bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative non‐radioactive in situ hybridization of preproenkephalin mRNA with digoxigenin‐labeled cRNA probes
Authors: Robbins Elaine , Baldino Frank , Roberts‐Lewis Jill M. , Meyer Sheryl L. , Grega Debra , Lewis Michael E.
Published: May 2005

Non‐radioactive detection of mRNA with in situ hybridization histochemistry has emerged as an important new technology for the study of gene expression. Quantitative in situ hybridization studies have generally relied upon counting of autoradiographic grains in the emulsion overlying cells containing hybridized, radioactively labeled probe. However, such high resolution studies require tedious grain counting over individual cells, frequently in addition to weeks of exposure to nuclear emulsion. The present report describes a quantitative, non‐radioactive approach to the detection of a specific mRNA in the brain with the advantages of comparatively rapid tissue processing and computerized image analysis. The validity of this approach was tested by measuring the haloperidol‐induced increase in the level of preproenkephalin mRNA in striatal sections of the rat brain using an RNA probe labeled with digoxigenin‐11‐UTP. Detection of probe hybridized to tissue sections was carried out enzymatically following complex formation with an antidigoxigenin‐alkaline phosphatase conjugate. Using computerized image analysis, it was found that chronic treatment of rats with haloperidol resulted in a 50 ± 6% increase in striatal neuronal optical density, a value in good agreement with previous studies using low‐resolution radioactive methods, showing a 30–80% increase in striatal preproenkephalin mRNA hybridization signal.
Serotonin‐like immunoreactivity in Merkel cells and their afferent neurons in touch domes from the hairy skin of rats
Authors: English Kathleen B. , Wang Zuo‐Zhong , Stayner Naida , Stensaas Larry J. , Martin Hervé , Tuckett Robert P.
Published: May 2005

Immunoreactivity to serotonin was observed in Merkel cells as well as the afferent type I nerves terminating upon them in touch domes excised from the belly skin of rats. Type I nerves were strongly immunoreactive and could be traced through the dermis of the domal papilla. Merkel cell immunoreactivity was sometimes seen in the entire cell, but was often localized in the Merkel cell cytoplasm adjacent to nerve terminals and may have been in the terminals themselves. Domes were fixed by immersion in 4% paraformaldehyde‐lysine‐sodiumm‐periodate (PLP) fixative at 4°C for 2.5–3 hours and cryoprotected in 30% sucrose overnight. Sections were processed with the avidin‐biotin complex peroxidase (ABC), peroxidase‐antiperoxidase (PAP), and indirect immunofluorescence techniques with rabbit antiserum generated against serotonin.
EST! EST!! EST!!!
Authors: Hartl Daniel L.
Published: May 2005

A recently published study(1) has identified a set of candidate genes for human diseases based on findings from Drosophila. Each human expressed sequence tag (EST) in a large database was compared with all known Drosophila genes. After eliminating matches between genes of already known function, the remaining sequences were mapped in the human genome. In each region, the phenotypes of all known human diseases were compared with the phenotypes of known Drosophila mutations in order to identify candidate genes for the human diseases. Are the correspondences real or coincidental?
Human physiology in space
Authors: Vernikos Joan
Published: May 2005

The universality of gravity (1g) in our daily lives makes it difficult to appreciate its importance in morphology and physiology. Bone and muscle support systems were created, cellular pumps developed, neurons organised and receptors and transducers of gravitational force to biologically relevant signals evolved under 1g gravity. Spaceflight provides the only microgravity environment where systematic experimentation can expand our basic understanding of gravitational physiology and perhaps provide new insights into normal physiology and disease processes. These include the surprising extent of our body's dependence on perceptual information, and understanding the effect and importance of forces generated within the body's weightbearing structures such as muscle and bones. Beyond this exciting prospect is the importance of this work towards opening the solar system for human exploration. Although both appear promising, we are only just beginning to taste what lies ahead.
Application of intracellular ATP determination in lymphocytes for HLA‐typing
Authors: Beckers B. , Lang H. R. M. , Weber W. , Pranada F. , Schweizer K.
Published: May 2005

The amount of adenosine triphosphate (ATP) in human lymphocytes was determined using a technique based on light emission from a bioluminescent reaction with luciferin‐luciferase. The amount of ATP changed when cells were incubated in the presence of specific HLA antisera and complement. For determination of intracellular ATP a modified method was applied, which was based on reduction of extracellular ATP by the addition of ATPase. The results of titration of an anti‐human lymphocyte serum using the bioluminescence assay were in agreement with the results of fluorescence vitality staining. Bioluminescent HLA‐determination in 57 cell samples each tested with 5 different antisera also gave good agreement (95.8%) with the conventional method. From these experimental data the calculated ATP content per lymphocyte was 0.135 ± 0.058 pg ATP.
A chemiluminometric method for the determination of urea in serum using a three‐enzyme bioreactor
Authors: Tabata Masayoshi , Murachi Takashi
Published: May 2005

