bluuebunny/crossref_metadata_2025 huggingface dataset:
Bioluminescent assay of ATPase activity in embryonic material using firefly luciferase
Authors: Hanocq‐Quertier J. , Baltus E. , Schram E.
Published: May 2005

The continuous bioluminescent assay of ATP has been adapted to the study of Mg2+‐dependent ATPases, including the (Na+,K+) pump, in amphibian tissues.

A discrete bioluminescent assay procedure for ATPase has also been developed. Components of the firefly luciferase assay reagent modify the observed ATPase activity but this can be circumvented by performing discrete instead of continuous measurements of enzyme activity. In assays with commercial ATPase preparations the continuous bioluminescent assay procedure gave ATPase activities 2.2‐fold lower than obtained with the discrete procedure. In Xenopus oocyte or egg homogenates, in contrast, the total ATPase activity measured is stimulated eight times by the luciferase reagent, mainly through an unexplained activation of a Mg2+‐independent ATPase. In other tissues, such as Xenopus brain homogenates, both the continuous and discrete monitoring procedures are equally suitable for the determination of ATPase activity.
Lipopolysaccharide‐dependent enhancement of adherence‐mediated chemiluminescence response of polymorphonuclear leukocytes
Authors: Dwenger A. , Schweitzer Gertraud , Funck Monika
Published: May 2005

Adherence of resting polymorphonuclear leukocytes to nylon fibre increased the chemiluminescence response (CL) from 99400 to 910300 cpm/25000 PMNL. This effect could be amplified by lipopolysaccharide (LPS) priming of granulocytes in a dose‐dependent fashion. The results of nylon fibre adherence experiments suggest an in vitro model that might approximate certain conditions of in vivo PMNL‐endothelial adherence and respiratory burst activation, and these reactions of polymorphonuclear leukocytes may contribute to the pathomechanisms of the Adult Respiratory Distress Syndrome.
Chemiluminescent determination of choline in cerebrospinal fluid and red blood cells
Authors: Molini G. , Verzé S. , Tosca P. , Zerbi F. , Savoldi F.
Published: May 2005

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).
Determination of sterilization effectiveness by measuring bacterial growth in a biological indicator through firefly luciferase determination of ATP
Authors: Webster Joann J. , Walker Billy G. , Ford Sharon R. , Leach Franklin R.
Published: May 2005

A bioluminescence procedure for measurement of microbial ATP allows a rapid determination of the effectiveness of autoclave sterilization. This determination is achieved faster than detection of acid production in a biological indicator via a pH indicator. Bacterial outgrowth from spores on test strips of the biological indicator was detected by measurement of ATP using the firefly luciferase reaction. A measureable increase in ATP was found after 5 hours of incubation of a biological indicator that had been treated under sterilizing conditions that produced 75% sterility of the biological indicator as measured by acid production. This is a marked improvement over the 24‐48 hours of incubation currently required.
Extra‐weak chemiluminescence of drugs. VIII. Extra‐weak chemiluminescence arising from the amino‐carbonyl reaction
Authors: Kurosaki Yuichiro , Sato Hideaki , Mizugaki Michinao
Published: May 2005

Extra‐weak chemiluminescence (CL) from amino‐carbonyl reactions of L‐lysine with various sugars and aldehydes in aqueous solution was examined. Amongst the aldehydes and sugars tested, glycolaldehyde and D‐arabinose produced the highest CL intensity. The CL of the amino‐carbonyl reaction reached a maximum after about 60 minutes. The CL was pH dependent, and a linear relation between CL intensity and hydroge‐ion concentration was demonstrated. Low oxygen levels inhibited CL and no CL was produced in nitrogen purged solutions. Addition of cupric or ferrous ion, decreased the CL. The involvement of free radical intermediates was demonstrated by ESR. Our findings suggest that the CL of the amino‐carbonyl reaction arises from free radicals derived from melanoidines or their intermediates. CL should prove useful for evaluating the stability of crude drugs extracted from natural resources that contain various amino acid derivatives protein and sugar components.
Bioluminescent flow sensors: L‐lactate dehydrogenase activity determination in serum
Authors: Girotti Stefano , Bassoli Cinzia , Cascione Maria Loredana , Ghini Severino , Carrea Giacomo , Bovara Roberto , Roda Aldo , Motta Roberto , Petilino Roberto
Published: May 2005

The catalytic activity of serum L‐lactate dehydrogenase (LDH), was determined by monitoring the NADH produced by LDH with bacterial bioluminescent enzymes immobilized on a nylon coil.

