A bioluminescent assay for the sensitive detection of proteases

A bioluminescent general protease assay was developed using a combination of five luminogenic peptide substrates. The peptide-conjugated luciferin substrates were combined with luciferase to form a homogeneous, coupled-enzyme assay. This single-reagent format minimized backgrounds, gave stable signals, and reached peak sensitivity within 30 min. The bioluminescent assay was used to detect multiple proteases representing serine, cysteine, and metalloproteinase classes. The range of proteases detected was broader and the sensitivity greater, when compared with a standard fluorescent assay based on cleavage of the whole protein substrate casein. Fifteen of twenty proteases tested had signal-to-background ratios >10 with the bioluminescent method, compared with only seven proteases with the fluorescent approach. The bioluminescent assay also achieved lower detection limits (≤100 pg) than fluorescent methods. During protein purification processes, especially for therapeutic proteins, even trace levels of contamination can impact the protein's stability and activity. This sensitive, bioluminescent, protease assay should be useful for applications in which contaminating proteases are detrimental and protein purity is essential.     

Simultaneous extraction and combined bioluminescent assay of NAD+ and NADH

A new method for extracting pyridine nucleotides from tissue samples at room temperature that allows the simultaneous extraction of both the oxidized and reduced nucleotide when using a 70% buffered ethanol solution as the extractant has been developed. The extraction efficiencies for NAD⁺ and NADH were 91 and 102%, respectively. The extraction method was followed by a combined bioluminescent assay of both nucleotides. A bacterial bioluminescent system, which included luciferase and low levels of a NADH-specific oxidoreductase, was used to produce a constant light intensity directly proportional to the amount of NADH in the tissue extract sample. When the NADH had been measured, the NAD⁺ present in the extract was enzymatically converted to NADH by the addition of alcohol dehydrogenase, after which the second increase in light level was recorded. The sensitivity of the bioluminescent assay presented here is 5 × 10^?14 mol NADH or NAD⁺ per assay.     

Effect of low-level alpha-radiation on bioluminescent assay systems of various complexity.

This study addresses the effects of low-level alpha-radiation on bioluminescent assay systems of different levels of organization: in vivo and in vitro. Three bioluminescent assay systems are used: intact bacteria, lyophilized bacteria, and bioluminescent system of coupled enzyme reactions. Solutions of 241Am(NO3)3 are used as a source of alpha-radiation. It has been shown that activation processes predominate in all the three bioluminescent assay systems subjected to short-term exposure (20-55 h) and inhibition processes in the systems subjected to longer-term exposure to radiation. It has been found that these effects are caused by the radiation component of 241Am3+ impact. The intensity of the 241Am3+ effect on the bioluminescent assay systems has been shown to depend on the 241Am3+ concentration, level of organization and integrity of the bioluminescent assay system. The bioluminescent assay systems in vivo have been found to be highly sensitive to 241Am3+ (up to 10(-17) M).     

Bioluminescent assay for sphingolipid ceramide N-deacylase using <Emphasis Type="Italic">Vibrio harveyi</Emphasis> dark mutant M-17

A new bioluminescent assay method for the activity of sphingolipid ceramide N-deacylase (SCDase: EC 3.5.1.69) as well as ceramidase (CDase: EC 3.5.1.23) was developed using bioluminescent marine bacteria. Enzymatically synthesized ceramide (N-myristoyl sphigosine, C14:0-18:l) and commercial SCDase were used in this demonstration, and myristic (tetradecanoic, C14:0) acid produced by the SCDase hydrolysis was quantified using Vibrio harveyi M-17, a dark mutant of V. harveyi. The in vivo light intensity of M-17 was stimulated up to thousands fold in the presence of myristic acid, was used for this assay. SCDase activity with as little as 10 μU and 5 nM of myristic acid production were detected in less than one min. The assay worked well for the determination of Km and chromatographic fraction assay.     

