Development and characterization of the NanoOrange protein quantitation assay: a fluorescence-based assay of proteins in solution

We developed a sensitive fluorescence assay for the quantitation of proteins in solution using the NanoOrange reagent, a merocyanine dye that produces a large increase in fluorescence quantum yield upon interaction with detergent-coated proteins. The NanoOrange assay allowed for the detection of 10 ng/mL to 10 micrograms/mL protein with a standard fluorometer, offering a broad, dynamic quantitation range and improved sensitivity relative to absorption-based protein solution assays. The protein-to-protein variability of the NanoOrange assay was comparable to those of standard assays, including Lowry, bicinchoninic acid, and Bradford procedures. We also found that the NanoOrange assay is useful for detecting relatively small proteins or large peptides, such as aprotinin and insulin. The assay was somewhat sensitive to the presence of several common contaminants found in protein preparations such as salts and detergents; however, it was insensitive to the presence of reducing agents, nucleic acids, and free amino acids. The simple assay protocol is suitable for automation. Samples are briefly heated in the presence of dye in a detergent-containing diluent, allowed to cool to room temperature, and fluorescence is measured using 485-nm excitation and 590-nm emission wavelengths. Therefore, the NanoOrange assay is well suited for use with standard fluorescence microplate readers, fluorometers, and some laser scanners.     

Microplate fluorescence assay for the quantification of double stranded DNA using SYBR Green I dye

A high-throughput, 96-well microplate fluorescence assay (MFA) was developed for DNA quantification using the double-stranded DNA-binding dye SYBR Green I. Samples mixed with SYBR Green I in the wells of a microtiter plate produced fluorescence in proportion with DNA concentration which was measured using a fluorescence plate reader. The performance characteristics of the assay were compared with spectrophotometric quantification based on ultraviolet absorption and the Hoefer DyNA Quant assay utilizing the fluorescent dye, Hoechst 33258. The MFA accurately quantified different types of DNA over a broad linear dynamic range of concentrations (0.25–2,500 pg/μl), and was not affected by a variety of contaminants in the assay mixture.     

High-performance liquid chromatographic assay with fluorescence detection for the determination of cephaeline and emetine in human plasma and urine

A high-performance liquid chromatographic assay method for the quantitation of ipecac alkaloids (cephaeline and emetine) in human plasma and urine is described. Human plasma or urine was extracted with diethylether under alkaline conditions following the addition of an internal standard. Concentrations of alkaloids and internal standard were determined by octadecylsilica chromatographic separation (Symmetry C₁₈ columns, plasma analysis; 15 cm×4.6 mm I.D., 5 μm particle size, urine analysis; 7.5 cm×4.6 mm I.D., 5 μm particle size). The mobile phase consisted of buffer (20 mmol/l 1-heptanesulfonic acid sodium salt, adjusted to pH 4.0 with acetic acid)–methanol (51:49, v/v). Eluate fluorescence was monitored at 285/316 nm. The lowest quantitation limits of cephaeline and emetine were 1 and 2.5 ng/ml, respectively, in plasma, and 5 ng/ml in urine. Intra- and inter-day relative standard deviations were below 15%. The assay is sensitive, specific and applicable to pharmacokinetic studies in humans.     

Optimization of a phagocyte microplate chemiluminescent assay

Chemiluminescent assays have been used to quantify phagocytic activity since 1972. In recent years these assays have been adapted to the 96-well microplate format as new luminometers have been developed. In this report we describe the optimization of a lucugenin enhanced phagocyte chemiluminescent assay using a Titertek Luminoskan. Factors such as cell concentration, serum concentration in the opsonization of the zymosan used and lucigenin concentration were all optimized in our assay. In addition we have found that some of the unique features of the Luminoskan, continuous microplate agitation during the assay and microplate temperature control up to 43°C, also significantly enhanced the chemiluminescent response.     

