Precise construction of oligonucleotide–Fab fragment conjugate for homogeneous immunoassay using HaloTag technology

The use of oligonucleotide–protein conjugates enables the development of novel types of bioanalytical assays. However, convenient methods for producing covalent and stoichiometric oligonucleotide–protein conjugates are still rare. Here we demonstrate, for the first time, covalent conjugation of DNA oligonucleotide to Fab fragments with a 1:1 ratio using HaloTag self-labeling technology. The oligonucleotide coupling was carried out while the Fab was attached to protein G matrix, thereby enabling straightforward production of covalent conjugates. Furthermore, it allowed convenient purification of the product because the unreacted components were easily removed before the elution of the high-purity conjugate. The prepared conjugate was employed in a homogeneous immunoassay where prostate-specific antigen was used as a model analyte. Switchable lanthanide luminescence was used for detection, and the obtained limit of detection was 0.27 ng/ml. In the future, the developed method for covalent conjugation and successive purification in protein G column could also be applied for introducing other kinds of modifications to Fab fragments in a simple and site-specific manner.     

Carbostyril derivatives as antenna molecules for luminescent lanthanide chelates

Luminescent lanthanide complexes consisting of a lanthanide-binding chelate and organic-based antenna molecule have unusual emission properties, including millisecond excited state lifetimes and sharply spiked spectra, compared to standard organic fluorophores. We have previously used carbostyril (cs124, 7-amino-4-methyl-2(1H)-quinolinone) as an antenna molecule (Li and Selvin, J. Am. Chem. Soc., 1995) attached to a polyaminocarboxylate chelate such as DTPA. Here, we report the chelate syntheses of DTPA conjugated with cs124 derivatives substituted on the 1-, 3-, 4-, 5-, 6-, and 8-position. Among them, the DTPA chelate of cs124-6-SO(3)H has similar lifetime and brightness for both Tb(3+) and Eu(3+) compared to the corresponding DTPA-cs124 complexes, yet it is significantly more soluble in water. The Tb(3+) complex of DTPA-cs124-8-CH(3) has significantly longer lifetime compared to DTPA-cs124 (1.74 vs 1.5 ms), indicating higher lanthanide quantum yield resulting from the elimination of back emission energy transfer from Tb(3+) to the antenna molecule. Thiol-reactive forms of chelates were made for coupling to proteins. These lanthanide complexes are anticipated to be useful in a variety of fluorescence-based bioassays.     

An enzymatic fluorometric assay for L-lysine in plasma and tissue

A simple, specific, and reliable method has been developed for the determination of L-lysine in blood plasma and tissue. The L-lysine in the sample is decarboxylated enzymatically, and fluorescamine is added to a pentan-1-ol extract of the cadaverine formed. This produces a stable product which is measured fluorometrically.     

Targeted molecular imaging agents for cellular-scale bimodal imaging

Molecular imaging is a powerful tool that has the ability to elucidate biochemical mechanisms and signal the early onset of disease. Overexpression of the peripheral benzodiazepine receptor (PBR) has been observed in a variety disease states, including glioblastoma, breast cancer, and Alzheimer's disease. Thus, the PBR could be an attractive target for molecular imaging. In this paper, the authors report cellular uptake and multimodal (MRI and fluorescence) imaging of PBR-overexpressing C6 glioblastoma (brain cancer) cells using a cocktail administration approach and a new PBR targeted lanthanide chelate molecular imaging agent.     

Rapid homogeneous PCR assay for the detection of Chlamydia trachomatis in urine samples

Chlamydia trachomatis infection is the most common bacterial sexually transmitted disease and a major public health problem worldwide. Fast and sensitive point-of-care diagnostics including non-invasive sample collection would be of value for the prevention of C. trachomatis transmission. The aim of this study was to develop a fast, reliable, non-invasive and easy-to-use homogenous PCR assay for the detection of C. trachomatis. Bacteria were concentrated from urine by a simple and fast centrifugation-based urine pretreatment method. Novel automated GenomEra technology was utilized for the rapid closed-tube PCR including time-resolved fluorometric detection of the target using lanthanide chelate labeled probes. We have developed a rapid C. trachomatis assay which provides qualitative results in 1 h with diagnostic sensitivity and specificity of 98.7% and 97.3%, respectively. The novel assay can be performed with minimal laboratory expertise and without sophisticated DNA-extraction devices and has performance comparable to current gold standard assays.     

Direct electrochemical determination of glucose oxidase in biological samples

An electrochemical method for the quantitation of glucose oxidase in murine plasma and tissues has been developed. Instead of oxygen, this method uses benzoquinone as an artificial cosubstrate of glucose oxidase. The quantitative detection of the enzymatically produced hydroquinone by controlled-potential amperometry allows measurement of glucose oxidase concentrations in biological samples. The use of an internal standard corrects for all possible interfering effects. We demonstrated a 10-fold increase in sensitivity, as well as the ability to work in turbid media, in comparison to spectrophotometric methods.     

