A spin-reduction assay for lipoxidase.

Spin-label signals are reduced when the nitroxide is present in a lipoxidase reaction mixture. This spin reduction can be used as an assay for enzyme activity in turbid systems where the conventional uv assay cannot be used. Data taken with the spin-reduction assay show that phospholipid preparations containing cholesterol are more resistant to oxidation by lipoxidase than are cholesterol-free preparations.     

A rapid assay for dihydropteroate synthase activity suitable for identification of inhibitors

The enzymes 6-hydroxymethylpterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) catalyze sequential steps in folate biosynthesis. They are present in microorganisms but absent in mammals and therefore are especially suitable targets for antimicrobials. Sulfa drugs (sulfonamides and sulfones) currently are used as antimicrobials targeting DHPS, although resistance to these drugs is increasing. The most widely used assay that measures activity of these enzymes, to assess new inhibitors in vitro, is not amenable to automation. This article describes a simple, coupled, enzymatic spectrophotometric assay where the product of the DHPS reaction, dihydropteroate, is reduced to tetrahydropteroate by excess dihydrofolate reductase (DHFR) using the cofactor NADPH. The oxidation of NADPH is monitored at 340 nm. The activity of both HPPK and DHPS can be measured in this assay, and it has been used to measure kinetic parameters of wild-type and sulfa drug-resistant DHPS enzymes to demonstrate the utility of the assay. It is a sensitive and reproducible assay that can be readily automated and used in multiwell plates. This NADPH-coupled microplate photometric assay could be used for rapid screening of chemical libraries for novel inhibitors of folate biosynthesis as the first step in developing new antimicrobial drugs targeting the folate biosynthetic pathway.     

The bacterial tryptophan reverse mutation assay with Escherichia coli WP2

The Escherichia coli WP2 tryptophan reverse mutation assay detects trp(-) to trp(+) reversion at a site blocking a step in the biosynthesis of tryptophan prior to the formation of anthranilic acid. The different WP2 strains all carry the same AT base pair at the critical mutation site within the trpE gene. The assay is currently used by many laboratories in conjunction with the Ames Salmonella assay for screening chemicals for mutagenic activity. In general the WP2 strains are used as a substitute for, or as an addition to Salmonella strain TA102 which also carries an AT base pair at the mutation site. The assay is also recommended together with the Ames assay for data submission to regulatory agencies. National and international guidelines have been established for performing these mutagenicity assays.The E. coli WP2 assay procedures are the same as those described elsewhere in this volume for the Ames Salmonella assay (Mortelmans and Zeiger, 2000) with the exception that limited tryptophan instead of limited histidine is used. This chapter is an addendum to the previous chapter and the reader should refer to the previous chapter for details regarding experimental procedures and assay design.     

A liver micronucleus assay using young rats exposed to diethylnitrosamine: methodological establishment and evaluation

We have developed a simple liver micronucleus assay using young rats (up to 4 weeks old) to assess cytogenetic damage of chemicals in liver cells. Diethylnitrosamine (DEN) was used as a model rodent hepatocarcinogen in this study. Compared to the partial hepatectomy method most commonly used for the liver micronucleus assay, the present assay method showed equal or even higher practicability. The young rat liver micronucleus assay was also characterized for rat strain differences, sampling time after treatment, single treatment vs. split treatment, age of animals, administration route, and staining method. Although based on one model chemical, we propose an acceptable protocol for the micronucleus assay using young rat liver as follows: Up to 4-week-old rats should be used; oral or intraperitoneal administration can be used; single or repeated treatment protocols can be applied; sampling time is 3-5 days after the last treatment; hepatocytes are prepared by the collagenase perfusion method; and cells are stained with the AO-DAPI double staining method.     

Fluorometric assay for urea in urine, plasma, and tubular fluid

The variation in the sensitivity of proteins to some commonly used protein assay procedures was estimated by a calculation of the ideal titer per unit weight of protein for a sample of 350 proteins. On the basis of such calculations, the variation expected of nitrogen-based assay procedures is expected to be small, and such procedures are considered to give a more consistent quantification of a variety of proteins than other commonly used assay procedure.     