A flow injection chemiluminometric assay for urea has been developed based on a minicolumn bioreactor packed with immobilized enzyme‐bearing glass beads. The reactor contains immobilized urease, L‐glutamate dehydrogenase and L‐glutamate oxidase, aligned in this order (upstream to the downstream). When the sample is introduced into the bioreactor, urea is first hydrolysed by urease to produce ammonia, which is then converted into L‐glutamate by L‐glutamate dehydrogenase. L‐Glutamate is finally oxidized by L‐glutamate oxidase to produce hydrogen peroxide, which is quantified by measuring chemiluminescence emitted upon admixing with luminol and potassium ferricyanide. One assay cycle is completed within 1 minute. The method is sensitive (detection limit 0.5 nmol) and is linear in the range 0–30 mmol/l. It can be readily applied to the determination of urea in human serum, and requires no blank corrections for ammonia and/or L‐glutamate present in serum samples.
Chemiluminescent assay of lipid hydroperoxides
Authors: Belghmi Khalid , Nicolas Jean‐Claude , de Paulet André Crastes
Published: May 2005

The addition of luminol plus a catalyst such as peroxidase or a heme prosthetic group to a solution containing a small quantity of lipid hydroperoxides results in a flash of chemiluminescence, the intensity of which is a function of the hydroperoxide concentrations. Various protocols for lipid hydroperoxide assays have been described and we have studied conditions to increase their sensitivity and specificity.

Plasma lipid hydroperoxide determinations require an extraction, since compounds present in plasma interfere with light emission. Moreover, the sensitivity of the assay is by the presence of hydrogen peroxide in the medium, which causes high background values. Catalase does not act on lipid hydroperoxides and can be used to eliminate hydrogen peroxide from the reaction medium. The determination requires a blank tube in which hydroperoxides are destroyed by incubating the sample with haematin plus ascorbate. The increase in the chemiluminescence of the assay tube caused by the presence of lipid hydroperoxides is then compared to the value obtained for an internal standard.
In memoriam Dr Marlene Deluca
Authors: Kricka Larry J. , Leach Franklin R.
Published: May 2005

The untimely death of Marlene DeLuca in 1987 has deprived the scientific community of an outstanding expert on bioluminescence. Earlier in that year she was honoured as thethirty‐ninth recipient of the Otto Mitchell Smith Lectureship Award at Oklahoma State University, Stillwater, Oklahoma.

On 20 March 1987 Dr DeLuca presented a scientific lecture entitled ‘Firefly Luciferase‐Mechanism of Action, Cloning, and Expression of the Active Enzyme’ and a popular lecture at the banquet that evening entitled ‘Light and Life’. She was selected for her excellence in research, her oral presentation ability, and her personableness. Marlene was the first woman so honoured.

To honour Dr Otto M. Smith the Alpha Delta Chapter of Phi Lambda Upsilon, a national chemistry honorary organization, inaugurated The Otto Mitchell Smith Lectureship in 1948 at Oklahoma State University. Former awardees include Nobel Laureates H. C. Brown, Stanford Moore, and Arthur Kornberg and the following prominent biochemists/molecular biologists: Robert A. Alberty, University of Wisconsin; Daniel E. Koshland, Brookhaven National Laboratory; Sol Spiegelman, University of Illinois; Carl Djerassi, Stanford University; and John T. Edsall, Harvard University. The lectureship honours Dr O. M. Smith, who was Director of the Research Foundation, professor, and Head of the Departments of Chemistry and Chemical Engineering.

As a tribute to Dr DeLuca's outstanding contibution to bioluminescence we reproduce here the edited text of her Otto Mitchell Smith Lectureship and a selected bibliography of her work on firefly bioluminescence.
Enhanced chemiluminescence in the peroxidase‐luminol‐H2O2 system: Anomalous reactivity of enhancer phenols with enzyme intermediates
Authors: Hodgson Martin , Jones Peter
Published: May 2005

Phenols which markedly enhance chemiluminescence in the horseradish peroxidase catalysed oxidation of luminol by hydrogen peroxide show anomalously high reactivity (by factors of ∼102 compared with published Hammett correlations) in the reduction of the enzyme intermediates, Compound I and Compound II. The results support the hypothesis that efficient production of phenoxy radicals from phenols is a necessary criterion for chemiluminescence enhancer action.
Incorporation of enhanced chemiluminescent reactions into fully automated enzyme immunoassays
Authors: Thorpe G. H. , Kricka L. J.
Published: May 2005

Enhanced chemiluminescent quantitation of horseradish peroxidase labels has been incorporated into assays performed on the ES 600 automatic immunoassay analyser. The rapid end‐point produced excellent dose‐response curves and potential savings in total assay times of up to 40%.
Studies on chemiluminescence in the enzymatic and autoxidative transformation of 3,4‐dihydroxyphenylalanine into eumelanins
Authors: Villablanca Miguel , Indig Guilermi , Slawinska Danuta , Slawinski Janusz
Published: May 2005

