MXPA00005800A - Continuous format high throughput screening - Google Patents

Abstract

Continuous format high throughput screening (CF-HTS) using at least one porous matrix allows the pharmaceutical industry to simultaneously screen large numbers of chemical entities for a wide range of biological or biochemical activity. In addition, CF-HTS is useful to perform multi-step assays.

Description

CLASSIFICATION OF HIGH PRODUCTION OF CONTINUOUS FORMAT TECHNICAL FIELD OF THE INVENTION The high-throughput continuous format (CF-HTS) classification using at least one porous matrix allows the pharmaceutical industry to simultaneously classify large numbers of chemical entities for large-scale biological or biochemical activity. In addition, CF-HTS is useful for performing multi-step tests.

BACKGROUND OF THE INVENTION Biochemical and biological assays are designed to test the activity on a wide scale of systems ranging from protein-protein interactions, enzyme catalysis, small molecule-protein binding, to cellular functions. In the "high production classification" (HTS), these types of tests are used to test a large number of chemical entities in order to discover previously unknown biological or biochemical activities of the chemical entities.

Homogeneous Tests Against Heterogeneous All the different types of biological tests can be divided into two main classes: homogeneous tests and heterogeneous tests. In homogeneous assays, all reagents are aggregated together and the results are measured or interpreted without any additional manipulation. For example, cells developed in a Petri dish can be exposed to a chemical. If the chemical is toxic to the cells, a clearing zone will indicate toxicity by simple observation. Another example is to use cells that express a protein that changes the color of cells. In the case of cells expressing beta-galactosidase (ß-gal) that develop on agar containing x-gal, the cells become more or less blue depending on how much ß-gal protein is expressed. In this way a homogeneous assay can be constructed for any biological step that affects the expression of a reporter gene such as the beta-galactosidase gene. Yet another example of a homogeneous assay uses a substrate that changes color or bloom when processed by an enzyme. Finally, technologies such as Cinylation Closeness Assays (SPA) by Amersham directly measure the binding of a labeled radio ligand to a protein or any ligand binding substance attached to beads containing a scintillator. All the above examples are homogeneous tests since they do not require any step other than the addition of reagents before the final detection, measurement, or reading of the signal. Heterogeneous trials, on the other hand, require at least two steps that, because they are inherently incompatible to a certain degree, can not be combined in a single step. For example, many heterogeneous assays require adding the reagents in a certain order (for example, when some reagents may interfere with previous steps for the assay but are required to complete the last steps). Common examples of this include assays where signal development reagents are added to indirectly report the presence or concentration of a reaction product. Another common step in heterogeneous trials is a washing step. The excess test reagents should usually be added at the beginning during the test, but they need to be washed before the subsequent steps so that the reactions can proceed without a high background signal. For example, in a radioligand binding assay, a labeled ligand is first incubated with a protein that is bound to a solid surface, but that only a small fraction of the ligand actually binds to the limited number of protein sites. After incubation, the excess bound ligand must be washed before making an accurate measurement of the bound radioactive ligand. Washing can be achieved through a variety of alternative methods, including filtration, evaporation, precipitation / phase separation, and / or centrifugation. Many biological and biochemical processes can only be measured through heterogeneous methods. further, in spite of the existence of ways of adapting other biological and biochemical processes to homogeneous methods, these other processes work with a more effective cost and / or with reactivations easily more available through heterogeneous methods. A variety of methods and equipment for homogeneous techniques such as SPA (Amersham), Fluorescence Polarization (Jolley and others), and Time Resolution Fluorescence (Packard and others) to name just a few, are commercially available. However, using these types of methods this incurs additional cost and time. For many trials, heterogeneous methods are more established and easier to develop quickly. For this reason the use of heterogeneous methods such as ELISA, filter binding, RIA, luciferase cell assays, etc., continues to be very broad. Some of these methods will be described in more detail later.

High Production Classification (HTS) Over the years, the pharmaceutical industry has relied heavily on HTS from collections of chemical compounds to find drug candidates. The HTS describes a method in which many discrete compounds are tested in parallel so that large numbers of "pruase" compounds are classified for biological activity simultaneously or almost simultaneously. At present, very widely established techniques use 96-well microtiter plates. In this format, 96 independent tests are performed simultaneously on an individual plastic plate of 8 cm x 12 cm containing 96 reaction cavities. These cavities typically require assay volumes ranging from 50 to 500 μl. In addition to the plates, many instruments, materials, pipettes, robots, plate washers and plate readers are commercially available to adjust the 96 cavity format to a wide variety of homogeneous and heterogeneous assays. Currently, efforts to improve HTS have focused on the manufacture of smaller cavities (miniaturization). As the cavity size is reduced, the number of cavities on each plate can be increased to provide a more parallel test. In addition, by reducing test volumes, the cost of reagents per test can also be reduced. In addition, since more tests can be run in parallel with smaller assay volumes, more compounds can also be tested simultaneously to find drug candidates. Thus, miniaturization has marginally improved the 96 cavity technology by providing a 384 cavity format (96 x 4). See Comley et al., J. Biomol, Screening, 2 (3) 1 p. 171-78 (1997). In fact, even higher density formats have been reported, including a 9600 cavity format. However, miniaturization has inherent costs and complexities. These costs and complexities refer directly to the three primary components of the miniaturization of a classification format. First, it should be possible to make the test containers (tubes, cavities, depressions, etc.) smaller. Secondly, it must be possible to accurately fill all the necessary test reagents in more and smaller cavities (usually achieved through robots that handle liquids that simultaneously supply reagents to many cavities). Third, it must be possible to "read" the results of the tests in the high density array. Given the requirements of parallel independent trials, each component provides challenges and limits how much minlaturing is reliable or cost-effective. For example, a smaller, newer format may require a completely different method of supplying reagents, or may require a reading instrument that has the resolution, sensitivity, and engineering conditions that is compatible with the newer miniaturized format. As the size of each cavity is reduced, there is the ability to manufacture the container arrangement, to dispense reagents in smaller amounts, and to read each test sample also becomes more difficult, time consuming and very expensive. In addition, a smaller sample size also increases the statistical variability of sample "a" sample V due to inaccuracies inherent in supplying smaller volumes of reagents and measuring weaker sample signals. reduces beyond a certain point, factors such as evaporation and surface tension add an even greater cost and complexity to implement the new miniaturized formats.

For a quantum hurdle in HTS technology, the industry greatly yearns for the possibility of "free form testing" or testing that has no physical barrier between samples. Typically, small test droplets are considered in a cavity-free format, which at present no one has reported the use of a free-format test in HTS with standard discrete composite collections.

Combinatorial Collections of Classification-Gel Penetration Methods. With the advent of combination chemistry, millions of chemical entities can be rapidly produced in solid supports (usually pearls). Although the 96-cavity format is being used to classify collections based on pearls, this format is generally considered ineffective, since (1) each pearl carries only small amounts of a chemical entity; (2) the number of compounds that will be treated is extremely large; and (3) the pearls are difficult to manipulate in 96-well "96-well" criss-crossing cups. " "" "" "" To avoid the problems inherent in the classification of combination collections through the 96-cavity format, some have reported the use of simple homogeneous tests that can be described as "free-form". As an example, an assay using pigment (melanocyte) cells in a simple homogeneous assay for combination peptide collections was reported by Jayawickreme et al., In Proc. Nat'l Acad. Sci. (USA). vol. 191, p. 1614-18 (March 1994). According to the authors, they placed cells under agarose in Petri dishes, then placed beads carrying combination compounds on the surface of the agarose and then partially released the compounds from the beads. The active compounds were visualized as areas of dark pigment since, as the compounds diffused locally into the gel matrix, the active compounds caused the cells to change color. Another recent example is from Daniel Chelsky's "Strategies for Combinatorial Libraries Screening: Novel and Traditional Approaches ", reported in First Annual Conference of The Society for Biomolecular Screening in Philadelphia, PA (November 7-10, 1995) Chelsky placed a simple homogeneous enzyme assay for carbonic anhydride inside a gel agarose, so that the enzyme in the gel could cause a color change through the gel.Then, the beads carrying the combination compounds through a photo-labeler were placed inside the gel and the compounds were partially released to the gel. through d "ultraviolet light. The compounds that inhibited the enzyme were observed as local zones of inhibition having less color change. Finally in Molecular Diversity, V. 2, p. 57-63 (1996), Salmon, and others, reported methods similar to that of Jayawickreme et al., Where combination collections were classified for compounds that had cytotoxic effects on cancer cells grown on agar. The three examples are variations of gel assays tested over time for antibacterial or anti-cancer agents, and are also similar to familiar immunological assays in which the antigen / antibody interaction in a gel is measured. Although these gel penetration assays were very well suited to classify pearl-based combination collections, no one has reported the extension of this format to heterogeneous assays or to collections based on non-pearls. Conventional knowledge discouraged researchers from testing samples in a continuous format that could allow samples to mix. Between the type of limited trials reported and the aspects related to the samples running together in a continuous format, only pearl-based collections have been tested. Due to these limitations, the researchers believed that the 96-cavity format was more suitable for the heterogeneous assay and the non-pearl-based collection classification. It may be desirable to conduct heterogeneous assays in a "fixation" format. In addition, "it may be desirable to test discrete compounds in a free-form fixation.

