A reproducible and affordable method of conducting luciferase assay.

Commercially available glow luciferase assay kits are widely popular and convenient to use. However, concerning high-throughput screening, commercial kits are limited by huge running costs. As an alternative to commercial luciferase assay kits, this study presents a cost-effective and efficient methodology of performing a simple and rapid laboratory flash luciferase assay. The proposed luciferase assay method has a versatile use ranging from screening lysates in a microplate reader for quantitative assay as well as screening live cells qualitatively or quantitatively under an imaging system.

In Vitro Assessment of Prebiotic Activity.

Bifidogenic effect is a main target for the assessment of prebiotic activity. pH-controlled batch processes of bifidobacteria and fecal microbiota are herein presented. Growth of bifidobacteria, carbohydrate breakdown and consumption, organic acid production, and activity of specific glycosyl hydrolases involved in the hydrolysis of di-, oligo-, or polysaccharides are exploited to study and compare substrate preference of bifidobacteria for candidate prebiotics.

Comparison of four low-cost carbapenemase detection tests and a proposal of an algorithm for early detection of carbapenemase-producing Enterobacteriaceae in resource-limited settings.

Rapidly progressing antibiotic resistance is a great challenge in therapy. In particular, the infections caused by carbapenem-resistant Enterobacteriaceae (CRE) are exceedingly difficult to treat. Carbapenemase production is the predominant mechanism of resistance in CRE. Early and accurate identification of carbapenemase-producing carbapenem-resistant Enterobacteriaceae (CP-CRE) is extremely important for the treatment and prevention of such infections. In the present study, four phenotypic carbapenemase detection tests were compared and an algorithm was developed for rapid and cost-effective identification of CP-CRE. A total of 117 Enterobacteriaceae (54 CP-CRE, 3 non-CP-CRE, and 60 non-CRE) isolates were tested for carbapenemase production using modified Hodge test (MHT), modified carbapenem inactivation method (mCIM), Carba NP test (CNPt), and CNPt-direct test. The overall sensitivity/specificity values were 90.7%/92.1% for MHT, 100%/100% for mCIM, 75.9%/100% for CNPt, and 83.3%/100% for CNPt-direct. OXA-48-like enzymes were detected with 93.2% sensitivity by MHT and >77.3% sensitivity by two Carba NP tests. MHT could only detect half of the NDM carbapenemase producers. CNPt-direct exhibited enhanced sensitivity compared to CNPt (100% vs 25%) for detection of NDM producers. Considering these findings we propose CNPt-direct as the first test followed by mCIM for rapid detection of CP-CRE. With this algorithm >80% of the CP-CRE could be detected within 24 hours from the time the sample is received and 100% CP-CRE could be detected in day two. In conclusion, mCIM was the most sensitive assay for the identification of CP-CRE. CNPt-direct performed better than CNPt. An algorithm consisting CNPt-direct and mCIM allows rapid and reliable detection of carbapenemase production in resource-limited settings.

Erythrocyte transketolase activity coefficient (ETKAC) assay protocol for the assessment of thiamine status.

Vitamin B1 (thiamine) is an essential nutrient that acts as a cofactor for a number of metabolic processes, particularly in energy metabolism. Symptoms of classic thiamine deficiency are recognized as beriberi, although clinical symptoms are nonspecific and recognition of subclinical deficiency is difficult. Therefore, reliable biomarkers of thiamine status are required. Thiamine diphosphate is a cofactor for transketolase, including erythrocyte transketolase (ETK). The ETK activity assay as an indirect, functional marker of thiamine status has been used for over 50 years. The ETK activity assay provides a sensitive and specific biomarker of thiamine status; however, there is a lack of consensus over the cutoffs for deficiency, partly due to a lack of assay harmonization. Here, we provide a step-by-step protocol for the measurement of ETK activity and the calculation of the ETK activity coefficient, including detailed explanations of equipment and chemicals required and guidance for quality control procedures. Harmonization of the protocol will provide the basis for the development of internationally recognized cutoffs for thiamine insufficiency. The establishment of quality control materials and a quality assurance scheme are recommended to provide reliability. This will ensure that the ETK activity assay remains an important method for the assessment of thiamine status.

qDNase assay: A quantitative method for real-time assessment of DNase activity on coated surfaces.