The catechol oxidase‐catalysed and autoxidative transformation of 3,4‐dihydroxyphenylalanine (DOPA) to eumelanin have been studied by oxygen consumption, energy transfer, absorption and fluorescence spectroscopy. Formation of transient dopachrome (λmax = 480 nm) and dopalutin (λex = 423 nm, λem = 491 nm) have been found in the enzymatic and autoxidative reaction. In the enzymatic reaction, neither a photon emission with quantum yield Φ > 10−13 nor energy transfer to triplet and singlet energy acceptors (sensitizers such as anthracene derivatives, xanthene dyes and chlorophyll‐a) in water and micellar solutions have been found. The autoxidative reaction is chemiluminescent (Φ = 10−9), the emission occurring in the 400‐600 nm range. The excitation energy is not transferred to sensitizers. The effect of various enzymes and traps of active oxygen species as well as the spectral distribution of chemiluminescence indicate that there is no emission from oxygen dimoles. Carbonates and active species of oxygen are shown to participate in the chemiexcitation reaction.
A comparison of chemiluminescence immunoassay and radioimmunoassay for the detection and quantification of methyltestosterone residues in meat samples
Authors: Van Peteghem C. H. , Van Look L. J. , De Guesquière A.
Published: May 2005

A chemiluminescence immunoassay (CLIA) for methyltestosterone is compared with a radioimmunoassay (RIA) employing the same antiserum, raised against methyltestosterone‐3‐CMO‐BSA and using N‐(4‐aminobutyl)‐N‐ethylisoluminol conjugate of MT and [1,2‐H3]methyltestosterone as tracers.

Muscle tissue from slaughtered animals was selected as the matrix. After enzymatic digestion and diethylether extraction only a limited sample clean‐up on Lipidex‐5000 and on Bond Elut C18 was required. Both methods had similar limits of detection, sensitivities, limits of quantification and speed of analysis (working‐load and availability of results).
Novel poly‐substituted aryl acridinium esters and their use in immunoassay
Authors: Law Say‐Jong , Miller Thomas , Piran Uri , Klukas Carol , Chang Steve , Unger John
Published: May 2005

A new series of stable acridinium ester conjugates have been developed for use as non‐isotopic labels in immunoassay. They have proved to be a flexible alternative to radioimmunoassay. We present data showing the successful development of immunoassays in sandwich, competitive and receptor formats. In addition, hydrophilic acridinium ester analogues have been synthesized, encapsulated in liposomes, and utilized as labels in immunoassay. The potential of this technology is discussed.
Luciferin derivatives in bioluminescence‐enhanced enzyme immunoassays
Authors: Miska Werner , Geiger Reinhard
Published: May 2005

Ultrasensitive bioluminescence immunoassays for the determination of peptides and proteins (illustrated with human urinary kallikrein, bradykinin and the determination of human urinary kallikrein antibody titres) have been developed. The usable ranges of the standard curves are from 5 pg to 5000 pg per litre. The relative intra‐assay coefficients of variation of the tests were between 2% and 6%, and the inter‐assay coefficients of variation between 4% and 12%.
T4 and ultrasensitive TSH immunoassays using luminescent enhanced xanthine oxidase assay
Authors: Baret A. , Fert V.
Published: May 2005

The use of xanthine oxidase in immunoanalysis has never been reported. We describe here a procedure in which the xanthine oxidase dependent luminescence of luminol is enhanced in the presence of Fe–EDTA complex, providing an highly sensitive assay (3 amol of enzyme) and a long‐term signal.

This specific amplification has been applied to T4 and ultrasensitive TSH solid phase immunoassays, with T4–XO and anti‐TSH monoclonal antibody‐XO conjugates as tracers. The performances of these assays are at least equivalent to those obtained with iodinated tracers, using the same solid phases and the same calibrators. The major advantages of these immunoassays are: (1) the long‐term signal which can be repeatedly recorded over several days, (2) the high detection sensitivity, (3) the long‐term stability of the luminescence reagent and (4) the stability of the conjugates.
Luminescent immunoassay using isoluminol derivatives
Authors: Messeri Gianni , Orlandini Alessandro , Pazzagli Mario
Published: May 2005

Isoluminol derivatives (ID) were early employed in the preparation of tracers for immunoassay. Their wide use was mainly due to their high quantum efficiency, low molecular weight, well‐known chemical structure, low cost and high stability. Moreover the light efficiency of some ID may be modified by specific binding to the antibody, thus allowing the development of homogenous immunoassays requiring no bound/free separation step. Some ID are commercially available under both the amino terminal and carboxyl terminal form for conjugation to respectively carboxy derivative of steroid and amino residues of protein. The linking reaction can be commonly carried on via active esters chemistry and can be easily accomplished within one day. Steroid conjugates can then be rapidly purified by silica gel thin layer chromatography, and protein conjugates by gel filtration on a short disposable column. In the field of steroid studies, isoluminol derivative conjugates were prepared for the immunoassay of almost all compounds of clinical interest. When dealing with protein, both antigens and antibody were labelled in this way, resulting in highly specific activity tracers for competitive and non‐competitive immunoassays. Recently the labelling of streptavidin with amino‐buty‐ethyl‐isoluminol allowed the development of very sensitive immunoassay methods which take advantage of the biotin‐avidin system.
An investigation on the catalytic mechanism of enhanced chemiluminescence: Immunochemical applications of this reaction
Authors: Vlasenko S. B. , Arefyev A. A. , Klimov A. D. , Kim B. B. , Gorovits E. L. , Osipov A. P. , Gavrilova E. M. , Yegorov A. M.
Published: May 2005