COMPENDIUM OF THE INVENTION This invention describes the classification of high production continuous format (CF-HTS), which successfully implants the concept of free format for any test, homogeneous or heterogeneous, that can be achieved in a 96-cavity format. In addition, CF-HTS is also useful for classifying combination collections with heterogeneous assays, not just homogeneous assays. In addition, CF-HTS can analyze discrete compounds without the costs and complexities associated with miniaturization. Aspects related to the potential for reagents and test results to run together during the subsequent steps proved to be unfounded. One embodiment of the invention relates to approving samples for a biological or biochemical activity: a) introducing multiple test samples into or onto a porous test matrix that optionally contains one or more test components; b) using at least one matrix to introduce one or more test components to the assay, wherein the matrix may or may not be the porous test matrix; and c "" "performing the '' s '' from: '" ~ "'" "~ i) washing any matrix used in the test to remove an excess amount of the test sample, test component or a combination of the same; ii) by contacting any matrix used in the "test with an additional reagent in a bulk solution or as a liquid." Another embodiment refers to testing sample for a biological or biochemical activity by introducing multiple test samples in or on a matrix of porous assay that optionally contains one or more test components, and using at least two additional matrices to be tested, Still further, another embodiment refers to simultaneously testing more than 96 test samples for biological or biochemical activity by introducing more than 96 test samples in or on a porous test matrix that optionally contains one or more test components and perform the assay.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the gELISA test samples on a non-porous matrix by contacting one side of the porous gel matrix containing the assay reagent, which in turn contacts a non-porous matrix carrying an additional assay reagent . * "- - .- .. - Figure 2 illustrates the removal of the sample matrix of the gELISA test and the porous gel matrix, followed by washing and addition of liquid or solution reagents to form the report complex in the non-porous reagent matrix Figure 3 illustrates the visualization of the gELISA assay by contacting the report complex matrix with a porous gel matrix containing the report substrate Figure 4 illustrates how a ligand matrix is applied on the surface of filter placed on the filter surface carrying the cells Figure 5 illustrates which filter surface that carries the cells can be produced when the test is visualized Figure 6 shows the result of a VanA test using varying concentrations of A known dose-dependent inhibitor Figure 7 shows the result of an EF-3 assay using varying concentrations of a known inhibitor Figure 8 illustrates an experiment control for the gELISA assay for protein-protein interaction. Figure 9 illustrates the result of "inhibition" of gELISA of the protein-protein interaction. Figure 10 shows the dose-dependent binding of labeled ITAM radio for immobilized LCK. Figure 11 illustrates the pixel values against the concentration of 1TAM to show typical "curve" of receptor-ligand binding. Figure 12 illustrates a control experiment for the radiolabelled IL-8 ligand-cell interaction. Figure 13 illustrates the "inhibition" of the IL-8-cell ligand interaction. Figure 14 illustrates an assay for ds inhibitors. neuraminidase.

Figure 15 illustrates a simultaneous assay of 10,080 discrete compounds for the inhibition of neuraminidase.