DNase coatings show great potential to prevent biofilm formation in various applications of the medical implant, food and marine industry. However, straightforward and quantitative methods to characterize the enzymatic activity of these coatings are currently not available. We here introduce the qDNase assay, a quantitative, real-time method to characterize the activity of DNase coatings. The assay combines (1) the use of an oligonucleotide probe, which fluoresces upon cleavage by coated DNases, and (2) the continuous read-out of the fluorescent signal within a microplate fluorometer format. The combination of these two properties results in a real-time fluorescent signal that is used to directly quantify the activity of DNase coatings. As a proof of concept, bovine DNase I coatings were immobilized on titanium by means of chemical grafting and their activity was estimated at 3.87 × 10-4 U. To our knowledge, the qDNase assay provides the first approach to report the activity of a DNase coating in absolute DNase activity units. This assay will not only serve to compare existing DNase coating methods more accurately, but will also enable the rational design of new DNase coating methods in the future.

Enzymatic assays for the assessment of toxic effects of halogenated organic contaminants in water and food. A review.

Halogenated organic compounds are a particular group of contaminants consisting of a large number of substances, and of great concern due to their persistence in the environment, potential for bioaccumulation and toxicity. Some of these compounds have been classified as persistent organic pollutants (POPs) under The Stockholm Convention and many toxicity assessments have been conducted on them previously. In this work we provide an overview of enzymatic assays used in these studies to establish toxic effects and dose-response relationships. Studies in vivo and in vitro have been considered with a particular emphasis on the impact of halogenated compounds on the activity of relevant enzymes to the humans and the environment. Most information available in the literature focuses on chlorinated compounds, but brominated and fluorinated molecules are also the target of increasing numbers of studies. The enzymes identified can be classified as enzymes: i) the activities of which are affected by the presence of halogenated organic compounds, and ii) those involved in their metabolisation/detoxification resulting in increased activities. In both cases the halogen substituent seems to have an important role in the effects observed. Finally, the use of these enzymes in biosensing tools for monitoring of halogenated compounds is described.

Assessment of mitochondrial respiratory chain enzymes in cells and tissues.

Measurement of the individual enzymes involved in mitochondrial oxidative phosphorylation (OXPHOS) forms a key part of diagnostic investigations in patients with suspected mitochondrial disease, and can provide crucial information on mitochondrial OXPHOS function in a variety of cells and tissues that are applicable to many research investigations. In this chapter, we present methods for analysis of mitochondrial respiratory chain enzymes in cells and tissues based on assays performed in two geographically separate diagnostic referral centers, as part of clinical diagnostic investigations. Techniques for sample preparation from cells and tissues, and spectrophotometric assays for measurement of the activities of OXPHOS complexes I-V, the combined activity of complexes II+III, and the mitochondrial matrix enzyme citrate synthase, are provided. The activities of mitochondrial respiratory chain enzymes are often expressed relative to citrate synthase activity, since these ratios may be more robust in accounting for variability that may arise due to tissue quality, handling and storage, cell growth conditions, or any mitochondrial proliferation that may be present in tissues from patients with mitochondrial disease. Considerations for adaption of these techniques to other cells, tissues, and organisms are presented, as well as comparisons to alternate methods for analysis of respiratory chain function. In this context, a quantitative immunofluorescence protocol is also provided that is suitable for measurement of the amount of multiple respiratory chain complexes in small diagnostic tissue samples. The analysis and interpretation of OXPHOS enzyme activities are then placed in the context of mitochondrial disease tissue pathology and diagnosis.