The LDH reaction of L‐lactate with NAD took place in a flow‐through mixing coil that preceded the bioluminescent detector coil. The response was linear from 1 to 5000 U/l at 37°C and from 3 to 2000 U/l at 25°C. The intra‐ and inter‐assay reproducibility (CV%) were less than 10% and recovery range was 92% to 110%. The results agreed well with those obtained with a spectrophotometric method.
A sensitive CCD image system for detection of chemiluminescent reactions
Authors: Jansen Eugène H. J. M. , Buskens Carin A. F. , van den Berg Rijk H.
Published: May 2005

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.
The effect of quenchers on the chemiluminescence of luminol and lucigenin
Authors: Bottu G.
Published: May 2005

The chemiluminescence of luminol and lucigenin is often used to detect the production of reactive oxygen derivatives by phagocytic cells. Also, several quenchers and enzyme inhibitors are used to determine which oxygen derivatives are responsible for the observed effects. In the present work we have assessed the reliability of dimethylthiourea and cysteamine (OH. quenchers), desferrioxamine (iron chelator) and diethyldithiocarbamate (superoxide dismutase inhibitor). They all react with CIO− and are also strong inhibitors of the luminescence of luminol catalysed by horseradish peroxidase (HRP); cysteamine and diethyldithiocarbamate also react with H2O2. NaN3 is an inhibitor of myeloperoxidase and a quencher of singlet O2, but we found that under certain conditions it can amplify the the luminescence of luminol triggered by CIO− or Fenton's reagent. A complex of copper and penicillamine that had been proposed as an \documentclass{article}\pagestyle{empty}\begin{document}$ {\rm O}_{\rm 2} ^{\bar .} $\end{document} quencher, quenches all luminescent reactions studied. On the other hand, we were able to confirm the relative specificity of other quenchers: taurine for CIO−, benzoate for OH. and mannitol for both OH. and ‘crypto‐OH.’.
Measurement of phagocyte chemiluminescence using a microtitre plate luminometer
Authors: Blair Andrea L. , Cree Ian A. , Beck J. Swanson
Published: May 2005

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.
Luciferase cDNA from Japanese firefly, Luciola cruciata: Cloning, structure and expression in Escherichia coli
Authors: Tatsumi Hiroki , Masuda Tsutomu , Kajiyama Naoki , Nakano Eiichi
Published: May 2005

We have cloned the cDNA for luciferase from lantern poly(A)+ RNA of a Japanese firefly, Luciola cruciata (Genji botaru in Japanese). This cDNA directed the synthesis of enzymatically active luciferase under the control of the lac promoter in Escherichia coli. The amino acid sequence predicted from the cDNA sequence shows that Genji firefly luciferase consists of 548 amino acids and has a molecular weight of 60,024. Considerable sequence homology was found upon the comparison of the Genji and North American firefly luciferases.
Properties and cellular localization of a luciferin binding protein in the bioluminescence reaction of Gonyaulax polyedra
Authors: Morse D. , Fritz L. , Pappenheimer A. M. , Hastings J. W.
Published: May 2005

A luciferin binding protein LBP involved in the bioluminescence reaction of Gonyaulax polyedra was purified and used for antibody production. Luciferin bound to LBP is fluorescent and can be used as a marker in living cells, allowing the localization of LBP in cortical organelles to be visualized. In cell sections, the same peripheral localization was observed using anti‐LBP and immunofluorescence microscopy. The amount of LBP is ten‐fold greater from cells from in night phase compared to those from in day phase, as determined both by immunoblots of cell extracts, and in vivo fluorescence. These changes correlate with the circadian changes in bioluminescence of living cells.
Chemiluminescent immunoassay (CLIA) for urinary albumin
Authors: Bagazgoitia F. Javier , Garcia J. Luis , Ibarra J. M. , Patiño R. , Diéquez Carlos , Weeks Ian , Woodhead J. Stuart
Published: May 2005

A simple chemiluminescent immunoassay (CLIA) for urinary albumin has been developed based on the use of a chemiluminescent acridinium ester‐labelled human albumin and a commercially available antiserum. It includes two incubation steps and a second polyethylene glycol‐assisted antibody separation. The sensitivity of detection is 0.016 mg/l, the assay working range is 0.1‐5 mg/l, and the inter‐assay CVs are ≤ 15%. Using 10− and 50‐fold sample dilutions in assay buffer, a wide working range (1‐250 mg/l) is obtained covering normal and pathological conditions. Timed overnight urine samples (bed rest conditions) were collected on three consecutive days for each patient. Albumin excretion rate (AER) was 4.7 ± 2.7 μg/min (x ± SD), range 1‐15.9 μg/min in 36 healthy subjects (17♂, 19♀, ages 4‐56 years), with day‐to‐day variations of 28.5 ± 20% (x ± SD), range 3.3‐76.1%. The use of an acridinium ester as a chemiluminescent (CL) label overcomes the disadvantages of short shelf‐life and health and safety hazards associated with radioisotopes. Results compare favourably with those obtained using a commercially available RIA kit.
Comparison of the lux systems in Vibrio harveyi and Vibrio fischeri
Authors: Miyamoto C. , Boylan M. , Cragg L. , Meighen E.
Published: May 2005

Comparison of the nucleotide sequences and gene organization of the lux systems from Vibrio harveyi and Vibrio fischeri has demonstrated that the location and order of the lux structural genes are highly conserved whereas considerable divergence has arisen in the location and/or presence of lux regulatory genes in the two marine bacteria. The order of the lux structural genes (luxCDABE) are identical in the two bacteria with the three fatty acid reductase genes (luxCDE) required for aldehyde biosynthesis flanking the luciferase genes (luxAB). Complementation in trans of the upstream V. fischeri DNA containing the luxC and luxD structural genes with the downstream luxA, B and E genes of V. harveyi resulted in luminescent Escherichia coli that did not require any exogenous aldehyde. These results indicate that the light‐emitting systems are very similar in the two bacteria.