DETAILED DESCRIPTION OF THE INVENTION The central idea in addition to CF-HTS is to place test samples in the context of a porous matrix. The method comprises placing one or more test components within, on top of, or at the bottom of a matrix, a gel, a plastic sheet, a filter or other forms of easily manipulated solid support. When the samples are introduced into the porous matrix, they diffuse sufficiently slowly, so that the tests can be carried out without the test samples running together. In this way, the CF-HTS format separates the test samples through diffusion instead of an impermeable barrier. If the tests are allowed to run too much, the test samples and the results will finally run together. However, when carefully controlled, the CF-HTS permits a 'very' high density of com positions that are 'simultaneously' classified, even individually, without the need to fill individual cavities or reaction vessels with solvents or solvents. test components. In addition, by manipulating the matrices that carry the reaction components, even complex heterogeneous assays can be carried out in this Tormato. The manipulation of matrices for heterogeneous assays is completely unprecedented and makes CF-HTS as flexible as the 96-cavity format in its ability to classify a wide range of biological or biochemical processes. further, CF-HTS achieves the types of advantages anticipated by miniaturization without the concomitant disadvantages and has unique advantages. CF-HTS employs a wide variety of matrices and test components. The matrices include, but are not limited to, gels composed of agarose, acrylamide, or other gelatinous materials, membranes, filters and plastics. The dies may be composed of materials including, but not limited to, polystyrene, polypropylene, other plastics, paper fiber, glass, fiberglass, silica, polycarbonate, polyester, polyvinylidene chloride, and polyethylene. The matrices can be waterproof solids, porous solids such as filters or gels. Test components include, but are not limited to, macromolecules such as nucleic acids, proteins and other synthetic or natural macromolecules; cells, cell lysates, biological extracts, organelles and other complex biological entities "and" small molecules "such as pH regulators, salts, inhibitors, substrates, peptides, dyes, nucleotides, cofactors, ions and solvents A CF-HTS assay is an assay where multiple test samples or compounds are separated through diffusion rather than through impermeable barriers.The critical component is the introduction of multiple test samples (greater than 1) into or on a porous test matrix that optionally contains one or more test components.The porous test matrices containing components are prepared by adding, mixing, emptying, supplying or soaking components in the matrix.The porous matrices are also prepared by coupling, coating, joining, fixing, linking, conjugating or joining test components on or on a surface of a matrix.Also, a porous matrix is also pre to form a thin film of solution or liquid on a bed of cells, enzymes or other immobilized test component. Porous test matrices are used to control the order and / or duration of component addition, and the degree of mixing and diffusion when the test components are combined. CF-HTS can also use non-porous matrices. Non-porous matrices are prepared by coupling, coating, attaching, attaching, bonding, conjugating or joining test components or sample tests onto a surface of the non-porous matrix. The use of non-porous matrices in CF-HTS spatially fixes one or more d Tos' test components. '- - - When the test components are introduced on the surface of a matrix, the test components are bound through covalent or non-covalent, specific or non-specific interactions with matrices that are not derivatized, derivatized or otherwise pretreated to facilitate the binding of test components After the binding, the test component is spatially fixed, so that it is immobilized for the purposes of the test. The test must be capable of diffusing through a matrix to reach the test components, and / or subcomponents or products of the test components must be able to diffuse through a matrix to reach the test samples. Porous matrix containing the test samples is used in any or more of the following steps. 1) Bring the surface of the porous matrix in contact with at least one other matrix (porous or non-porous), so that the samples and / or one or more of the test components can diffuse through the adjoining surface. 2) Separating two or more matrices to interrupt the interaction of components and / or samples. 3) Bringing the surface of two or more matrices in contact so that the test components can interact. '* "' 4 ')' * Washing, rinsing or pouring a liquid with pH regulators or other solvents to remove unbound and / or non-specifically bound test components. 5) Providing, emptying, adding or soaking test components in solution with a matrix or filtering said components through a matrix. 6) Forming images, reading, scanning, detecting or otherwise visualizing the radiometric, fluorescent, spectrophotometric or electromagnetic signals present on or in one or more matrices. The CF-HTS provides many advantages over the prior art. The absence of different cavities eliminates the need to simultaneously and accurately supply test components or reagents into the cavities, rather, the test components are mixed and mixed through homogeneous bulk handling. Since the test components are prepared as a homogenous bulk solution or matrix, there is a variation from sample to minimum statistical sample. Through comparison, the presence of cavities in the 96-cavity format creates large variations from sample to sample. In addition, CF-HTS provides an extremely high density classification of large numbers of compounds. Even if the blows observed "run together" to a limited degree, you only need to retest the compounds that are located near the blow. In this way, if you can reduce 10,000 samples of préba "to 50 candidates" by the end of the visualization, you can easily reduce the 50 candidates to the active compounds with the technology of 96 old cavities By dispensing and drying discrete compounds on plastic sheets in highly packaged arrangements, then applying them to CF-HTS, all critical miniaturization emissions can be directed.This format does not require innovations in plastic or other materials to achieve miniaturization, since miniaturization is achieved simply by limiting the amount of sample that is diffused into the matrix.This format does not require microfluids to supply test reagents, since the entire trial is presented, essentially in "a giant cavity", in Where all reagents and solutions are handled in bulk, only the test samples need to be supplied through microfluids. This is a much smaller statistical variation in this format, since one only needs to look for localized zones of heterogeneity in the otherwise homogeneous matrix. You do not need to read and compare many different cavities. In addition to all the anticipated benefits of miniaturization (cost, production, use of reagent, use of test compound), CF-THS also provides amazing benefits such as the ability to handle most steps of bulk testing. A central aspect of CF-HTS is the observation that components and test samples do not diffuse rapidly in "form the foam even on the adjoining surfaces" in re matrices: 'For example, when an agarose gel is placed on a plastic plate, there is important liquid on the adjoining surface on the surface of the gel. When an interaction between a test component in the gel and a test component on the plate is required (as in the ELISA example), it is critical that the component in the gel be able to diffuse out of the gel and onto the plate . However, CF-HTS requires that the concomitant lateral diffusion be considerably slower, so the interaction on the plate is located near the original site of the gel-based component. These same principles apply to any contiguous matrix-matrix surface between gels, filters or surfaces (or any other matrix) in any combination. The realization that this diffusion behavior is controllable and is generally applicable to all adjoining matrix surfaces is unprecedented and contrary to conventional desires. A preferred method for introducing samples or test compounds into a matrix (such as a moistened gel or filter) is to fill and dry small volumes of each sample on a surface, so that the surface of a plastic sheet in a disposition so that two shows can be mixed or overlapped, and each one is in a specific place. When the plastic sheet is placed on a matrix, the samples dissolve and diffuse towards the matrix in locations corresponding to their predefined locations in the initial layout. "" "A 'm all' alternative * to, provide" samples "in an" arrangement "is to deliver the samples on a porous matrix such as a filter, where the volume of each sample supplied is low enough so that the samples do not overlap within the matrix.After contact with another porous matrix containing more liquid, the compounds diffuse to start the assay.A preferred method for introducing pearl-based combination compounds into a matrix is to supply the beads randomly or in an orderly arrangement on a surface, such as a surface of a plastic sheet The beads can then be treated to release (separate) the test compounds if they are covalently bound to the beads through a labile linker (photoseparation) and gas phase acid separation which are well known in the art.) Each compound is then non-covalently associated with the area occupied by its origin bead, and the dry compounds can then be introduced into or onto a matrix. An alternative method for introducing discrete compounds into a matrix is to soak or otherwise covalently bind each compound in or on the beads. Using this method, a large number of compounds can be mixed together once on the beads so that each bead still has a unique compound therein. Then, the bead mixes can be easily spread on a surface to be introduced into a matrix. This procedure completely eliminates the need for a more liquid "dé p" équúéñó "vorum" eñ. "'' * 'When the initial arrangement of samples entered into a matrix in a CF-HTS classification is high density, so that a particular area of activity spatially covers the initial location of more than one sample, then each These samples are potentially the source of the observed activity.For higher initial densities, there will be more candidate compounds for each zone since multiple compounds will be present in a particular area.The compounds can diffuse together, but each will continue to have its own spatial gradient. and it will not be quantitatively mixed anywhere, therefore, the center of the zone will remain correlated to the precise location of the active compound in the initial layout.In practice, impacts are rare enough that retesting multiple samples ensures The identification of active compounds for each active zone is trivial. The alternative of the invention is to introduce physical barriers (thus making the non-continuous format in the rigorous use of the word) in the matrices of an assay to limit the distance that the samples can be disseminated. For example, two gels containing an enzyme and substrate, respectively, each can be cut with a mesh ("kitchen cutter") so that each gel is subdivided into many discrete locations.

Afterwards, the two gels can be kept in contact, so that the substrate and the enzyme can diffuse The test samples can then be introduced into each piece of gel subdivided, so that the tests are completely independent without any diffusion between the samples. essays. This modality effectively eliminates some of the advantages of CF-HTS by introducing statistical deviations between the test samples and setting the volume and thus limiting the signal for high density arrays. But, this modality could cut the benefit of heterogeneous analysis based on matrix since the test components do not need to be supplied in a large number of parallel reaction vessels, and eliminates the partial mixing of the samples. gELISA Tests with enzyme-linked immunosorbent (ELISA) are heterogeneous assays, which detect the binding between ligands in solution and immobilized receptors. ELISA requires Many reagent mixing and washing steps are difficult to perform in a 96-cavity format, and a greater difficulty can be ascertained when the cavities are reduced from the 96-cavity format to the 384-cavity format. The inventors have applied the CF-HTS method to detect binding inhibition between ligand and immobilized receptor targets (gELISA). A receptor is any molecule that can bind to another molecule. Non-limiting examples are proteins, peptides, nucleic acids, carbohydrates and complexes for the previous examples.

"" "" '' A receitor is immobilized on the basis of vain matrices' receptor matrix) including, but not limited to, plastic surfaces (e.g., petri dishes or plastic (Nunc) plates), membranes or filters having a high target binding capacity (eg, nitrocellulose, nylon , or PVDF (Millipore, Corning Costar, Schleicher &Schuell, BioRad) or membranes derivatized such as SAM membranes (Promega)). A porous ligand matrix (e.g., agarose gel or porous membrane) is prepared so that the ligand for the immobilized receptor is stocked on or in the matrix. The compounds or test samples are supplied directly on the ligand matrix, or alternatively, on or in a test sample matrix (for example, polystyrene (Tekra), polyvinylidene (for example from Dow Brands) or other flexible plastic sheet or membrane.The test matrix is contacted with the ligand matrix and the samples are allowed to diffuse into the ligand matrix.After a suitable incubation period, the ligand matrix is contacted with the receptor matrix, allowing the ligand and the samples to contact and potentially react with the receptor via diffusion (Figure 1 shows the binding R of immobilized receptor to the biotinylated ligand Lß.) During incubation, the ligands will bind to the immobilized receptor Unless a sample compound inhibits ligand / receptor binding, after a suitable incubation period, the receptor matrix is removed and washed with a regulator. of pH suitable for Ve move 'ligand' and 'samples' laughed' un í'd'á's "and" joined "ho" specifically. "The receptor matrix is then rinsed in a solution containing test reagents that will interact with the ligand (eg, an antibody, avidin or streptavidin in the case of a biotinylated ligand) and has the ability to be detected either directly (eg, through fluorescence or radioactivity) or indirectly (eg, horseradish peroxidase (HRP) ), alkaline phosphatase (AP), or beta-galactosidase conjugate). (Figure 2 shows a conjugate of avydine-HRP, AHRP bound to biotinylated ligands). After a suitable incubation, the receptor matrix is removed from the solution and washed to remove the unbound or non-specifically bound reagent. In the case of direct signal detection, the matrix is image-formed using the appropriate method (e.g., spectrophotometric scanners, CDD cameras, film, phosphor imagers, or scintillation detection devices). Indirect signals (eg HRP or AP) require an additional signal development reaction, achieved by supplying substrates or other necessary reaction components in or on a porous substrate matrix and placing this matrix in contact with the receptor matrix. The enzyme (eg, HRP or AP) then reacts with the substrate (Figure 3). Alternatively, a precipitation substrate is introduced in solution instead of in a matrix.

