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Results for “quantitative complementation”

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PubMed1986

The role of complement in inflammation during experimental pneumococcal meningitis.

The mechanism whereby an effective bactericidal inflammatory reaction develops in the subarachnoid space is not clearly defined. While normal cerebrospinal fluid is deficient in complement, immunoglobulin and leukocytes, these serum components appear in cerebrospinal fluid (CSF) during the course of bacterial meningitis. Using a rabbit model of pneumococcal meningitis we examined the role of the alternate complement pathway in three early events important to the defense of the subarachnoid space: leukocyte chemotaxis, phagocyte mediated bacterial killing, and clearance of bacterial components from the cerebrospinal fluid space. Rabbits treated with cobra venom factor to deplete complement were inoculated intracisternally with encapsulated (type II or XIX) pneumococci. Following complement depletion, there was a dramatic (at least 100-fold) decrease in the LD50 for these strains. Nevertheless, complement depletion did not affect the magnitude of CSF leucocytosis or the rate of clearance of bacterial particles from CSF. A short delay in the appearance of leukocytes in CSF was found in the absence of complement. The major effect of complement depletion, however, was to diminish the efficiency of leukocyte mediated killing of encapsulated bacteria in the CSF. Although the short delay in the onset of leukocytosis in the complement depleted animals is consistent with a chemotactic role of complement in the normal animal, the quantitatively normal leukocytosis in the complement depleted rabbits clearly indicates that important chemotaxins other than complement function in CSF. Inhibition of leukocytosis by indomethacin and diclofenac suggests that metabolite(s) of the arachidonic acid pathway may perform such a chemotactic role. A major role of complement in the defense of the subarachnoid space appears to be as an opsonin needed for the effective bactericidal activity of leukocytes. It is the lack of this function that best explains the greatly decreased LD50 value of encapsulated pneumococci in the complement depleted animal.

Animals↗
PubMed2001

Estrogen receptor analysis in primary breast tumors by ligand-binding assay, immunocytochemical assay, and northern blot: a comparison.

Estrogen receptor (ER) status is an important parameter in breast cancer management. In this study, ER protein contents established by two conventional techniques were confronted to ER mRNA level, to analyze whether the latter may be introduced in routine assay. Eighty-seven breast tumor samples were examined. ER amounts were determined by ligand-binding assay (LBA) and by computer-assisted immunocytochemical assay (ICA), ER mRNA was analysed and quantified by northern blot. Seventy-seven percent of tumor samples examined were positive for ER mRNA and they all expressed the 6.7-kb receptor signal. No trace of small-sized ER mRNA variants was detected in any sample. Following akaike information criterion (AIC) discriminant analysis, a simple linear correlation was found between ER mRNA levels and ER amounts provided by LBA. This was not observed when either mRNA or LBA values were compared to ICA values. These latter were found to rapidly reach a plateau at increasing mRNA or LBA values. In conclusion, our data points to the linear correlation between ER amounts determined in breast tumors at both protein and mRNA levels by quantitative methods; they also indicate that the semi-quantitative computer-associated ICA may complement rather than replace these quantitative methods.

Adult↗
PubMed1987

Competitive enzyme-linked immunoassay for the quantitation of platelet-associated immunoglobulins (IgG, IgM, IgA) and complement (C3c, C3d) with polyclonal and monoclonal reagents.

A competitive enzyme-linked immunoassay (CELIA) was developed for the quantitation of platelet-associated immunoglobulins and complement proteins. The use of unlabeled polyclonal rabbit or monoclonal antibodies to human immunoglobulins and enzyme-labeled anti-mouse or anti--rabbit IgG (double-step technique) resulted in a higher sensitivity compared to the single-step technique using only enzyme-labeled anti-human immunoglobulin antibody preparations. Sensitivity and results obtained by both techniques were compared. The range of normal values for platelet-associated IgG, IgM, IgA, C3c and C3d was assessed upon a large number of normal blood donors. When platelet-associated IgG was concomitantly assayed with polyclonal and monoclonal anti-IgG by the double-step technique on platelets obtained from normal donors and thrombocytopenic patients, identical results were obtained with both reagents. Problems related to the quantitation of immunoglobulins on platelets with different assays and antibody preparations are discussed.