Homogeneous time-resolved fluorescence quenching assay (LANCE) for caspase-3

In addition to kinases and G protein-coupled receptors, proteases are one of the main targets in modern drug discovery. Caspases and viral proteases, for instance, are potential targets for new drugs. To satisfy the current need for fast and sensitive high-throughput screening for inhibitors, new homogeneous protease assays are needed. We used a caspase-3 assay as a model to develop a homogeneous time-resolved fluorescence quenching assay technology. The assay utilizes a peptide labeled with both a luminescent europium chelate and a quencher. Cleavage of the peptide by caspase-3 separates the quencher from the chelate and thus recovers europium fluorescence. The sensitivity of the assay was 1 pg/microl for active caspase-3 and 200 pM for the substrate. We evaluated the assay for high-throughput usage by screening 9600 small-molecule compounds. We also evaluated this format for absorption/distribution/metabolism/excretion assays with cell lysates. Additionally, the assay was compared to a commercial fluorescence caspase-3 assay.     

A simple assay for measuring cellobiose dehydrogenase activity in the presence of laccase.

The commonly used assay for measuring cellobiose dehydrogenase (CDH) activity, based on the reduction of dichlorophenol-indophenol (DCIP), has been adapted to measure this enzyme activity in the presence of laccase, which is often formed concurrently with CDH by a number of fungi. Laccase interferes with the assay by rapidly reoxidizing the reduced form of DCIP and can mask CDH activity completely. It can be conveniently and completely inhibited by 4 mM fluoride in the assay, while CDH activity is only slightly affected by the addition of this inhibitor. The modified assay enables the detection of low CDH activities even in the presence of very high excesses of laccase. It should be useful for screening culture supernatants of wood-degrading fungi for CDH since the assay is rapid and uses inexpensive and nontoxic reagents. Furthermore, it might be used for the detection of other enzyme activities which are assayed by following the reduction of quinones or analogue compounds such as DCIP.     

Utilization of fluorescence polarization and time resolved fluorescence resonance energy transfer assay formats for SAR studies: Src kinase as a model system

High-throughput screening (HTS), a major component of lead identification, often utilizes fluorescence-based assay technologies. For example, HTS kinase assays are formatted using a variety of fluorescence-based assay technologies including, but not limited to, dissociation enhanced lanthanide fluoroimmunoassay (DELFIA), time-resolved fluorescence resonance energy transfer (TR-FRET), and fluorescence polarization (FP). These assays offer tremendous advantages such as a nonradioactive format, ease of automation, and excellent reproducibility. Fluorescence-based assays frequently used for lead identification can also be useful for structure activity relationship (SAR) studies during lead optimization. An important issue when assessing an assay to be used for SAR is the ability of the assay to discriminate high-affinity small molecule inhibitors (pM-nM) from low-affinity inhibitors (microM-mM). The purpose of this study was to utilize HTS-friendly assay formats for SAR by developing TR-FRET, FP, and DELFIAassays measuring Src kinase activity and to define the theoretical lower limit of small molecule inhibitor detection achievable with these assay formats. The authors show that 2 homogeneous assay formats, TR-FRET and FP, allowed for the development of Src kinase assays with a lower limit of detection of K(i) = 0.01 nM. This study indicates that assay technologies typically used for HTS can be used during lead optimization by providing quantitative measurements of compound activity critical to driving SAR studies.     

Direct high-performance liquid chromatography determination of diastereomeric oxprenolol glucuronides

The β-blocking agent oxprenolol is used therapeutically as the racemate. In humans and animals it is metabolized i.a. to ether glucuronide diastereomers. A stereoselective HPLC assay was developed to determine directly, without hydrolysis to their parent enantiomers, the oxprenolol glucuronides in biological samples. The glucuronide standards for this direct assay are prepared by incubation of rabbit liver microsomes with RS-oxprenolol. The glucuronides obtained are purified and concentrated with solid-phase extraction, and their concentration is measured by an indirect method, i.e. HPLC assay of the oxprenolol enantiomers after enzymatic hydrolysis with β-glucuronidase. The direct assay involves separation by HPLC using a C₁₈-reversed-phase column, with UV detection at 274 nm; nalorphine is used as internal standard. On injection onto the column, without previous hydrolysis, the limit of detection is 20 ng for both glucuronides. The assay is sensitive, accurate and reproducible. The method is suitable for the assay of glucuronides in liver microsomal incubates and plasma.     

