CytostarT Technology

Cytostar-T™ scintillating microplates from Amersham International plc are standard 96-well format, sterile, tissue culture treated microplates. The Cytostar-T plate is a polystyrene plate with a transparent base coated with scintillant. Upon addition of radioactive tracer to cells grown in the bases of the wells, the scintillant generates light when the tracer is bound to the cell membranes or taken up by the cell due to the close proximity of the radioactive isotope to the scintillant at the base of the plate and can be counted in a standard plate counter [70]. The free radiolabel in the medium is physically too far from the scintillant to trigger a light reaction. Homogeneous cell based assays can be done in the Cytostar-T plate because there is no need for separation of the free radiolabel from the cell bound radiolabel.

1. Instrumentation

The Topcount (Packard) and MicroBeta (Wallac) scintillation plate counters capable of measuring 96- and 384-well microtiter plates are available. The signal is enhanced considerably in these instruments with new counting modes, high efficiency count mode for the Topcount, and paralux counting for the MicroBeta.

2. Applications

Cytostar-T plate cell-based assays can be done in homogeneous mode with radioisotopes (beta-emitters, 3H, 14C, 35S, and 45Ca). Some of the cell-based homogeneous assays that have been done in Cytostar-T include receptor radioligand binding assays with intact cells, amino acid uptake into cells, DNA synthesis monitoring in cells in response to drug treatment, and apoptosis measurements [69].

3. Comments

Cytostar-T can be used for assays with live cells plated on the bottom of the well. As with the FlashPlate assays, the Cytostar-T assays can be used with weak beta emitters in homogeneous assay mode. When strong beta emitters or 125I are used, the plates need to be washed to remove unbound radioactivity.

C. Scintillation Proximity Assay

The Scintillation Proximity Assay (SPA) was first described by Hart and Greenwald in an immunoassay using two polymer beads coated with antigen, one coated with fluorophore and the other with 3H [71]. Antibody agglutination brings many of the 3H beads into close proximity to the fluorophore beads and excites them, and after very long incubations they can be counted in a scintillation counter. Udenfriend et al. have improved on this with microbeads containing a fluorophore and coated with antibody [72]. 125I labeled antigen binds to the antibody on the beads and by its proximity the emitted short-range electrons of the 125I excite the fluorophore in the bead, which can be measured in a scintillation counter without separation of unbound antigen. Amersham International further developed SPA radioisotopic assay technology. SPA is a homogeneous radioisotope assay technology that can be utilized for a variety of biological assays. In SPA the target of interest is immobilized to a small scintillant containing microspheres or fluoromicrospheres (SPA beads) approximately 5 |m in size. The flu-oromicrosphere consists of a solid scintillant-polyvinyltoluene core coated with polyhydroxy film, which reduces the hydrophobicity of the particle. Generic SPA beads to which proteins such as antibodies, streptavidin, receptors, and enzymes or small molecules such as glutathione or copper ions are chemically linked to the coating on the bead are available with Amersham.

Assays are done in aqueous buffers with beta emitters such as 3H, 14C, 35S, and 33P isotopes and 125I. When 3H atom decays, it releases a P-particle with an average energy of 6 keV and a mean path length of 1.5 |m in water. The path lengths of 14C, 35S, and 33P isotopes and 125I are 58, 66, 126, and 17.5 |m, respectively. If a 3H P-particle meets a scintillant molecule within 1.5 |m of the particle being released, it will have sufficient energy to excite the scintillant into emitting light. On the other hand, if the P-particle of 3H travels longer distances of more than 1.5 | m, it will not have enough energy to cause scintillation. In SPA, if a radioactive molecule is bound to the SPA bead directly or through a molecule coupled to the bead, it is brought into close proximity for the emitted radiation to stimulate the scintillant to emit light (Fig. 22). Unbound radioactive molecules are too far away from the scintillant of the bead, the energy released is dissipated in the solution before reaching the bead, and no light is produced [73]. The amount of light produced is proportional to the amount of radioactive molecules bound to the SPA bead and is easily measured in a scintillation counter. Thus the bound radioactive molecules only produce the scintillation signal but not the unbound, hence there is no need for separation of free radioisotope in the assay.

Spa Assay Principle
Figure 22 Principles of SPA. Radiation energy from the radiolabeled ligand bound to the acceptor molecule on the SPA bead is absorbed by bead fluor and generates a signal on the bead, whereas unbound radioligand does not stimulate the bead. (Courtesy of Amer-sham.)