However, the lux regulatory systems in these two bacteria appear to have clearly diverged. In V. harveyi, an open reading frame of >40 codons does not exist within 600 bp of the start of luxC in contrast to the luxl regulatory gene present in the V. fischeri lux system. An open reading frame of 615 bp is present farther upstream of luxC with the same direction of transcription and approximate location as the luxR regulatory gene of V. fischeri; however, apparent homologies do not exist between the two genes. The similarities in organization of the lux structural genes and the differences in the existence and/or location of lux regulatory genes in the two Vibrio species raises the question of how these marine bacteria can have a similar growth‐dependent regulation of luminescence expression.
Chemistry, clones, and circadian control of the dinoflagellate bioluminescent system. The Marlene DeLuca memorial lecture
Authors: Hastings J. Woodland
Published: May 2005

Bioluminescence in the dinoflagellate Gonyaulax polyedra occurs as brief bright flashes, originating from many (∼400) small (∼0.5 μm) cytoplasmic organelles which protrude into the acidic vacuole, and are thus surrounded by the tonoplast. Biochemically, the substrate is unusual; it is an open chain tetrapyrrole, highly unstable to air but protected in the cell at pH̃ 8 by virtue of a luciferin binding protein (LBP). This molecule is a dimer of 72 kDa subunits which, upon a decrease in pH, releases luciferin to react with oxygen in the luciferase (∼140 kDa) catalysed luminescent reaction. cDNAs for both luciferase and LBP have been isolated and cloned, and the identity of LBP was confirmed by hybrid selection and in vitro translation of the message. The tenfold circadian (day to night) change in the amount of LBP, which parallels the in vivo rhythm of luminescence, is due to de novo synthesis and subsequent degradation of the protein each day. The LBP mRNA levels, as determined by in vitro translations and by Northern hybridizations, do not vary over the daily cycle, indicating that circadian control of bioluminescence in this species is mediated at the level of translation.
Bioluminescent click beetles revisited
Authors: Wood Keith V. , Lam Y. Amy , McElroy William D. , Seliger Howard H.
Published: May 2005

In studying beetle bioluminescence in the early 1960s, Dr McElroy and his colleagues found that the Jamaican click beetle, Pyrophorus plagiophthalamus, was capable of emitting different colours of light. They further found that the luciferin substrate used by this beetle was the same as that in the firefly, demonstrating that the different colours of bioluminescence were due to differences in the structure of the luciferases. We have recently cloned cDNAs from this beetle species which code for at least four different luciferases. The luciferases are distinguishable by their different colours of bioluminescence when expressed in Escherichia coli. The sequence differences between these different luciferases are few, so the amino acids responsible for the different colours of emission must also be few. Through the construction of hybrid luciferases, by rearranging fragments of the original cDNA clones, we have identified some of these amino acid determinants of colour.
High specific activity chemiluminescent and fluorescent markers: Their potential application to high sensitivity and ‘multi‐analyte’ immunoassays
Authors: Ekins Roger , Chu Frederick , Micallef Jacob
Published: May 2005

The sensitivities of immunoassays relying on conventional radioisotopic labels (i.e. radioimmunoassay (RIA) and immunoradiometric assay (IRMA)) permit the measurement of analyte concentrations above ca 107 molecules/ml. This limitation primarily derives, in the case of ‘competitive’ or ‘limited reagent’ assays, from the manipulation errors arising in the system combined with the physicochemical characteristics of the particular antibody used; however, in the case of ‘non‐competitive’ systems, the specific activity of the label may play a more important constraining role. It is theoretically demonstrable that the development of assay techniques yielding detection limits significantly lower than 107 molecules/ml depends on:

the adoption of ‘non‐competitive’ assays designs;

the use of labels of higher specific activity than radioisotopes;

highly efficient discrimination between the products of the immunological reactions involved.

Chemiluminescent and fluorescent substances are capable of yielding higher specific activities than commonly used radioisotopes when used as direct reagent labels in this context, and both thus provide a basis for the development of ‘ultra‐sensitive’, non‐competitive, immunoassay methodologies. Enzymes catalysing chemiluminescent reactions or yielding fluorescent reaction products can likewise be used as labels yielding high effective specific activities and hence enhanced assay sensitivities.

