Chemiluminescent assay of co-factors

Bioluminescent methods are widely used for the assay of the co-factors, NADH and ATP. Although the bioluminescent method is highly sensitive, the enzymes used are unstable and expensive. Therefore a chemiluminescent method would be valuable in clinical routine assay. We have developed a chemiluminescent method for the assay of NADH using the 1-methoxy-5-methylphenazinium methyl sulphate (1-MPMS)/isoluminol(IL)/microperox-idase(m-POD) system. In order to increase the sensitivity of this method, enzymatic cycling system was coupled to the chemiluminescent assay of NADH. Alcohol dehydrogenase and malate dehydrogenase were used as the cycling enzyme. The standard curve was obtained in the range from 3 × 10^?14 to 5 × 10^?12mol/assay. The detection limit of NADH was 30fmol/assay which was comparable to that of the bioluminescent method using bacterial luciferase. Two chemiluminescent methods for the assay of ATP have been developed. Method 1 is the system using hexokinase/G6PDH and 1-PMS/IL/m-POD, and method 2 is the system based on the enzymatic cycling reaction of ATP using hexokinase/pyruvate kinase. Method 2 is 1000/fold more sensitive than the method 1. The detection limit of ATP was 10 fmol/assay.     

Chemiluminescent assay for detection of viable microorganisms

The redox reaction between quinone and viable microorganisms produces active oxygen species. In this study, the production rates of active oxygen species were determined by a luminol chemiluminescent assay, and the luminescence intensity was found to be proportional to the viable cell number. The high sensitivity of the luminol chemiluminescent assay was achieved with Mo-ethylenediaminetetraacetate complex and menadione or coenzyme Q1. The detectable cell densities of bacteria and yeasts were found to be approximately several thousand colony-forming units (CFU/ml) when assays were performed with a 96-well microplate luminometer. The chemiluminescent assay requires 10 min for incubation of quinone and microorganisms and 2s for photon counting. Single Escherichia coli was detected after 4h of cultivation and centrifugation (5 min x 2). This simple chemiluminescent assay is expected to be useful for the rapid detection of viable bacteria and yeast.     

Application to Mammalian Tissues of the Chemiluminescent Method for Detecting Acetylcholine

Abstract: It is now possible to extend to mammalian tissues the chemilumi-nescent acetylcholine assay. Mammalian tissue extracts must be treated with oxidants (which is not necessary for electric organ extracts). The assay can then be performed as previously described (acetylcholinesterase hydrolyses acetylcholine; choline oxidase converts choline to betaine and H₂O₂, which gives off light in the presence of luminol and peroxidase). It is also shown that release experiments can be performed on mammalian tissue slices (mouse caudate nucleus) after the slice is washed in oxygenated saline solutions.     

Perturbations of the Briggs-Rauscher oscillating system by iron-phenanthroline complexes

Perturbations of the oscillatory regime of the Briggs-Rauscher (BR) reaction by the iron(II)-phenanthroline (ferroin) complex were observed in batch conditions. The path of the perturbation (immediate quenching of oscillations - inhibition time - resumption of oscillations) is similar to that provoked by antioxidant free-radical scavengers; however, perturbations occur in a ferroin concentration range much lower than that in which antioxidants act. To investigate this effect, reactions between the iron(II)-or iron(III)-phenanthroline (ferriin) and reagents or intermediates of the BR system were studied in detail. Results of these studies showed that the interaction of the ferroin/ferriin couple with iodine production is responsible for the observed perturbation. A mechanistic interpretation involving HOO radical is proposed.     

Chemiluminescent determination of choline in cerebrospinal fluid and red blood cells

The concentration of choline in the cerebrospinal fluid (CSF) of patients affected by primary dementia and in red blood cells (RBC) of depressed patients before and after treatment with lithium salts was determined using a chemiluminescent assay. The mean CSF concentration of choline was found to be 60 pmoles/ml (SD = 20 pmoles/ml) and this was lower than values obtained previously by spectrophotometric-colorimetric methods. Mean RBC choline concentrations before and after therapy with lithium salts were 20 nmoles/ml (SD = 16 nmoles/ml and 328 nmoles/ml (SD = 206 nmoles/l) respectively and these are similar to those reported previously (obtained by chemiluminescent and non-chemiluminescent methods).     