Assay for cellobiose dehydrogenase in the presence of laccase

The currently used assay for cellobiose dehydrogenase (CDH), an enzyme produced by many wood degrading fungi, lacks specificity and can give false results. The presence of laccase interferes with the standard assay. We have developed an assay for CDH that is insensitive to the presence of both laccase and other phenoloxidases. The method is based on the decrease of reducing end groups in lactose determined by the DNS method. Ferricyanide is present as electron acceptor. Advantages and drawbacks of CDH assay methods are discussed     

Comparison of sensitivity between real-time detection of a TaqMan assay for Batrachochytrium dendrobatidis and conventional detection

A sensitive and quantitative TaqMan assay for the causative agent of chytridiomycosis in amphibians (Batrachochytrium dendrobatidis) has been developed and is routinely used in diagnostic laboratories. We assessed whether the real time detection of the TaqMan assay was as sensitive as the detection of the PCR product by agarose gel electrophoresis and ethidium bromide staining. We found, for practical purposes, that gel-based detection of the diagnostic fragment produced by means of the TaqMan assay or by conventional PCR that used a different polymerase and reaction mix was as sensitive as the real-time detection of the TaqMan assay. We recommend the qualified use of conventional PCR amplification combined with agarose gel electrophoresis and ethidium bromide staining for studies where only prevalence data are required, funding for equipment is limited or the acquisition of a real-time system is not cost effective.     

An Optimized SYBR Green I/PI Assay for Rapid Viability Assessment and Antibiotic Susceptibility Testing for Borrelia burgdorferi

Lyme disease caused by Borrelia burgdorferi is the most common tick-borne disease in the US and Europe. Unlike most bacteria, measurements of growth and viability of B. burgdorferi are challenging. The current B. burgdorferi viability assays based on microscopic counting and PCR are cumbersome and tedious and cannot be used in a high throughput format. Here, we evaluated several commonly used viability assays including MTT and XTT assays, fluorescein diacetate assay, Sytox Green/Hoechst 33342 assay, the commercially available LIVE/DEAD BacLight assay, and SYBR Green I/PI assay by microscopic counting and by automated 96-well plate reader for rapid viability assessment of B. burgdorferi. We found that the optimized SYBR Green I/PI assay based on green to red fluorescence ratio is superior to all the other assays for measuring the viability of B. burgdorferi in terms of sensitivity, accuracy, reliability, and speed in automated 96-well plate format and in comparison with microscopic counting. The BSK-H medium which produced a high background for the LIVE/DEAD BacLight assay did not affect the SYBR Green I/PI assay, and the viability of B. burgdorferi culture could be directly measured using a microtiter plate reader. The SYBR Green I/PI assay was found to reliably assess the viability of planktonic as well as biofilm B. burgdorferi and could be used as a rapid antibiotic susceptibility test. Thus, the SYBR Green I/PI assay provides a more sensitive, rapid and convenient method for evaluating viability and antibiotic susceptibility of B. burgdorferi and can be used for high-throughput drug screens.     

Tetrazolium [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction by mycoplasmas.

We investigated 22 mycoplasma and acholeplasma species for their ability to reduce tetrazolium salts by using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The test results were evaluated visually, as well as spectrophotometrically, by using an enzyme-linked immunosorbent assay reader. Our results were very similar to the results obtained when the tetrazolium salt reduction assay described by Aluotto et al. was used. However, the MTT reduction assay appeared to be better because it is faster, more objective and sensitive, easier to evaluate, and less expensive; in addition, it allows quantitative determinations. By using regression analysis a linear correlation between formazan production and the number of colony-forming units was demonstrated for all of the species investigated, indicating that the MTT assay can also be used for growth, toxicity, or chemosensitivity tests for the mycoplasma species that are capable of reducing tetrazolium salts.     

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.     

Microtiter assay for glutamine synthetase biosynthetic activity using inorganic phosphate detection

A microtiter assay was developed for the improved detection of inorganic phosphate released from adenosine 5'-triphosphate (ATP) in the glutamine synthetase biosynthetic assay. In this assay, ascorbic acid replaces the traditionally used ferrous sulfate to reduce the phosphomolybdate complex. As a result, increased color development, linearity, and sensitivity are achieved. Additionally, in the microtiter format, multiple sets of kinetic experiments can be rapidly performed in parallel. The color that forms is rendered highly stable by the addition of sodium citrate. However, the commonly used sodium arsenite in this solution has been omitted, making the assay less hazardous. The assay is linear to 100 nmol Pi in the presence of 10mM ATP.     

A sensitive immunochromatographic assay using gold nanoparticles for the semiquantitative detection of prostate-specific antigen in serum

