A comparison of whole blood neutrophil chemiluminescence measured with cuvette and microtitre plate luminometers

Results obtained by measuring human whole blood neutrophil chemiluminescence (CL) using the BioOrbit 1251 cuvette luminometer and the Immunotech LM‐01T microtitre plate luminometer are compared in this study. Opsonized zymosan, phorbol myristate acetate, N‐formyl–Met–Leu–Phe and calcium ionophore A23187 were used as activators. The CL response of neutrophils to their stimulation with the individual types of activators tested was fully detectable using either type of the luminometers. The kinetic curves of CL activity obtained from both the cuvette and the microtitre plate luminometers had similar characteristics. The only insignificant difference observed when comparing the kinetic curves was in the rates of the CL reactions. The peak CL response of activated neutrophils was reached faster when using the luminometer BioOrbit 1251 than with the luminometer Immunotech LM‐01T. A likely reason for this difference is the mode of transporting samples during the measurement, inducing different degrees of agitation. However, although this fact needs to be considered when interpreting results, both types of luminometer can be fully utilized in both research and clinical laboratories. Copyright © 2002 John Wiley & Sons, Ltd.     

A new luminometer for sensitive quantification by chemiluminescence of specific proteins in microtitre plates and on blot membranes

Generally applicable technologies are described, which depend on the use of chemiluminescence and a new type of a versatile luminometer, that can measure also weak light in microtitre plates (microplates) and on blotting membranes that is especially useful for dot blot immunoassay. Applications are described using alkaline phosphatase conjugated antibodies against IgG and IgE and the reagent Lumi-Phos 530. This chemiluminescence offers advantages over the use of radioactive isotopes, densitometry and light reflection measurement on membranes and also ELISA, for sensitive quantification of e.g. specific proteins. Special procedures are described for the first time that with the mentioned reagent in agarose gel allows specific and very sensitive quantification of proteins on dot blot membranes. The luminometer, which has temperature control, is very sensitive, precise and allows efficient protocols for various assays. It thus fulfils many of the requirements for good quantification, is time-saving and in addition brings significant improvements due to very low detection limits and large linear concentration ranges that can be measured with excellent regression coefficients r² often about 0.999).     

Model components of luminol chemiluminescence generated by PMNL

The production of activated oxygen species (AOS) by neutrophils (PMNL) is thought to play a key role in the host defence against invading microorganisms. However, the oxygen metabolites are toxic not only to the invading bacteria but also to the surrounding tissue. The oxidative metabolites production can be evaluated by means of chemiluminescent methods. In this study, the possibility of a new analytical approach for quantitative assessment of chemiluminescent kinetics (AOS generation) of isolated PMNL was estimated.

Based on the assumption that the kinetics of luminol-amplified chemiluminescence (LCL) of stimulated PMNL possesses a time-probabilistic nature, this kinetics was described with three components. These components, obtained from different investigated systems, were analyzed and a conclusion was made that the first and the second component represent the processes resulting in extra-and intracellular myeloperoxidase (MPO)-dependent light emission (AOS generation), respectively. The second component was found to be completely dependent on the stimulus ingestion. The third component was not completely MPO-dependent and complicated for interpretation. This component was weakly dependent on the stimulus ingestion, and presents at least some intracellular processes different from those presented by the second component.

A conclusion is made that the examined approach for analysis of LCL kinetics allows an assessment of extra-and intracellularly generated quantities of AOS by stimulated PMNL. The assessment could be done for emitting systems in which no additional modificators are used.     

Measurement of phagocyte chemiluminescence using a microtitre plate luminometer

The use of chemiluminescence techniques to study the interaction between bacteria and phagocytes has been useful for examining the extent to which serum factors, such as opsonins, are important in internalization of the organisms and the response of the cell to phagocytosed bacteria. However, such methods have been limited by the number of experiments which can be performed at one time using most commercial luminometers. However, the recent introduction of the Amerlite microtitre plate luminometer allows the measurement of chemiluminescence responses in 96-well microtitre plates. Using this instrument, lucigenin-enhanced chemiluminescence can be detected from as few as 5000 cells (polymorphonuclear leukocytes or monocytes) per well with a 1:10 ratio of cells to zymosan particles opsonized with 10% serum. The opsonic capacity of up to 100 sera can be measured in triplicate wells in a single experiment using four microtitre plates and polymorphonuclear leukocytes prepared from less than 40 ml freshly obtained venous blood. We are currently using this technique to investigate the effect of serum opsonins on the interaction between normal human polymorphonuclear leukocytes and monocytes with mycobacteria of three species (Mycobacterium leprae, M. tuberculosis, and M. aviumintracellulare). Other possible applications of this method are discussed.     