1. Instrumentation

Topcount NXT (Packard) and MicroBeta (Wallac) scintillation plate counters capable of measuring 96- and 384-well microtiter plates are available.

2. Applications

SPA microsphere beads have been prepared from hydrophobic polymers such as poly(vinyl toluene) (PVT) and inorganic scintillators such as yttrium silicate (YSi) beads [73]. The capacity of the YSi bead is higher than that of the PVT bead. The YSi bead is more dense than the PVT bead. Before developing an assay the compatibility of radioligands with SPA beads has to be tested. Also, the microplates have to be screened for low nonspecific binding of the radioli-gand. SPA has been applied to a wide variety of different assays including radio-immunoassays (RIAs), receptor binding assays, protein-protein interaction assays, enzyme assays, and DNA-protein and DNA-DNA interaction assays [74]. SPA beads coated with protein A or secondary antibody captures the antibody-antigen complex and is quantitated in the radioimmunoassays (RIAs).

RIAs are used in clinical and pharmacological studies to measure drugs, the second messengers, prostaglandins, steroids, and other serum factors.

SPA beads with wheat germ agglutinin (WGA), polyethylimine WGA-PVT beads, or polylysine coated YSi beads have been used for several membrane receptors including neuropeptide Y, galanin, endothelins, nerve growth factor, TGFa, TGFP, Ach, EGF, insulin, angiotensins, P-adrenoceptors, somatostatin, bFGF, dopamine, and interleukin receptors [66,67]. SPA also has been utilized in designing screens for nuclear receptor binding assays with the ligand binding domain of the nuclear receptor expressed as a fusion protein with His-tag (His6 or Hisi0), radioligand, and nickel-SPA beads. The protein-protein interaction assays that have been developed using SPA consist of SH2 and SH3 binding domains, Fos-Jun, Ras-Raf, selectin, and integrin adhesion assays [65,66]. SPA has been used for protein-DNA binding interaction assays such as binding of transcription factor NF-kB to DNA.

Enzyme assays can be grouped into three main formats (Fig. 23). (1) Signal removal. As in the case of hydrolytic enzymes such as proteases, nucleases, phos-pholipases, and esterases, the radiolabeled substrate is linked to the streptavidin-SPA bead via biotin, and the enzyme action cleaves the radiolabel from the biotin-ylated portion of the molecule, resulting in a decrease in the signal. (2) Signal addition. As in the case of synthetic enzymes such as transferases, kinases, and polymerases, the acceptor substrate is linked to the SPA bead through biotinyla-tion, and the donor substrate is radiolabeled. The action of the enzyme transfers the radiolabel to the acceptor molecule on the bead from the donor, resulting in an increase in the signal. (3) Product capture. In this assay format, the radiolabeled product of the reaction is captured by biospecific recognition to antibody as in the case of PTK. In the PTK assay the phosphorylated product but not the substrate is captured specifically by antiphosphotyrosine antibody that binds to protein A or secondary antibody coated onto the bead [73]. SPA has also been used for quantification of PCR using biotinylated PCR primers and [3H] dNTPs. The biotinylated [3H] DNA produced is captured onto streptavidin coated beads [73].

3. Comments

SPA is a very widely used homogeneous assay format for many biological assays. SPA is applicable to HTS and can be adapted for automation. Assays are routinely done in 96-well plates and for increased throughput in 384-well plates. Assays in 384-well plates reduce the radioactive waste generated and the cost of the reagents. The most commonly used radioisotopes in SPA are 3H and 125I. Recently, 33P is also being used in protein kinase assays. Though the path lengths of 35S and 14C are similar, due to the low specific activity 14C-labeled compounds (~ 60 mCi/mmol) have not been utilized that much in SPA. When 35S or 33P is

14c Labeled Compounds

Figure 23 SPA enzyme assay formats. Schematic representation of the three enzyme assay formats. (1) When a radiolabeled compound is added to a substrate attached to a SPA bead, the result is a signal increase. (2) When a radiolabeled substrate attached to a SPA bead is cleaved, releasing the radiolabel will result in a signal decrease. (3) When a substrate forms a radiolabeled product in the reaction, the product can be captured onto the antibody coated SPA bead, resulting in signal increase. (Courtesy of Amersham.)