Under any visualization method, the ligand / receptor binding areas will produce a visible reaction, while "" "" the 'areas' where 'union' gives "? igarid? Vrécépfor was inhibited nd will produce a visible reaction.

Cell / Ligand Binding CF-HTS can also be used to detect inhibition of ligand / cell receptor binding In the traditional assay, a test compound, radiolabelled ligands and cells expressing the corresponding receptor, such as an antibody, are combined. cavity. Then, sufficient time is provided to allow the receptor to bind to the ligand, if said binding has not been inhibited by the test compound. Any of the unbound and non-specifically bound components are removed from the cells, and the amount of radioactivity associated with the cells is measured. The inventors have adopted the CF-HTS method to detect the inhibition of ligand binding to cells. Cells expressing the desired receptor are developed or plated on a matrix (cell matrix) such as, but not limited to, gels, filters, or membranes (e.g., Transwell tissue culture membranes (Corning Costar) or chemotaxis membranes (Neuto Probé)). A porous matrix (e.g., agarose gel or porous membrane) is prepared, so that the ligand labeled for the receptor is stocked on the matrix (ligand matrix). The test compounds or samples are supplied directly on the ligand or its cell matrix, or alternatively on or in another matrix (for example, polystyrene Tekra), phosphorus or sulfuric acid. plastic flexibl b "merfibran'ár sample matrix." The sample matrix is brought into contact with the ligand matrix allowing the sample to diffuse into the ligand matrix, after a suitable incubation period, the matrix The ligand is contacted with the cell matrix, preferably on the non-cell side of the matrix, and allows the ligand and sample to be contacted and reacted with the receptor via diffusion (Figure 4). Incubation, labeled ligands will bind to immobilized cells unless a sample inhibits ligand / cell binding After incubation, the cell matrix is separated from the ligand matrix and washed with a pH regulator suitable for rowing see the ligands and samples not bound and not specifically bound. The cell matrix is imaged using the appropriate method (e.g., spectrophotometric scanners, CCD cameras, films, phosphor imagers, or scintillation detection devices) (Figure 5 illustrates the development of the assay on a film) . As shown above, CF-HTS achieves all the desired benefits for "free format" assays, and can be applied to all different types of biological or biochemical assays, under all different types of formats, and with all different reagents and equipment. Due to its broad applicability, this is best illustrated by the following examples. However, these examples illustrate the preferred embodiment of the present invention and "do not" "limit the returns" or "the specification." The technical examples will readily appreciate that changes and modifications to the specified embodiments can be made. do without departing from the scope and spirit of the invention Finally, all citations herein are incorporated by reference.

EXAMPLES Example 1 Two Step Colorimetric Gel Test to Detect Phosphate Generated by Vancomycin Resistant Enzyme VanA VanA is a key enzyme in vancomycin resistance, and catalyzes the binding of D-Alanine to D-Alanine or D-Alanine to D-lactate. Traditionally this enzyme was analyzed generating color from phosphate that was released when the enzyme was active (VanA activity hydrolyzes ATP to ADP and phosphate). Scientists know that D-cycloserine inhibits VanA in a dose-dependent manner, and the use of this inhibitor as a positive control against other potential inhibitors.

} Enzyme Gel An enzyme gene was prepared by adding the VanA enzyme to melt 1% agarose (high melting agarose, Gibco BRL) at 45 ° C in 50 mm HEPES ((N- [2-hydroxyethyl) piperazine-N '- [2- "efaphsulfonic acid]');" 2d "" ~ mrVf "MgC? 2, '" '20' m'lví '"KClT "p'H" 7.3"a" final VanA concentration of 2 μM This agar mixture was then emptied into a BioRad gel casting apparatus and allowed to solidify at room temperature. 2-8 ° C for 30 minutes Substrate Gel A substrate gel was prepared by adding ATP, D-alanine and D-lactate to molten agarose to bind each component at 1 mM, 1.5 M and 1.7 mM, respectively, and prepare the gel as described for the enzyme gel .

Sample Matrix A series of 1 μl aliquots of serial dilutions of 5000, 2000, 1000, 500, 200, 100 μM D-cycloserine, a known inhibitor used as a control sample, in 1: 1 ethanol-water It was filled on a piece of polyvinylidene chloride (PVDC) film and allowed to dry for 10 minutes.

Incubation of the Enzyme with the Substrate in Presence of Inhibitor The enzyme gel was contacted with the sample matrix for 5 minutes. Then the substrate gel was placed on top of the enzyme gene and allowed to incubate for 15-20 minutes. Subsequently, the two gels separated. During incubation, one expects the phosphate to be produced through the gel as the enzyme catalyses the binding of the substrates, with the exception of the areas where D-cycloserine was concentrated enough to inhibit the reaction. .

Test Visualization A phosphate detection cocktail consisting of freshly prepared 0.15% malachite green and 1.4% ammonium molybdate in 1.33N HCl was emptied and evenly distributed over the enzyme and substrate gels. These reagents react with phosphate and generate enormously dark shades of green color as a function of increasing phosphate concentrations. The color was allowed to develop for 5-10 minutes (Figure 6 illustrates the color development on the gene where the inhibitor represents varying amounts from 5 nanomoles to 0.5 nanomoles). The green gels were photographed using a Stratagene Eagle Eye CCD camera. As expected, the degree to which the zones of inhibition looked less green correlated with the aggregate inhibitor concentration. This assay, therefore, can be used to classify VanA inhibitors by arranging beads or mixed combination compounds on any other surface which is then contacted with the gel assay. This test also demonstrates that the gel sorting format can be handled for multi-step reactions. This aspect is necessary for this format to be useful with a wide scale of trials, since many trials may require multiple steps. In. this ca.so, he "essay of" Van A is a two step essay of enzymatic activity followed by the development of color. The homogeneous versions (single pass) of this assay are not easily reliably due to the color development reagents and conditions that interfere with VanA activity and are also incompatible with the delivery on an agar gel. Therefore, the spatial and temporal separation of these two steps having first an enzyme gel assay followed by a solution phase color development step is desirable.

Example 2 Two step gel test to detect phosphate generated by the ATPase activity of Strep Factor 3 Elongation stimulated by Ribosomes binding When the fungal elongation factor 3 (EF-3) interacts with ribosomes, the phosphatase activity is stimulated. The inventors have applied CF-HTS to analyze this activity. An enzyme gel containing EF-3 (a highly temperature sensitive enzyme) and starch ribosomes in an EF-3 assay pH regulator was prepared. A substrate gel containing 1 mM ATP was also prepared in the assay pH regulator. Both gels contained 2% dimethyl sulfoxide in low melting agarose (Gibco BRL), and were prepared at 37 ° C and allowed to settle for 30 minutes at 4 ° C. Serial dilutions of Pilu-L-lysine, an EF-3 inhibitor used as a 'sample' control, were placed on top of the PVDC film and dried (sample matrix). conducted as in Example 1 by preincubating the enzyme gel with the inhibitors on the sample matrix, then the enzyme gel was placed in contact with the substrate gel for 20 minutes, as in Example 1, the enzyme and The substrate was stained with a green malachite / ammonium molybdate development cocktail, then the enzyme gel was imaged with a CCD camera, and the inhibitor spots appeared as lighter areas on the green background. illustrates the development of color on the gel where the inhibitor represents varying amounts of 5 picomoles at 200 picomoles.The results show the dose dependent signal of the inhibitor, indicating that the compounds can be classified in this test to discover new s EF-3 inhibitors. Example 2 demonstrates the utility of CF-HTS at one with the complexity introduced by the presence of organelles or other crude biological mixtures or extracts.

