activation of Complement (C) in solution can be caused either by denatured Ig molecules and by polymeric aggregates of same or more specifically by antigen-antibody complexes. As a quality criteria, e.g. for i.v.-IgG preparations, it is agreed by the majority of people concerned that the activation of C via the classical pathway might not be unspecifically initiated. Therefore, the generally used assay systems are based on quantitating the degree of consumption of C undergoing an antigen-independent, 'frustrated' activation via the classical pathway. There are principally two different types of test modifications which were investigated: a) C is being kept constant and protein is diluted or b) protein is being kept constant and C is titrated. When a group of differing IgG preparations or IgG fragments was assayed for its so-called 'anticomplementary' activity in various already described test methods, the particular test results from the individual test methods could not unequivocally be compared with each other because some of the methods had not been optimized with regard to the amount of reagents or test substance used; only a rough estimate of the data was obtained. The value of the not standardized assays remains, therefore, questionable reasonably good and reproducible results are obtained when the amount in particular of C added as well as its quality have been adequately optimized and standardized. This allows a refined differentiation of various IgG preparations by a reliable and unadjusted quantitation of C consumption. If for example, inappropriate and not optimized rations of C amboceptor (e.g. slight C excess) was used, a suppressed C consumption was found. It seems advisable to accept prophylactic testing of anticomplementary activity for quality control as one out of several other in vitro and in vivo parameters which potentially might predict safety for the patient, the physician, and also for the producer. However, it is agreed that, irrespective of in vitro tests, in vivo testing in animals and clinical proof of safety is undoubtedly needed.

Animals↗
PubMed1978

Quantitation of serum complement components and plasma C3d in patients with malignant lymphoma: relation to the stage of the tumor and circulating immune complexes.

Total hemolytic complement activity (CH 50) and complement component levels were measured in 27 patients with Hodgkin's disease and 31 patients with non-Hodgkin malignant lymphoma. CH 50 values were higher than normal in almost all the patients. Increased levels of serum C4, C3 and factor B were observed in 62%, 31% and 19% of these patients, respectively. However, plasma concentration of C3d, a breakdown product of C3, was elevated in 29% of the cases. The hypercatabolism of C3 was not closely associated with the presence of circulating immune complexes, as assessed by the C1q binding assay, nor with the presence of general symptoms in the patients. On the contrary, it appeared to be in relation with the extent of the malignant disease.

Adolescent↗