A particular advantage of fluorescent labels (albeit one not necessarily confined to them) lies in the possibility they offer of revealing immunological reactions localized in ‘microspots’ distributed on an inert solid support. This opens the way to the development of an entirely new generatio of ‘ambient analyte’ microspot immunoassays perrnitting the simultaneous measurement of tens or even hundreds of different analytes in the same small sample, using (for example) laser scanning techniques. Early experience suggests that microspot assays with sensitivities surpassing that of isotopically based methodologies can readily be developed.
1,2‐Dioxetanes: Novel chemiluminescent enzyme substrates. Applications to immunoassays
Authors: Bronstein Irena , Edwards Brooks , Voyta John C.
Published: May 2005

We have synthesized and studied two 1,2‐dioxetane‐based chemiluminescent enzyme substrates: 3‐(2′‐spiroadamantane)‐4‐methoxy‐4‐(3″‐phosphoryloxy)phenyl‐1,2‐dioxetane (AMPPD), and, 3‐(2′‐spiroadamantane)‐4‐methoxy‐4‐ (3″‐β‐D′‐galactopyrano‐yloxy)phenyl‐1,2‐dioxetane (AMPGD), which can be activated to chemiluminescence at 470 nm by alkaline phosphatase and βD‐galactosidase, respectively. In addition, we observed that certain macromolecules enhance the luminescence of AMPPD. For example, the addition of 0.1% bovine serum albumin amplifies the luminescent signal and improves the detection limit for alkaline phosphatase by approximately one order of magnitude under certain conditions. This effect is due to the presence of a hydrophobic microenvironment provided by the enhancer which ‘stabilizes’ the dephosphorylated AMPPD emitter.

Alkaline phosphatase catalysed chemiluminescence from AMPPD is constant for a prolonged period of time. Using AMPPD we were able to detect 0.01 attomole quantities of alkaline phosphatase immobilized on membrane supports and imaged on photographic film and, in solution, measured in a luminometer.

AMPPD and AMPGD offer alternatives to colorimetric and fluorescent subsrates for alkaline phosphatase and β‐D‐galactosidase labels used in enzyme immunoassays. The simplicity and sensitivity of this chemiluminescent readout allowed the development of rapid clinical assays (e.g. β‐hCG, LH, TSH and others).
The development of non‐separation time‐resolved fluoroimmunoassays for the measurement of urinary metabolites
Authors: Barnard Geoff , Kohen Fortune , Mikola Heikki , Lovgren Timo
Published: May 2005

We describe the principles of a new generation of sequential or simultaneous time‐resolved fluoroimmunoassays, namely, simple, rapid, liquid‐phase non‐separation procedures which may be applied to the measurement of urinary steroid and drug metabolites. As an example, a method for the measurement of estrone‐3‐glucuronide in undiluted urine is reported. This method has a similar sensitivity, specificity and accuracy to a conventional separation fluoroimmunoassay or radioimmunoassay but in terms of speed, convenience, precision, reliability and clinical utility the new method has many advantages. The labelled antigen is a novel fluorescent europium chelate covalently linked to estrone‐3‐glucuronide. The antibody‐binding reaction involves the incubation of the labelled antigen (2ng) with a limited concentration of polyclonal or monoclonal antibodies to estrone‐3‐glucuronyl‐6‐BSA and an aliquot of standard or sample (undiluted urine; 10 μl) in microtitre wells. After a 10 min incubation, the fluorescence which emanates from the antibody‐free label is measured in a time‐resolved fluorometer and is proportional to the concentration of estrone‐3‐glucuronide in the standard or sample. The method may be applied for the monitoring of ovarian function in women.
A new luminescence immunoassay for thyrotropin using coated tubes: Evaluation and comparison with immunoradiometric assay
Authors: Pilo A. , Zucchelli G. C. , Ferdeghini M. , Masini S. , Calvo S.
Published: May 2005

A recently available chemiluminescence immunoassay (LIA‐MAT) for TSH, set up by Byk‐Sangtec Diagnostica, has been evaluated and compared with IRMA methods. The system LIA‐MAT uses two monoclonal antibodies: one coated on tubes and the other labelled with isoluminol. To compute the within‐assay precision profile, we estimated the response error relationship from all the duplicates (420) of eleven experiments. The CV of the response was 4–5% from the maximal response until 10,000 RLU (corresponding to about 1 μlU/ml); in the lower response range the CV worsened up to 8%. The sensitivity, derived from the precision profile, was 0.052 μlU/ml similar to that found in IRMA‐MAT (0.044 μlU/ml); the working range extended from 0.33 to 100 μlU/ml. Results from LIA‐MAT in the concentration range 1–30 μlU/ml were compared with the consensus mean produced by users of IRMA methods (IRMA‐MAT, IRMA‐Behring, Maiaclone Serono) participating in an inter‐laboratory survey; a good correlation with IRMA techniques was found. The distribution of TSH determinations produced by LIA‐MAT on 62 low concentration sample (<0.3 μlU/ml) from patients unresponsive to TRH test, was found similar to that observed for the kit IRMA Boots‐Celltech assumed as reference.
Lipid peroxidation during the oxidation of haemoproteins by hydroperoxides. Relation to electronically excited state formation
Authors: Cadenas Enrique
Published: May 2005

The formation of electronically excited states during hydroperoxide metabolism is analysed in terms of recombination reactions involving secondary peroxyl radicals and scission of the OO bond of peroxides by haemoproteins, mainly myoglobin. Both processes may be sequentially interrelated, for the cleavage of H2O2by metmyoglobin leads to the formation of a strong oxidizing equivalent with the capability to promote peroxidation of polyunsaturated fatty acids. The decomposition of lipid hydroperoxides by ferryl‐hydroxo complexes, as that formed during the oxidation of metmyoglobin by H2O2, is a source of peroxyl radicals, the recombination of which proceeds with elimination of a conjugated triplet carbonyl or singlet oxygen.
Formation of electronically excited states during the interaction ofp‐benzoquinone with hydrogen peroxide
Authors: Brunmark Anders
Published: May 2005