Direct analysis of nitrocatechol-type glucuronides in urine by capillary electrophoresis–electrospray ionisation mass spectrometry and tandem mass spectrometry

Direct, quantitative capillary electrophoresis–electrospray ionisation mass spectrometric (CE–ESI-MS) and tandem mass spectrometric (CE–ESI-MS–MS) methods are described for the quantitation of 3-O-glucuronides of E- and Z-entacapone isomers (EEG and EZG) and tolcapone (TG) in urine. 3-O-Glucuronide of nitecapone was used as internal standard. Good separation of glucuronides was achieved with 20 mM ammonium acetate as separation solution at pH 6.84. Stacking was used to increase the sensitivity of the method by introducing samples in 5 mM ammonium acetate. CE–ESI-MS and CE–ESI-MS–MS methods are linear with correlation coefficients better than 0.9983 and 0.9982, and repeatable with relative standard deviations below 9 and 14%, respectively. The limit of detection (LOD) in CE–ESI-MS at signal-to-noise ratio 3 is 100 ng/ml for EEG and EZG and 250 ng/ml for TG. The CE–ESI-MS–MS method was the more sensitive; LOD was 7 ng/ml for all compounds, without any concentration of the sample.     

Quantitative liquid chromatographic–tandem mass spectrometric determination of reserpine in FVB/N mouse plasma using a “chelating” agent (disodium EDTA) for releasing protein-bound analytes during 96-well liquid–liquid extraction

A sensitive, specific, accurate and reproducible analytical method employing a divalent cation chelating agent (disodium EDTA) for sample treatment was developed to quantitate reserpine in FVB/N mouse plasma. Samples pretreated with 40 μl of 2% disodium EDTA in water were extracted by a semi-automated 96-well liquid–liquid extraction (LLE) procedure to isolate reserpine and a structural analog internal standard (I.S.), rescinnamine, from mouse plasma. The extracts were analyzed by turbo ionspray liquid chromatography–tandem mass spectrometry (LC–MS–MS) in the positive ion mode. Sample preparation time for conventional LLE was dramatically reduced by the semi-automated 96-well LLE approach. The assay demonstrated a lower limit of quantitation of 0.02 ng/ml using 0.1-ml plasma sample aliquots. The calibration curves were linear from 0.02 to 10 ng/ml for reserpine. The intra- and inter-assay precision of quality control (QC) samples ranged from 1.75 to 10.9% for reserpine. The intra- and inter-assay accuracy of QC samples ranged from −8.17 to 8.61%. Reserpine and the I.S. were found to be highly bound to FVB/N mouse plasma protein. This is the first report of disodium EDTA employed as a special protein-bound release agent to recover protein-bound analytes from plasma. These matrix effects and the effects of pH in the HPLC mobile phase on the sensitivities of LC–MS–MS are discussed in this paper.     

Bicinchoninic acid (BCA) assay in low volume

The BCA assay is a colorimetric method for estimating protein concentration. In 96-well plates, the relationship between protein content and absorbance is nearly linear over a wide range; however, performance is reduced in lower volume. To overcome this limitation, we performed the BCA assays in opaque, white 384-well plates. These plates emit fluorescence between 450–600 nm when excited at 430 nm; thus, their fluorescence is quenched by the BCA chromophore (λ_max 562 nm). This arrangement allowed accurate determination of protein content using only 2 μL of sample. Moreover, soluble flourescein could replace the white plates, creating a homogenous format.     

Use of a fluorescence microplate reader for the detection and characterization of metal-assisted peptide hydrolysis

Metal ions and complexes that hydrolyze peptides under nondenaturing conditions of temperature and pH hold great promise for use in protein structural studies. However, the extreme stability of the peptide amide bond has placed limits on the number of reagents available. In addition, the development of new cleavage strategies has been hindered by the fact that no facile procedure exists for the detection and characterization of metal-assisted peptide hydrolysis. Here we describe a rapid assay in which a microplate reader is used to detect fluorescence produced by the reaction of fluorescamine with hydrolyzed peptides. We have employed this assay to detect Zn(II) and Pd(II)-assisted peptide hydrolysis in multiple samples and in each case have extended our approach to a successful analysis of reaction kinetics. Aliquots from multiple time points are treated with fluorescamine in a single 96-well plate. Because the plate is scanned in a microplate reader in only 58 s, the assay is very convenient compared to conventional approaches which rely on NMR and HPLC to monitor individual reactions. Using our assay, rate constants and half-lives are easily derived from the kinetic data by means of linear regression curve fits of triplicate runs.     