Chemiluminescent immunoassay (CLIA) for the detection of brucellosis and tularaemia antigens

The detection of brucellosis and tularaemia infection agents is of particular interest for medical practice. The possibility of using enhanced chemiluminescence reactions for the determination of these agents is studied in this work. Light intensity depends on both the conjugate concentration used and the conditions at which the adsorption was performed. Optimal conditions for these test-systems were: ～ 20 μg/mL of Ig and 200 μg/mL (titre 1:20) of conjugate. As is seen from the chemiluminescent and spectrophotometric results the lowest determined concentrations are 10 and 30 ng/mL (for brucellosis) and 1 and 5 ng/mL (for tularaemia), respectively. Calibration curves in the antigen concentrations ranging from 10 to 2500 ng/mL (for brucellosis) and from 1 to 500 ng/mL (for tularaemia) are observed. Optical density depends linearly on the logarithm of the antigen concentration from 30 to 5000 ng/mL (for brucellosis) and from 5 to 250 ng/mL (for tularaemia). The results obtained permit the conclusion that the chemiluminescence method can be used in enzyme immunoanalysis for brucellosis and tularaemia antigens.     

Chemiluminescence (CL) in blood samples reflects leukocyte activation (LA) during anaphylactoid reactions in dogs

Inflammation is accompanied by leukocyte activation (LA). We decribe a simple ex vivo technique for studying LA that might help to find new LA inhibitors for the treatment of pathologic events related to LA. Arterial and venous blood samples obtained from six permanently catheterized beagle dogs ?60, 0, +15 min and +23 h after i.v. challenge with C 48/80, and also blood samples from six normal beagles, were minimally diluted 1:2.5 with buffer. Total leukocyte counts (LC), and luminol amplified CL, induced by opsonized zymosan (C₃-Z), were estimated. Blood samples from dogs elicited CL responses of almost 1/10 the magnitude of erythrocyte-free human leukocytes, whereas blood samples from rats reacted three orders of magnitude less. Obviously quenching of CL by accompanying erythrocytes in blood samples from dogs is not important, for CL correlated almost linearly with the CL in differently diluted samples. In arterial, but not in venous samples from catheterized dogs, absolute CL and LC, both were significantly depressed (p < 0.05) 15 min after C 48/80 challenge. CL/10⁶ leukocytes was augmented twofold. All leukocyte deviations returned to pre-values 23 h post-challenge.     

Chemiluminescent immunoassay of thyroxine enhanced by microchip electrophoresis

A homogeneous chemiluminescent immunoassay of thyroxine (T4) enhanced by microchip electrophoresis separation has been developed. The method deployed the competitive immunoreaction of T4 and horseradish peroxidase (HRP)-labeled T4 (HRP-T4) with anti-T4 mouse monoclonal antibody (Ab). HRP-T4 and the HRP-T4-Ab complex were separated and quantified by using microchip electrophoresis (MCE) with chemiluminescence (CL) detection. Highly sensitive CL detection was achieved by means of HPR-catalyzed luminol-H₂O₂ reaction. Due to the effective MCE separation, the CL analytical signal was less prone to sample matrix interference. Under the selected assay conditions, the MCE separation was accomplished within 60 s. The linear range for T4 was 5-250 nM with a detection limit of 2.2 nM (signal/noise ratio = 3). The current method was successfully applied for the quantification of T4 in human serum samples. It was demonstrated that the current MCE-CL-enhanced competitive immunoassay was quick, sensitive, and highly selective. It may serve as a tool for clinical analysis of T4 to assist in the diagnosis of thyroid gland functions.     

A sensitive CCD image system for detection of chemiluminescent reactions

A new ultra-sensitive light detection system is described for the detection of chemiluminescence reactions. The system consists of a CCD camera equipped with a two-stage image intensifier coupled to an image analysis system with real-time filtering capabilities. The sensitivity was determined with the enhanced chemiluminescence system using horseradish peroxidase as label. Additional possibilities for further increase in sensitivity and the potential use in immuoassays and other applications are discussed.     

