Marcel

2. Applications of HTRF

Essentially three types of labeling of macromolecules (direct, indirect, and semi-direct) can be done for HTRF/Lance assays similar to labeling for FP [27]. In direct labeling, the donor lanthanide chelate and the acceptor allophycocyanin, rhodamine, or fluorescein are directly labeled on molecule(s) involved in the reaction. In indirect labeling, these donor and acceptor fluorophores are labeled to macromolecules involved in the secondary interactions such as antibody-secondary antibody binding or biotin-streptavidin interaction. Semidirect labeling is a combination of direct and indirect labeling. HTRF/Lance assays can be successively developed for reactions involving macromolecular interactions such as DNA-DNA, RNA-DNA, DNA-protein, protein-protein, and receptor-ligand, for reactions involving hydrolysis of macromolecules such as nucleases or proteases, for reactions involving synthesis of macromolecules such as polymerases, and for other protein modification reactions such as kinases [28-30].

PTK Assay. In the PTK assay, a biotin-peptide substrate is phosphory-lated by PTK action. Eu-PY antibody binds to the phosphorylated biotin-peptide [32,33]. The biotin-peptide binds to XL665 or CY5 labeled streptavidin (APC-streptavidin) (Fig. 13). Due to the close proximity of the two fluorophores, the energy from Eu3+ is transferred to APC-streptavidin. Typically, with the best PY antibody, a signal-to-noise ratio of 20 or more is obtained [35]. In the indirect method, PY antibody (not labeled with Eu3+) binds to the phosphorylated biotin-peptide and the complex binds to a generic reagent, Eu-protein G or Eu-protein A (Fig. 13). FRET can occur from Eu to APC conjugated to streptavidin, though the signal will be somewhat lower (signal-to-noise ratio ~ 10) than the above direct method [34]. Nevertheless, the use of generic reagents provides an easy assay development with comparable signal response.

The applications for HTRF/TR-FRET technology have been rapidly growing. They include competitive immunoassays to measure hormones like prolactin, PhCG [35], tumor necrosis factor (TNF) a [36], receptor-ligand binding interactions, e.g., TNF receptor 1 (TNFRl)-TNFa binding in which TNFR1 is labeled with lanthanide chelate and the ligand with acceptor. When TNFR1 is labeled with Eu chelate biotin-TNFa is used as ligand and APC-streptavidin as acceptor (Fig. 14), and with TNFR1 labeled with Tb chelate, rhodamine labeled TNFa is used as ligand. The IC50 obtained for TNFa is 3.9 and 3.5 nM, respectively. IL-2 receptor a-IL-2 interaction in which IL-2 is labeled with Eu3+ and a monoclonal antibody for IL-2 receptor a is labeled with Cy5 [36]; EGF-EGF receptor interaction in which EGF is labeled with Eu3+ and a monoclonal antibody against the nonbinding region of EGF receptor is labeled with XL-665 [29]; protein-protein interactions, e.g., jun-fos heterodimerization is measured by incubating biotinyl-ated jun peptide with XL665-fos peptide and (Eu)K-streptavidin [29]; DNA-hybridization assay in which one oligo nucleotide strand was labeled with Eu3+

Figure 13 Schematic representation of TR-FRET protein tyrosine kinase assays. In the direct assay, phosphorylated biotinyl peptide is conjugated to Eu3+-PY antibody and energy is transferred to APC-streptavidin bound to the phosphorylated biotinyl peptide. In the indirect assay, Eu3+-protein A or Eu3+-secondary antibody binds to the PY antibody conjugated to the phosphorylated biotinyl peptide and transfers energy to APC-streptavidin bound to the phosphorylated biotinyl peptide. (Courtesy of Wallac.)

Figure 13 Schematic representation of TR-FRET protein tyrosine kinase assays. In the direct assay, phosphorylated biotinyl peptide is conjugated to Eu3+-PY antibody and energy is transferred to APC-streptavidin bound to the phosphorylated biotinyl peptide. In the indirect assay, Eu3+-protein A or Eu3+-secondary antibody binds to the PY antibody conjugated to the phosphorylated biotinyl peptide and transfers energy to APC-streptavidin bound to the phosphorylated biotinyl peptide. (Courtesy of Wallac.)

at the 5' end and the complementary strand was labeled with biotin at the 5' end and coupled to XL-665 labeled streptavidin [38]; helicase assay in which one DNA oligonucleotide strand is labeled at the 3' end either with CY5 or tetramethylrhodamine and the other strand is labeled at the 5' end with Eu or Tb chelate, respectively [39].