Development and clinical application of a bioluminescence enzyme immunoassay for oxytocin

The development of a highly sensitive analytical method for oxytocin could be useful in the diagnosis and treatment of autistic spectrum disorder. We previously developed a colorimetric enzyme immunoassay (EIA) for plasma oxytocin measurement. In this study, we developed a method to measure oxytocin concentrations using a higher sensitivity bioluminescent EIA. Biotinylated oxytocin bridged with five lysine residues was used in a competitive format. The standard curve range for oxytocin was 1.0 to 1000 pg/assay. In addition, there was good correlation between the colorimetric and bioluminescent immunoassays in terms of measured oxytocin concentration (r = 0.9665, n = 48). The bioluminescent EIA for plasma oxytocin was more rapid and provided higher sensitivity than the colorimetric immunoassay, making it suitable for clinical application.     

Chemiluminescent assay of co-factors

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

Method for assessment of functional affinity of antibodies for live bacteria

A rapid and convenient method for assessment of functional affinity of antibodies against live bacteria is described. When a combination of immunomagnetic separation (IMS) with bioluminescent or fluorescent genetic labelling of the cells was employed, the method showed good correlation with plate count. However, the use of reporter bacteria allowed results to be obtained within 1 h compared with days using conventional methods. Due to its lower detection limit, the bioluminescent assay performed better than the fluorescent assay. Antibody affinities for Escherichia coli O157:H7 and Salmonella enteritidis were examined at different environmental conditions such as pH 3-7, temperature 4-25 degrees C, and sodium chloride concentrations 0-5% and compared with sensitivities of ELISA.     

Bioluminescent methods of analysis in microbiology

The prospects for application of bioluminescent ATP-metry in microbiology are considered. A bioluminescent assay is proposed to analyse biomass by measuring the content of intracellular ATP by means of immobilized firefly luciferase after ATP extraction with dimethylsulfoxide. The assay can be used for plotting the growth curves of microorganisms and for determining the sensitivity of microorganisms to antibiotics. The detection limit of the assay is 700 cells per ml of the measured solution.     

Influence of phage population on the phage-mediated bioluminescent adenylate kinase (AK) assay for detection of bacteria

AIMS: The effect of phage concentration on the activity of adenylate kinase (AK) released from the cells lysed during infection was investigated in order to optimize a bioluminescent phage-mediated method for bacterial enumeration. METHODS AND RESULTS: The number of bacteria lysed by phages specific to Salmonella enteritidis and E. coli was determined using a bioluminescent method for the detection of AK released. In order to optimize the assay, the effect of phage concentration and time of infection on the amount of AK released was investigated. The release of AK was greatest at a multiplicity of infection (moi) of 10-100. CONCLUSION: The amount of AK released from Salmonella enteritidis and E. coli G2-2 cells by specific phages, SJ2 and AT20, respectively, depended on the type of bacteria, the stage of growth, the nature of phage, moi and time. SIGNIFICANCE AND IMPACT OF THE STUDY: An assay is described which allows detection of E. coli and Salmonella Enteritidis within 2 h at levels of 103 cfu ml-1.     

Use of bacterial luciferase for determining monoamine oxidase activity

A bioluminescence assay is proposed for measuring monoamine oxidase activity in different biological specimens (platelets, mitochondria). The assay is based on the bioluminescent reaction catalysed by bacterial luciferase and coupled to monoamine oxidase. Two modifications of the bioluminescence assay were used. In the first case, the bioluminescent system was added to monoamine oxidase preincubated with the substrates, while in the second case, all the components of the coupled enzymatic systems were directly mixed in a cell. The proposed bioluminescence assay is simple, highly sensitive and rapid, and could be especially useful for biomedical examinations.     