An optimized algorithm with improved turnaround time for detection of carbapenemase-producing Enterobacterales using the NG Test CARBA 5 in a routine laboratory.

Introduction. Rapid and reliable detection of carbapenemase-producing Enterobacterales (CPE) from surveillance cultures is critical in supporting a good infection control programme. We implemented a new algorithm for CPE detection incorporating the NG Test CARBA 5 in January 2019.Aim. Our goals were to compare turnaround time (TAT), costs and staff requirements between the old and new algorithm, and to evaluate the performance of the CARBA 5 test directly on colonies grown on CARBA Smart agar.Methodology. We analysed and compared the TAT of CPE surveillance cultures processed using the old and new CPE screening algorithm. The total actual reagent costs and staff requirements for the new CPE algorithm were compared with the estimated costs and staff requirements of the old CPE algorithm.Results. Of 197 isolates included in the evaluation of the new algorithm, 64 were positive for carbapenemases by both CARBA 5 and Xpert Carba-R assay. Of the 133 that were negative, two were found to harbour NDM and IMI genotypes. Significant improvements in TAT were achieved with 88.7 % of cultures with CPE, reported on the same day as growth was observed on CARBA Smart agar compared to none in the old algorithm. The new algorithm incurred lower costs and, based on our workload, the new algorithm is estimated to save 28.9 man-hours annually.Conclusion. CARBA 5 performs well on colonies growing on CARBA Smart agar and significant improvements in TAT can be achieved without incurring additional costs or staff requirements.

A Simple and Direct Assay for Monitoring Fatty Acid Synthase Activity and Product-Specificity by High-Resolution Mass Spectrometry.

De novo fatty acid synthesis is a pivotal enzymatic process in all eukaryotic organisms. It is involved in the conversion of glucose and other nutrients to fatty acyl (FA) chains, that cells use as building blocks for membranes, energy storage, and signaling molecules. Central to this multistep enzymatic process is the cytosolic type I fatty acid synthase complex (FASN) which in mammals produces, according to biochemical textbooks, primarily non-esterified palmitic acid (NEFA 16:0). The activity of FASN is commonly measured using a spectrophotometry-based assay that monitors the consumption of the reactant NADPH. This assay is indirect, can be biased by interfering processes that use NADPH, and cannot report the NEFA chain-length produced by FASN. To circumvent these analytical caveats, we developed a simple mass spectrometry-based assay that affords monitoring of FASN activity and its product-specificity. In this assay (i) purified FASN is incubated with 13C-labeled malonyl-CoA, acetyl-CoA, and NADPH, (ii) at defined time points the reaction mixture is spiked with an internal NEFA standard and extracted, and (iii) the extract is analyzed directly, without vacuum evaporation and chemical derivatization, by direct-infusion high-resolution mass spectrometry in negative ion mode. This assay supports essentially noise-free detection and absolute quantification of denovo synthetized 13C-labled NEFAs. We demonstrate the efficacy of our assay by determining the specific activity of purified cow FASN and show that in addition to the canonical NEFA 16:0 this enzyme also produces NEFA 12:0, 14:0, 18:0, and 20:0. We note that our assay is generic and can be carried out using commonly available high-resolution mass spectrometers with a resolving power as low as 95,000. We deem that our simple assay could be used as high-throughput screening technology for developing potent FASN inhibitors and for enzyme engineering aimed at modulating the activity and the product-landscape of fatty acid synthases.

Low-concentration trypsin detection from a metal-enhanced fluorescence (MEF) platform: Towards the development of ultra-sensitive and rapid detection of proteolytic enzymes.