Temporally and spectrally resolved imaging microscopy of lanthanide chelates.

The combination of temporal and spectral resolution in fluorescence microscopy based on long-lived luminescent labels offers a dramatic increase in contrast and probe selectivity due to the suppression of scattered light and short-lived autofluorescence. We describe various configurations of a fluorescence microscope integrating spectral and microsecond temporal resolution with conventional digital imaging based on CCD cameras. The high-power, broad spectral distribution and microsecond time resolution provided by microsecond xenon flashlamps offers increased luminosity with recently developed fluorophores with lifetimes in the submicrosecond to microsecond range. On the detection side, a gated microchannel plate intensifier provides the required time resolution and amplification of the signal. Spectral resolution is achieved with a dual grating stigmatic spectrograph and has been applied to the analysis of luminescent markers of cytochemical specimens in situ and of very small volume elements in microchambers. The additional introduction of polarization optics enables the determination of emission polarization; this parameter reflects molecular orientation and rotational mobility and, consequently, the nature of the microenvironment. The dual spectral and temporal resolution modes of acquisition complemented by a posteriori image analysis gated on the spatial, spectral, and temporal dimensions lead to a very flexible and versatile tool. We have used a newly developed lanthanide chelate, Eu-DTPA-cs124, to demonstrate these capabilities. Such compounds are good labels for time-resolved imaging microscopy and for the estimation of molecular proximity in the microscope by fluorescence (luminescence) resonance energy transfer and of molecular rotation via fluorescence depolarization. We describe the spectral distribution, polarization states, and excited-state lifetimes of the lanthanide chelate crystals imaged in the microscope.     

The product-selective blot: a technique for measuring enzyme activities in large numbers of samples and in native electrophoresis gels

A method termed "product-selective" blotting has been developed for screening large numbers of samples for enzyme activity. The technique is particularly well suited to detection of enzymes in native electrophoresis gels. The principle of the method was demonstrated by blotting samples from glutaminase (EC 3.5.1.2) or glutamate synthase (EC 1.4.7.1) reactions into an agarose gel embedded with ion-exchange resin under conditions favoring binding of product (glutamate) over substrates and other substances in the reaction mixture. After washes to remove these unbound substances, the product was measured using either fluorometric staining or radiometric techniques. Glutaminase activity in native electrophoresis gels was visualized by a related procedure in which substrates and products from reactions run in the electrophoresis gel were blotted directly into a resin-containing "image gel." Considering the selective-binding materials available for use in the image gel, along with the possible detection systems, this method has potentially broad application.     

A Simple Preparation of N-Phosphorylated Lysine and Arginine

An improved specific synthesis of N-ε-phospholysine is described. Phosphorylation under basic conditions of the copper chelate of L-lysine with phosphorus oxychloride and subsequent removal of the copper ion affords N-ε-phospholysine as the sole phosphorylated product. Similar treatment of L-arginine yields N-ω-phosphoarginine as the sole product which is identical to that produced enzymatically.     

Time-resolved fluorescence-based assay for rapid detection of Escherichia coli

Fast and simple detection of pathogens is of utmost importance in health care and the food industry. In this article, a novel technology for the detection of pathogenic bacteria is presented. The technology uses lytic-specific bacteriophages and a nonspecific interaction of cellular components with a luminescent lanthanide chelate. As a proof of principle, Escherichia coli-specific T4 bacteriophage was used to infect the bacteria, and the cell lysis was detected. In the absence of E. coli, luminescent Eu³⁺–chelate complex cannot be formed and low time-resolved luminescence signal is monitored. In the presence of E. coli, increased luminescence signal is observed as the cellular contents are leached to the surrounding medium. The luminescence signal is observed as a function of the number of bacteria in the sample. The homogeneous assay can detect living E. coli in bacterial cultures and simulated urine samples within 25 min with a detection limit of 1000 or 10,000 bacterial cells/ml in buffer or urine, respectively. The detection limit is at the clinically relevant level, which indicates that the method could also be applicable to clinical settings for fast detection of urine bacteria.     