The reaction betweenp‐benzoquinone and H2O2in slightly alkaline solutions yields three major quinoid products that accumulate in the reaction mixture: (a) 2,3‐epoxy‐p‐benzoquinone, (b) 2‐hydroxy‐p‐benzoquinone and (c)p‐benzohydroquinone. The reaction is accompanied by photoemission, probably originating from excited triplet 2‐hydroxy‐p‐benzoquinone. These products originate from hydrogen peroxide and hydroxide nucleophilic addition to the C2C3double bond, as well as secondary redox interactions. The hydroxy substituent and the epoxide ring exert a substantial influence on the electronic distribution in thep‐benzoquinone molecule leading to a decrease in the half‐wave potential, as compared to the parentp‐benzoquinone.

The generation of electronically excited states is the result of reactions secondary to the nucleophilic additions involving 2‐hydroxy‐p‐benzosemiquinone, H2O2and hydroxyl radical. The process involves the primary oxidation of 2‐hydroxy‐p‐benzosemiquinone by hydrogen peroxide, followed by oxidation of the semiquinone by hydroxyl radical leading to the formation of the electronically excited quinone. The decay of the excited triplet to the ground state is accompanied by photoemission with maximal intensity at 485–530 nm. Thermodynamic calculations along with an observed increase of photoemission intensity in anaerobiosis point to the triplet (n, π*) multiplicity of the excited state. The efficiency of chemiluminescence could be calculated as 10−8photons/2‐hydroxy‐p‐benzoquinone molecule formed.

Photoemission arising from thep‐benzoquinone/H2O2reaction was inhibited efficiently by addition of GSH to the reaction mixture. This may be due to deactivation of the triplet quinone by a 2‐glutathionyl‐p‐benzohydroquinone adduct, involving thioether α‐hydrogen atom‐transfer to the triplet ketone.
Low‐level chemiluminescence during lipid peroxidations and enzymatic reactions
Authors: Nakano Minoru
Published: May 2005

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.
The lipoxygenase pathway and chemiluminescence in horse eosinophilic leukocytes
Authors: Müller T. , Chavaillaz P. A. , Jörg A. , Grob M. , Peterhans E.
Published: May 2005

It was shown in several cell types that the dual lipoxygenase and cyclooxygenase inhibitor eicosatetraynoic acid but not the cyclooxygenase inhibitor acetylsalicylic acid suppressed luminol‐dependent chemiluminescence. Since lipoxygenase is known to generate chemiluminescence in vitro, these observations were interpreted as evidence for a direct contribution of the lipoxygenase pathway to light emission in intact cells.

We have investigated a possible contribution of the lipoxygenase to the chemiluminescence of horse eosinophils by directly comparing the formation of the byproduct chemiluminescence with the formation of stable end‐products of the lipoxygenase pathway, leukotrienes and HETEs. Azide as well as eicosatetraynoic acid almost completely inhibited chemiluminescence stimulated by the calcium ionophore A23187 but had less effect on the formation of leukotrienes. The tumour‐promoting ester, phorbol myristate acetate, stimulated chemiluminescence in an azide‐ and eicosatetraynoic acid‐sensitive manner and failed to evoke the production of leukotrienes. Azide, but also eicosatetraynoic acid inhibited the luminol‐dependent chemiluminescence generated by isolated eosinophil peroxidase in the presence of H2O2.

Our results argue against a direct role of the lipoxygenase pathway in the generation of light in horse eosinophilic leukocytes but do not exclude that product(s) of this pathway may be involved in stimulus‐response coupling.
Introduction to beetle luciferases and their applications
Authors: Wood Keith V. , Lam Y. Amy , McElroy William D.
Published: May 2005

All beetle luciferases have evolved from a common ancestor: they all use ATP, O2, and a common luciferin as substrates. The most studied of these luciferases is that derived from the firefly Photinus pyralis, a beetle in the superfamily of Cantharoidea. The sensitivity with which the activity of this enzyme can be assayed has made it useful in the measurement of minute concentrations of ATP. With the cloning of the cDNA coding this luciferase, it has also found wide application in molecular biology as a reporter gene. We have recently cloned other cDNAs that code for luciferases from the bioluminescent click beetle, Pyrophorus plagiophthalamus, in the superfamily Elateroidea. These newly acquired luciferases are of at least four different types, distinguishable by their ability to emit different colours of bioluminescence ranging from green to orange. Unique properties of these luciferases, especially their emission of multiple colours, may make them additionally useful in applications.
The regulatory control of the bacterial luminescence system—A new view
Authors: Ulitzur S.
Published: May 2005