The mechanism of peroxidase‐catalysed oxidation of luminol by H2O2 was studied. The stopped‐flow technique was used to measure the rate constants for the reactions between the oxidized forms of peroxidase with luminol and the following substrates: p‐iodophenol, p‐bromophenol, p‐clorophenol, o‐iodophenol, m‐iodophenol, luciferin, and 2‐iodo‐6‐hydroxybenzothiazole. The correlation between kinetic parameters and the degree of enhancement was established. The effect of charged synthetic polymers and specific antibodies on the peroxidase activity in the enhanced chemiluminescent reaction. Novel homogenous methods of luminescent immunoassay (LIA) for (1) antibodies to insulin, (2) insulin and (3) antibodies to trinitrophenyl group are proposed on the basis of regulatory facilities of the enhanced chemiluminescent reaction. Based on the enhanced chemiluminescent reaction a peroxidase flow‐injection assay was developed and successfuly tested in the flow‐injection enzyme immunoassays for human IgG and for thyroxin (T4). The immunoassay proposed has a detection limit of 10−9M for IgG and 10−11M for T4, the overall time of the assay being 5–15 min.
Near infrared emission of singlet oxygen generated in the dark
Authors: Khan Ahsan U.
Published: May 2005

Singlet oxygen generation is reported from (1) enzymatic reaction and (2) electron transfer reactions of the superoxide anion measured directly with an ultrasensitive near‐IR emission spectrophotometer by monitoring the O2(1Δg) → O2(3Σg−) transition at 1268 nm. Near‐IR emission spectra from the myeloperoxidase and lactoperoxidase enzymatic systems show only emission of singlet oxygen at 1268nm. The lipoxygenase/Na–linoleate enzymatic reaction exhibits two emissions, 1268 nm and 1288 nm. The latter emission is identified as originating from a peroxy radical. Spectral and kinetic data giving evidence of singlet oxygen generation is obtained from the reaction of potassium superoxide solubilized by 18‐crown‐6‐ether in acetonitrile with a series of organometallic coordination compounds.
Hydroxycoumarins as sensitizers and reactants of chemiluminescent oxidative reactions
Authors: Slawinska Danuta , Slawinski Janusz
Published: May 2005

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 (λmax = 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.
Oxidative stress in the rat heart, studies on low‐level chemiluminescence
Authors: Ursini Fulvio , Barsacchi Renata , Pelosi Gualtiero , Benassi Antonio
Published: May 2005

Detection of ultraweak chemiluminescence (CL) emission from the surface of the organ is a sensitive and non‐disruptive tool to evaluate the oxidative stress in rat heart. Indeed, an increased photon emission rate can be observed when cellular antioxidants such as glutathione or vitamin E are depleted, or when organic hydroperoxides are infused. We used CL recording to demonstrate in rat heart that: (i) different diets may lead to different heart sensitivity to an oxidative stress; and (ii) post‐ischaemic reoxygenation induces an oxidative stress. CL emission induced by an oxidative stress is accompanied by an increased release of eicosanoids. However, while non‐steroid anti‐inflammatory drugs (aspirin, indomethacin and ibuprofen) prevented eicosanoid release, these compounds dramatically enhanced hydroperoxide‐dependent CL. The nature of this phenomenon is still obscure, but the increase of steady‐state concentration of excited species caused by anti‐inflammatory drugs seems to be pathophysiologically relevant, since in all our experimental conditions tissue damage was proportional to CL emission rate.
Localization of the luminol‐dependent chemiluminescence reaction in human granulocytes
Authors: Dahlgren Claes , Follin Per , Johansson Agneta , Lock Ronny , Orselius Kristina
Published: May 2005

The granulocyte luminol‐dependent chemiluminescence (CL) reaction is linked to the enzyme myeloperoxidase reacting with products of the respiratory burst activation. The results presented in this paper, show that the light generated in granulocytes originate both from intracellular and extracellular reactions; however, depending on the stimulus used the one or the other will dominate the activity measured. Furthermore, lysosomal fusion is proposed to be required for the intracellular CL reaction.
A rapid chemiluminescent DNA hybridization assay for the detection of Chlamydia trachomatis
Authors: Clyne J. M. , Running J. A. , Stempien M. , Stephens R. S. , Akhavan‐Tafti H. , Schaap A. P. , Urdea M. S.
Published: May 2005

With an estimated 3–4 million new cases per year, human infections from Chlamydia trachomatis are probably the most prevalent sexually transmitted disease (STD) in the United States. Diagnosis of Chlamydia is usually conducted by tissue culture methods. Direct immunofluorescence and ELISA tests have become available, but there remains a need for a test with better specificity and sensitivity. In response to this need, we have developed a rapid DNA hybridization assay using synthetic oligonucleotide probes to detect the presence of the Chlamydia trachomatis specific 7.4 kb plasmid. The assay involves solution phase hybridization of unlabelled probes, rapid capture of the probe‐target duplex onto a microtitre dish surface, a new signal amplification technique that employs chemically cross‐linked oligonucleotides, and an alkaline phosphatase labelled probe. Signal is obtained by reacting the labelled probe‐target complex with an enzyme triggerable dioxetane substrate. Detection of the chemiluminescent output is performed either with a luminometer or by exposure to instant film. All 15 serovars of Chlamydia trachomatis react positively while organisms known to co‐inhabit the human urogenital tract react negatively.
A review of bioluminescent ATP techniques in rapid microbiology
Authors: Stanley Philip E.
Published: May 2005

Use of firefly luciferase to assay adenosine triphosphate (ATP) extracted from microorganisms provides an easy means to enumerate microbes within minutes. The small amount of light produced is proportional to ATP and thus microbial number.