A novel bifunctional europium chelate applied in quantitative determination of human immunoglobin G using time-resolved fluoroimmunoassay

The authors demonstrate herein a novel time-resolved fluoroimmunoassay (TRFIA) protocol for quantification of human IgG with the new bifunctional chelate Eu(TTA)₃(5-NH₂-phen) (ETNP) labeling the goat anti-human IgG. The immunoassay was conducted by following the typical procedure for sandwich-type immunoreactions. Goat anti-human IgG was immobilized on aldehyde-modified glass slides. The human IgG analyte was first captured by the primary antibody and then sandwiched by a secondary antibody labeled with the chelate ETNP. The experimental procedure was simple to follow and gave desirable levels of sensitivity and low limits of detection. To the best of our knowledge, this is the first application of the new chelate, ETNP, in an immunoassay. In comparison to typical organic, fluorescent compounds and other lanthanide fluorescent chelates used in immunoassay, the detection sensitivity of our method using ETNP chelate in the solid phase was greatly improved and a concentration of human IgG about 5 μg/L could be detected under optimal conditions. The main result of this work shows that the new chelate ETNP can be applied as a powerful fluorescent labeling material for constructing ultrasensitive TRFIAs. The detection of human IgG, using ETNP as the chelate, is a model example of the effectiveness of this immunoassay. Many other types of antigen–antibody immunoassays should be possible using the protocol described herein.     

Labeling of proteins and oligopeptides with luminescent lanthanide(III) chelates.

Synthesis of a building block that allows introduction of photoluminescent europium(III) and samarium(III) chelates to synthetic oligopeptides on solid phase using standard Fmoc chemistry is described. Upon completion of the oligopeptide synthesis, these conjugates were converted to the corresponding lanthanide(III) chelates by treatment with appropriate lanthanide(III) salt. Also synthesis of a new terpyridine-based europium(III) chelate designed for solution phase protein labeling is demonstrated.     

Biospecific nanoparticles for multiplex phosphorescence analysis (PHOSPHAN)

We have developed a technology for the production of polymeric nanoparticles containing the incorporated phosphorescent label (europium ions–naphthoyltrifluoroacetone complexes) and streptavidin that is covalently bound on the surface. The aggregation-stable biospecific nanoparticles (40–60 nm in diameter) include up to 2000 molecular tags/particle and retain biological activity and stable phosphorescence for at least 20 months. They can be used in phosphorescence analysis (PHOSPHANTM)-based biochip technology as an effective detector system to record phosphorescence from microzones (microarrays) printed on the well bottoms of standard polystyrene microplates. The creation of a dense monolayer on the surface of a microzone requires up to 10⁸ particles/microarray, or 10⁹ particles/mm² of area; this is in good agreement with theoretical estimates. The detection limit is as low as 300–400 phosphorescent nanoparticles per a microzone with an area of ~0.1 mm². It has been demonstrated in the model of thyroid stimulating hormone (TSH) detection in filter paper dried blood that the newly developed detector system is five times more sensitive than the conventional methods of multiplex PHOSPHAN (with Pt-coproporphyrin phosphorescent label) and lanthanide immune fluoroassay (with fluorescent Eu³⁺ chelate complexes registered in the enhancement solution). The sensitivity of phosphorescent nanoparticle-based detector system is as low as 6.8 × 10⁵ molecules/1.5 μL sample, which corresponds to a TSH concentration of 1.5 × 10^–14 M.     

Micellar electrokinetic chromatography separation and laser-induced fluorescence detection of the lipid peroxidation product 4-hydroxynonenal

4-Hydroxnonenal (HNE) is a product of lipid peroxidation in biological systems that causes a variety of harmful biological effects. A method for identifying HNE based on derivatization with the fluorescent reagent dansylhydrazine (5-(dimethylamino)naphthalene-1-sulphonehydrazine (DNSH) followed by micellar electrokinetic chromatography separation laser-induced fluorescence detection has been developed. The derivatization reaction has also been investigated for significant experimental parameters and rat brain homogenates with induced lipid peroxidation have been analysed for HNE contents. The limit of detection (3 S/N) was 30 nM or 0.3 fmol in the injected sample.     

A robust and convergent synthesis of dipeptide-DOTAM conjugates as chelators for lanthanide ions: new PARACEST MRI agents

A generally applicable synthetic approach to dipeptide-DOTAM conjugates has been developed which is based on the peralkylation of 1,4,7,10-tetraazacyclododecane (cyclen) with protected N-iodoacetyl dipeptides. Standardized procedures were used for the alkylation, metalation, and purification of the resultant lanthanide complexes. Using this approach, we have been able to rapidly and reliably prepare and screen five different ligands each with up to six lanthanide ions. This preliminary investigation has identified several paramagnetic compounds with strong chemical exchange saturation transfer (PARACEST) properties in water at physiological temperature and pH. Extension of the synthetic approach to a wide variety of amino acids is possible.     