Bioluminescent Assay of Total and Brain-Specific Creatine Kinase Activity in Cerebrospinal Fluid

Abstract: A bioluminescent assay based on the firefly luciferase reaction has been used for determination of creatine kinase activity in CSF. Activities as low as 0.1 U/L can be measured. The coefficient of variation at an activity level of 0.3–0.4 U/L was between 5 and 6%. The assay conditions optimized for serum specimens can be used for CSF. The adenylate kinase activity is almost completely inhibited, which simplifies the procedure. The creatine kinase (CK) isoenzyme distribution was obtained using the bioluminescent assay in combination with immunoinhibition or ion exchange chromatography. All specimens contained both MM and BB activity, but no MB was found. The study indicates that the bioluminescent assay is useful in the determination of CK isoenzymes in CSF. The clinical importance of the observed CK levels will be reported in a separate communication.     

Specific protein-binding reactions monitored with ligand-ATP conjugates and firefly luciferase

A new method was developed to monitor specific protein binding reactions with an ATP-labeled ligand and firefly luciferase. The ligand, 2,4-dinitrobenzene, was covalently coupled to four ATP derivatives and three of these conjugates were measured quantitatively at nanomolar levels with firefly luciferase. Incubation of the conjugates with antibody to the 2,4-dinitrophenyl residue diminished the peak light intensities produced in the bioluminescent assay, whereas incubation with immunoglobulin from a nonimmunized rabbit did not affect light production. Therefore, the antibody-bound ligand-ATP conjugates were inactive in the bioluminescent assay and levels of unbound conjugate could be measured in the presence of the bound form. The firefly luciferase was used to monitor competitive binding reactions between the antibody, the conjugates, and N(2,4-dinitrophenyl)-β-alanine.     

Methodological problems of direct bioluminescent ADP assay in platelets and erythrocytes

We have developed a method for ADP bioluminescent measurement in platelets and erythrocytes which complements our previous method for ATP assay. When the different parameters of the system under investigation are taken into account, a linea range between 10(-9) and 10(-7) g/ml can be obtained without incubation or troublesome extraction. This makes the method easy and useful for identifying any disease-induced alterations in ATP and/or ADP levels in these blood cells. The data obtained correlate well with those of a bioluminescent method requiring extraction with ethanol/EDTA and incubation, giving the reference intervals of 3.5-5.5 mumol/10(11) PLT for ATP determination and 1.9-3.7 mumol/10(11) PLT for ADP determination in platelets, and 3.2-3.8 mumol/g Hgb for ATP determination and 0.56-0.73 mumol/g Hgb for ADP in erythrocytes. This assay was applied to quality control on blood bags in transfusion centers and proved to be a rapid and reliable method for testing the viability of stored blood cells.     

Bioluminescent immunoassay for alpha-fetoprotein

A bioluminescent immunoassay of alpha-fetoprotein is described. It uses monoclonal antibodies labeled with glucose-6-phosphate dehydrogenase and polyclonal antibodies coimmobilized on Sepharose with bioluminescent enzymes from marine bacteria. The bioluminescent reaction which occurs in the immunosorbent is proportional to the amount of alpha-fetoprotein in the assay. The protocol is simple and rapid, and no separation step is required to remove the excess labeled antibodies. The assay can be performed directly on 25 microliters serum and it is as sensitive as other immunometric assays.     

A highly sensitive and rapid method for the detection of DNA fragments using the photoprotein obelin as a reporter

The recombinant Ca²⁺-activated photoprotein obelin was used as a reporter protein in a solid-phase bioluminescent hybridization DNA assay. Oligonucleotide probes were immobilized on the surface of polymer methacrylate beads or microbiological plates of different types. A 30-mer oligonucleotide or its derivative with the biotin residue on the 3′-terminus, as well as a denatured double-stranded PCR fragment of the hepatitis C virus with the sequence of the 30-mer oligonucleotide was used as a DNA template. The probe in the hybridization complex was labeled by the elongation of the chain using a Taq DNA polymerase in the presence of biotinylated deoxyuridine triphosphate. The results of the bioluminescent assay were compared with the results of colorimetric analysis obtained with alkaline phosphatase as a reporter protein. It was shown that the use of the bioluminescent obelin label substantially accelerates the DNA detection procedure, provides a high sensitivity of the assay (no less than 10^?15 mol of DNA template), and ensures a quantitative determination of the amount of DNA template in the tested sample.     