3. Comments

The HTRF/Lance assay is a nonradioactive, homogeneous, sensitive assay without any separation steps. With appropriate delay time in HTRF/Lance, the back-

Lance Assay Htrf

Figure 14 TR-FRET TNFa binding assay. (A) Schematic representation of energy transfer in the TNFa-Eu3+ APC Lance assay system. TNFR1 was labeled with Eu, TNFa was biotinylated, and APC bound streptavidin was used in this assay. (B) TNFa competition binding curve using Tb/Rh Lance assay system gave an IC50 for a TNFa of 3.5 nM. The TNFa competition binding curve using the Eu/APC Lance assay system gave an IC50 for a TNFa of 3.9 nM, which is very close to that obtained with the Tb/Rh system. (Courtesy of Wallac.)

Figure 14 TR-FRET TNFa binding assay. (A) Schematic representation of energy transfer in the TNFa-Eu3+ APC Lance assay system. TNFR1 was labeled with Eu, TNFa was biotinylated, and APC bound streptavidin was used in this assay. (B) TNFa competition binding curve using Tb/Rh Lance assay system gave an IC50 for a TNFa of 3.5 nM. The TNFa competition binding curve using the Eu/APC Lance assay system gave an IC50 for a TNFa of 3.9 nM, which is very close to that obtained with the Tb/Rh system. (Courtesy of Wallac.)

ground interference can be minimized, and the emission of acceptor excited during the excitation of donor can be eliminated, and the pure energy transfer signal from acceptor can be measured. Energy transfer from lanthanide donor to allo-phycocyanin acceptor occurs when they are in proximity, giving long-lived decay at acceptor emission, the signal being specific for the biomolecular interaction. When acceptor (XL665) is not in proximity to the donor [Eu]K or lanthanide chelates a short-lived signal at 665 nm occurs that is discriminated by a time-delayed measurement. The interference from colored and fluorescent compounds in the assay can be eliminated by measuring the ratio of specific acceptor signal to the donor (Eu)K or lanthanide chelate signal. HTRF/Lance is a homogeneous assay technology that can be used in 96-, 384-, and 1586-well plate format and is amenable to automation, thus suited well for HTS and uHTS. The reagents are environmentally safe and are stable. One of the limitations of this assay is that two interacting biomolecules have to be conjugated to a donor and an acceptor fluorophore if the generic labeled reagents cannot be used, which can be complex and problematic. There is a limited choice of donors and acceptors.

E. Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a statistical physics based new analytical technology that extracts quantitative information from the spontaneously fluctuating fluorescence molecules of small molecular ensembles [40]. FCS monitors interactions of molecules present at minuscule concentrations in femto-liter volumes and thus offers the highest potential as the detection technique in the nano scale in the determination of molecular interactions in solution, on cell surfaces, or in the cells using homogeneous assays. In FCS, a sharply focused laser beam illuminates a very small volume element (typically femtoliter). Single molecules diffusing through the illuminated confocal volume produce bursts of fluorescent light quanta during the entire course of their journey (Brownian motion), and each individual burst is recorded in a time-resolved manner by a highly sensitive single-photon detector and analyzed using autocorrelation techniques [41,42]. This FCS autocorrelation function gives information on concentration, the diffusion time of all the individual molecules (related to the size and shape of the molecule), and the brightness of each molecule. Thus autocorrelation of the time-dependent fluorescence signal allows differentiation of slow and faster diffusing particles, and binding and catalytic activity can be directly calculated from the diffusion times and the ratio of faster and slower molecules.

1. Instrumentation

The Confocor is a commercial instrument developed by a joint venture between Evotec and Carl Zeiss. EVOscreen™ platform is a modular, miniaturized uHTS based on FCS and Evotec's proprietary FCS-related single molecule detection technology (FCS plus) and reader Confocor™ (discussed in greater detail in this volume, Chapter 22).

2. FCS Applications

Evotec in parternership with SmithKline Beechem and Novartis have developed several FCS based assays representing various classes of target proteins [42]. FCS can be used for mass-dependent and mass-independent fluorescence assays.