The average bacterium contains around 10−15 g ATP per cell. Present reagents permit detection of 103 cells per tube. Luminometers currently on the market detect about 10−12 g ATP.

Proper extraction of ATP from the microbes is an essential part of any protocol, as is the removal of non‐microbial ATP from, for example, somatic cells also present in samples. The technique may be applied to a wide range of samples, for example food and beverages and clinical samples such as urine.

The ATP assay gives a global measure of microbial numbers, i.e. it is not species specific unless a species separation step is included in the protocol.
Coupled reactions for the determination of analytes and enzymes based on the use of luminescence
Authors: Roda A. , Girotti S. , Ghini S. , Carrea G.
Published: May 2005

Immobilized enzymes are widely used in the clinical laboratory in the assay of several analytes and enzymes. The use of immobilized enzymes makes these reagents recoverable and re‐usable, and in most cases increases their stability and catalytic activity. In conjunction with bioluminescent enzymes (firefly and bacterial luciferases) and chemiluminescent catalyst (peroxidase) we set up high‐sensitive flow methods based on the use of nylon tube coil or epoxy methacrylate column as solid support.

All the NAD(P)/NAD(P)H‐dependent dehydrogenases (bacterial luciferase), ATP‐dependent enzymes (firefly luciferase) and oxidases producing H2O2 (peroxidase) can be immobilized and a large variety of analytes have been sensitively measured. As an alternative format we also reported a dry chemistry method in which all the enzymes, substrates and cofactors are ready to use, supported on dry cellulose disks. Methodological problems such as flow conditions, stability, pH, ionic strength and analytical performances are also reported.
Detection of chemiluminescence in lipid peroxidation of biological systems and its application to HPLC
Authors: Miyazawa Teruo , Saeki Rie , Inaba Humio
Published: May 2005

A combined system of chemiluminescence detection and high performance liquid chromatography (CL–HPLC) was developed to determine primary peroxidation products in biological tissues, such as phosphatidylcholine hydroperoxide (PCOOH). The CL–HPLC assay consists of separation of lipid classes with HPLC and detection of hydroperoxide‐specific chemiluminescence. Hydroperoxides react with heme compounds to produce oxidants as suggested by our early studies on tissue low‐level chemiluminescence in which singlet molecular oxygen is generated as one of the excited species in several biological systems involving free radical events. In the CL–HPLC method, a cytochrome c–luminol mixture was used as a hydroperoxide‐specific luminescent reagent, and the quantification of hydroperoxide was performed by detecting chemiluminescence due to the luminol oxidation caused by the oxidant produced during the lipid hydroperoxides with heme. The detection limit of PCOOH was 10 pmole hydroperoxide–O2. PCOOH in normal human blood was found to be 10–500 pmol/ml plasma and significantly higher levels of PCOOH were observed in some hospitalized patients.
The use of a CCD imaging luminometer in the quantitation of luminogenic immunoassays
Authors: Leaback D. H. , Haggart R.
Published: May 2005

We describe the application of a CCD Imaging Luminometer (CCDIL) for the detection and quantitation of rapid, simultaneous, peroxidase‐linked chemiluminescent immunoassays of multiple samples of human serum alphafetoprotein (AFP). Results from some analogous immunoassays (total IgE, and thyroid stimulating hormone (TSH)) are included for comparison.

Values for precision and antigen concentration obtained using the CCDIL and colorimetric versions of the immunoassays, on human serum samples, were in good agreement.

The flexibility of the CCDIL is demonstrated; its ability to detect and quantitate antigen (particularly AFP) on a variety of solid phases is indicated.

The work on the AFP immunoassays illustrates not only the flexibility of the CCDIL for sample presentation on a variety of solid phase systems, but also some relative merits of such systems.
Bioluminescence in oceanology
Authors: Gitelson I. I. , Levin L. A.
Published: May 2005

For analytical purposes bioluminescence can be used in three main ways: 1. luminescence measurement of bioluminescent system components isolated in vitro; 2. determination of luminous organisms' reaction to the in vivo test‐action; 3. measurement of bioluminescence in marine ecological systems.

The majority of the reports of this Symposium are dealing with the first two topics. The aim of our presentation is to draw attention to the third one. The possibilities of bioluminescent analysis are wider than its traditional scheme of applications in the laboratory, when the emitting system is withdrawn from a native source and is placed in a cuvette of the light measuring device. The reverse scheme is also possible, i.e. the device can be introduced into light emitting system such as a marine biocenosis–the community of the sea inhabitants–where we obtain a highly sensitive and rapid means of gaining the information on the vital activity of marine ecosystems, i.e. their spatial structure, rhythms, man's influence upon them, etc. The present communication will consider the possibilities of this form of bioluminescent analysis.
Methodological problems of direct bioluminescent ATP assay in platelets and erythrocytes
Authors: Girotti Stefano , Ferri Elida , Cascione Maria Loredana , Comuzio Sergio , Mazzuca Augusto , Orlandini Alessandro , Breccia Alberto
Published: May 2005