On-line chemiluminescence determination of mitoxantrone by capillary electrophoresis

A novel capillary electrophoresis (CE) with chemiluminescence (CL) detection method for the determination of mitoxantrone (MTX) has been developed, which based on the CL reaction of potassium ferricyanide with luminol in sodium hydroxide medium sensitized by MTX. Under optimum analytical conditions, MTX is determined over the range of 7.0 × 10⁻⁸–1.0 × 10⁻⁶ M with a detection limit of 1.0 × 10⁻⁸ M. The relative standard deviation (RSD) was 3.7%, 2.6% and 3.0% for 7.0 × 10⁻⁸, 5.0 × 10⁻⁷ and 1.0 × 10⁻⁶ M MTX (n = 11), respectively. In laboratory-built CE–CL apparatus, the proposed method has been applied to determination of MTX in commercial drug and spiked in human urine and plasma with satisfactory results.     

Facile determination of DNA-binding nuclear factor-kappaB by chemiluminescence detection

A simple, rapid, and sensitive method for the assay of a sequence-specific DNA-binding protein, nuclear factor-kappaB (NF-kappaB), has been developed by using a DNA-detectable chemiluminogenic reagent and a centrifugal filter that distinguishes different molecular sizes. After the formation of a complex between NF-kappaB and DNA, the unbound DNA is separated from the complex by the centrifugal filter. The amount of the bound NF-kappaB is estimated by chemiluminescence detection of the bound DNA. This detection is performed within 2 min at room temperature by the use of a chemiluminogenic reagent, 3',4',5'-trimethoxyphenylglyoxal, which selectively recognizes guanine moiety in oligonucleotides or DNAs. This method does not require any labeled probes or antibodies and can determine a concentration as low as 5 nM of DNA-binding NF-kappaB. The sensitivity is nearly the same as that of other methods such as gel shift assay using fluorescence-labeled probes and enzyme-linked immunosorbent assay. Therefore, the current method provides a convenient tool for surveying various DNA-binding proteins.     

Use of a microtitre plate chemiluminescence reader to study surface phagocytosis by human monocytes

Human mononuclear cells were separated from freshly obtained peripheral venous blood by density centrifugation and the number of monocytes present estimated by volume spectroscopy. The mononuclear cells were then placed directly into the wells of a microtitre plate and incubated for one hour at 37°C to promote adherence of the monocytes to the plastic wells. Non-adherent cells were then removed by washing, thus avoiding the need to treat the monocytes with EDTA or other reagents during cell preparation. The time course and dynamics of the chemiluminescence response of adherent monocytes towards opsonized zymosan was similar to those seen using non-adherent cells. The ability of adherent monocyte preparations to produce chemiluminescence following incubation for varying periods with T-lymphocyte conditioned medium was investigated. The use of a microtitre plate chemiluminescence reader allows several plates to be assayed over the 24-hour period and since small numbers of cells are required, many cultures can be analysed in one experiment. This technique (Patent applied for) promises to be a powerful tool for dissecting the cellular events which occur during macrophage activation and examining the effect of various lymphokines on the ability of monocytes to produce a chemiluminescence response.     

Studies on PVP hydrogel-supported luminol chemiluminescence: 2. Luminometer calibration and potential analytical applications.

The use of poly(N-vinyl-2-pyrrolidone) (PVP) hydrogel-supported luminol chemiluminescence (CL) for the automatic determination of hydrogen peroxide and the quantification of the antiradical capacity of Trolox is described. The hydrogel containing luminol and hemin is prepared directly on a 96-well microplate and can be stored for up to 3 months without significant decrease in CL quantum yields. Furthermore, this system can also be used as a secondary light standard for the calibration of microplate luminometers.     

Surface phagocytosis of Staphylococcus epidermidis and Escherichia coli by human neutrophils: serum requirements for opsonization and chemiluminescence

Abstract We examined the serum requirements for surface phagocytosis of Staphylococcus epidermidis and Eschericia coli and for the subsequent chemiluminescent response of human neutrophils. Substantial surface phagocytosis of S. epidermidis occured in the absence of opsonins, although the presence of 10% pooled or heat-inactivated serum significantly increased phagocytosis. There was no significant difference between these opsonins, indicating that surface phagocytosis of S. epidermidis did not require complement, Unopsonized E. coli were not as readily phagocytized as S. epidermidis (33% versus 57%). In contrast to S. epidermidis optimal phagocytosis of E. coli required complement as 10% heat inactivated donor serum (HHS) was significantly less effective as an opsonin than 10% pooled healthy donor serum (PHS). The time kinetics for phagocytosis of each organism were similar, with most of the phagocytosis iluminescent response of neutrophils produced discrepant results. Maximal chemiluminescence was observed when neutrophils were stimulated with bacteria opsonized in PHS. The response to HHS-opsonized bacteria was less, and chemiluminescence to unopsonized bacteria was only marginally higher than the control, even though there was relatively good phagocytosis. These results define the opsonic requirements for surface phagocytosis of S. epidermidis and E. coli and indicate that although complement may not be required for phagocytosis, it is necessary for generation of a maximal oxidative burst, and thus may be essential for efficient intracellular killing.     