Antibodies, Monoclonal↗
PubMed1997

Decreased C4 complement component serum levels correlate with the degree of emphysema in patients with chronic bronchitis.

Patients with COPD who fulfill the diagnostic criteria of chronic bronchitis have been shown to exhibit lower serum levels of complement components C3 and C4 than healthy subjects, and this may indicate sustained complement activation as a result of recurrent respiratory tract infections. Since activation of complement leads to influx of inflammatory cells into the lung parenchyma with subsequent release of elastases and oxidants that cause damage to elastic lung tissue, we postulated that there might be a quantitative relationship between complement consumption and degree of elastic tissue destruction. In this study, we tried to investigate possible correlations between serum levels of C3 and C4 and degree of emphysema among patients with COPD of the bronchitic type. We studied 20 patients with chronic bronchitis aged 68+/-1 years (mean+/-SEM) without significant fluctuations of serum C3 and C4 levels over a 3-month period by performing detailed lung function tests, recording of emphysema score in chest radiogram, and the incidence of infective exacerbations during the past 3 years. Measured C3 and C4 serum levels were 124+/-9 and 28.5+/-2 mg/dL, respectively, lower than the respective levels in control subjects (141+/-3 and 39+/-2 mg/dL, respectively). Significant correlations were observed between levels of C4 and (1) incidence of respiratory tract infections during the past 3 years (r=-0.747, p<0.001), (2) radiologic emphysema score (r=-0.936, p<0.001), and (3) various functional indexes, such as midexpiratory flow rate, percent of predicted (r=0.629, p<0.01), forced expiratory flow rate at 50% of vital capacity, percent of predicted (r=0.606, p<0.01), residual volume/total lung capacity ratio (r=-0.651, p<0.01), and the exponential constant of static pressure-volume curve (r=-0.606, p<0.01). These results suggest that patients with chronic bronchitis with the lowest levels of C4 are those experiencing more frequent respiratory infections, tend to have more signs indicative of emphysema in their chest radiograph, have a more prominent small airways dysfunction and gas trapping, and present a greater defect in lung elastic recoil.

Aged↗
PubMed1987

A prospective study on infection with cytomegalovirus in renal allograft recipients immunosuppressed with cyclosporine A and low dose prednisone.

To investigate the course of infection with cytomegalovirus (CMV) in renal allograft recipients treated with cyclosporine A, 10 patients were followed for 1 year after transplantation. Virus cultures from blood, urine and throat washings were performed employing a quantitative technique. Complement-fixing and IgM antibodies to CMV were measured at scheduled intervals. The incidence 90% and course of CMV infections were found not to differ from those reported in patients receiving conventional immunosuppressive therapy. The quantitative virus cultures showed a consistent pattern with viremia most prominent at the beginning of an infection, and the highest concentration found in the one patient who developed symptoms of viral disease. It is suggested that information about the concentration of virus in a specimen will improve the diagnostic value of virus cultures in this group of patients.

Adolescent↗
PubMed1991

Complement activation and depletion during LDL-apheresis by heparin-induced extracorporeal LDL-precipitation (HELP).

The heparin-induced extracorporeal elimination of low density lipoproteins (LDL) is a well-established clinical procedure to markedly reduce cholesterol levels. The biocompatibility of this artificial filter system (HELP) was investigated by quantitation of representative complement proteins within the extracorporeal circuit using established ELISA procedures, based on monoclonal antibodies recognizing exclusively either native (C6, C7) or activated proteins (act.C3, C5a, TCC). HELP was found to be a self-limiting extracorporeal system with respect to complement activation, since act.C3 and TCC, generated mainly at the plasma filter, were partially adsorbed to the following HELP specific filters to concentrations which were lower than those obtained before the plasma filter. C5a, which increased 14.5-fold at the plasma filter was not eliminated by the following filters; however, elevated levels were not found in the patients at the end of apheresis and no leucocytopenia was observed.

Adult↗
PubMed1979

A rapid and simplified technique for the assay of chicken hemolytic complement.

The technique described is a modification of a qualitative hemolytic radial diffusion technique. The test involves the use of sensitized sheep erythrocytes that have been incorporated into agarose. Tube dilutions were made of chicken serum and samples of each dilution were placed into wells cut in the agarose. The test is quantitative for hemolytic complement in that the highest dilution showing visible hemolysis of sensitized erythrocytes in agarose is determined to be the endpoint for that serum sample. The test as compared with the standard tube assay was determined to be less sensitive by approximately one dilution. The advantages of speed, simplicity, and cost more than offset the decrease in sensitivity of the test.

