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Results for “quantitative complementation”

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PubMed1985

A quantitative method for assessing the third complement factor (C3) attached to the surface of opsonized Pseudomonas aeruginosa: interrelationship between C3 fixation, phagocytosis and complement consumption.

A direct enzyme-linked immunoassay is described for the determination of C3 that becomes attached to the surface of bacteria upon incubation in serum. The assay uses horseradish peroxidase-conjugated rabbit antiserum specific for human C3. In this study various Pseudomonas aeruginosa strains, Escherichia coli and Staphylococcus aureus were opsonized under different conditions and the amount of C3 fixed to the bacterial surface was measured directly. The extent of C3 fixation was compared with the percentage of phagocytosis by human polymorphonuclear leukocytes and with the complement consumption in the fluid phase. It was demonstrated that C3 fixation closely correlated with the percentage of phagocytosis but not with complement consumption. The method can be performed with relatively simple equipment and could be used in routine laboratories in order to determine the extent of opsonization of Pseudomonas aeruginosa and other bacteria and to detect opsonic defects in the sera of patients.

Complement Activation↗
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↗
PubMed1957

Quantitative studies on the properdin-complement system. II. Kinetics of the reaction between properdin and zymosan.

The reaction of human, bovine, and pig properdin with excess zymosan exhibits an induction period followed by a rapidly accelerating formation of the properdin-zymosan (PZ) complex. Two methods for determining the quantity of PZ complex are given, one using human serum lacking properdin, the other using a fraction of pig serum containing complement components, C'1, C'3 and C'4.

Animals↗
PubMed1954

The combined actions of chloramphenicol and of bactericidal antibody plus complement on Salmonella typhosa.

Quantitative determinations, employing turbidimetric growth assays, have been made of the inhibitory actions of choramphenicol, and of specific antibody plus complement, separately, and in combination. In experiments with the Ty2 strain of S. typhosa the combined activities of the two groups of reagents have been predicted, within 3 per cent, from the activities of the separate components, on the hypothesis of joint, independent action (additive inhibitions). A fourfold increase in resistance to chloramphenicol of the 0901 strain of S. typhosa has been shown to have little, if any, effect on its susceptibility to the bactericidal action of anti-O antibody and complement. Indirect evidence indicates that the latter may not be a general effect but may vary with the particular strain.

Anti-Bacterial Agents↗
PubMed1980

A new method for the simultaneous quantitation of platelet-bound immunoglobuin (IgG) and complement (C3) employing an enzyme-linked immunosorbent assay (ELISA) procedure.

An ELISA technique for the quantitation of platelet-bound IgG and C3 is described. This is an antiglobulin consumption assay using commercial horseradish peroxidase conjugated anti-IgG and anti-C3 antisera. The greater the amount of antiglobulin consumed by the reaction with platelets the less is available to bind to an immune adsorbent in the form of human serum-coated polystyrene balls. This test can be calibrated by adding known quantities of IgG of C3 to the specific enzyme-linked antibody and the unbound fraction of antiserum is quantitated by allowing it to react with a chromogenic substrate for the enzyme and measuring the intensity spectrophotometrically, thus establishing an inverse relationship with the amount of antigen. Platelets from normal donors and those with non-immunological thrombocytopenia gave values of 1.3--15.5 ng IgG/10(6) platelets and 0.22--0.96 ng C3/10(6) platelets which are in accordance with the normal ratio of these proteins in normal serum. Fifteen patients in whom immune destruction of platelets weas suspected had excess platelet-bound IgG ranging from 30 to 450 ng IgG/10(6) platelets. In none of these patients could excess platelet-bound C3 be demonstrated. Compared to the antiglobulin consumption test we have found this test to be superior both technically and in terms of sensitivity and reproducibility.

Blood Platelets↗
PubMed2003

Locating a protein-protein interaction in living cells via split Renilla luciferase complementation.