Luminol-enhanced chemiluminescence by Listeria monocytogenes: A test for rapid assessment of antimicrobial agents

Abstract Listeria monocytogenes produces chemiluminescence in brain heart infusion broth at 37°C in the presence of carbonate ions and acetaldehyde. This phenomenon can be enhanced by the use of luminol rather than acetaldehyde. Furthermore, there is direct relationship between the extent of growth and the level of luminescence which culminates at the end of the exponential growth. This property was used to study the susceptibility of this bacterium to two antiseptics, cetrimonium bromide and chlorhexidine, and to two antibiotics, ampicillin and chloramphenicol. Inhibition of chemiluminescence was proportional to the antimicrobial agents' concentrations and was complete at their minimal inhibitory concentrations.     

线粒体电子传递链电子漏的化学发光测定

本实验用差速离心法分离正常大鼠肝脏和心肌线粒体 ,以lucigenin (探测超氧阴离子 )与luminol (探测过氧化氢 )为探剂 ,用化学发光法测定METC电子漏的生成。在反应体系中加入外源底物 ,其发光强度明显高于空白对照 (体系中无线粒体 )。在肝线粒体体系中 ,无论是lucigenin还是luminol诱发的发光 ,琥珀酸底物引起的发光强均要高于丙酮酸 /苹果酸引起的发光强度。在心肌线粒体 luminol体系中也有与肝线粒体相似的结果 ,在心肌线粒体 lucigenin体系中 ,加入外源底物丙酮酸 /苹果酸诱发的发光强度高于琥珀酸诱发的发光强度     

The increasing effect of some synthetic peptides on luminol-dependent chemiluminescence of mouse blood

Some synthetic peptides increased the luminol-dependent chemiluminescence of mouse blood during phagocytosis. It is suggested that the levels of antimicrobial activity of the neutrophil peroxidase system can be raised very quickly (within some dozen of seconds) by these peptides. This raises the possibility of finding a new approach to the therapy for infectious diseases.     

Correlation between hydroperoxide-induced chemiluminescence of the heart and its function

The isolated perfused rat heart emits a spontaneous ultraweak chemiluminescence. When the perfusion is stopped, light emission decreases, indicating the dependency of this phenomenon on aerobic metabolism. Emitted chemiluminescence was markedly enhanced following perfusion with 0.05 mM H₂O₂ or cumene hydroperoxide or tert-butyl hydroperoxide; substitution of O₂ for N₂ in the gassing mixture of the perfusion media significantly lowered photon emission. Lipid peroxidation, which is known to be associated with chemiluminescence, was evaluated by HPLC analysis of peroxidized and unperoxidized heart phosphatidylcholines. During hydroperoxide perfusion, coronary flow and heart rate progressively decreased, while lactic dehydrogenase was released after complete cardiac arrest. The resultant morphology of this damage corresponds to the so-called ‘stone heart’, a pattern already described in both human and experimental pathology.     

Automation and computerization of chemiluminescence: A new methodological approach in the study of human phagocytes

Peripheral blood phagocytic cells (PMNLs) are activated by contact with opsonized particles. Metabolic activation of PMNLs is associated with a remarkable increase in the respiratory burst and generates high energy oxygen compounds which are responsible for the bactericidal activity of PMNLs and for their ability to produce luminol-dependent chemiluminescence (CL). The CL phenomenon is measured by an automated and computerized photoluminometer (Berthold LB950) in whole blood stimulated with opsonized zymosan. This whole blood method of CL measurement has been applied to the study of the phagocytic process and to the investigation of cellular and humoral abnormalities in several pathologies, indicating this assay as a simple, rapid and reliable test.     

The suitability of different microtitre plates for detection of antibody to virus antigens by indirect ELISA

To optimize enzyme linked immunosorbent assays (ELISAs) for the detection of virus-specific antibodies, a range of commercially available microtitre plates was evaluated for their ability to bind virus antigen. Rinderpest virus and foot-and-mouth disease virus were investigated as target antigens. Binding capacity for antigen, binding ratios (attachment of specific antibody versus that of non-immune antibody) and the variation in the results of the tests within and between plates were measured. Binding capacity was found to be greater with rinderpest virus (RPV) antigen than with foot-and-mouth disease virus (FMDV) antigen, although higher binding ratios were obtained with FMDV antigen. Variation within and between plates was generally less with RPV antigen than with FMDV antigen. One plate could not be said to out-perform the other plates in all tests. For our purpose, that is the detection of monoclonal antibody production against a variety of virus antigens, a number of plates were found to be suitable (e.g. Dynatech M129B, Flow 77-172-05 and Nunc 4-39454). The differences in the performances of the microtitre plates with these two virus antigens highlights the need for consideration of the solid phase as part of the standardization procedures for ELISAs.     