Chemiluminescent oxidation of ribose catalyzed by horseradish peroxidase in presence of hydrogen peroxide

The reaction of ribose with horseradish peroxidase in the presence of H₂O₂ is accompanied by light emission. The detection of horseradish peroxidase Compound II (FeO²⁺) indicates that the enzyme participates in a normal peroxidatic cycle. Hydrogen peroxide converts horseradish peroxidase into Compound I (FeO³⁺) which in turn is converted into Compound II by abstracting a hydrogen atom from ribose forming a ribosyl radical. In aerated solutions oxygen rapidly adds to the ribosyl radical. Based on the spectral characteristics and the enhancement of the chemiluminescence by chlorophyll-a, xanthene dyes, D₂O and DABCO, it is suggested that the excited species, apparently triplet carbonyls and ¹O₂, are formed from the bimolecular decay of the peroxyl radicals via the Russell mechanism.     

Continuous Determination by a Chemiluminescent Method of Acetylcholine Release and Compartmentation in Torpedo Electric Organ Synaptosomes

Abstract: The detection of acetylcholine (ACh) with a chemiluminescent procedure enables one to follow continuously the release of transmitter from stimulated synaptosomes and to study the compartmentation of ACh in resting and active nerve terminals. A compartment of ACh liberated almost entirely by a single freezing and thawing could be directly measured and compared with a compartment of ACh resistant to several cycles of freezing and thawing but liberated by a detergent (60–70% of the total). It is the compartment liberated by freezing and thawing that is reduced when synaptosomes are stimulated. Up to half the total synaptosomal ACh content is readily releasable provided the calcium entry is maintained, or if a strong releasing agent such as the venom of Glycera convoluta is used. In addition, it is shown that synaptosomes contain only negligible amounts of choline, and that the proportion of the two ACh compartments is not influenced by changing extracellular calcium just before their determination.     

Low-level chemiluminescence during lipid peroxidations and enzymatic reactions

Low-level chemiluminescence during lipid peroxidation and enzymatic reaction have been analysed by a filter type spectrometer. Tyrosine and tryprophan residues in proteins were found to be emitters in the visible region during their enzymatic oxidation. The natural chemiluminescence from fertilization of sea urchin eggs was found to have originated from tyrosine – cation radical mediated reaction in ovo-peroxidase – membrane protein – H₂O₂ system.     

Acetylcholine Release from Isolated Synaptic Vesicles Related to Ionic Permeability Changes: Continuous Detection with a Chemiluminescent Method

The effect of ionic permeability changes on acetylcholine (ACh) release from isolated cholinergic synaptic vesicles of Torpedo was studied using a chemiluminescent method for continuous ACh detection. Vesicles rendered freely permeable to potassium by valinomycin lost most of their ACh content in K+ media, if the accompanying anion was permeant; it thus appeared that ACh leakage occurred as the result of internal osmotic changes. Upon addition of ionophores that catalyse monovalent cation/H+ exchange (gramicidin D or a mixture of valinomycin plus protonophore FCCP), a rapid but transient ACh release was observed. Surprisingly, nigericin which also catalyses K+/H+ exchange, had no effect on ACh release. The divalent cation ionophore A23187 promoted ACh release only when calcium (and not magnesium) was introduced into the external medium in a millimolar concentration range. As the simultaneous addition of the protonophore FCCP and A23187 decreased this calcium-dependent ACh leakage, a releasing effect of A23187 through Ca2+/H+ exchange is suspected. The present results emphasise the role of internal protons for ACh retention inside synaptic vesicles.     

Chemiluminescence of the Fenton reaction and a dihydroxybenzene-driven Fenton reaction

A dihydroxybenzenes(DHB)-driven Fenton reaction was found to be more efficient than a simple Fenton reaction based on OH radical and activated species production. The reason for this enhanced reactivity by [Fe DHB] complexes is not well understood, but results suggest that it may be explained by the formation of oxidation species different from those formed during the classic Fenton reactions. In previous work, greater concentrations, and more sustained production of OH over time were observed in DHB driven Fenton reactions versus neat Fenton and Fenton-like reactions. In this work, chemiluminescence (CL) was monitored, and compared to OH production kinetics. The CL of the DHB-driven Fenton reaction was shorter than that for sustained production of OH. CL appears to have been caused by excited Fe(IV) species stabilized by the DHB ligands initially formed in the reaction. Formation of this species would have to have occurred by the reaction between OH and Fe(III) in a DHB complex.     