A quantitative fluorescence-based microplate assay for the determination of double-stranded DNA using SYBR Green I and a standard ultraviolet transilluminator gel imaging system

Various assays are available for quantification of DNA in solution, but none has been described that is both sensitive and specific for double-stranded (ds) DNA and features practical properties such as low dye and equipment costs, speed, and highly parallel microplate formats. Here we show that quantitative and sensitive measurement of ds DNA in solution is achieved using a 96-well microplate SYBR Green I assay and a standard uv transillumination-based gel-imaging system for detection. Specific detection of ds DNA was obtained over a broad concentration range of 0.5-500 ng using a single low dye concentration of up to 1/6250. Measured SYBR Green I fluorescence was not significantly affected by pH variation (4-10), assay volume (50-250 microliter l), and time (4-15 min), and measurements were appreciably compatile with commonly encountered concentrations of contaminating salts, organics, detergents, and other substances. ds DNA yielded up to 13-fold higher fluorescence compared to single-stranded DNA or RNA, but this ratio was dependent somewhat on GC content and fragment size. Of note, linear ds DNA fluoresced significantly stronger than supercoiled plasmid DNA. Our method should be broadly applicable for sensitive, rapid, and inexpensive ds DNA quantification in the average molecular biology laboratory.     

Quantification of daunorubicin and daunorubicinol in plasma by capillary electrophoresis

Capillary electrophoresis (CE) with laser-induced fluorescence detection was applied to quantify daunorubicin and daunorubicinol in plasma. Separation was carried out in a 47 cm×50 μm I.D. fused-silica capillary, with a running buffer, pH 5 containing 60 μM spermine and 70% acetonitrile. Sample preparation was done either by protein precipitation with acetonitrile or by liquid–liquid extraction. The assay can be applied in a concentration range from 40 mg/l down to 2 μg/l for daunorubicin and from 1 mg/l to 2 μg/l for daunorubicinol. Precision and accuracy were between 2.9 and 14.5% (n=6) on 1 day and between 1.0 and 14.7% from day to day (n=6) for both analytes. Thus, the CE method enables precise and accurate quantification of daunorubicin and daunorubicinol in small sample volumes over a wide concentration range.     

A homogeneous time-resolved fluorescence resonance energy transfer assay for phosphatidylserine exposure on apoptotic cells

A simple, “mix-and-measure” microplate assay for phosphatidylserine (PtdSer) exposure on the surface of apoptotic cells is described. The assay exploits the fact that annexin V, a protein with high affinity and specificity for PtdSer, forms trimers and higher order oligomers on binding to membranes containing PtdSer. The transition from soluble monomer to cell-bound oligomer is detected using time-resolved fluorescence resonance energy transfer from europium chelate-labeled annexin V to Cy5-labeled annexin V. PtdSer detection is achieved by a single addition of a reagent mix containing labeled annexins and calcium ions directly to cell cultures in a 96-well plate, followed by a brief incubation before fluorescence measurement. The assay can be used to quantify PtdSer exposure on both suspension cells and adherent cells in situ. This method is simpler and faster than existing annexin V binding assays based on flow cytometry or microscopy, and it yields precise data with Z’ values of 0.6-0.7.     

Identification of Vibrio cholerae serotypes in high-risk marine products with non-gel sieving capillary electrophoresis