For spatial and quantitative kinetic analysis of protein-protein interactions (PPIs) in living mammalian cells, a method was developed in which PPI-induced complementation of split Renilla luciferase triggers spontaneous emission of luminescence using a cell membrane permeable substrate, coelenterazine. This split Renilla luciferase complementation readout was shown to work for locating a PPI between the tyrosine-phosphorylated peptide (Y941) of IRS-1 and the SH2 domain of PI3K among insulin signaling pathways in living Chinese hamster ovary cells overexpressing human insulin receptors (CHO-HIR). It was thereby found that the insulin-stimulated interaction occurred near the plasma membrane in the cytosol.

Amino Acid Sequence↗
PubMed1989

Complement receptor (CR1) and IgG or IgA on erythrocytes and in circulating immune complexes in patients with glomerulonephritis.

This study reports the quantitative analysis of complement receptor (CR1) molecules on erythrocyte surface, the amount of immunoglobulin-containing material (IgG-IC and IgA1-IM) on the erythrocyte surface, and the concentrations of circulating immune complexes (IgG-CIC and IgA-CIC); also reported are the HLA phenotypes of 44 patients affected by various forms of glomerulonephritis (including 20 primary IgA nephropathy, 11 membranous glomerulonephritis, 9 lupus nephritis and 4 renal vasculitis). Erythrocyte CR1 molecules were found to be decreased (P less than 0.02) and erythrocyte IgG-IC were less than in controls (P less than 0.025) in lupus nephritis patients, whereas IgG-CIC were significantly greater (P less than 0.02). In patients affected by primary IgA nephropathy, mean erythrocyte CR1 concentrations were significantly decreased (P less than 0.02). Patients with impaired renal function had mean erythrocyte CR1 values significantly greater than those with normal renal function (P less than 0.002). Immunoglobulin-containing material on the erythrocyte surface was not significantly increased, whereas the serum concentrations of both IgA-CIC and IgG-CIC were significantly increased (P less than 0.02). In membranous nephropathy erythrocyte CR1 molecules were quantitatively similar to control data and no increase in CIC was observed. Conversely, erythrocyte IgG-IC were significantly increased (P less than 0.01). No significant relationship among erythrocyte CR1 molecules, erythrocyte surface-associated immunoglobulins, CIC, and HLA phenotype was observed in any patient group.

Adult↗
PubMed1977

Complementation analysis of measles virus temperature-sensitive mutants.

Two sets of independently isolated measles virus temperature-sensitive mutants were quantitatively tested for complementation. Analysis of the nine possible combinations of representative mutants indicated that only one pair of mutants is noncomplementing. Thus, the measles virus mutants studied to date define five complementation groups.

Genetic Complementation Test↗
PubMed1985

Deleterious effects of cardiopulmonary bypass. A prospective study of bubble versus membrane oxygenation.

A number of hematologic and immunologic parameters that reflect erythrocyte and platelet damage and host defense mechanisms against infection were studied in 20 patients undergoing cardiopulmonary bypass during coronary operations. The patients were randomly assigned to a group in which a bubble oxygenator or a hollow-fiber membrane oxygenator was used. Hemolysis, thrombocytopenia, and significant release of beta thromboglobulin occurred in patients from the bubble oxygenator group and, to much lesser extent, in patients from the membrane oxygenator group. Polymorphonuclear leukocytes and monocytes from bubble oxygenator patients demonstrated increased generation of reactive oxygen species in the resting state and in the presence of the stimulating agents N-formyl-methionyl-leucyl-phenylalanine, concanavalin A, and opsonized zymosan, as compared with cells from membrane oxygenator patients. No difference was found between bubble and membrane oxygenator patients in the time of occurrence or intensity of leukopenia during bypass, of leukocytosis at the end of bypass, nor in the rate of complement activation, as assessed by quantitation of plasma C3a antigen. Complement activation was dependent on the alternative pathway. Immunoglobulin M concentration significantly decreased during bypass in both groups of patients. The serum opsonizing capacity for endotoxin and serum bactericidal activity for Serratia marcescens were decreased in both groups, mainly because of hemodilution, although they were additionally affected by bubble oxygenation. Several deleterious hematologic consequences of cardiopulmonary bypass can be minimized by the use of a membrane oxygenator. However, complement activation remains a potential risk factor even in membrane oxygenator patients and requires further investigation to obtain better hemocompatible materials for cardiopulmonary bypass circuits.

Blood Bactericidal Activity↗