Mathematical modeling of a single-cell enzyme assay

A quantitative assay of beta-galactosidase activity in single cells of Saccharomyces cerevisiae has been developed using a fluorogenic substrate and flow cytometry [reported in Wittrup & Bailey, Cytometry, 9,394 (1988)]. The beta-galactosidase activity is expressed in yeast from the Escherichia coli lacZ gene under the control of the yeast GAL10 promoter, and is used as a marker for multicopy plasmid content. A nonfluorescent fluorogenic substrate is enzymatically cleaved by intracellular beta-galactosidase to form a fluorescent product. The accumulation of fluorescent product in single cells was found to depend on bulk substrate concentration and single-cell enzyme activity in a fashion that could not be described by a Michaelis-Menten kinetic rate form. It has been demonstrated that diffusion limitation rather than enzyme activity can determine the level of single-cell fluorescence under certain assay conditions, and a mathematical model has; been formulated which accounts for substrate and product diffusion. Guided by the mathematical model, the assay conditions were modified to allow measurement of single-cell enzyme activity rather than diffusion rates.     

Bioanalytical strategy used in development of pharmacokinetic (PK) methods that support biosimilar programs

The development of biosimilar products is expected to grow rapidly over the next five years as a large number of approved biologics reach patent expiry. The pathway to regulatory approval requires that similarity of the biosimilar to the reference product be demonstrated through physiochemical and structural characterization, as well as within in vivo studies that compare the safety and efficacy profiles of the products. To support nonclinical and clinical studies pharmacokinetic (PK) assays are required to measure the biosimilar and reference products with comparable precision and accuracy. The most optimal approach is to develop a single PK assay, using a single analytical standard, for quantitative measurement of the biosimilar and reference products in serum matrix. Use of a single PK assay for quantification of multiple products requires a scientifically sound testing strategy to evaluate bioanalytical comparability of the test products within the method, and provide a solid data package to support the conclusions. To meet these objectives, a comprehensive approach with scientific rigor was applied to the development and characterization of PK assays that are used in support of biosimilar programs. Herein we describe the bioanalytical strategy and testing paradigm that has been used across several programs to determine bioanalytical comparability of the biosimilar and reference products. Data from one program is presented, with statistical results demonstrating the biosimilar and reference products were bioanalytically equivalent within the method. The cumulative work has established a framework for future biosimilar PK assay development.     

Development of inductively coupled plasma-mass spectrometry-based protease assays

Rapid, sensitive, and quantitative assays for proteases are important for drug development and in the diagnosis of disease. Here an assay for protease activity that uses inductively coupled plasma-mass spectrometry (ICP-MS) detection is described. Peptidic α-chymotrypsin substrates were synthesized containing a lanthanide ion chelate at the N terminus to provide a distinct elemental tag. A biotin label was appended to the C terminus of the peptide, allowing separation of uncleaved peptide from the enzymatic digestion. The enzyme activity was determined by quantifying the lanthanide ion signal of the peptide cleavage products by ICP-MS. Biotinylated substrates synthesized include Lu-DTPA-Asp-Leu-Leu-Val-Tyr∼Asp-Lys(biotin) and Lu-DTPA-βAla-βAla-βAla-βAla-Gly-Ser-Ala-Tyr∼Gly-Lys-Arg-Lys(biotin)-amide. Parallel assays with a commercially available fluorogenic substrate (Suc-AAPF-AMC) for α-chymotrypsin were performed for comparison. Using the ICP-MS assay, enzyme concentrations as low as 2 pM could be readily detected, superior to the detection limit of an assay using the α-chymotrypsin fluorogenic substrate (Suc-AAPF-AMC). Furthermore, we demonstrated the use of this approach to detect chymotrypsin activity in HeLa cell lysates.     

A liquid chromatographic-tandem mass spectrometric method for determination of artesunate and its metabolite dihydroartemisinin in human plasma

A bioanalytical method for the analysis of artesunate and its metabolite dihydroartemisinin in human plasma using high throughput solid-phase extraction in the 96-wellplate format and liquid chromatography coupled to positive tandem mass spectroscopy has been developed and validated. The method was validated according to published FDA guidelines and showed excellent performance. The within-day and between-day precisions expressed as RSD, were lower than 7% at all tested concentrations including the lower limit of quantification. Using 50 microl plasma the calibration range was 1.19-728 ng/ml with a limit of detection at 0.5 ng/ml for artesunate and 1.96-2500 ng/ml with a limit of detection at 0.6 ng/ml for dihydroartemisinin. Using 250 microl of plasma sample the lower limit of quantification was decreased to 0.119 ng/ml for artesunate and 0.196 ng/ml dihydroartemisinin. Validation of over-curve samples in plasma ensured that accurate estimation would be possible with dilution if samples went outside the calibration range. The method was free from matrix effects as demonstrated both graphically and quantitatively.