Vibrio cholerae, a natural inhabitant of the marine environment, poses a threat to human health, and its new epidemic variants have been reported. A method of multiplex polymerase chain reaction–capillary electrophoresis–laser-induced fluorescence (PCR–CE–LIF) detection has been developed to detect and identify V. cholerae in marine products sensitively, rapidly, and reliably. Four sets of primers were selected to amplify genus-specific VCC gene, O139 serogroup-specific O139 gene, O1 serogroup-specific O1 gene, and ctxA gene associated with the CT toxin of enterotoxigenic V. cholerae. The PCR products were detected using CE–LIF with SYBR Gold serving as the DNA fluorescent dye. The parameters of PCR and the separation conditions of CE–LIF were optimized. Under the optimal conditions, V. cholerae was detected and four serotypes were identified simultaneously within 8 min. The alignment analysis showed that the PCR products had good agreement with the published sequences from GenBank, indicating that the primers selected in this study had high specificity and the PCR results were reliable. The proposed method could detect 5 to 20 cfu/ml V. cholerae. The intraday precisions of migration time and peak area of DNA marker and PCR products were in the ranges of 1.60–2.56% and 1.60–6.29%, respectively. The specificity results showed that only five standard bacteria used in this study showed the specific peaks when the target bacteria were mixed with seven other common intestinal pathogenic bacteria at the same concentration. The assay was applied to 71 high-risk marine products, and different serotypes of V. cholerae could be identified sensitively and reliably.     

Improvement in the high-performance liquid chromatography malondialdehyde level determination in normal human plasma

We report a very rapid and simple isocratic reversed-phase HPLC separation of malondialdehyde (MDA) in normal human plasma without previous purification of the MDA–2-thiobarbituric acid (TBA) complex. The separation of MDA–TBA complex was performed using a 250×4.6 mm Nucleosil-5C18 column with a mobile phase composed of 35% methanol and 65% 50 mM sodium phosphate buffer, pH 7.0. Samples of 50 μl (composed of 100 μl plasma mixed with 1.0 ml of 0.2% 2-thiobarbituric acid in 2 M sodium acetate buffer containing 1 mM diethylenetriaminepentaacetic acid, pH 3.5, and 10 μl of 5% 2,6-di-tert.-butyl-4-methylphenol in 96% ethanol, incubated at 95°C for 45 min [K. Fukunaga, K. Takama and T. Suzuki, Anal. Biochem., 230 (1995) 20] were injected into the column. The MDA–TBA complex was eluted at a flow-rate of 1 ml/min and monitored by fluorescence detection with excitation at 515 nm and emission at 553 nm. Analysis of groups of normal male and female volunteers gave plasma levels of MDA of 1.076 nmol/ml with a coefficient of variation of about 58%. No significant statistical differences were found between male and female groups, and no correlation was discovered on the age.     

A high-throughput,homogeneous microplate assay for agents that kill mammalian tissue culture cells

Screens for cytostasis/cytoxicity have considerable value for the discovery of therapeutic agents and the investigation of the biology of apoptosis. For instance, genetic screens for proteins, protein fragments, peptides, RNAs, or chemicals that kill tissue culture cells may aid in identifying new cancer therapeutic targets. A microplate assay for cell death is needed to achieve throughputs sufficient to sift through thousands of agents from expression or chemical libraries. The authors describe a homogeneous assay for cell death in tissue culture cells compatible with 96- or 384-well plates. In combination with a previously described system for retroviral packaging and transduction, nearly 6000 expression library clones could be screened per week in a 96-well plate format. The screening system may also prove useful for chemical screens.     

Semi-automated 96-well solid-phase extraction and gas chromatography–negative chemical ionization tandem mass spectrometry for the trace analysis of fluprostenol in rat plasma

Semi-automated 96-well plate solid-phase extraction (SPE) was used for sample preparation of fluprostenol, a prostaglandin analog, in rat plasma prior to detection by gas chromatography–negative chemical ionization tandem mass spectrometry (GC–NCI-MS–MS). A liquid handling system was utilized for all aspects of sample handling prior to SPE including transferring of samples into a 96-well format, preparation of standards as well as addition of internal standard to standards, quality control samples and study samples. SPE was performed in a 96-well plate format using octadecylsilane packing and the effluent from the SPE was dried in a custom-made 96-well apparatus. The sample residue was derivatized sequentially with pentafluorobenzylbromide followed by N-methyl-N-trimethylsilyltrifluoroacetamide. The derivatized sample was then analyzed using GC–NCI-MS–MS. The dynamic range for the method was from 7 to 5800 pg/ml with a 0.1-ml plasma sample. The methodology was evaluated over a 4-day period and demonstrated an accuracy of 90–106% with a precision of 2.4–12.9%.     