Figure 23 SPA enzyme assay formats. Schematic representation of the three enzyme assay formats. (1) When a radiolabeled compound is added to a substrate attached to a SPA bead, the result is a signal increase. (2) When a radiolabeled substrate attached to a SPA bead is cleaved, releasing the radiolabel will result in a signal decrease. (3) When a substrate forms a radiolabeled product in the reaction, the product can be captured onto the antibody coated SPA bead, resulting in signal increase. (Courtesy of Amersham.)

used in SPA, for best results the samples are centrifuged to bring the beads to the bottom of the well or CsCl is added to increase the density of the reaction medium, allowing the beads to float to the top for reducing background. The count time in scintillation counters for a 96- or 384-well plate is approximately 10 or 40 min, respectively, and this may restrict the throughput to some extent. The signal-to-noise ratio is generally lower than with the conventional assays but may be adequate for use in HTS. Other critical issues associated with SPA are color quench and detection efficiency of scintillation counting. The availability of many protein and other biomolecule coated generic SPA beads and SPA assay kits from Amersham makes assay development for HTS convenient. The radioactive waste is reduced and does not require any special equipment. However, for SPA use in HTS, a technology transfer agreement has to be obtained from Amers-ham, which is expensive.

D. LEADseeker™ Homogeneous Imaging System

The LEADseeker homogeneous imaging system is being developed by Amersham in collaboration with Imaging Research Inc. This proprietary system combines imaging instrumentation and specialized software with radioactive proximity reagents that are at least 10 times more sensitive than SPA [75]. The LEADseeker™ Radiometric Imager system consists of a CCD camera and uses europium yttrium oxide (YO:Eu) or europium polystyrene (PST:Eu) particles, which exhibit emission at 615 nm. The LEADseeker™ imaging beads have an emission maximum of 615 nm and show very little quenching with colored (yellow) compounds. These beads are available with streptavidin, WGA, glutathione, protein A, and nickel coatings, which produce higher light output than the SPA bead. The assays have been developed for 96- and 384-well plates. This system is capable of capturing the signal from an entire 384-well plate in a single exposure within 10 min. The camera reads the density of the image in grey scales over a total range of 216 levels. The assays are currently developed for higher density plates such as 1536-well plates.

1. Instrumentation

Amersham is developing LEADseeker instrument in collaboration with Imaging Research Inc. for performance partnership partners. The main features of the LEADseeker consists of a camera with a cooled CCD chip that has an imaging area of 1024 X 1024 pixels for high-resolution imaging. Shadowing, which is a common problem with standard lenses used in imaging, is overcome in the LEADseeker with the use of the telecentric Borealis lens, which captures light more efficiently from the full area of the plate. The LEADseeker™ will be extended to nonradiometric applications with a multimodality imager that will be capable of reading fluorescence, luminescence, and color. Fluorescent assays will be based on proprietary cyanine fluors (500-800 nM) that will be applicable to including steady-state, FP, FRET, and TRF assays.

2. Applications

The LEADseeker™ can be used for assays that can be performed with the SPA, the difference being the use of more sensitive Eu-complexed polystyrene or yt trium oxide beads in place of SPA beads. Thus the assays can be converted from macro to micro assays using LEADseeker™ technology. Some of the assays that have been tested include reverse transcriptase, EGF binding, GTPyS binding, and extracellular response kinase 1 [75].

3. Comments

The LEADseeker is a homogeneous radioactive/nonradioactive imaging technology suitable for HTS. A CCD camera images the signals from a 384-well plate, and imaged for less than 10 min, and with development of a 1536-plate imager, will allow screening > 100,000 compounds a day. This technology is being developed in partnership with major pharmaceutical companies. Color quench is overcome by the new bead types. When available, this technology will be a rapid homogeneous radiometric and nonradiometric uHTS, which will save reagents and shorten screening times.

V. OTHER METHODS A. Surface Plasmon Resonance

Surface plasmon resonance (SPR) has become a popular method for looking at biomolecular interactions. SPR occurs when surface plasmon waves are excited at the sensor surface consisting of thin metal such as gold coated onto a glass support [76,77]. SPR is a phenomenon that occurs between incoming photons and electrons in the sensor surface. The light energy at a particular wavelength and angle of incidence is transferred to the electrons in the metal surface, causing alterations in the reflected light. The resonance (nonreflectance) angle is dependent on the refraction index in the vicinity of the metal surface, which in turn is dependent on the mass concentration. Molecules attaching to the sensor surface with gold film cause changes in the refractive index close to the surface, resulting in a change in the SPR signal. Biomolecular binding events cause further changes in the refractive index that is detected as changes in the SPR signal. In the BIA-core (biomolecular interaction analysis), the shift in the resonance angle with time is measured. SPR technique can be used to precisely measure the kinetics of macromolecular interactions.