EXAMPLE 3 Indirect Color Detection Using gELISA of Pro tein-Pro tein Interaction Inhibitors As discussed above, ELISA is commonly commonly used to detect the inhibition of ligand-receptor interactions when the receptors are immobilized in "d" -mecrotifution cavities. The 'Tig-rece'pi? R' pairs used in ELISA can contain any pair of binding molecules from proteins or other macromolecules to small molecules. These assays are complex, multistep assays that require immobilization of the receptor, incubation of the receptor with ligand, washing to remove the non-specifically retained unwanted ligand that might otherwise cause a high signal background, binding display reagents (for example, a ligand-specific antibody conjugated to a reporter enzyme) to the receptor-bound ligand and generate a visible signal by providing substrates for the reporter enzyme. It is evident that the complexity of ELISA has led the HTS industry to conclude that ELISA can not be adapted in a free-form assay. However, the inventors have adapted this complex multi-step assay to the CF-HTS format to analyze a variety of protein-protein, ligand-protein interactions and other ligand binding interactions. The urokinase-type plasminogen activator (uPA) binds to its corresponding receptor (uPAR). The uPA / uPAR interaction has been implicated in the metastasis of several types of cancers. The inventors have adapted a traditional uPA / uPAR ELISA to CF-HTS using purified receptors and ligands.

UPAR tint Plastic plates were coated (7.5 cm x 11.5 cm, from Nunc, Inc: Napervrifé, IL) overnight With '15 ri l of '118 nWI cfe'uFÁ'R "purified at regulated outlet at its pH with phosphate (PBS) (Life Technologies, Grand Island, NY) at a pH of 7.4 and at 4 ° C. After coating the plastic layer overnight, the uPAR solution was decanted, and the remaining binding sites on the plastic plate were blocked by adding 15 ml of PBS containing 1% casein (w / v) and incubating for 2 hours. hours at room temperature (RT).

After blocking, the blocking solution was decanted and the plastic plate was washed 5 times with 20 ml of washing pH buffer consisting of 0.05% Tween-20 (polyoxyethylene sorbitan monolaurate) in PBS. After washing, the plastic plate was dried for 10 minutes at room temperature. This was controlled so that the plastic plate could then be used in the test described further ahead to avoid over drying of the matrix which can lead to loss of activity. ß-uPA matrix For the purposes of the trial, the uPA used was labeled with biotin (ß-uPA). Gels containing ß-uPA were prepared by soaking ß-uPA on agar and avoiding high temperatures (as opposed to casting on molten agar in Examples 1 and 2). The agar was prepared by first mixing 0.1 g of agarose (Sigma, St. Louis, MO) with 10 ml of PBS, heating until melted, and then casting in an 8 x 7 x 0.075 cm3 gel apparatus (Bio-Rad, Hercules , CA). After solidification (either at room temperature or at 4 ° C), the gels were soaked in "tea" at night "" 4"° C" 'rf "15' * ml of B-úP" A (about 10 nM) in assay pH buffer consisting of PBS, 0.05% Tween-20 and 0.1% casein (both from Sigma, St. Louis, MO) .The gel was dried for 20 minutes at room temperature just before of use.

Sample Matrix In the absence of a known small molecule inhibitor for uPA / uPAR binding, non-biotinylated uPA (Pro-uPA) was used as a control sample inhibitor of ß-uPA / uPAR binding. Aliquots of 5 microliters of 0, 0.003, 0.01, 0.3, 1 and 3 μM pro-uPA in pH-regulating assay were supplied on a PVDC film (Dowbrands LP, Indianapolis, IN) and dried for 2 hours at room temperature .

Incubation of uPAR with Pro-uPA and ß-uPA The sample matrix was placed on one side of the ß-uPA gel with spots of dry pro-uPA in contact with the gel surface of ß-uPA. The pro-uPA was allowed to diffuse in the gel for 10 minutes at room temperature. Subsequently, the other side of the ß-uPA gel was placed on the plastic plate to allow ß-uPA (acting as a ligand) and pro-uPA (acting as a competitive inhibitor) to interact with the uPAR on the surface of the plastic plate. The binding / competition reaction was incubated for "20" 'minutes "" to "t mperatur ambienté." After the incubation, the plastic plate was separated from the gel, and rapidly washed 4 times with 20 ml of regulator. Wash pH. A solution of avidin-horseradish peroxidase conjugate (avidin-HRP) was prepared by diluting avidin-HRP (Sigma, St. Louis, MO) from 1 to 25,000 in assay pH buffer and adding 15 ml to the plastic plate. The reaction was incubated for 10 minutes at room temperature followed by decanting the avidin-HRP solution and washing the plastic plate as before. The plastic plate was allowed to dry for 20 minutes. The avidin in avidin-HRP binds specifically to biotin, so that only the areas of the matrix that exhibit the "ligand / receptor" junction finally exhibited color development. Areas of the matrix where the binding of "ligand / receptor" is competitively inhibited by pro-uPA will not exhibit color.

Color Development ___ __ _ The color development gel containing a colorimetric HRP substrate (OPD gel) was prepared by dissolving two tablets of o-phenylenediamine hydrochloride (OPD) in 7 ml of diluent (both from Abbott equipment No. 6172-30, Abbott Labs, Abbott Park, IL) and combining this solution with an agarose solution made by melting 0.1 g of agarose in 3 ml of water. The final 10 ml mixture was placed in an 8 x 7 x 0.075 mini-protein II gel apparatus and allowed to solidify at 4 ° C for 15 minutes. The gel was transferred from the glass plates of the "géT" cylinder to "rested" either to "PVDC '5 to" a flexible plastic sheet and allowed to dry for 10 minutes at room temperature. The gel was then transferred to another PVDC or plastic sheet and the other side allowed to dry for 10 minutes. Then the gel or PVDC was placed on the plastic plate to begin the color development. At various times during the OPD incubation, the plastic plate was placed on top of a 440 nm bandpass filter (Omega Optical, Inc., Brattleboro, VT) which in turn was placed on the of a fiber optic diffusion plate illuminated by a Fiber-Líte light source (both from Dolan-Jenner Industries, Inc., Lawrence, MA). The resulting images were acquired with a CCD camera (Eagle Eye system, Stratagene, La Jolla, CA).

Control Experiment Figure 8 illustrates a control experiment for Example 3. For Figure 8A, β-uPA agarose squares were soaked in solutions of various concentrations (1 cm2) as indicated below each table (except for the solution 50 nM, which was marked to the left of the agarose box). After the agarose squares were incubated in an immobilized uPAR plastic plate, they were removed. Then the plastic plate was washed and the areas of the matrix were of binding occurrence of ß-uPA / uPAR was visualized as described above. Figure 8B shows a graph of the average pixel value (less background) "for" each frame (as "deferred" using the digital image of the CCD camera with NIH image analysis software) against the concentration of ß-uPA in each agarose frame at various times during OPD color development The ß-uPA supplied from the agarose gel showed a typical receptor-ligand binding curve with a maximum average binding (Kd) around 3-5 nM which is consistent with values reported for this reaction in the standard ELISA assay and other assays that measure this parameter Figure 8 demonstrates that the indirect colorimetric signal generated by gELISA is quantitatively dependent on the degree of ligand-receptor interaction.

Results of the Pro-uPA / ß-uPA Competition Figure 9 demonstrates the inhibition of ß-uPA / uPAR binding through pro-uPA. The spots in Figure 9A indicate areas where ß-uPA / uPAR binding was inhibited. Therefore, avidin-HRP did not bind in these areas. Consequently, the HRP did not react with the substrates in the OPD gel to generate the color development. Figure 9 is an alternative way to display these same CCD image data, which improves the ability of the human eye to see the quantitative titration.

EXAMPLE 4 Direct Radiometric Detection of Interaction Inhibitors Protein-Protein T cell activation is a component of the body's immune response. For downstream events that occur during T cell activation, p56lck (LCK, a protein) must interact with the ITAM region (immunoglobulin-related tyrosine-based activation motif) of the cytoplasmic domains of the T cell antigen receptor. Compounds that inhibit this protein-protein interaction are potentially immunosuppressive. The inventors have used CF-HTS to analyze this protein-protein interaction, where LCK is immobilized to a membrane.

LCK Matrix Biotinylated LCK is immobilized on a biotin capture membrane (SAM membrane) (Promega Corp., Madison, Wl) by flooding an 11 cm x 12 cm strip with 5 ml of 3 μMLCK in PBS containing 5 mM DTT ( dithiothreitol) for 10 minutes at room temperature, after which, the pH regulator was removed. This was controlled so that the SAM membrane could be used right after (in minutes) in the assay described below.