Separation and identification of neuropeptide Y, two of its fragments and their degradation products using capillary electrophoresis–mass spectrometry

This paper describes the development of an analytical method for the separation and identification of neuropeptide Y (NPY) and two important NPY fragments by capillary electrophoresis (CE) and mass spectrometry (MS). A satisfactory separation and the highest sensitivity were obtained with formic acid at high concentrations (250 mM, pH 2.75). The addition of 25 or 50 mM triethylamine (TEA) improved the separation. When applying full scan CE–MS, the separated peptides could be detected and identified using the spectra of each peak. The use of TEA as an additive to the formic acid slightly decreased the sensitivity but was compensated by the improved efficiency. The best compromise for optimal separation and MS detection was found to be 50 mM formic acid to which 50 mM TEA was added. CE–MS could be used for identification of the decomposition products of NPY. Decomposition products with one amino acid difference, which could not be distinguished with CE–UV, could be distinguished with CE–MS.     

Improved and simplified liquid chromatographic assay for adefovir, a novel antiviral drug, in human plasma using derivatization with chloroacetaldehyde

A rapid and simplified chromatographic assay is reported for the quantification of adefovir (PMEA) utilizing derivatization with chloroacetaldehyde. Adefovir is isolated from plasma using protein precipitation with trichloroacetic acid; next, the fluorescent 1,N⁶-etheno derivative is directly formed at 98°C in the buffered extract with chloroacetaldehyde. This derivative is analyzed using isocratic ion-pair liquid chromatography and fluorescence detection at 254 nm for excitation and 425 nm for emission. In the evaluated concentration range (10–1000 ng/ml) precisions ≤5% and accuracies between 95 and 117% were found, using a 0.2-ml volume of plasma. The lower limit of quantification is 10 ng/ml with a intra-assay precision of 16%. The currently reported bioanalytical method is 20–25-fold more sensitive than previously published assays.     

Immunological analysis of methamphetamine antibody and its use for the detection of methamphetamine by capillary electrophoresis with laser-induced fluorescence

An accurate, simple and rapid immunoassay is demonstrated for the detection of methamphetamine in urine by capillary electrophoresis (CE) with laser-induced fluorescence (LIF). An aminobutyl derivative of methamphetamine was conjugated with proteins, and used as an immunogen to produce antibodies for the assay. The methamphetamine derivative was also labeled with fluorescein isothiocyanate (FITC) to compete with free methamphetamine in the sample for the antibody binding site. Levels of free and antibody-bound FITC-labeled methamphetamine were monitored by performing CE–LIF using an untreated fused-silica column. This competitive immunoassay used antiserum instead of purified antibody or antibody fragment, yet was found to have good precision with a sensitivity of lower than 20 ng/ml. Various antibodies were also screened, and cross-reactivity of anti-MA antibody with methamphetamine analogues were also investigated. The results indicate that CE–LIF-based immunoassay is a powerful tool for the screening and characterization of antibody and may have possible applications in the detection of abused drugs in urine.     

Measuring beta-galactosidase activity in bacteria: cell growth, permeabilization, and enzyme assays in 96-well arrays.

We describe a high-throughput procedure for measuring beta-galactosidase activity in bacteria. This procedure is unique because all manipulations, including bacterial growth and cell permeabilization, are performed in a 96-well format. Cells are permeabilized by chloroform/SDS treatment directly in the 96-well blocks and then transferred to 96-well microplates for standard colorimetric assay of beta-galactosidase activity as described by Miller [J. H. Miller (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY]. Absorbance data are collected with a microplate reader and analyzed using a Microsoft Excel spreadsheet. The beta-galactosidase specific activity values obtained with the high-throughput procedure are identical to those obtained by the traditional single-tube method of Miller. Thus, values obtained with this procedure may be expressed as Miller units and compared directly to Miller units reported in the literature. The 96-well format for permeabilization and assay of enzyme specific activity together with the use of 12-channel and repeater pipettors enables efficient processing of hundreds of samples in an 8-h day.