Determination of uric acid in human urine and serum by capillary electrophoresis with chemiluminescence detection

A simple and sensitive method based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of uric acid (UA). The sensitive detection was based on the enhancement effect of UA on the CL reaction between luminol and potassium ferricyanide (K₃[Fe(CN)₆]) in alkaline solution. A laboratory-built reaction flow cell and a photon counter were deployed for the CL detection. Experimental conditions for CL detection were studied in detail to achieve a maximum assay sensitivity. Optimal conditions were found to be 1.0 × 10⁻⁴ M luminol added to the CE running buffer and 1.0 × 10⁻⁴ M K₃[Fe(CN)₆] in 0.2 M NaOH solution introduced postcolumn. The proposed CE-CL assay showed good repeatability (relative standard deviation [RSD] = 3.5%, n = 11) and a detection limit of 3.5 × 10⁻⁷ M UA (signal/noise ratio [S/N] = 3). A linear calibration curve ranging from 6.0 × 10⁻⁷ to 3.0 × 10⁻⁵ M UA was obtained. The method was evaluated by quantifying UA in human urine and serum samples with satisfactory assay results.     

Rate constants of oxidation of unsaturated fatty esters studied by chemiluminescence

This study deals with oxidation kinetics of three unsaturated fatty esters: methyl oleate, methyl linoleate and methyl linolenate at temperatures ranging from 90 to 150 °C. The reaction was monitored by chemiluminescence. A kinetic model was derived from a simple mechanistic scheme, in which initiation is due to hydroperoxides decomposition, whereas propagation results from the abstraction of the most labile hydrogen and termination results only from the bimolecular combination of peroxyl radicals. Analysis of induction period duration indicated that hydroperoxides mainly decompose by a bimolecular process. The model well predicts the main features of the experimental chemiluminescence curves. Kinetic parameters of the three unsaturated fatty esters were assessed from inverse method and discussed.     

Characterization of Candida albicans antigenic determinants by two-dimensional polyacrylamide gel electrophoresis and enhanced chemiluminescence

The use of a two-dimensional polyacrylamide gel electrophoresis joined with Western blotting allowed us to investigate the reactivities of antibodies present in sera from mice and humans to antigens of Candida albicans blastoconidia. The analysis of the antibody response in the two models studied and the comparison between the antibody response in infected and noninfected individuals showed that the infection by C. albicans produces changes in the antibody response which may be of relevance in the serodiagnosis of invasive candidiasis. These changes include the induction of antibodies against new antigens, the disappearance of antibodies against a group of antigens and variations in the reactivity of antibodies directed to a different group of antigens. The technique used resolved the isoforms of several antigens including enolase. It is concluded that the antibody response in humans and mice with candidiasis is not homogeneously directed to all the isoforms of an antigen.     

Effect of antioxidants on chemiluminescence produced by reactive oxygen species

Luminol chemiluminescence was used to evaluate the scavenging of superoxide, hydroxyl and alkoxy radicals by four antioxidants: dipyridamole, diethyldithiocarbamic acid, (+)catechin, and ascorbic acid. Different concentrations of these compounds were compared with well-known oxygen radical scavengers in their capacity to inhibit the chemiluminescence produced in the reaction between luminol and specific oxygen radicals. Hydroxyl radicals were generated using the Fenton reaction and these produced chemiluminescence which was inhibited by diethyldithiocarbamate. Alkoxy radicals were generated using the reaction of tert-butyl hydroperoxide and ferrous ion and produced chemiluminescence which was inhibited equally by all of the compounds tested. For the determination of superoxide scavengers we describe a new, simple, economic, and rapid chemiluminescence method consisting of the reaction between luminol and horseradish peroxidase (HRP). With this method it was found that 40 nmol/l dipyridamole, 0.18 μmol/l ascorbic acid, 0.23 μmol/l (+)catechin, and 3 μmol/l diethyldithiocarbamic acid are equivalent to 3.9 ng/ml superoxide dismutase (specific scavenger of superoxide) in causing the same degree of chemiluminescence inhibition. These results not only indicated that the antioxidative properties of these compounds showed different degrees of effectiveness against a particular radical but also that they may exert their action against more than one radical.