Mass-Dependent Assays. Ligand-receptor binding assays can be performed by FCS at the molecular level with membrane bound receptors in live cells on cell surfaces, membrane preparations, or cell derived vesicles and nuclear receptors based either on fluorescence intensity or on FP [41,42]. Screens based on FCS have been used for EGF receptors, acetylcholine receptors, and thyroid receptors [41,42]. FCS protease assays with fluorescent casein substrate based on total intensity and with fluorescent biotinylated peptide substrate based on FP have been reported [41,42]. Enzyme assays such as PTK, PTP [42], proteinprotein interactions such as SH2-phosphotyrosine binding [42], DNA/protein interactions such as topoisomerase-DNA binding, thyroid hormone receptor-DNA binding [41,42], and DNA/DNA interactions as in template-primer association have been investigated with FCS [42].

Mass-Independent Assays. Assays based on fluorescence intensity changes have also been developed by FCS. In these assays, the fluorescence of the substrate is quenched, and in the product of the reaction the quenching is relieved, increasing the molecular brightness and the total fluorescence intensity. A quenched fluorescent protease substrate, tetramethylrhodamine (TMR)-quenched fluorescence peptide, or streptavidin-quenched rhodaminegreen (RhGn)-peptide, when cleaved by protease, leads to the relief of quenching, which results in an increase in fluorescence intensity, apparent particle number, and mean confocal intensity [42]. In the RNA-ligand binding assay, association of a RhGn labeled ligand to RNA quenches the RhGn fluorescence of the ligand due to environmental effects on the dye and reduces the confocal fluorescence intensity and apparent particle number [42]. The binding of TMR labeled chemo-kine to chemokine receptor membrane vesicles increases the cumulative brightness of TMR-chemokine bound vesicles, which can be monitored by FCS [42]. FCS can also be used for Ca2+ uptake functional assays for 7-transmembrane receptor [42].

A dual-color fluorescence cross-correlation spectroscopy method suitable for binding and fast catalytic rate study was developed that does not depend on diffusion properties as with conventional FCS [43,44]. Based on dual-color fluorescence cross-correlation spectroscopy, RAPID FCS (rapid assay processing by integration of dual-color FCS) has been developed, which combines short analysis times with the development of fast and flexible assays resulting in sensitive, homogeneous, fluorescence based assays to measure molecular fragmentation and assembly resulting from a reaction/interaction [44]. This further extends the scope of FCS for uHTS.

3. Comments

FCS can be used in a microvolume configuration (1-10 |L assay volumes) and is adaptable for uHTS. The read times for vesicle assays are 5-10 sec and for solution assays 1-2 sec. FCS assays are based on the analysis of the molecular dynamics and the reaction kinetics of fluorescence labeled molecules that undergo temporal changes in their diffusion properties and determine the concentration of interaction parameters. A large number of simultaneously derived molecular parameters are obtained in FCS that allow selection of the most robust signal change for an assay. FCS can be used for several types of assays and for detection techniques monitoring intensity, particle number, polarization, energy transfer, and lifetimes, increasing the scope of this technology. FCS can thus be used for most of the target classes of assays encountered in drug discovery. Though FCS was first described more than 25 years ago, the application of FCS technology to HTS is very recent, and there are no commercial FCS readers available other than that being developed by Evotec (Confocor). Access to the technology is semiexclusive to the consortium partners, limiting the accessibility of this technology. Also, the limited screening data that is in the public domain is from presentations at conferences and a few review articles [41,42]. FCS could prove to be a promising technology for uHTS in the future.

F. Fluorescent Reporter Assays

Cell based assays are either used in conjunction with in vitro assays or in place of in vitro biochemical assays to examine output of specific cellular process. Reporter genes have been used in drug discovery for transcriptional studies as well as for characterization of receptor function and metabolic regulation [45]. Agonist activation of a receptor or a ligand-gated ion channel produces changes in the transcription of a number of genes that can be readily measured by using gene fusions. A reporter gene construct consists of an inducible transcriptional element that controls the expression of a reporter gene. Generally, a strong promoter (constitutively not active) that is controlled by a desired response element that is regulated by receptor activation is fused to the coding region of a reporter protein such as green fluorescent protein, P-lactamase, luciferase, P-galactosi-dase, chloramphenicol acetyltransferase, or secreted alkaline phosphatase (see Chapter 10).