Direct bioluminescent ATP determination in platelets and erythrocytes involves the study of different parameters which are discussed here. Some parameters are linked to the bioluminescent reaction and to the analyte (ATP); others have regard to the biological matrix. The composition of bioluminescent reagents and the preparation and conservation of the ATP standard, also in the presence of excipients, are among the first given. Matrix problems involve cell characteristics related to age and form, lysis resistance and the possible formation of aggregates (platelets) that may inhibit the complete release of ATP. For these reasons we used the most efficient ATP release agent with the lowest inhibitory effect on luciferase. The data obtained correlate well with a bioluminescent method requiring extraction with ethanol/EDTA, and therefore more time, for ATP determination in platelets and erythrocytes.
Unusual luminescent properties of odd‐ and even‐substituted naphthyl‐derivatized dioxetanes
Authors: Edwards B. , Sparks A. , Voyta J. C. , Bronstein I.
Published: May 2005

With the advent of enzymatically induced chemiluminescence and improved instrumentation for luminometry, ultrasensitive detection of a wide variety of analytes is now possible using standard immunoassay and DNA probe formats. Model molecular orbital calculations and literature precedent suggest that the singlet efficiencies observed upon decomposition of dioxetanes appended with donor substituted aromatic moieties are dependent on substitution pattern. We have recently discovered, in a series of 3‐(2′‐spiroadamantane)‐4‐methoxy‐4‐acetoxynaphth‐2′‐yl‐1,2‐dioxetanes, that enzymatic generation of a nonconjugated, charge transfer excited state results in luminescence of markedly different properties than that observed from an isomeric, conjugated excited state. An example of the former type, 3‐(2′‐spiroadamantane)‐4‐methoxy‐4‐(7″‐acetoxy)naphth‐2′‐yl‐1,2‐dioxetane (1) emitting at 550 nm, not only provides an increase in ΦCL but exhibits a dramatic bathochromic shift of 80–110 nm from the 460 nm emission of the conjugated isomer 3‐(2′‐spiroadamantane)‐4‐methoxy‐4‐(6″‐acetoxy)naphth‐2′‐yl‐1,2‐dioxetane (2).

These developments, along with the attendant glow‐type luminescence kinetics displayed during the enzymatic decomposition of the new ‘odd‐pattern’ dioxetane, allow the design of simple protocols capable of simultaneous or ‘multichannel’ detection of several analytes.
Fusion of luxA and luxB and its expression in E. coli, S. cerevisiae and D. melanogaster
Authors: Almashanu S. , Musafia B. , Hadar R. , Suissa M. , Kuhn J.
Published: May 2005

Luciferase from Vibrio harveyi is encoded by two adjacent genes, luxA and luxB. The two genes were fused by replacing a segment extending from near the end of luxA into the N‐terminal end of luxB by a synthetic oligonucleotide. The construction removed the TAA stop codon at the end of luxA, the intervening region of 26 base pairs, and the initial methionine of luxB. A Smal site was included at the junction between the two genes and an Aatll site was created near the end of luxA without altering its amino acid sequence. In Escherichia coli the fused luxAB gene could be expressed to produce functional luciferase that gave about 20% of the activity in cells without the fusion.

An out‐of frame ATG exists close to and preceding the ATG of the luxA gene. This was removed and the entire fused gene bracketed by several restriction enzyme sites.

The fused luxAB gene was successfully expressed in Saccharomyces cerevisiae and Drosophila melanogaster by transferring it to appropriate plasmid vectors.
The effect of detergents on firefly luciferase reactions
Authors: Simpson W. J. , Hammond J. R. M.
Published: May 2005

The reaction rate of ATP‐limited firefly luciferase‐catalysed reactions, is affected by the presence of detergents. Anionic detergents inhibit luciferase activity without causing significant enzyme inactivation during the reaction. Cationic detergents increase reaction rate several‐fold with a sharply defined optimum concentration of detergent for the effect. However, cationic detergents inactivate firefly luciferase during the reaction, resulting in a continuously decreasing reaction rate. Under such conditions, peak light intensity must be used as an indication of initial reaction rate. The inactivation rate increases with increasing detergent concentration. Non‐ionic and zwitterionic detergents increase reaction rate over a broad range of detergent concentrations. Enzyme stability during the reaction is not affected by non‐ionic detergents and only affected by zwitterionic detergents at high detergent concentration. Cyclodextrins, which can increase reaction rates of some chemiluminescent reactions, have little effect on firefly luciferase activity.

Assays for ATP using firefly luciferase must be internally standardized by the constant addition technique in which a known amount of ATP is added to the test sample, since external calibration of such assays, by reference to a previously prepared standard curve, can lead to imprecision when detergents are present.
Chemiluminescent 10‐methyl‐acridinium‐9‐(N‐sulphonylcarboxamide) salts. Synthesis and kinetics of light emission
Authors: Mattingly Phillip G.
Published: May 2005

10‐Methyl‐acridinium‐9‐(N‐sulphonylcarboxamide) salts are prepared via acylation of sulphonamides with acridine‐9‐carboxylic acid chloride and subsequent N‐alkylation with methyl triflate. Substituents on the sulphonamide component were varied to show the effect of steric and electronic factors on the kinetics of light output. The lifetime of the chemiluminescence light output ranged from 1 to 50 seconds for the 15 compounds reported. The long‐term stability of the new compounds was superior to the phenyl ester counterparts.
Luminol‐dependent chemiluminescence in bovine eosinophils and neutrophils: Differential increase of intracellular and extracellular chemiluminescence induced by soluble stimulants
Authors: Freiburghaus Jürg , Jörg Andreas , Müller Thomas
Published: May 2005