We have recently shown that the transcription of the PR lux operon for Vibrio fischeri luminescence is positively controlled by the htpR (σ32) protein. It was suggested that the LexA protein might negatively control the lux genes. This paper extends these findings. It was found that Escherichia coli cells that contain the entire lux operon (pChv1) in RecA or LexA mutants which are unable to remove the LexA protein are considerable dimmer than the wild‐type strain. Mutants that do not make LexA or from a weakly bound LexA are very bright. The role of σ32 protein was studied on luxR‐luxl genes that are fused to β‐galactosidase. The addition of V. fischeri inducer brings about the formation of β‐galactosidase activity in htpR+ but not in htpR− strains of E. coli/pMJ3. Similar to the effect of starvation on the induction of luminescence in marine bacteria and in E. coli/pChv1 cells, β‐galactrosidase activity in such constructs is preferentially induced by low nutrient concentrations. A new model for the regulatory control of the V. fischeri luminescence system is discussed.
Mutants of luminous bacteria selected for bioluminescent toxicity tests
Authors: Wagner A. , Winkler U. , Lümmen P.
Published: May 2005

Mutants of the luminescent bacterial strain NRRL B‐11177 were isolated with pleiotropic hypersensitivity towards hydrophobic antimicrobial agents. SDS‐PAGE analyses of outer membrane proteins and lipopolysaccharides revealed that the outer membrane structure of the ahs‐mutants was altered. QSAR analysis showed that the inhibitory effect of chloro‐substituted phenols on bioluminescence of the ahs‐mutants depended on their hydrophobicity. The effect of chlorinated phenols and detergents on bioluminescence was increased in the ahs‐mutants. The potential use of these mutants in bioluminescent toxicity tests was discussed.
Bacteriuria testing by the ATP method as an integral part in the diagnosis and therapy of urinary tract infection (UTI)
Authors: Lundin A. , Hallander H. , Kallner A. , Lundin U. Karnell , Österberg E.
Published: May 2005

Rapid tests for bacteriuria have the highest value, if the test result is available while the patient is with the doctor. At the bacteriological laboratory rapid testing of samples obtained by mail may be cost‐effective but is of little clinical value. In a previous study performed at a health care centre using conventional urine culture as a reference the ATP test came out as the most reliable one among several rapid bacteriuria tests. The present study was performed to see how the ATP test could be fitted into the routine of the health care centre. Female patients with UTI symptoms were asked to deliver a urine sample to the health care centre laboratory and to wait for the result before seeing the doctor. After having the symptoms confirmed the doctor based the diagnosis on the ATP value. A low ATP value ruled out UTI and a high ATP value confirmed UTI. In patients with an intermediary ATP value (10–50 nmol/I) a positive nitrite test was used to confirm UTI. Only those patients with intermediary ATP values and negative nitrite test had to wait for conventional urine culture. Thus in most patients the decision on antibiotic therapy or not was based on clinical symptoms and ATP results only. Antibiotics (trimethoprim) were given as single dose or as a conventional 7‐day regime in a double‐blind comparison. The correlation between the ATP method and conventional culture was good. Although results of the present study are promising the ATP test as performed is too complicated to become widely accepted at health care centres. However, the dipstick version of the ATP test at present being developed will make the method ideally suited for rapid bacteriuria testing at health care centres and similar doctor's surgery situations.
Determination of adenosine triphosphate in human semen to estimate the fertilizing potential and to quantify sperm antibodies
Authors: Comhaire Frank H. , Vermeulen Lutgart , Monsieur Lieve , Hinting Aucky
Published: May 2005

The measurement of the ATP content of fresh semen is as accurate as the estimation of sperm motility by conventional methods in discriminating between semen of fertile versus subfertile men. The ATP content of frozen thawed donor semen is correlated with the probability of conception per cycle of insemination. Exact quantification of cytotoxic sperm antibodies in serum is possible with the adenosine‐triphosphate‐release‐cytotoxicity test, since measurement is free of the bias of microscopic examination. The procedure has been simplified by testing only one serum dilution and calculating the ‘sperm toxicity index’.
Luciferase of Luciola mingrelica fireflies. Kinetics and regulation mechanism
Authors: Ugarova N. N.
Published: May 2005

Biochemical properties, spectral parameters of bioluminescence and reaction kinetics for Luciola mingrelica firefly luciferase are described and analysed. The kinetic scheme of the enzymatic process is proposed and discussed. Allosteric regulation of luciferase activity by ATP and its analogues is considered and binding Mg2+ to luciferase shown to increase its activity. Regulation mechanism of luciferase activity by phospholipids is analysed and choline‐containing phospholipids shown to be specific luciferase activators. Some properties of firefly luciferae and the luciferase synthesized during firefly mRNA translation in frog oocytes are compared.
New approaches to the preparation and application of firefly luciferase
Authors: Filippova N. Yu. , Dukhovich A. F. , Ugarova N. N.
Published: May 2005

Allowing for the lipid nature of firefly luciferase we have developed a new method for obtaining high‐activity and high‐stability enzyme preparations for bioluminescent microassay. The method includes the step of differential centrifugation in presence of stabilizing additives which entails a partial purification of the enzyme and its essential stabilization likely due to the fact that luciferase retains its lipid environment which plays an important role in catalysis. The resultant luciferase preparation is stable in solution at 4 °C for 2‐3 months and allows the detection of down to 10−11M ATP.