Animals↗
PubMed1975

The antigenicity of myelin encephalitogenic protein: production of antibodies to encephalitogenic protein with deoxyribonucleic acid--encephalitogenic protein complexes.

Rabbits immunized with monkey encephalitogenic protein (EP) complexed to deoxyribonucleic acid (DNA) formed complement-fixing antibodies that reacted well with EP from monkey, bovine, and human brain and the large EP of rat brain. Reactivity with the small EP of rat brain was much less. The antibodies, analyzed by the quantitative micro-complement fixation test, were of the 7S class, were produced in high titer, and failed to react with histone, lysozyme, or acidic liver extracts. Immunohistochemical studies revealed that the antibodies bound to central nervous system white matter from human, rat, guinea pig, and mouse as well as human peripheral nerve myelin. These findings demonstrate that the immunogenicity of EP is greatly enhanced by attachment to DNA and perhaps other negatively charged molecules. This method makes available anti-EP with which to investigate immunochemically the antigenic and conformational features of EP.

Animals↗
PubMed1983

The binding properties of human complement component C1q. Interaction with mucopolysaccharides.

Quantitative measurements have been made of the interaction of human complement subcomponent C1q with mucopolysaccharides. The binding of C1q to heparin was quantitatively examined by utilizing an assay that employs a 125I-labeled low molecular weight heparin glycosaminoglycan (LMW-Hep) (Mr = 8500). Two classes of binding sites were detected. The first class of sites bound 2.02 mol of LMW-Hep/mol of C1q with a Kd of 76.6 nM. The second class of sites complexes with 12 mol of LMW-Hep with a Kd of 1.01 microM. The higher affinity-binding site for LMW-Hep could be assigned to the collagenous region of C1q (C1q-c); 2.2 mol of 125I-LMW-Hep were bound/mol of purified isolated C1q-c with a Kd = 381 nM. In contrast, the isolated C1q globular region did not bind to 125I-LMW-Hep. The binding of LMW-Hep to C1q and the C1q-c region was confirmed by fluorescence polarization experiments; C1q and C1q-c bound 2.3 and 2.02 mol of fluorescamine-labeled LMW-Hep/mol of protein, respectively. A variety of mucopolysaccharides were able to inhibit interaction of C1q with 125I-LMW-Hep, the most effective being heparan sulfate and dermatan sulfate. LMW-Hep (2.5 nM) inhibited the ability of C1q (0.5 nM) to recombine with C1r (1.4 nM) and C1s (1.6 nM) to form hemolytically active C1. At 250 mM, LMW-Hep inhibited the hemolytic activity of reconstituted C1. The ability of mucopolysaccharides to interact with purified C1q suggests a role for such molecules in the regulation of the first component of complement.

Centrifugation, Density Gradient↗
PubMed1999

Rapid onset monoclonal gammopathy in cutaneous lupus erythematosus: interference with complement C3 and C4 measurement.

Accurate nephelometric immunoassay requires both the analyte and the calibration standard to have the same molecular mass. Alteration in the size of the analyte can affect light scatter and yield erroneous results. We report a case where an autoantibody, a monoclonal IgM with immunoconglutinin activity, interfered with the nephelometric quantitation of plasma complement components C3 and C4.

Adult↗
PubMed1993

Vaccination and the role of capsular polysaccharide antibody in prevention of recurrent meningococcal disease in late complement component-deficient individuals.

This study sought to quantitate the response of late complement component-deficient (LCCD) individuals to the meningococcal vaccine, to examine antibody persistence, and to investigate the contribution of these antibodies to meningococcal killing in complement- and phagocyte-dependent bactericidal assays. The mean concentration of antibody to group A and C capsular polysaccharide after vaccination was similar in 8 LCCD, 11 family members, and 7 unrelated normal individuals. LCCD individuals had a greater percentage decline in antibody concentration to group C polysaccharide and had lower concentrations of antibody to group Y polysaccharide 2.0-2.5 years after vaccination than did normal and heterozygous deficient persons. Antibody to subcapsular antigens was minimally effective in mediating complement-dependent killing and had no effect in the opsonophagocytic assay. In contrast, antibody to capsular polysaccharide promoted meningococcal killing in both assays. These data support vaccination as a preventive measure in LCCD individuals and lend credence to the idea that these individuals are critically dependent on capsular antibody for protection against meningococcal disease.