Luminol chemiluminescence was used to detect activation of the respiratory burst oxidase in bovine eosinophils and neutrophils. Extracellular and intracellular chemiluminescence were measured by supplementing the medium with horseradish peroxidase and catalase, respectively. Pure bovine eosinophils (> 90%), maximally stimulated with 1 nmol/l phorbol 12‐myristate‐13‐acetate (PMA) showed ten times more extracellular luminol‐dependent chemiluminescence (CL) than maximally stimulated pure bovine neutrophils (> 96%). Extracellular CL from eosinophils was preferably induced over intracellular CL by both PMA (27‐fold difference) and platelet‐activating factor (PAF) at 2 μmol/l (9‐fold difference), but not by calcium ionophore A23187 (15 μmol/l).

Time course information was used in the following experiments to distinguish between the mode of action of various stimulants. A progressively longer lag period was observed in eosinophil suspensions treated with decreasing doses of PMA, whereas platelet‐activating factor induced a dose‐dependent increase in the maximum response with no change in time to peak CL. The time course of extracellular CL was almost identical to intracellular CL for all stimulants tested, providing no evidence to suggest that extracellular CL stems from a different enzyme system than intracellular CL.

Eosinophils generated most extracellular CL when stimulated with PMA, whereas neutrophils were most efficiently stimulated with A23187, which induced intracellular CL in eosinophils as well as in neutrophils. This accords with the greater tendency of neutrophils to ingest and kill microorganisms, whereas eosinophils are armed to destroy large extracellular targets.
Bilirubin chemiluminescence induced by the attack of active oxygen species
Authors: Watanabe Haruo , Nagoshi Toshiyuki , Agatsuma Shinichi , Kobayashi Masaki , Inaba Humio
Published: May 2005

Ultraweak chemiluminescence (CL) from bilirubin occurs in the presence of triplet oxygen and is stimulated by the addition of aldehydes. Active oxygen species also enhance bilirubin CL, in the absence of aldehydes. An inhibitory effect of active oxygen scavengers on the CL indicated that active oxygens generated from the decomposition of added hydrogen peroxide or from the xanthine‐xanthine oxidase reaction contributed to the CL from bilirubin molecules. However, the contribution of singlet oxygen to the CL disappeared in the presence of formaldehyde. This suggested that the scission of tetrapyrrole bonds via a dioxetane intermediate or the production of triplet carbonyls from the oxidation of aldehydes by singlet oxygen was not involved in the CL, at least in the presence of formaldehyde. The spectrum of CL induced by the generation of active oxygen was the same as that from the aldehyde‐enhanced CL reaction. We propose that the formation of a hydroperoxide (and/or hydroxide) bilirubin intermediate, but not a dioxetane, may be involved in the excitation of bilirubin molecules for CL.
Effect of antioxidants on chemiluminescence produced by reactive oxygen species
Authors: Pascual Carlos , Romay Cheyla
Published: May 2005

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.
Comparison of properties of commercially available crystalline native and recombinant firefly luciferases
Authors: Ford Sharon R. , Hall Marliese S. , Leach Franklin R.
Published: May 2005

Commercially available crystalline native and recombinant firefly luciferases were compared. The two types of luciferase had indistinguishable responses to variation in ATP and luciferin concentrations and to omission of reaction components. The time courses of light production, the responses to nucleotide analogues, and the stability of the enzymes under several storage conditions were identical. The native enzyme had a slightly greater specific activity and was more sensitive to trypsin degradation. These differeces are probably attributable to differences in conformation.
Mechanism for iron control of the Vibrio fischeri luminescence system: Involvement of cyclic AMP and cyclic AMP receptor protein and modulation of DNA level
Authors: Dunlap Paul V.
Published: May 2005

Iron controls luminescence in Vibrio fischeri by an indirect but undefined mechanism. To gain insight into that mechanism, the involvement of cyclic AMP (cAMP) and cAMP receptor protein (CRP) and of modulation of DNA levels in iron control of luminescence were examined in V. fischeri and in Escherichia coli containing the cloned V. fischeri lux genes on plasmids. For V. fischeri and E. coli adenylate cyclase (cya) and CRP (crp) mutants containing intact lux genes (luxR luxlCDABEG), presence of the iron chelator ethylenediamine‐di (o‐hydroxyphenyl acetic acid) (EDDHA) increased expression of the luminescence system like in the parent strains only in the cya mutants in the presence of added cAMP. In the E. coli strains containing a plasmid with a Mu dl(lacZ) fusion in luxR, levels of β‐galactosidase activity (expression from the luxR promoter) and luciferase activity (expression from the lux operon promoter) were both 2‐3‐fold higher in the presence of EDDHA in the parent strain, and for the mutants this response to EDDHA was observed only in the cya mutant in the presence of added cAMP. Therefore, cAMP and CRP are required for the iron restriction effect on luminescence, and their involvement in iron control apparently is distinct from the known differential control of transcription from the luxR and luxlCDABEG promoters by cAMP‐CRP. Furthermore, plasmid and chromosomal DNA levels were higher in E. coli and V. fischeri in the presence of EDDHA. The higher DNA levels correlated with an increase in expression of chromosomally encoded β‐galactosidase in E. coli and with a higher level of autoinducer in cultures of V. fischeri. These results implicate cAMPCRP and modulation of DNA levels in the mechanism of iron control of the V. fischeri luminescence system.
Mechanoluminescence: An assay for lymphocyte analysis in neoplasia
Authors: Orel Valeri E. , Alekseyev Sergei B. , Grinevich Yuri A.
Published: May 2005