Sensor surfaces can be functionalized either directly to capture different target molecules or to do affinity capture of the target molecules. SPR of macro-molecules uses Au SPR film and a flow cell that houses a chip coated with a thin layer of Au colloidal particles. The sensor chip CM5 has a carboxymethyl-ated dextran matrix surface to which ligand can be immobilized through covalent derivatization through amine, thiol, aldehyde, or carboxyl groups. Different types of sensor surfaces are available that can be used for different assays; the sensor chip CM5 (carboxymethylated dextran matrix surface) with immobilized ligand can be used for studying the interaction with target molecules or affinity capture of an alternative molecule that interacts with the target molecule; the sensor chip SA (streptavidin surface), which captures large biotinylated DNA fragments, is used in nucleic acid interactions; the sensor chip NTA (NTA coated sensor surface) through nickel chelation captures histidine tagged biomolecules that can be used in receptor binding assays; the sensor chip HPA (hydrophobic surface) to which membranes or liposomes containing receptors can be coated are used in receptor binding studies.

When light is reflected off the surface of the Au particle, the angle of reflection gives information about the mass bound to the matrix. A solution containing compounds that interact with the molecule bound to the chip (e.g., ligand to receptor or antigen to antibody) is passed over the surface of the chip. As these molecules interact, there is an effective change in surface roughness of a few nanometers on Au-SPR films that is easily detected. This allows a rapid and direct measurement of the binding kinetics of a broad variety of interactions in real time. Despite its sensitivity, this technique is limited, and it is not applicable to small molecule measurements.

1. Instrumentation

The single channel Biacore probe is used for fast detection and concentration of target biomolecules. In the Biacore-probe, SPR occurs in the gold film at the tip of a sensor probe. The multichannel Biacore X, Biacore 2000, and Biacore 3000 can be used to study biomolecular binding events in real time, allowing direct assessment of kinetic constants. Flow cells use as little as 5 |L sample. The Biacore X is a manual system with one continuous flow pump and two flow cells. The Biacore 2000 and Biacore 3000 are automated systems with two autosam-plers and continuous flow pumps and four flow cells on one sensor, which allow immobilization of four different molecules; four different interactions can be monitored simultaneously.

2. Comments

SPR based assays are homogeneous assays though lower throughput without angle scanning. The sensitivity of Biacore technology is sufficient for detection and characterization of binding events involving low-molecular-weight compounds and their immobilized protein targets. Biacore systems measure real-time binding events, with accurate determination of kinetic constants. Automation reduces the analysis times and increases throughput. Multiple interactions can be screened on a large array sensor simultaneously using imaging technology with a CCD camera for detection. As this technology develops it will be a powerful tool for HTS to measure the binding of small molecule compounds to their drug targets directly.

B. CLIPR System

The Chemiluminescence Imaging Plate Reader (CLIPR) a product of Molecular Devices, is an ultra high throughput luminometer system for 96-, 384-, 864-, and 1536-well microplates. The instrument can be used in HTS mode for cell based assays and SPA assays in microplates [78].

1. Instrumentation

CLIPR integrates a high sensitivity CCD camera, a telecentric lens, a high precision positioning mechanism, and a computer system with software for instrument and record data. The CLIPR system can be loaded manually, can have a plate stacker, or can be integrated to a linear robot line. The imaging plate reader system reads plates in under a second and it is possible to do kinetic studies.

C. Infrared Thermography

To measure thermogenesis in a cell culture, infrared imaging system thermogene-sis was reported [79]. The infrared imaging system was shown to be a rapid, very sensitive (0.002°C), and effective method for measuring thermogenesis in cell culture in vitro. Cells grown in a 96- or 384-well plate are equilibrated in an incubator at 37°C, compounds are added, equilibration at 37°C is done for 10 min, and the heat generation is measured by imaging in the infrared thermography system. Thermogenesis increased in yeast expressing the mitochondrial uncoupling protein-2 after treating with an uncoupler of mitochondrial respiration and in adipocytes treated with rotenone, an inhibitor of mitochondrial respiration or ß-adrenergic receptor agonists [79].

1. Instrumentation

Commercial systems are not available in the market. A custom-made infrared thermography system consists of a thermo electrically cooled Agema Thermovision 900 Infrared System AB (at a focal distance of 6 cm), equipped with a SW Scanner and a lens (40° X 25° lens) that detects a 2-5.4 micron spectral response. The data analyzer consists of OS-9 advanced systems and ERIKA 2.00 software from Agema Infrared Systems. The sensitivity of this infrared thermography system is 0.002°C, and its robustness (96- as well as 384-well plates) makes this system very useful for HTS assays in detection of altered thermogenic responses in various cell types.