ITAM matrix *. An agarose gel containing the radiolabelled ITAM peptide (ITAM *) was prepared by mixing 0.1 g of agarose with 10 ml of pH regulator, heating until complete. and then casting in a gel apparatus of 8 x 7 x 0.075 cm. The ITAM * was added either just before casting or alternatively soaked in the gel after solidification to a final concentration of 10 mM.

Sample Matrix _ ^ _ ___. . The test samples that will be sorted are dispensed onto a plastic or PVDC surface and dried to form the sample matrix.

Incubation of ITAM *, LCK and the Test and Display Samples The sample matrix was contacted with one side of the ITAM * gel, so that the test samples could diffuse into the ITAM * gel. Subsequently, the other side of the ITAM * gel was contacted with the SAM membrane on which LCK had been immobilized. After incubation for 15-45 minutes, the SAM membrane was removed, washed and imaged with a phosphor or film imager. Inhibitors of the ITAM-LCK interaction are indicated by the lower radioactivity zones that correspond to the lower signal intensity on the image.

Control Experiment for the Union of ITAM * / LCK Agarose gels were prepared by mixing 0.1 g of agarose "cbh" ~ 10 'ñ? l regulator of "p'H'," "" calé 'tá "" d' "till" and then "casting" in a gel apparatus of 8 x 7 x 0.075 cm . After solidification, circles with a diameter of 1 cm were drilled in the gel and soaked in 400 μl of 0.1, 0.3, 1, 3, 10, and 20 nM of 125 I labeled ITAM (Amersham, Ariington Heights, IL) in pH regulator overnight at 4 ° C. Gel circles were removed from the solution and allowed to dry for 20 minutes at room temperature. Then, they were placed on the SAM membrane immobilized with LCK and incubated for 45 minutes at room temperature. The gels were removed and the membranes were washed 4 times with pH regulator. After drying, the SAM membranes were imaged using a phosphor imaging. Figure 10 indicates a dose-dependent binding between 125 | -ITAM and LCK immobilized on the SAM membrane. Figure 11 shows a graph of the average value of the pixel (less background) for 125 I-ITAM gel (as determined by analyzing the digitalized image of the phosphor imaging with ImageQuant Molecular Dynamics software) against the concentration of 125 I-ITAM in the gel circle. The 125I-ITAM supplied from the gel to immobilized LCK showed a typical receptor-ligand binding curve.

EXAMPLE 5 Whole Cell Report Gene Assay a Gel Format / Combination Filter Kidney cells, known as HEK cells, were transfected with a plasmid containing a cyclic AMP response element (CREB) promoter fused to a luciferase gene (luciferase report gene from Promega). When the transfected cells were treated with forskolin, expression of the luciferase reporter gene was induced. Then, when a biological pH regulator containing the luciferase substrate (Promega beetle luciferin) and appropriate cofactors (20 mM Tricine pH 7.8, 0.1 mM EDTA, 33 mM DTT, 0.3 mM Coenzyme A, 0.5 mM Adenosine Trifosphate and 1 mM MgCl2) was added to the HEK cells, a photon emission was generated that could be detected through conventional instrumentation. The inventors have aed this assay to CF-HTS.

Cell Matrix Cells in culture were treated with trypsin, transferred to a Corning / Costar TRANSWELL ™ membrane (3 micron polycarbonate filter with a plastic support ring) and incubated overnight at 37 ° C, 5% of C02 in the presence of tissue culture medium. Then, the medium was removed from the membrane and the filter to which the cells were attached was dried with air for 15 minutes and used immediately afterwards in the following steps.

Induction Matrix A gel containing the luciferase expression inducer was prepared by adding 12 μl of a 10 mM su material of forscholine (Sigma ethanol su material) in 6 ml of a 1% agarose gel with a low melting temperature The gel was solidified at room temperature with forscohna at a final concentration of 20 μM.

Sample Matrix Samples that can block induction of forskolin were dispensed in discrete locations over high density PVDC.

Incubation of Reagents and Detection of Inhibition The inhibitory side of PVDC was incubated with the inducer gel. Then, the gel containing the forscholine inducer was placed on the non-cell side of the cell matrix prepared above. These were incubated together at 37 ° C at 5% CO2 for 20 minutes. Then, the forskolin gel was removed and the cell matrix was incubated at 37 ° C at 5% C02 for a further 4 hours for maximum expression of the luciferase construct. To detect the enzymatic activity of luciferase (or its inhibition), the cell matrix filter was physically removed from its plastic support ring and placed in a petri dish. The petri dish was Tn Unció 'with substrate' of luciferása (Promega beetle luciferina) in a biological pH regulator with appropriate co-factors to generate light as a signal. Since the signal is localized within the immobilized cells expressing luciferase, inhibitors of the initial induction step resulted in lower emission photo areas EXAMPLE 6 CF-HTS Gel / Filter to Detect Directly Inhibitors of Ligand-Cell Interactions Ligand / receptor binding on cell surfaces initiates signal pathways in cells that ultimately lead to functional responses (eg, cell proliferation or secretion of biologically active substances). To regulate the biological response of cells in disease states, it is generally sought to inhibit ligand binding to cell surfaces. A common method for evaluating inhibitors of ligand / cell receptor binding is to assess the ability of the inhibitor to reduce the binding between the radiolabelled natural ligand and the cell. This involves incubation of the cells with radioligand and inhibitor, followed by removal of the unbound and non-specifically bound radioligand by washing, and then measuring the amount of bound radioactivity. Interleukin-8 (IL-8) is a chemotactic chemokine "involved in the infl uction through the urinary receptors of several cell types." The inventors have developed a ligand-receptor cell assay of CF-HTS to evaluate the inhibitors of this interaction.

Cell Matrix = HEK cells (ATCC, Bethesda, MD) were plated by expressing the IL-8a receptor on a cavity membrane filter with a diameter of 75 mm (Corning Costar Corp, Cambridge, MA) at a density of approximately 20 million cells per plate. The membrane filter was allowed to bind overnight in pH buffer (RPMI from Life Technologies, Grand Island, NY) containing 10 mM Hepes (Sigma, St. Louis, MO) at a pH of 7.2 and at 37 ° C. After the cells were attached to the filter, the medium was removed, and the cells were washed with fresh pH buffer to remove any unbound cells. The filters were inverted down the cell and placed at an angle to allow drainage of the excess medium, and then dried for 20 minutes. This was controlled so that the cell matrix could be immediately used in the assay described below.

Ligand Matrix The ligand matrix was prepared by soaking IL-8 labeled with 1251 (Amersham, Inc., Ariington Heights, IL) on an agarose gel made by mixing 0.1 g of agarose with 10 ml of pH buffer, heating to melt and then cast in a gel apparatus of 8 x 7 x 0.075 cm. The gels were soaked overnight at room temperature, while they were slowly mixed on a rotating platform (New Brunswick Scientific Co., Inc., Edison, NJ). After soaking, the gels were dried for 20 minutes at room temperature just before use. This was controlled so that the gel could be immediately used in the assay.

Sample Matrix In the absence of a known inhibitor of IL-8 receptor binding / cell, non-radiolabeled IL-8 (Genzyme Corp., Cambridge, MA) was used as a control sample inhibitor to observe inhibition of the binding of 125I-IL-8 to HEK cells. One microliter of 0.03, 0.1, 0.3, 1, 3, 10, and 100 μM of IL-8 was filled on a plastic sheet such as PVDC and dried for 1 hour under vacuum at room temperature.