We have studied the mechano‐electrochemical activation of optical emission (mechanoluminescence, ML) from the surface of lymphocytes. In C57BL/6 mice with B16 melanoma at the terminal stage of tumour growth and after immunotherapy with thymic agents, there is a correlation between light emission and the value of lymphocyte surface charge and titres of thymic serum factor (FTS).
Luminescence from the yeast Candida utilis and comparisons across three genera
Authors: Tilbury R. N. , Quickenden T. I.
Published: May 2005

Weak luminescence was detected from oxygenated liquid cultures of the yeast Candida utilis during two stages of its growth cycle. The first period of emission occurred during the exponential phase of growth and comprised an ultraviolet band (270‐390 nm; ca 19 photons s− 1 cm−2 of culture surface) and a visible band (450‐620 nm; ca 68 photons s− 1 cm− 2). The second period of emission occurred late in the stationary phase of growth and was comprised almost entirely of a visible region band (450‐620 nm; 6.8 × 102 photons s− 1 cm− 2). No luminescence was observed when the yeast was grown anaerobically. These observations are compared with those previously obtained for two other yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe. The ratios of the intensities of blue/red emissions in the stationary phase luminescences correlated with the ratio of the saturated/unsaturated lipid content for the three yeasts. This result provided further support for the claim that the stationary phase luminescence arises from the reactions associated with lipid peroxidation. A number of previously suggested sources of the exponential phase luminescence are discussed and rejected. Oxidative side reactions accompanying protein synthesis remain a possible source of that emission.
Separation of pH, dilution, lonic strength and chemical matrix effects for biological monitoring of urines with the Microtox® test using nicotine, cotinine and reference urines
Authors: Chou Chin C. , Hee Shane S. Que
Published: May 2005

The aim was to investigate the factors influencing light emission from Photobacterium phosphoreum in the Microtox® test to interpret bioassay results for urine. Four reference urines were assessed as reference materials for the bioassay. Nicotine and cotinine were investigated as urinary markers for tobacco exposure. The optimum luminescence conditions were: 1.85%–3.25% NaCl, 0.33–0.58 mol/L ionic strength, and pH 5.8–6.7. Low pH values and high concentration of toxic trace metals were important factors in this study. Unexpacted toxicity for a Standard Reference Material was attributed to zinc contamination. Nicotine and cotinine together exhibited antagonistic effects in 2% saline but this could not be observed in the urines because of substantial urine toxicity. Thus practical urinary biological monitoring with the Microtox® test necessitates excretion of metabolites and compounds that are much more toxic than the urine components. Also, separation of the effects of physical factors like pH, ionic strength and dilution is essential before chemical toxicity effects can be assigned. This is the first report of Microtox® EC50 values for nicotine and cotinine. The results have application to environmental samples since analyses are often uncontrolled relative to pH, ionic strength and dilution.
A survey of some commercially available kits and reagents which include bioluminescence or chemiluminescence for their operation: Including immunoassays, hybridization, labels, probes, blots and ATP‐based rapid microbiology. Products from more than forty companies
Authors: Stanley Philip E.
Published: May 2005

This survey was compiled between January and November 1992 from public domain information requested by the author from companies specializing in these products. It includes individual sections giving the name and address of each company together with brief details of their (a) kits used for assay of certain analytes in the routine laboratory and (b) reagents used mainly as research tools. This survey is a companion to a previous survey about 90 commercially available luminometers (Stanley PE. J Biolumin Chemilum 1992; 7: 77–108 and 157–69).
Chemiluminescent enzyme immunoassay for alpha‐fetoprotein using β‐D‐galactosidase as label and 5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐galactopyranoside as substrate
Authors: Arakawa H. , Ikegami T. , Maeda M. , Tsuji A.
Published: May 2005

We developed a sensitive chemiluminescent sandwich‐type enzyme immunoassay (CLEIA) of alpha‐fetoprotein (AFP) using b̃‐D‐galactosidase (b̃‐gal) as a label and 5‐bromo‐4‐chloro‐3‐indolyl‐b̃‐D‐galactopyranoside as a substrate. The CL‐EIA for AFP was performed using two monoclonal antibodies, one antibody is labelled with b̃‐gal, the other is coated onto the inside surface of a polystyrene tube. The detection limit for AFP was 0.5 ng/mL, equivalent to 10 pg/assay tube. The coefficient of variation for within and between assay imprecision were 2.0%−4.9% (n = 10) and 4.4%−9.8% (n = 5), respectively. AFP values in serum determined by this method correlated well with those obtained by radioimmunoassay (n = 26, r = 0.99). This sensitive AFP assay can be performed within 4 h and can be used as a routine assay in clinical diagnosis.