1. Green Fluorescent Protein

Green fluorescent protein (GFP), a fluorescent protein originally isolated from the jellyfish Aequoria victoria, is a 238 amino acid protein that attains the fluorescent state spontaneously; the fluorescence is stable [46]. The wild GFP is relatively low in fluorescence intensity, has multiple absorption and emission maxima, and has about 4 hr lag time between the expression of protein and attaining full fluorescence. These drawbacks of GFP are remedied with the development of new and improved GFP mutants with different spectral properties, increased brightness of fluorescence, and mammalian cell compatible cloning vectors. Because of different spectral properties of mutant GFPs, it is possible to follow two GFPs in the same cell, and also they can be used in FRET assays to study proteinprotein interactions and other FRET based assays. A FRET protease assay be tween two linked variants of the GFP was described [46]. The C-terminus of a red-shifted variant of GFP (RSGFP4) was fused to the N-terminus of a protein linker containing a Factor Xa protease cleavage site, and the N-terminus of a blue-variant of GFP (BFP5) was fused to the C-terminus of the protein linker. In the gene product, energy transfer occurs from BFP5 to RSGFP4. With Factor Xa protease action the protein linker is cleaved and the two GFPs dissociate, resulting in a decrease in energy transfer. The emission ratio of the BFP5 (450 nm) and RSGF4 (505 nm) increases with the protease activity because FRET decreases. A similar chymotrypsin assay using G-protein FRET peptide in 3546-well Nano well assay plates was reported [47].

2. P-Lactamase Reporter Assays

P-lactamase from E. coli is a 29 kD product of the ampicillin-resistant gene Amp that hydrolyzes pencillins and cephalosporins. The recent report of a membrane-permeant ester derivative CCF2/AM (6-chloro 7-hydroxy coumarin and fluorescein conjugated at 7 and 3' positions of cephalosporin, respectively), a P-lacta-mase substrate, provides a quantitative measure of gene transcription and thus allows the development of a homogeneous transcription activation HTS assay with P-lactamase reporter [48]. Excitation of coumarin at 409 nm by FRET results in the emission from fluorescein of a green fluorescence (520 nm). When P-lactamase cleaves fluorescein on the 3' position, it disrupts FRET and results in the emission of coumarin at 447 nm, i.e., blue fluorescence appears while the green fluorescence of fluorescein is quenched. Thus each molecule of P-lactamase attacks many substrate molecules and changes the fluorescence of substrate molecules from green to blue by disrupting FRET. The response element or promoter with P-lactamase reporter has to be introduced into a clonal cell line with stable expression of the receptor of interest. Jurkat cells stably transfected with Mj muscarinic receptor and nuclear factors of activated T-cell (NF-AT)-P-lactamase reporter gene with a cytomegalovirus promoter, when treated with carbachol, induced P-lactamase activity as measured by the conversion of green fluorescence to blue fluorescence after loading cell-permeable P-lactamase substrate CCF2/ AM. The induction of P-lactamase activity (the appearance of blue cells) depended on carbachol concentration and time of incubation [48]. The measurement of P-lactamase activity as an emission ratio at 450/530 nm improves accuracy. A cell based G-protein coupled receptor assay using the P-lactamase reporter gene system in a 3456-well Nano well assay plate was described [48]. This functional activity assay with P-lactamase reporter holds great promise for screening for receptors and ligand-gated ion channels. The main drawback of reporter gene technology is the limitation on using reporter genes in recombinantly expressed cells (heterologous expression is available to consortium partners of Aurora Bioscience Corp. and other institutions that license the reporter assay technologies).

G. Fluorescence Imaging

In fluorescence imaging technology, unlike in a plate reader wherein the fluorescence signal is read one well at a time, all the wells of a microtiter plate (96-, 384-, 1536-well plates) are read simultaneously by imaging in a CCD camera capable of recording kinetics in the subsecond range.

1. High Content Screening

High content screening (HCS) is analysis of cells using fluorescence based reagents with the ArrayScan system to extract spatial and temporal information of target activities within cells [49]. HCS yields information that will permit more efficient lead optimization before the in vivo testing. There are two types of HCS: (1) using fixed cells with fluorescent antibodies, ligands, and/or nucleic acid probes, and (2) using live cells with multicolor fluorescent indicators and biosensors. Two additional parameters can be measured simultaneously with the availability of two more channels of fluorescence in the ArrayScan system.

Instrumentation. The ArrayScan™ System is being developed by Cel-lomics along with Carl Zeiss, Jena. It is a tabletop instrument (optics with a spatial resolution of 0.68 |M) with subcellular resolution from many cells in a field within the well of a microtiter plate. The ArrayScan II automatically scans a microtiter plate, acquiring multicolor fluorescence image datasets of fields of cells at a preselected spatial resolution. The CellChip™ System is a miniaturized chip-based screening platform being developed by Cellomics.