A Bioluminescent γ-Aminobutyrate Assay for Monitoring Its Release from Inhibitory Nerve Endings

Abstract: A bioluminescent GABA assay is described. The principle of the procedure is based on the action of GABASE (GABA-aminotransferase plus succinic semialdehyde dehydrogenase), coupled to the detection of succinic semialdehyde and NADH, using Photobacterium luciferase. The method was used for monitoring GABA release from depolarized brain slices.     

Bioluminescent assays using glucose-6-phosphate dehydrogenase: application to biotin and streptavidin detection

A streptavidin-glucose-6-phosphate dehydrogenase (G6PDH) conjugate was synthesized and its properties were studied, along with those of biotin-G6PDH conjugates. Two bioluminescent assays were used. Streptavidin was assayed in two steps: streptavidin samples were first incubated with a small amount of biotin-G6PDH and then with biotinylated rabbit gamma-globulins. The complex was immobilized on a bioluminescent immunoadsorbent. In the single-step biotin assay, free biotin was allowed to compete with biotin linked to rabbit gamma-globulins for binding to streptavidin-G6PDH in the presence of the bioluminescent immunoadsorbent. Neither assay required washing or separation steps and the sensitivity was 0.2 ng for streptavidin and 100 fg for biotin. Different applications are described: studies of biotin reactivity when linked to probes in solution or immobilized, and quantitation of biotin in biotinylated DNA probes and oligonucleotides.     

A bioluminescent assay for measuring glucose uptake

Identifying activators and inhibitors of glucose uptake is critical for both diabetes management and anticancer therapy. To facilitate such studies, easy-to-use nonradioactive assays are desired. Here we describe a bioluminescent glucose uptake assay for measuring glucose transport in cells. The assay is based on the uptake of 2-deoxyglucose and the enzymatic detection of the 2-deoxyglucose-6-phosphate that accumulates. Uptake can be measured from a variety of cell types, it can be inhibited by known glucose transporter inhibitors, and the bioluminescent assay yields similar results when compared with the radioactive method. With HCT 116 cells, glucose uptake can be detected in as little as 5000 cells and remains linear up to 50,000 cells with signal-to-background values ranging from 5 to 45. The assay can be used to screen for glucose transporter inhibitors, or by multiplexing with viability readouts, changes in glucose uptake can be differentiated from overall effects on cell health. The assay also can provide a relevant end point for measuring insulin sensitivity. With adipocytes and myotubes, insulin-dependent increases in glucose uptake have been measured with 10- and 2-fold assay windows, respectively. Significant assay signals of 2-fold or more have also been measured with human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and skeletal myoblasts.     

Bioluminescent assay of ATPase activity in embryonic material using firefly luciferase

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.     

A bioluminescent assay for 12-alpha-hydroxy bile acids using immobilized enzymes

A bioluminescent assay for 12-alpha-hydroxy bile acids was developed using enzymes coimmobilized onto Sepharose 4B. The immobilized enzymes used were a bacterial 12-alpha-hydroxysteroid dehydrogenase, bacterial luciferase, and NADPH:FMN oxidoreductase or bacterial diaphorase. The assay was specific for 12-alpha-hydroxy bile acids and the lower limit of detection was 4 pmol/0.5 ml assay volume with a linear range of 4 to 2000 pmol. Intraassay precision was from 7.8 to 8.2%. Values obtained with this assay showed good agreement with those obtained by gas-liquid chromatography. The system using diaphorase was not stable at 4 degrees C in the absence of added thiol compounds, but could be stabilized by the addition of glutathione (0.5 mM). The assay is a convenient, a rapid, and an extremely sensitive method for the measurement of 12-alpha-hydroxy bile acid concentrations in the serum of patients or experimental animals.