A new method has been offered for luciferase immobilization on film carriers precoated with a phospholipid layer. By sorption of the enzyme on such carriers, the samples of immobilized luciferase have been obtained suitable for constructing chemiluminescent biosensors, in the form of luciferase‐containing films. There are many‐fold applications for detection of ATP micro‐quantities.
Chemiluminescent assay of co‐factors
Authors: Tsuji Akio , Maeda Masako , Arakawa Hidetoshi
Published: May 2005

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.
On the mechanism of peroxyoxalate chemiluminescence. Quenched chemiluminescence as a detection method in HPLC
Authors: Gooijer C. , van Zoonen P. , Velthorst N. H. , Frei R. W.
Published: May 2005

Several analytes such as the inorganic anions bromide, iodide, sulphite and nitrite and organic compounds as substituted anilines and sulphur compounds cause quenching of peroxyoxalate chemiluminescence. A detection method for liquid chromatography based on the quenching phenomenon has been developed. It makes use of an immobilized luminophore, i.e. 3‐aminofluoranthene covalently bound via an alkyl‐spacer on controlled pore glass, packed in the detector cell.

The mechanism behind the quenching has been elucidated by investigating the roles of luminophores (both in the liquid and in solid state) and oxalates in peroxylate CL with respect to quenchers. Most probably the quencher destroys the radical ion pair produced after electron transfer in the last stage of the CIEEL reaction scheme, thus preventing the formation of electronically excited luminophore.
Effects of serine protease inhibitors on oxyradical burst from phagocytizing neutrophils—analysis by chemiluminescence counting and its microscopic imaging
Authors: Suematsu Makoto , Houzawa Shigenari , Miura Soichiro , Nagata Hiroshi , Kitahora Tetsuji , Morishita Tetsuo , Oshio Chikara , Tsuchiya Masaharu
Published: May 2005

Reaction difference of oxyradical generation and luminol‐dependent photoemission of zymosan‐ and phorbol ester‐treated neutrophils were investigated using a conventional photomultiplier and ultrasensitive photonic imaging technique. Zymosan‐treated cells released a concentrated photonic burst corresponding to the cellular distribution. In contrast, phorbol ester‐treated cells produced a negligible level of photoemission, and the additional application of Ca2+ ionophore enhanced the photonic burst, which was gradually spread out into extracellular space. Serine protease inhibitors did not attenuate PMA‐induced chemiluminescence but did attenuate zymosan‐induced chemiluminescence. This suggests the involvement of serine protease in the respiratory burst of phagocytizing neutrophils.
Design of luminescence photobiosensors
Authors: Blum Loïc J. , Gautier Sabine M. , Coulet Pierre R.
Published: May 2005

The potential of immobilized enzyme membranes in biosensors has been explored in our group for several years. Although part of our work has been mainly devoted to electrochemical transducers and oxidases for the design of enzyme electrodes, the demand for ultrasensitive and highly selective sensors led us to consider the use of luminescent enzyme systems associated to optical transduction. When considering the need for operational and reliable biosensors in biotechnology, immobilization and stability of the sensing element still remain, in most cases, an unavoidable problem. We recently proposed a very fast and reliable procedure for preparing enzymatic membranes from Pall (Biodyne Immunoaffinity membranes) supplied in a pre‐activated form. Both the firefly and bacterial systems as well as peroxidase for the chemiluminescent determination of various analytes, could be bound to such a support.

Based on this approach, a fibre‐optic sensor with immobilized enzymes has been designed which permits bio‐ or chemiluminescent analysis of ATP, NADH or H2O2 respectively. With the NADH‐based system, other analytes could be detected using coupled dehydrogenases. This device appears very promising and includes the convenience of both the luminescence sensitivity as well as the handling of the biosensor design.
Stable‐light‐emitting Escherichia coli as a biosensor
Authors: Korpela Matti , Mäntsälä Pekka , Lilius Esa‐Matti , Karp Matti
Published: May 2005

We have studied possibilities for constructing Escherichia coli strains capable of producing stable light. Light production in E. coli is achieved by cloning the genes encoding bacterial luciferase from Vibrio harveyi. To gain the advantage of sensitive detection of light we transferred the genes under the control of a strong, regulatable promoter system.

Stabilization of light produced by E. coli clones was accomplished by finding the optimal plasmid construction and growth conditions as well as suitable measuring buffers. The adjustment of the luciferase synthesis for bioluminescence measurements to a high but not harmful level gives healthy cells and stable luciferase. Cultivation at 30 °C in an uninduced state was found to be the most important factor in getting stable‐light production. The overall cell metabolism being unstressed gives us the possibility of monitoring cell physiology and factors affecting it via bioluminescence reactions in vivo. To make the results easy to interpret the light emission has to be stable during a measurement period of one to several hours. In the case of the original light‐producing bacteria, Vibrio and Photobacterium strains it has not thus far been possible to find conditions where light emission would be stable for several hours. Based on our findings an automated biosensor system can be developed to monitor the effects of biologically active compounds against stable‐light‐producing bacteria.
A chemiluminescent immunoassay for the direct measurement of urinary 5α‐androstane‐3α, 17β‐diol‐glucuronide in urine
Authors: Pinzani P. , Franceschetti F. , Magini A. , Orlandini A. , Pazzagli M.
Published: May 2005