Applications. HCS can be very effectively applied to study drug-induced dynamic redistribution of intracellular constituents. In cells transfected with green fluorescent protein coupled to human glucocorticoid receptor (GFR-hGR), drug-induced cytoplasm to nuclear translocation of GFR-hGR was studied by HCS [49]. With HCS, translocation in each cell can be quantified. In addition, the availability of two more fluorescence channels in ArrayScan II allows the determination of two additional parameters in parallel such as other receptors or cellular processes. A high content screen has also been explored for multiparametric measurement of apoptosis, which provides information on parameters such as nuclear size and shape changes, nuclear DNA content, mitochondrial potential, and actin-cytoskeletal rearrangements during drug-induced programmed cell death [49].

Comments. HCS is a promising technology that can be applied for measurement of molecular events such as signal transduction pathways and effects on cell functions. It can be used with fixed cells to measure end points, with live cells continuous monitoring of the activities is possible. Availability of up to two additional fluorescence channels in ArrayScan II allows the measurement of two additional parameters simultaneously. HCS allows subcellular measurements, and data can be obtained for each individual cell in the field. The technology is in the early stages, and there is no published screening data.

2. Fluorescence Confocal Microscope Imaging

With the development of the confocal laser scanning microscope and many technological advancements in laser scanning techniques and digital imaging methods and photostable fluorescent dyes, it is possible to do multiple fluorescence labeling of biological specimens, live cell imaging, and multidimensional microscopy in addition to imaging fixed and fluorescently labeled biological specimens in single and multiple wavelength modes [50]. Confocal laser scanning microscopy, multiple fluorescence labeling, together with immunofluorescence and fluorescence in situ hybridization, have become powerful techniques to map gene expression, for the detection of DNA and RNA and the expression of proteins.

A fast fluorescence confocal microscope optimized for homogeneous cell based assays has been designed by SEQ Ltd. [51]. The instrument is capable of simultaneous two-color laser excitation and detection of three-color imaging. The cells (e.g., expressing the receptor of interest) adhering to the bottom of the plate are probed with a ligand labeled with fluorescent dye such as fluorescein and fluorescent dye LDS 751, a nonspecific nucleic acid stain. A cell by cell analysis of the binding activity can be made using a mask generated by LDS 751 emission. The overlay of this binary mask with a binding activity image yields a pseudocolor map of receptor activity. Real-time data processing for most assays can be obtained by image analysis. Other assays tested include trafficking of a transcription factor and agonist activated transient Ca2+ levels. This technology is under development, and so screening data are not available.

3. Fluorescent Imaging Plate Reader

The Fluorescent Imaging Plate Reader (FLIPR) was developed to perform high throughput quantitative optical screening for cell based fluorescent assays [52]. FLIPR measures fluorescence signals in all the wells of a microtiter plate simultaneously, with kinetic updates in the subsecond range. This permits the determination of transient signals such as the release of intracellular calcium using calcium indicators, calcium Green-1 and Fluo-3 [53]. It has also been used for measuring luminescence based luciferase reporter assays [54].

Instrumentation. FLIPR96 and FLIPR384 by Molecular Devices will perform optical measurements on all wells of a 96- or 96-and-384-well plate, respectively, at rates of up to once per second. FLIPR uses water-cooled argon-ion laser (5 W) illumination to excite the fluorescent dyes, and the emitted light is detected with a cooled charge coupled device (CCD) camera. The laser provides many discrete spectral lines spaced from 350 to 530 nm. If broader spectral coverage is needed, FLIPR can be fitted with a broad-band xenon arc lamp (300 to 700 nm). FLIPR384 contains interchangeable pipettor heads for simultaneous dispensing to either 96- or 384-well plates, and FLIPR96 contains pipettor head for a 96-well plate with an integrated washing station. Typical fluid volumes are 50-100 |L for FLIPR96 and 2-30 | L for FLIPR384. The instrument has precise temperature control. FLIPR has three primary configurations, manual, robot line, and stacker fed.

Applications. FLIPR can be used for measurements of intracellular calcium, intracellular pH, intracellular sodium, and membrane potential. FLIPR assays will be discussed in more detail in Chapter 7.