Adult↗
PubMed1989

[Synthetic inhibitors of serine proteinases. 35. Inhibition action of amidino- and guanidinophenyl derivatives against hemolysis initiated by complement activation].

Selected amidino- and guanidinophenyl derivatives which have already proved to be inhibitors of serine proteinases were tested on their inhibitory effect on complement activation. To quantitate the inhibitory action the decreasing rate of haemolysis of sensitized sheep erythrocytes was recorded continuously. Compounds exerting a more or less pronounced inhibitory effect on trypsin-like proteinases turned out to have a corresponding inhibitory influence on complement-mediated haemolysis. The inhibitory effect of some amidinobenzyliden derivatives on the complement activity was markedly higher than could be expected from their general antiproteolytic efficacy. Changing the basic structure into amidinophenyl amino acid amides with strong and selective antithrombin effect completely abolished the inhibitory influence on complement.

Amidines↗
PubMed2001

A monoclonal antibody cytolytic to androgen independent DU145 and PC3 human prostatic carcinoma cells.

BACKGROUND: While a range of therapeutic products is available for androgen-dependent prostatic cancer, no specific intervention modality exists for androgen-independent prostatic cancer. The objective of this research was to explore whether epitopes exist on androgen-independent prostatic DU145 cancer cells, which could be susceptible to cytotoxic action of specific antibodies. METHODS: Hybrid cell clones were developed by immunization of mice with DU145 cells and tested for immunoreactivity by solid phase EIA and cytotoxicity in vitro on DU145 in the presence of the complement, employing colorimetric quantitation by MTS (3- (4-, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-(4-sulfophenyl)-2H-tetrazolium). Binding and cytotoxicity studies were also carried out by flow-cytometry. RESULTS: Of 15 stabilized clones immunoreactive with DU145 cells, one monoclonal antibody (mAb 730) manifested cytotoxicity on DU145 cells. Approximately 80% of cells in the DU145 cell line were susceptible to lysis with this antibody at saturating levels. This figure corresponded quantitatively to the number of cells binding with this antibody as determined by Flow-cytometry. Staining with ethidium monoazide bromide (EMA) showed that the cell binding the antibody was also the one killed by the antibody in the presence of the complement. MAb 730 was also cytotoxic to PC3, another androgen-independent human prostatic cancer cell line. This antibody is devoid of classical autoantibody reactivities and does not react with normal human liver, thyroid, kidney, pancreas, and adrenal tissues, as determined by immunofluorescence. Also, it shows negative immuno-reactivity to benign glandular tissue but is observed to positively react with neoplastic prostate tissue. CONCLUSIONS: Epitopes exist on androgen-independent prostatic cancer cells that are susceptible to cytolysis by monoclonal antibodies and these could be investigated for potential immunotherapy.

Animals↗
PubMed1977

A solid phase fluorescent immunoassay for the quantitation of the C3 component of human complement.

A non-competitive method for the determination of the C3 component of human complement in serum is described. This procedure involves use of a specific antibody covalently attached to derivatized polyacrylamide beads and a fluorescently labeled specific antibody. Reproducible results were achieved for C3 in serum in the range of 20 mg/d1 to 195 mg/d1 within 2 h. C3 levels as low as 125 ng/m1 can also be measured. Fluorescent immunoassay and radial immunodiffusion were used to determine C3 levels in healthy adults. Good agreement was found between the two methods.

Animals↗
PubMed1977

A solid phase fluorescent immunossay for the quantitation of the C4 component of human complement.

A non-competitive method for the determination of the C4 component of human complement in serum is described. This procedure involves use of a specific antibody covalently attached to derivatized polyacrylamide beads and a fluorescently labeled specific antibody. Reproducible results were achieved for C4 in serum in the range of 10 mg/dl to 170 mg/dl within 2 h. C4 levels as low as 150 ng/ml can also be measured. Fluorescent immunoassay and radial immunodiffusion were used to determine C4 levels in healthy adults. Good agreement was found between the two methods.

Complement C4↗