Proteolytic enzymes, which serve to degrade proteins to their amino acid building blocks, provide a distinct challenge for both diagnostics and biological research fields. Due to their ubiquitous presence in a wide variety of organisms and their involvement in disease, proteases have been identified as biomarkers for various conditions. Additionally, low-levels of proteases may interfere with biological investigation, as contamination with these enzymes can physically alter the protein of interest to researchers, resulting in protein concentration loss or subtler polypeptide clipping that leads to a loss of functionality. Low levels of proteolytic degradation also reduce the shelf-life of commercially important proteins. Many detection platforms have been developed to achieve low-concentration or low-activity detection of proteases, yet many suffer from limitations in analysis time, label stability, and ultimately sensitivity. Herein we demonstrate the potential utility of fluorescein derivatives as fluorescent labels in a new, turn-off enzymatic assay based on the principles of metal-enhanced fluorescence (MEF). For fluorescein sodium salt alone on nano-slivered 96-well plates, or Quanta Plates™, we report up to 11,000x enhancement for fluorophores within the effective coupling or enhancement volume region, defined as ~100 nm from the silver surface. We also report a 9% coefficient of variation, and detection on the picomolar concentration scale. Further, we demonstrate the use of fluorescein isothiocyanate-labeled YebF protein as a coating layer for a MEF-based, Quanta Plate™ enzymatic activity assay using trypsin as the model enzyme. From this MEF assay we achieve a detection limit of ~1.89 ng of enzyme (2.8 mBAEE activity units) which corresponds to a minimum fluorescence signal decrease of 10%. The relative success of this MEF assay sets the foundation for further development and the tuning of MEF platforms for proteolytic enzyme sensing not just for trypsin, but other proteases as well. In addition, we discuss the future development of ultra-fast detection of proteases via microwave-accelerated MEF (MAMEF) detection technologies.

GABA enzymatic assay kit.

We developed an enzymatic assay system enabling easy quantification of 4-aminobutyric acid (GABA). The reaction of GABA aminotransferase obtained from Streptomyces decoyicus NBRC 13977 was combined to those of the previously developed glutamate assay system using glutamate oxidase and peroxidase. The three-enzyme system allowing GABA-dependent dye formation due to the oxidative coupling between 4-aminoantipyrine and Trinder's reagent enabled accurate quantification of 0.2 - 150 mg/L GABA. A pretreatment mixture consisting of glutamate oxidase, ascorbate oxidase and catalase eliminating glutamate, ascorbate, and hydrogen peroxide, respectively, was also prepared to remove those inhibitory substances from samples. Thus, constructed assay kit was used to measure the GABA content in tomato samples. The results were almost the same as that obtained by the conventional method using liquid chromatography-tandem mass spectrometry. The kit will become a promising tool especially for the on-site measurement of GABA content in agricultural products.

Enzymatic assay for urine lactose in the assessment of recent intravenous abuse of buprenorphine.

Urine samples were analyzed for lactose to investigate if elevated lactose concentrations indicate recent (< 48 hours) intravenous abuse of substances containing lactose as an excipient. Elevated lactose levels were found in samples given by patients who had recently injected substances intravenously, verified by fresh injection marks. Urine lactose assay can support clinical and toxicological findings when assessing substance abuse.

Novel Glycosylated Naphthalimide-Based Activatable Fluorescent Probe: A Tool for the Assessment of Hexosaminidase Activity and Intracellular Hexosaminidase Imaging.

The development of effective detection methods for hexosaminidase is of great importance for the rapid screening of potential inhibitors in vitro and for the early diagnosis of related diseases ex vivo. In this study, the activatable fluorescent probes that are based on naphthalimide decorated with ethylene glycol units were synthesized using N-acetyl-ß-d-glucosaminide as a hexosaminidase-responsive group. When exposed to this enzyme, the glucoside-linked naphthalimide moiety of 1c can be cleaved quickly with significant changes in both color (from colorless to yellow) and fluorescence (from blue to green). Probe 1c shows better water-solubility and fluorescence properties than common substrate 4-methylumbelliferyl N-acetyl-ß-d-glucosaminide. Furthermore, the response mechanism of 1c to hexosaminidase was evaluated using HPLC analysis and TD-DFT calculations. Molecular docking was performed to investigate the interaction mode. In addition, 1c has successfully achieved the straightforward rapid discovery of effective hexosaminidase inhibitors. Fluorescence imaging experiments indicate that 1c has good cell safety and can be employed as a useful tool for detecting intracellular hexosaminidase activity.