Comments. FLIPR, with its ability to take readings in all the wells of a plate simultaneously, enables the study of real-time kinetics. Real-time kinetic data gives additional pharmacological information for ranking relative potencies of drugs and gives information on the kinetics of the drug-receptor interaction. Functional response can be measured, thus providing the affinity, efficacy, and function of each drug; it can also distinguish full agonists, partial agonists, and antagonists within a single assay. FLIPR is a complex instrument. The user has to be familiar with all fine tunings needed to get the best results.

4. Fluorometric Microvolume Assay Technology

Fluorometric Microvolume Assay Technology (FMAT™), developed by PE Biosystems, uses Cy5 based fluorophores, and multiwell plates are scanned with a red, 633 helium/neon laser. In FMAT™, the laser is focused on the bottom of the well of a multiwell plate, and the fluorescence associated with each cell or bead is detected over the unbound and background fluorescence. The analysis algorithm ignores background fluorescence. Specific signal is detected as areas of concentrated fluorescence surrounding a cell or a bead, and the remaining background fluorescence is ignored in the final processing of the image data. FMAT is a homogeneous format for intact cell and bead based assays and does not require washing to remove unbound fluorophore. The FMAT™ system can be used with 96-well as well as higher density, 384- and 1536-well plates. The FMAT™ assays can be performed in a two-color format with two PMTs to determine more than one receptor binding assay on a single cell or multiple markers on a cell or a bead.

Instrumentation. The FMAT™ instrument is a fluorescence imager of a single-well plate of multiwell plates developed at Biometric Imaging. The excitation is from a 633 helium/neon laser and focused on a 1 X 1 mm area at the bottom of the well. The emission from the well is read in two PMTs at different wavelengths to measure the emission of different fluorophores to quantitate two different events on the same cell or bead. The FMAT™ system's optical platform yields population data in image format.

Applications. FMAT™ uses nonradioactive fluorescent tags and has been applied for cytotoxicity assays, functional assays such as ICAM-1 regulation by cytokines, and G-protein coupled receptor binding assays, nuclear hormone receptor assays, tyrosine and serine/threonine kinases, protein-nucleic acid interactions, and protein-protein interactions [55]. ELISA assays that are not HTS compatible, due to the number of wash steps and incubation steps involved, can be reformatted to bead based homogeneous assays with FMAT™. In a typical IL-8 fluorescent-linked immunosorbent assay (FLISA), secondary antibody (goat antimouse IgG) coated beads are complexed with monoclonal anti-IL-8 antibody and incubated with sample, biotinylated polyclonal anti-IL-8 antibody, and strep-tavidin labeled Cy5 (Fig. 15). IL-8 in the sample forms a sandwich by the matched antibody pair, and Cy5 labeled streptavidin binds to the biotin of the polyclonal antibody. The fluorescence associated with the bead complex is determined in the FMAT™ system over the unbound streptavidin Cy5 and background. FLISA uses 1% of capture antibody that is used in ELISA (coated on plate), thus reducing reagent. FLISA is a one-step incubation assay compared to the multiple wash and incubation steps with ELISA, with equal sensitivity as ELISA. FMAT™ can be used to develop multiplexed assays (simultaneous multiple assays) with different bead sizes or fluorophores within a single well. Though FMAT™ is a homogeneous assay because the imager reads one cell at a time, it will take several minutes reading high density 384- and 1536-plates, restricting throughput.

Figure 15 Schematic representation of FMAT interleukin-8 immunoassay. Goat anti-mouse beads coated with IL-8 monoclonal antibody bind IL-8 in the sample; then the second biotinylated IL-8 antibody (to a different epitope) will immunocomplex with IL-8. Streptavidin labeled with Cy5 will bind to biotin residue, and bead bound fluorescence is measured. The unbound fluorophone is ignored and does not give a signal. (Courtesy of PE Biosystems.)

Figure 15 Schematic representation of FMAT interleukin-8 immunoassay. Goat anti-mouse beads coated with IL-8 monoclonal antibody bind IL-8 in the sample; then the second biotinylated IL-8 antibody (to a different epitope) will immunocomplex with IL-8. Streptavidin labeled with Cy5 will bind to biotin residue, and bead bound fluorescence is measured. The unbound fluorophone is ignored and does not give a signal. (Courtesy of PE Biosystems.)

Goal antimouse IgG beads coated with monoclonal ;mti-IL-ï antibody

Bead bound fluorescence is measured: unbound fluorophores are ignored

Goal antimouse IgG beads coated with monoclonal ;mti-IL-ï antibody

Bead bound fluorescence is measured: unbound fluorophores are ignored l>

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