Incubation The sample matrix was placed on one side of the ligand matrix so that the dried IL-8 spots were contacted with the gel surface. The gel was inverted to allow the other side to dry for 10 minutes at room temperature and the "inhibitor" was allowed to diffuse into the gel. Subsequently, the gels were placed on the non-cell side of the cell matrix. The binding reaction was allowed to incubate for 45 minutes at room temperature. Afterwards, the gels were removed and the membrane side of the membrane was washed 4 times the pH regulator.The samples were allowed to dry completely before they were removed from their plastic support ring. The membranes were then imaged with either an X-ray film or a phosphor imaging (Molecular Dynamics, Sunnyvale, CA) Figure 12 shows a dose response for 125 I-IL-8 on agarose gels diffusing through a gel matrix to bind HEK cells expressing the IL-8a receptor Several agarose squares of 1 cm2 were soaked in the solutions of various concentrations of 25l-IL-8 as indicated. ligand were contacted with the cell matrix as described above.After incubation, the frames were removed from the cell matrix and the non-cell side of the membrane was washed with pH regulator. of phosphor images to locate 125l-IL-8 bound to cell. The data indicate that a direct radiometric reading in this gel-based cell assay is quantitatively dependent on the degree of ligand-receptor interaction. Figure 12 also indicates that the binding areas remain distinct and confirms that lateral diffusion of the signal is not a problem in the assay. Figure 13 illustrates the results of the competitive inhibition experiment. Inhibition of the binding between 125I-IL-8 and HEK cells is indicated by the light spots. It is also readily apparent what inhibition by labeled IL-8 is quantitatively dose dependent. These data indicate that the inhibitors, illustrated by unlabeled IL-8 in this case, can cross the cell matrix to reduce the binding between 1 5l-IL-8 and the cells.

EXAMPLE 7 Functional Cell Test Less Gel, Filter-based Changes in cell function are usually measured by observing the effect of test compounds or samples on report systems engineered into cells. Examples include looking at the effect on the synthesis of fluorescent intracellular proteins such as green fluorescent protein (GFP), extracellular proteins such as a receptor or adhesion molecule, or specific enzymes such as luciferase, chloramphenicol acetyltransferase, or β -galactosidase (Promega). First, the test compounds are incubated with the cells. Afterwards, the cells are allowed to express the report protein for a suitable period (may be minutes, hours or days) then, the level of the report protein is analyzed through direct methods (for example, GFP) or through indirect methods (for example, ELISA techniques with membrane-bound proteins). In addition, in the case of enzymes, the cell can be separated to extract the protein, then report and analyze the enzyme activity. Other functional cell assays measure the behavior or localization of specific molecules such as colorants or radiolabeled metabolites in response to stimulation of a receptor or changes in cell physiology such as membrane potential. An ELISA assay that measures the effect of the compounds on the expression of the intercellular adhesion molecule-1 (ICAM-1) can be formatted for CF-HTS as follows. Endotenial cells expressing ICAM-1 are plated on a polycarbonate chemotaxis membrane (Neuro Probé) at approximately 5,000 cells / mm 2. The cells are incubated overnight at 37 ° C in the medium. The medium is removed and the membranes are allowed to partially dry for 10 minutes at room temperature. The samples or compounds being tested for the induction of ICAM-1 are dried on plastic sheets and placed in contact with the non-cell side of the wet membrane. The compounds are allowed to interact with the cells for 1 hour at 37 ° C in a humidity chamber, and then the cells are bathed in the medium and incubated for 5 hours at 37 ° C. After allowing the cells to synthesize the induced protein, the medium is removed from the membrane and the cells are incubated in pH buffer containing the ICAM-1 antibody (Genzume, R &D).

Systems) either unconjugated or conjugated to isothiocyanate 'fluorescein (FITC)' or 'biotin.' After declines' incubation of 3? minutes at room temperature, the pH regulator was removed and the cells were washed several times to remove the unbound ICAM-1. In the case of the FITC conjugated antibody, the membrane is imaged using a CCD camera (Stratagene, Imaging Research) with an excitation of 484 nm and an emission of 520 nm. The compounds that stimulated the expression of ICAM-1 resulted in a high fluorescence zone due to the binding of the FITC-anti-ICAM-1 antibody. In the case of antibody conjugated with biotin, the cells were incubated in a pH regulator containing avidin-HRP for 10 minutes at room temperature. The regulator was removed and the bound avidin-HRP was washed. The membranes were then bathed in the pH buffer containing a precipitation HRP substrate such as diaminobecidine tetrachloride (Pierce) and observed for color development in areas where the underlying cells were induced to express ICAM-1. In the case of unconjugated ICAM-1 antibody, a secondary anti-anti-ICAM-1 antibody conjugated to the cells was reacted followed by development of the signal with the appropriate substrate with the conjugate. The images were captured through a CCD camera. All these variations (FITC, avidin, HRP, and anti-anti-ICAM-1) are alternative reports that should be given the same qualitative results, mainly, samples that affect the expression of ICAM-1 that can be correlated to areas of increased or reduced signal. "Observe that the 'continuous format' matrices in this case are a membrane and a plastic sheet, a gel for CF-HTS is not necessary.

EXAMPLE 8 CF-HTS Assay of Discrete Compounds for Inhibition of Neuraminidase Sample Matrix _ _ A collection of 528 structurally related, discrete compounds was tested through CF-HTS. A Packard Multiparous MP204 DT was used to dilute the compounds from jars in 96-well plates and to dilute and transfer the compounds onto plastic sheets. Compounds were initially diluted from 40 mM in DMSO in bottles at 4 mM in DMSO in 96-well plates. Then, they were diluted from 4 mM in DMSO to 200 μM in 50% EtOH / H 2 O in 96-well plates. These samples were transferred in duplicates of 1 μl onto plastic sheets of 8 cm by 8 cm with a separation averaging 5 mm between the samples (Bio-Rad cat # 165-2956) for a total of 192 samples per slide. Each 1 μl point, therefore, contained approximately 200 pmoles of a particular compound from the collection. As a "control," dilution of 2,3-dihydro-2-deoxy-N-acetyl neuraminic acid (DANA), a known neuraminidase inhibitor (Boehringer Mannheim # 528544) was then manually supplied to the compounds on each sheet. The sheets were dried in a vacuum oven so that each compound could be dried in its own place on the plastic.

Enzyme enzyme Before the assay, influenza neuraminidase enzyme was diluted 1,500 times from 25% glycerol, saline regulated at pH with phosphate on liquefied agar gel consisting of 1% agarose, 50 mM sodium citrate, pH 6.0, 10 mM calcium chloride at 40 ° C. The enzyme gel was emptied, 8 cm x 8 cm x 0.75 mm and solidified by reducing the temperature to 4 ° C.

Substrate Matrix A synthetic influenza neuraminidase substrate, 2'- (4-methylumbelliferyl) -alpha-DN-acetylneuraminic acid (Sigma cat.SM-d? ST ^) was diluted from 3 mM in 30 μM DMSO. in liquefied agar and was emptied in a similar manner to the enzyme gel described above.

Incubation and Detection The enzyme matrix was placed on the sample matrix on the side where the compounds of interest were dried. Then, the substrate matrix was stacked on top of the enzyme matrix *! The matrices were incubated at room temperature for 30 minutes. During this time, the extinguished fluorescent substrate and enzyme were diffused together through the two gels and the substrate was divided by the enzyme to produce an increase in fluorescence intensity. This was verified through excitation at 340 nm and emission at 450 nm. Compounds that were able to inhibit enzyme activity minimized the increase in fluorescence intensity. Since the gels increased the intensity of fluorescence in most locations, the areas containing enzyme inhibitors that were diffused into the gel from the plastic sheet were visible as darker areas having lower fluorescence. This was easily verified through a CCD camera with appropriate filters to control the emission and excitation wavelengths. The identification of the compounds showing zones of inhibition was determined through the location of the zone with respect to the inhibitory matrix. By comparing the fluorescence of the DANA control with the known amounts of each tested inhibitor, a quantitative IC50 estimate was made for each compound. The 520 compounds in a collection were tested in duplicate. The volume of total enzyme gel used was 33 ml, or 31.25 μl per test. In addition, all compounds were tested simultaneously, and the test conditions were constant as compared to the traditional 96-well test. In addition, as illustrated in Figure 14, the test was "TcT sensitive enough to detect inhibitors as weak as 100 μM." The IC 50 values estimated for the active compounds were in agreement with those observed for the same compounds tested by the assay. of 96 more expensive cavities, which required 200 μl per test. See table of quantitative gel test results. This example demonstrates that the test of higher density arrangements of compounds reduces cost and time. For example, even a smaller reduction in separation at 2.5 mm instead of 5 mm results in a 4-fold increase in the number of compounds tested per unit volume. In this experiment, this could lead to volume per compound tested below 10 μl. Moreover, the reagents are handled in a bulk form, without the need for low volume liquid handling equipment commonly used in miniaturized classification.