Lower creatinine concentration values and lower inter-laboratory variation among Swedish hospital laboratories in 2014 compared to 1996: results from the Equalis external quality assessment program.

Background Creatinine measurement for estimation of glomerular filtration rate (GFR) is a frequently used laboratory test. Differences in analytic creatinine methods have caused large inter-laboratory variation. International and national standardization efforts have been made in the last decade. Methods This study describes the results of the standardization efforts in Sweden by summarizing data for creatinine concentration in blood plasma in the Equalis quality assessment program during 1996-2014. Results Non-compensated Jaffe methods dominated in 1996-2001 (91 of 103 laboratories; 90%) and were then gradually replaced by either compensated Jaffe methods or enzymatic creatinine methods. In 2014 a majority of Swedish hospital laboratories (139 of 159; 87%) used enzymatic methods. The reported mean creatinine value by the Swedish laboratories was about 10 µmol/L higher than the isotope dilution mass spectrometry (IDMS) assured reference value in 2003, but consistent with the reference value from 2009 to 2014. The inter-laboratory CV was 7%-9% for creatinine values until 2007, and thereafter gradually decreased to about 4%-5% in 2014. Conclusions The introduction of enzymatic methods in Swedish laboratories has contributed to achieving a low inter-laboratory variation. Also, the reported values are lower for enzymatic methods compared to Jaffe methods, and the values obtained with enzymatic methods were consistent with IDMS certified values established at reference laboratories. Thus, many Swedish hospital laboratories reported 10 µmol/L lower, and more true, creatinine concentrations in 2012 than in 2003, which may cause bias in longitudinal studies.

Lactate Dehydrogenase Assay for Assessment of Polycation Cytotoxicity.

Cellular toxicity and/or cell death entail complex mechanisms that require multifaceted characterization. A detailed mechanistic assessment of cytotoxicity is essential for design and construction of more effective polycations for nucleic acid delivery. A single toxicity assay cannot stand alone in determining the type and extent of damage or cell death mechanism. In this chapter we describe a lactate dehydrogenase (LDH) assay for high-throughput screening that can be used as a starting point for further detailed cytotoxicity determination. LDH release is considered an early event in necrosis but a late event in apoptosis. An accurate temporal assessment of the toxic responses is crucial as late apoptosis may convert into necrosis as well as in situations where cell death is initiated without any visible cell morphological changes or responses in assays measuring late events, resulting in early ongoing toxicity being overlooked.

Set of Enzymatic Bioassays for Assessment of Soil Contamination.

A concept of the comprehensive assessment of soil contamination is proposed. According to it, the conclusion regarding the presence of toxic substances in the analyzed sample is based on the inhibition of enzymatic reactions responsible for various functions of a living organism, such as luminescence, respiration, etc. These functions are taken as test functions in classical bioassays with the use of living objects (luminous bacteria, daphnia, algae, and others). The regularities of the impact of different classes of toxicants on the activity of particular enzymes or coupled oligo-enzyme chains have been established. These enzyme reactions are selected as potential test objects: markers of contamination. Three enzyme systems with the maximal sensitivity to different classes of toxicants have been chosen for the set of enzymatic bioassays: butyrylcholinesterase, NAD(P)H:FMN-oxidoreductase + luciferase, and lactate dehydrogenase + NAD(P)H:FMN-oxidoreductase + luciferase. The possibility to use enzymes instead of living organisms in the bioassay of natural complex systems has been shown.

A method using angiotensin converting enzyme immobilized on magnetic beads for inhibitor screening.