We described a chemiluminescent immunoassay (CIA) for 5α‐androstane‐3α, 17β‐diol‐glucuronide (3α‐diol‐G) in human diluted urine. This method allowed the direct measurement in 1μl of urine avoiding the hydrolysis and extraction steps for sample pretreatment commonly used in routine methods. The hapten 3α‐diol‐G was synthesized by a Koenigs–Knorr reaction. The immunogenic complex, 3α‐diol‐G conjugated to bovine serum albumin (BSA), was employed to induce the formation of specific antibodies in New Zealand rabbits. In addition, the required chemiluminescent (CL) tracer was prepared. The characteristics of the antibody was determined as regard to specificity and sensitivity and the precision of the assay methods established. In 22 hirsute women affected by policystic ovarian syndrome we found 3α‐diol‐G values significantly (p < 0.01) higher (146.28 ± 73.77μg/g of creatinine; mean ± SD) than those observed in normal women (72.1 ± 32.58 μg/g of creatinine; mean ± SD).
Magic lite design and development
Authors: Woodhead J. Stuart , Weeks Ian
Published: May 2005

Chemiluminescence immunoassays have now achieved a recognized place in the diagnostic laboratory. The advantages of this non‐isotopic technology derive from the use of acridinium esters which can be used to label antigens and antibodies to high specific activities, as well as from optimized immunochemistry. The availability of simple, reliable instrumentation for chemiluminescence measurement together with a range of assay kits offers a logical alternative to traditional radioimmunoassay.
Extended standard curve stability on the CCD magic lite immunoassay system using a two‐point adjustment
Authors: Lynch Michael J.
Published: May 2005

Ciba Corning Diagnostics has developed a two‐point adjustment algorithm for use with the Magic Lite System which allows for extended stability of a full 7–10 point calibration curve over the life of a kit. This adjustment is accomplished by using a master calibration curve established during manufacturing along with two calibrators for each assay.

Most conventional non‐automated immunoassays contain anywhere from 6 to 10 calibrators which are included with each run of an assay.

Eliminating the need to run full standard curves and using the two‐point adjustment algorithm results in significant savings in both labour and reagent usage.
Isoluminol as a marker in direct chemiluminescence immunoassays for steroid hormones
Authors: de Boever J. , Kohen F. , Leyseele D. , Vandekerckhove D.
Published: May 2005

We have used isoluminol steroid hormone conjugates in competitive heterogeneous chemiluminescence immunoassays (ClA) for estradiol, progesterone and estriol in body fluids. The assays did not require prior extraction of the steroid hormone with an organic solvent. Polyclonal or monoclonal antibodies, covalently coupled to polyacrylamide microspheres or adsorbed onto the plastic surface of microtritation plates via an intermediate second antibody, were used as solid‐phase. The latter solid‐phase system allows rapid processing of incubation mixtures. Sensitivie and reliable direct CIAs for estradiol in serum, progesterone in serum and in saliva and for estriol in saliva have been developed and their clinical utility has been assessed. Sensitivities ranged from 0.54 to 0.04nmol/l, and precision (% CV) ranged from 10% to 16%.
A sensitive and rapid luminescence sandwich assay for antibodies to hepatitis‐B surface antigen (Anti‐HBsAg)
Authors: Missler Ulrich , Wood William Graham
Published: May 2005

A chemiluminescent assay for hepatitis‐B surface antigen is described which used an isoluminol derivative as the label. The assay is precise intra‐assay CV, 1.96‐2.45%; inter‐assay CV, 5.26‐8.11% and has a lower detection limit for hepatitis‐B surface antigen of 1.3U/I.
Optimization of an HPLC peroxyoxalate chemiluminescence detection system for some dansyl amino acids
Authors: Baeyens W. , Bruggeman J. , Dewaele C. , Lin B. , Imai K.
Published: May 2005

Bis(2,4,6‐trichlorophenyl) oxalate (TCPO)‐hydrogen‐peroxide‐generated chemiluminescence (CL) of four dansyl amino acids has been used as a model system for the optimization of a detection system in reversed‐phase high‐performance liquid chromatography. Dansylated alanine, glutamic acid, methionine, and norleucine were subjected to peroxyoxalate induced CL in a static system and in a flow system under various conditions with respect to TCPO (ethyl acetate) and hydrogen peroxide (acetone) concentrations, solvent composition and flow, using a two‐pump or a one‐pump post‐column reagent system. From the CL‐decay curve, the influence on the emission signal from the total flow rate in the detector was investigated. Special attention was focused on the mixing of the LC eluate and the reagent in order to combine an efficient collection of the emitted light using a 74μI flow cell (originally 10μI in the fluorescence detector) with minimal extra column band broadening. Therefore, a capillary fused‐silica tubing of about 100μm i.d. was inserted against the end‐frit of the column and brought through a mixing tee, in which the solutions of TCPO and hydrogen peroxide were added. The column end tubing ended in the flow cell and the LC eluate and the reagents were mixed when entering the flow‐cell. Average detection limits (S/N=2) of 200fmol injected dansylated amino acid could be reached. A comparison is made between the use of TCPO and DNPO (bis (2, 4‐dinitrophenyl) oxalate).