Hydroxycoumarins as sensitizers and reactants of chemiluminescent oxidative reactions

Chemiluminescence (CL) of 7-hydroxycoumarin, umbelliferone, a strongly fluorescing compound, the precursor of photoactive furocoumarins widely spread in plant kingdom and used in dye lasers has been examined in four oxidative systems producing active oxygen species: A–horseradish peroxidase + H₂O₂ + buffers pH = 3–10; B? K₃Fe (CN)₆ + H₂O₂ + buffers pH = 4–12; C? NaOCI + buffer pH = 3–10.8; and D? HCHO + H₂O₂ + K₂CO₃ (Na₂CO₃), the so-called Trautz–Schorigin reaction. In all these systems a 10–1000-fold increase of CL intensity and quantum yield and an appearance of the blue emission band (λ_max = 460–480 nm), typical of the fluorescence of hydroxycoumarin used have been observed. Spectrophotometric and fluorimetric measurements of the systems A–D indicate that the tested hydroxycoumarins - coumarin, umbelliferone and 6-glucoside umbelliferone or esculin – undergo slow (systems A and B) and fast (systems C and D) consumption, leading to the degradation of coumarins. These findings suggest that efficiently fluorescing hydroxy-coumarins may act both as sensitizers – secondary emitters of CL and/or reactants undergoing oxidative degradation.     

Chemiluminescent determination of copper in the serum

A procedure for chemiluminescent determination of copper in the blood serum ash is suggested using an indicator reaction between 4-aminophthalhydrazide and hydrogen peroxide permitting copper to be determined in a minimum (0.003 micrograms) concentrations in 1 ml of the blood serum.     

Chemiluminescent assays for choline and phospholipase-D using a flow injection system

A flow injection chemiluminescent method is described for the determination of choline. The method is based on the production of hydrogen peroxide from choline using on-line covalently bound immobilized choline oxidase column. The product is mixed downstream and detected via the cobalt catalyzed chemiluminescent oxidation of luminol. The detection limit is 1×10⁻⁷ mol/L, with rsd 1.8 to 2.8% in the range 2–10×10⁻⁵ mol/L. The sample throughput is 30 per hour. The method was applied to the determination of choline produced off-line from phosphatidylcholine using phospholipase-D isolated from cabbage. © 1997 John Wiley & Sons, Ltd.     

Kinetic determination of tryptophan by using the B-Z oscillating chemical system

A simple and rapid method was devised for determination of tryptophan, based on the Belousov-Zhabotinskii (B-Z) oscillating chemical system. Changes in oscillating period and amplitude were linearly proportional to the negative logarithm of l-tryptophan concentration over the range of 6.44 × 10⁻⁷–2.55 × 10⁻⁴ M, with the regression coefficients of near unity and a lower detection limit of 6.5 × 10⁻⁸ M. d-tryptophan was also examined although it is rarely found in most biological fluids, and perhaps not at all in natural proteins. The change of period against to negative logarithm of d-tryptophan concentration over the range of 4.9 × 10⁻⁵–8.24 × 10⁻⁴ M is linear. Because the optimum conditions for determination of l- and d-tryptophan are not the same, a little amount of d-tryptophan does not affect the determination of l-tryptophan. Various influences were studied and a possible mechanism of perturbation to the B-Z oscillator by tryptophan was also discussed. Spectrophotometry and fluorescence spectrophotofluorimetry were used for comparision and confirmation of the results.     

药物类过敏反应生物标志物探究

药物类过敏反应为非免疫球蛋白E(IgE)介导的急性过敏反应,与Ⅰ型超敏反应临床症状相似,首次接触药物即可发生,在临床药物过敏反应中占有相当大的比例.药物类过敏反应生物标志物可特异性反映药物类过敏反应的发生发展,监测其生物标志物可实现对该不良反应进行可靠的非临床评价和临床诊断,从而降低该不良反应的发生率.本文介绍了药物类过敏反应特点,重点阐述了组胺等几种药物类过敏反应生物标志物的研究现状,希望为药物类过敏反应的非临床评价以及临床诊断提供参考.