D. Nanoparticle Technologies

Highly luminescent semiconductor quantum dots (small nanoparticles made of zinc sulfide capped cadmium selenide) have been covalently coupled to biomo-lecules such as various antibodies or DNA probes for use in ultrasensitive biological detection [80,81]. The luminescent labels are ~ 20 times brighter and 100 times more stable against photobleaching and one-third wide in spectral line width compared to organic dyes such as rhodamine. Biomolecules are attached to different color nanoparticles. These biomolecule conjugates are water soluble and biocompatible. When cells are exposed to the different colored nanoparticles containing various antibodies, each antibody binds only to its specific antigen on the cell surface. Depending on the presence of types of antigen on the cell surface, those colored nanoparticles are captured and others are washed away. Spectral readings at different wavelengths give information on the types of antigens present and the amount of each antigen. Similarly, nanoparticles with different DNA probes can be used to identify a large number of gene sequences in blood and other biological samples.

Semiconductor nanocrystals labeled with fluorescent probes have a narrow, tunable, symmetrical emission spectrum and a broad continuous excitation spectrum; they are photochemically stable and may prove to be superior to existing fluorophores and may have many applications in several different assays [82]. These water soluble nanocrystals also have a long fluorescence lifetime (hundreds of nanoseconds), which can allow for time-gated detection of autofluorescence suppression. Several companies developing nanoparticle technologies are Quantum Dot, Auspex, Biocrystals, Nanomat, and Nanosphere.

E. Liquid Crystals

Liquid crystals are used to amplify and transduce receptor-mediated binding of proteins at the surface into optical outputs. Liquid crystal sandwiched between two gold films supporting self-assembled monolayers containing ligands, upon binding of proteins to the specific ligands, will change the surface roughness and trigger rapid changes in the orientations of 1-20 |m thick films of supported liquid crystals and changes the intensity of light transmitted through the liquid crystal, which can be further amplified and transduced into optical signals [83]. The orientations of liquid crystals are sensitive to a wide variety of physicochemi-cal properties of surfaces, which suggests that this approach can be used for the detection of binding of small molecules to proteins and protein aggregates to a surface. This approach does not need electroanalytical apparatus, provides spatial resolution of micrometers, and can be extended to assay the effect of spatially resolved chemical libraries on the ligand-receptor binding.

F. Microchip Technology

The HTS and uHTS assays use volumes of a few microliters (5-10 |L in a 1536-well plate) to several microliters (100 |L in a 96-well plate). Fluid dispensing, mixing, and evaporation are some of the major technical problems in reducing assay volumes to a few microliters or to submicroliter levels. Microchips are designed either for single or multiple use and consist of silicon and glass master chips combined with plastic injection molding or embossing produced by microfabrication technologies. Fluids are moved through microscopic channels by either electro-osmosis or electrophoresis (microfluidics). Microfluidic capillary electrophoresis has been successfully used in several different types of HTS enzyme assays in microchips.

Microchip technology has been used for enzyme assays and to determine the binding affinity of monoclonal antibody [84-86]. In a microchip based protein kinase A assay, fluorescein labeled Kemptide was used as substrate. The assay reagents were placed in wells on the microchip, aliquots of the reagents were transported by electro-osmosis into the network of etched channels, and enzyme reaction was performed. The phosphorylated fluorescein labeled Kemptide product was separated from the substrate by on-chip capillary electrophoresis, and kinetic constants for ATP and peptide substrates (Km) and the inhibition constant (K) for inhibitor H-89 were determined. This assay demonstrated the usefulness of microchips for performing enzyme assays. Thus microchip technology has potential for applications to immunoassays, nucleic acid assays, enzyme assays, and receptor-binding assays.

Microchip technology has been widely used in DNA analysis. A DNA chip is a small surface specked with thousands of dots of single stranded DNA of a gene or gene segment. Gene activity in the cells or tissues is measured by collecting mRNA from cells or tissues, converting it to cDNA, labeling it with a dye, and incubating it with a DNA chip. The cDNAs hybridize to complementary sequences on the chip and are identified. DNA array technology is widely used in various diseases including cancer. Microchips are now commercially available from Affymetrix Inc. (Santa Clara, CA), Caliper Technologies (Mountain View, CA), ACLARA BioSciences (Mountain View, CA) and others.

Was this article helpful?

0 0

Post a comment