Angiotensin converting enzyme (ACE), fusing with FLAG tag, was overexpressed in human embryonic kidney 293T cells. This recombinant FLAG-tagged ACE was immobilized on anti-FLAG antibody coated magnetic beads by affinity method in crude cell lysate for the first time. The enzyme-immobilized magnetic beads (ACE-MB), without further cleavage procedure, were used directly to establish a cost-effective and reliable method for screening ACE inhibitors by coupling with fluorescence detection. The enzymatic activity of the ACE-MB was validated based on its Michaelian kinetic behavior towards hippuryl-histidyl-leucine by UHPLC-MS/MS method firstly. Then, several conditions were optimized including amount of magnetic beads, incubation temperature and time in the procedure of ACE immobilization and amount of ACE-MB in the microplate operation. Moreover, this screening assay was validated with Z' factors between 0.71 and 0.81 using four known ACE inhibitors (captopril, lisinopril, fosinopril and fosinoprilat). The developed method was applied for the screening of ACE inhibitors from a small compound library of 45 natural products. As a result, epiberberine and fangchinoline with certain ACE inhibitory activities were screened out in the assay and validated. The results demonstrate the usefulness of this screening method using ACE immobilized on magnetic beads and the advantage of great efficiency with respect to both time and reagents for screening ACE inhibitors.

Inexpensive High-Throughput Screening of Kinase Inhibitors Using One-Step Enzyme-Coupled Fluorescence Assay for ADP Detection.

Protein kinases are attractive targets for both biological research and drug development. Several assay kits, especially for the detection of adenosine diphosphate (ADP), which is universally produced by kinases, are commercially available for high-throughput screening (HTS) of kinase inhibitors, but their cost is quite high for large-scale screening. Here, we report a new enzyme-coupled fluorescence assay for ADP detection, which uses just 10 inexpensive, commercially available components. The assay protocol is very simple, requiring only the mixing of test solutions with ADP detection solution and reading the fluorescence intensity of resorufin produced by coupling reaction. To validate the assay, we focused on CDC2-like kinase 1 (CLK1), a dual-specificity kinase that plays an important role in alternative splicing, and we used the optimized assay to screen an in-house chemical library of about 215,000 compounds for CLK1 inhibitors. We identified and validated 12 potent inhibitors of CLK1, including a novel inhibitory scaffold. The results demonstrate that this assay platform is not only simple and cost-effective, but also sufficiently robust, showing good reproducibility and giving similar results to those obtained with the widely used ADP-Glo bioluminescent assay.

Dual enzyme-assisted one-step isothermal real-time amplification assay for ultrasensitive detection of polynucleotide kinase activity.

A novel, simple, one-step and one-tube detection method was developed for ultrasensitive detection of polynucleotide kinase (PNK) activity on the basis of dual enzyme-synergistic signal amplification. This method was also demonstrated to work well for PNK inhibitor screening and endogenous PNK detection in cell lysates at a single-cell level.

Assessment and Impacts of Phosphorylation on Protein Flexibility of the α-d-Phosphohexomutases.

Enzymes in the α-d-phosphohexomutase (PHM) superfamily catalyze a multistep reaction, entailing two successive phosphoryl transfers. Key to this reaction is a conserved phosphoserine in the active site, which serves alternately as a phosphoryl donor and acceptor during the catalytic cycle. In addition to its role in the enzyme mechanism, the phosphorylation state of the catalytic phosphoserine has recently been found to have widespread effects on the structural flexibility of enzymes in this superfamily. These effects must be carefully accounted for when assessing other perturbations to these enzymes, such as mutations or ligand binding. In this chapter, we focus on methods for assessing and modulating the phosphorylation state of the catalytic serine, as well as straightforward ways to probe the impacts of this modification on protein structure/flexibility. This knowledge is essential for producing homogeneous and stable samples of these proteins for biophysical studies. The methods described herein should be widely applicable to enzymes across the PHM superfamily and may also be useful in characterizing the effects of posttranslational modifications on other proteins.