In prostate cancer screening, prostate-specific antigen (PSA) has been utilized as a valuable biomarker. There are routinely used procedures based on enzyme-linked immunosorbent assay (ELISA) for PSA detection. The procedures based on ELISA, however, are time consuming, complicated, and costly. We have developed a rapid, very simple, cost effective and sensitive immunochromatographic assay using gold nanoparticles and evaluated its applications for first screening of prostate cancer in serum samples. The sensitive immunochromatographic assay requires only 40 μL of the serum sample. The assay used is rapid and simple, that it totally takes approx 15 min to complete. The method for sensitive immunochromatographic assay has the other advantage of decreasing the antibody concentration that is used for the test line. In this study, we show the advantage to decrease the antibody concentration and the evaluation of our sensitive immunochromatographic assay for the semiquantitative detection of PSA in serum. The results obtained from 163 serum samples using sensitive immunochromatographic assay are compared with the results obtained using the chemiluminescent enzyme immunoassay (CLEIA) and normal immunochromatographic assay. The results obtained in the sensitive immunochromatographic assay correlated well with the values obtained in CLEIA. We concluded that our sensitive immunochromatographic assay is applicable to the first screening test for the diagnosis of prostate cancer. Our developed sensitive immunochromatographic assay is a promising candidate for diagnosis or research use, which may become commercially available in the near future.     

Activity assay of membrane transport proteins

Membrane transport proteins are integral membrane proteins and considered as potential drug targets. Activity assay of transport proteins is essential for developing drugs to target these proteins. Major issues related to activity assessment of transport proteins include availability of transporters, transport activity of transporters, and interactions between ligands and transporters. Researchers need to consider the physiological status of proteins (bound in lipid membranes or purified), availability and specificity of substrates, and the purpose of the activity assay (screening, identifying, or comparing substrates and inhibitors) before choosing appropriate assay strategies and techniques. Transport proteins bound in vesicular membranes can be assayed for transporting substrate across membranes by means of uptake assay or entrance counterflow assay. Alternatively, transport proteins can be assayed for interactions with ligands by using techniques such as isothermal titration calorimetry, nuclear magnetic resonance spectroscopy, or surface plasmon resonance. Other methods and techniques such as fluorometry, scintillation proximity assay, electrophysiologi-cal assay, or stopped-flow assay could also be used for activity assay of transport proteins. In this paper the major strategies and techniques for activity assessment of membrane transport proteins are reviewed.     

A sensitive, robust high-throughput electrochemiluminescence assay for rat insulin

In diabetes research, mouse and rat models are used for in vivo experiments, and quantification of insulin in serum samples under different pathophysiological conditions and after treatment with compounds is essential. There are few commercial radioimmunoassay and enzyme-linked immunosorbent assay (ELISA) assay kits to determine the rat/mouse plasma levels of insulin. However, reliability in insulin measurements using the available biological assays is a great concern. The authors report a robust, extremely sensitive electrochemiluminescence (ECL) insulin assay using the Origen technology platform. The assay performance, as judged by the Z' value of 0.82+/-0.03 and the signal-to-background (S/B) ratio of 133, suggests that this is a robust and reliable assay. The intra-assay and interassay variation is less than 5%. The dynamic range of detection for insulin is 5 pg to 5 ng in the ECL assays. Recovery of insulin was about 100% when different volumes of serum were spiked with exogenous insulin. These results suggest that the ECL insulin assay is an extremely sensitive, robust, nonradioactive homogeneous assay and can be used successfully to determine the insulin levels in rodent serum samples.     

Molecular identification of Pichia guilliermondii, Debaryomyces hansenii and Candida palmioleophila

Traditional phenotypic methods and commercial kits based on carbohydrate assimilation patterns are unable to consistently distinguish among isolates of Pichia guilliermondii, Debaryomyces hansenii and Candida palmioleophila. As result, these species are often misidentified. In this work, we established a reliable method for the identification/differentiation of these species. Our assay was validated by DNA sequencing of the polymorphic region used in a real-time PCR assay driven by species-specific probes targeted to the fungal ITS 1 region. This assay provides a new tool for pathogen identification and for epidemiological, drug resistance and virulence studies of these organisms.     

Adopting a practical statistical approach for evaluating assay agreement in drug discovery

The authors assess the equivalence of 2 assays and put forward a general approach for assay agreement analysis that can be applied during drug discovery. Data sets generated by different assays are routinely compared to each other during the process of drug discovery. For a given target, the assays used for high-throughput screening and structure-activity relationship studies will most likely differ in their assay reagents, assay conditions, and/or detection technology, which makes the interpretation of data between assays difficult, particularly as most assays are used to measure quantitative changes in compound potency against the target. To better quantify the relationship of data sets from different assays for the same target, the authors evaluated the agreement between results generated by 2 different assays that measure the activity of compounds against the same protein, ALK5. The authors show that the agreement between data sets can be quantified using correlation and Bland-Altman plots, and the precision of the assays can be used to define the expectations of agreement between 2 assays. They propose a scheme for addressing issues of assay data equivalence, which can be applied to address questions of how data sets compare during the lead identification and lead optimization processes in which assays are frequently added and changed.