Results of Cuantitavivo Gel Analysis Sample Kii (μM) Max. Estimated IC50s # Approximate (μM) of 96-well gel assay 34 100 > 100 35 80 > 100 36 80 42 37 100 > 100 38 100 > 100 39 '100 > 100 '40 40 32 41 100 > 100 42 40 50 43 100 > 100 44 > 200 > 100 45 100 > 100 46 100 > 100 47 10 7.5 48 100 > 100 49 100 > 100 50 100 > 100 As an example of development to demonstrate the benefits of this miniaturization, 10,080 discrete compounds were tested through CF-HTS in a total volume of 17 ml enzyme gel (less than 2 μl per test). A Packard Multipette kit was used to supply 30 ml of volume of each sample separated by 1 mm. The compounds were supplied in DMSO at a concentration of 5 mM, so that approximately 150 pmoles of each were supplied on the plastic. DANA was again used as a control inhibitor. In addition to the titration of the usual control outside the compound disposition, a control titration was included within the same composition as a blind test in which the control samples were treated in the same way as the 10,080 unknown when they were supplied on the same basis. plastic. How "before, the sheets were dried and used in a neuramin? Dasa trial." The 10,080 compounds were simultaneously classified in less than 1 hour through this method.Once again, no microfluids and / or handling were required. The high density used in this test did not interfere with the detection of inhibitors, and the high density did not complicate the identification of active compounds even when the fluorescent compounds (easily observed as brighter spots in the gel) were the closest of the active compounds, see Figure 15.)

Claims (31)

1. - A process for analyzing test samples for a biological or biochemical activity, comprising: a) introducing multiple test samples into or onto a porous test matrix that optionally contains one or more test components, b) using at least one test matrix for introducing one or more test compounds to the test, where the matrix may or may not be the porous test matrix, c) performing the additional step of: i) washing any matrix used in the assay to remove an excess amount of the test sample, test component or a combination thereof; ii) contacting any matrix used in the assay with an additional reagent in bulk solution or as a liquid.

2. The process according to claim 1, wherein the test samples are filled on the porous test matrix containing a ligand, another test matrix contains the immobilized receptor and the additional reagent is a visualization solution.

3. - The process according to claim 1, wherein the additional reagent detects the expression of a report gene in a cell assay.

4. The process according to claim 1, wherein a porous test matrix is a membrane that carries whole cells on a surface, and the additional step comprises adding a bulk solution containing a compound that is conjugated with a protein of report that is formed through a positive interaction between the cells and the test samples.

5. The process according to claim 1, wherein the additional reagent is a visualization agent.

6. The process according to claim 1, wherein the step of washing removes the excess test component that is used to visualize the test.

7. A process for analyzing test samples for a biological or biochemical activity, comprising: a) introducing multiple test samples into or onto a porous test matrix that optionally contains one or more test components, b) use at minus two additional matrices to perform the assay.

8.- A process for analyzing discrete test samples for a biological or biochemical activity, which comprises: a) entering more than 96 test samples in or on a porous test matrix that optionally contains one or more test components, b) perform the test.

9. The process according to claims 1, 7 or 8, further comprising the step of supplying the test samples on a test sample matrix.

10. The process according to claims 1, 7 or 8, wherein the porous test matrix contains a target macromolecule.

11. The process according to claims 1, 7 or 8, wherein the porous test matrix contains an enzyme.

12. The process according to claims 1, 7 or 8, wherein the porous test matrix contains mixtures or crude biological extracts.

13. The process according to claims 1, 7 or 8, wherein the porous test matrix contains organelles or cells.

14. The process according to claims 1, 7 or 8, wherein the porous test matrix is a filter or membrane.

15. The process according to claims 1, 7 or 8, wherein the porous test matrix is a thin film of liquid cover immobilized test components.

16. The process according to claims 1, 7 or 8, wherein an additional matrix contains an immobilized macromolecular target.

17. The process according to claims 1, 7 or 8, wherein an additional matrix contains a display agent.

18. - A process for simultaneously testing a multitude of samples of different substances for their ability to improve or inhibit a biological process, comprising: a) depositing in a disposition a small volume of each of more than 96 samples of different substances on a flat porous matrix containing or carrying a uniformly dispersed test reagent, so that each different substance is centered in its own distinct site and the identification of each substance can be determined from this deposition site, and b) observing the interaction of each substance with the assay reagent and correlating it to the ability of the substance to improve or inhibit the biological process.

19. A process for simultaneously testing a multitude of samples of different substances for their ability to improve or inhibit a biological process, comprising: a) depositing in a disposition a small volume of each of more than 96 samples of different substances on a flat matrix, so that each different substance is centered in its own different site and the identity of each substance can be determined from its deposition site, b) contact the matrix with a porous matrix containing or carrying a reagent uniformly dispersed test and allowing some of this distance to diffuse towards the porous matrix in a way that the spatial location of each diffused substance can be correlated to the site on the flat matrix where the substance was originally deposited, vc) conduct a test to determine the ability of each substance diffused to improve or inhibit the biological process.

20. A process for simultaneously testing a multitude of samples of different substances for their ability to improve or inhibit a biological process, comprising: a) depositing in a disposition a small volume of each of more than 96 samples of different substances on a flat matrix, so that each different substance is centered in its own distinct site and the identity of each substance can be determined from its deposition site, b) contacting the matrix with a first porous matrix containing or carrying a first test reagent uniformly dispersed and allowing some of each substance to diffuse into the porous matrix in a way that the spatial location of each diffused substance can be correlated to the site on the flat matrix where the substance was originally deposited, c) to put in contact the first porous matrix with a second matrix carrying or containing a second uniform test reagent dispersed and allowing the second test reagent to diffuse into the first porous matrix or allowing each substance and the first reagent to diffuse into the second matrix, in the latter case ensuring that the diffusion occurs in such a way that the location of each Substance in the second matrix can be correlated with the site on the flat matrix where the substantial one was originally deposited, and d) evaluate the ability of each substance to improve or inhibit the biological process.

21. A process for simultaneously testing a multitude of samples of different substances for their ability to improve or inhibit a biological process, comprising: a) depositing in a disposition a small volume of each of more than 96 samples of different substances on a first flat porous matrix, containing or carrying a first test reagent uniformly dispersed, so that each different substance is centered in its own distinct site and the identity of each substance can be determined from its deposition site, b) put in contact the first porous matrix with a second planar matrix carrying or containing a second uniformly dispersed test reagent and allowing the second test reagent to diffuse into the first porous matrix or allow each substance and the first reagent to diffuse towards or on the second matrix, in the latter case ensuring that the diffusion occurs in such a way that the cad site a substance in or on the second matrix can be correlated with the site on the first porous matrix where the substance was originally deposited, and c) evaluate the ability of each substance to improve or inhibit the biological process.

22. The process according to claims 18 or 19 or 20 or 21, wherein one of the uniformly dispersed test reagents is a macromolecule.

23. The process according to claim 18 or 19 or 20 or 21, wherein one of the uniformly dispersed test reagents is an enzyme.

24. The process according to claims 18 or 19 or 20 or 21, wherein one of the uniformly dispersed test reagents is a crude biological extract.

25. The process according to claims 18 or 19 or 20 or 21, wherein one of the uniformly dispersed test reagents are organelles.

26. The process according to claims 18 or 19 or 20 or 21, wherein one of the uniformly dispersed test reagents are whole cells.

27. The process according to claims 18 or 19 or 20 or 21, wherein a uniformly dispersed assay reagent is whole cells carried on a surface of a flat porous matrix.

28. The process according to claim 27, wherein said interaction is observed by contacting the porous matrix with a liquid solution or suspension of a reagent or porous matrix containing a reagent, which in any case aids the visualization of a protein whose expression in whole cells is the biological process being evaluated.

29. The process according to claim 21, wherein the first assay reagent is a labeled ligand and the second assay reagent is an immobilized receptor for said ligand.

30. The process according to claim 29, wherein the second planar matrix is washed to remove any labeled ligand that diffuses on or into the second planar matrix, but is not bound to the immobilized receptor.

31. The process according to claim 30, wherein the second planar matrix is contacted with a liquid solution or suspension of a reagent or a porous matrix containing a reagent, which in any case aids the visualization of the ligand. marked not removed by the washing step.