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WO2003006947A2 - Methode et composition de criblage cellulaire - Google Patents

Methode et composition de criblage cellulaire Download PDF

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Publication number
WO2003006947A2
WO2003006947A2 PCT/US2002/021339 US0221339W WO03006947A2 WO 2003006947 A2 WO2003006947 A2 WO 2003006947A2 US 0221339 W US0221339 W US 0221339W WO 03006947 A2 WO03006947 A2 WO 03006947A2
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WO
WIPO (PCT)
Prior art keywords
probe
cells
reporter
enzyme
probes
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Application number
PCT/US2002/021339
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English (en)
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WO2003006947A3 (fr
Inventor
Sharat Singh
Po-Ying Chan-Hui
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Aclara Biosciences, Inc.
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Application filed by Aclara Biosciences, Inc. filed Critical Aclara Biosciences, Inc.
Priority to AU2002316577A priority Critical patent/AU2002316577A1/en
Publication of WO2003006947A2 publication Critical patent/WO2003006947A2/fr
Publication of WO2003006947A3 publication Critical patent/WO2003006947A3/fr
Priority to US10/740,079 priority patent/US20040175765A1/en

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)

Definitions

  • the present invention relates to methods and compositions for screening of the effects of a cellular treatment on protein expression in cells, and especially, methods and compositions suitable for multiplexed screening.
  • the reporter gene produced a protein, typically an exogenous protein that is either directly assayable, e.g., the luciferase gene, or one that can act enzymatically on a substrate to produce a detectable reporter compound.
  • a protein typically an exogenous protein that is either directly assayable, e.g., the luciferase gene, or one that can act enzymatically on a substrate to produce a detectable reporter compound.
  • the reporter gene in this system is a /?-lactamase gene whose gene product, ⁇ -lactamase, can act on a chromogenic Mactam substrate to produce a product that can be detected by a green-to-blue change in fluorescence.
  • each probe in the set is cleavable by the enzyme into a substrate moiety and an electrophoretic tag (e-tag) reporter having a detection group and a separation modifier that confers on the e-tag reporter, a unique electrophoretic mobility with respect to the e-tag reporters derived from the other probes in the set.
  • the cells are incubated with the associated probes while exposing the cells to a potential regulatory stimulus.
  • the tags are obtained from the cells and electrophoretically separated. From the electrophoretic mobility and level of detection group of each separated e-tag reporter, the level of transcriptional response of each cell to the potential regulatory stimulus to which the cells were exposed is determined.
  • the incubating step may be carried out in separate wells, after which the cells are combined before obtaining the tags.
  • the potential regulatory stimuli may include one or more test compounds, and the exposing step includes adding the test compound(s) to the individual cell- containing wells.
  • the incubating step includes adding a different concentration of the test compound to each of a plurality of the wells.
  • the incubating step includes adding a different test compound to each of a plurality of the wells.
  • the cells in each of a plurality of wells are transfected with a different construct, and the incubating step includes adding the test compound to each of the plurality of the wells.
  • the incubating step is carried out in a single well containing a plurality of different cells, each containing a different probe.
  • the different cells in said well are each transfected with one of a plurality of different constructs, each comprising one of the promoters operatively linked to said coding sequence.
  • exemplary positions of the transport moiety are shown as T-i, T 2 , T3 and T 4 ;
  • the first detection group D- t is a cephalosporin;
  • the second detection group D 2 is a fluorescein;
  • exemplary positions of the separation modifier are shown as Mi and M 2 ;
  • the substrate is indicated as a four-member Mactam ring, labeled ⁇ .
  • the detection group is a fluorescent moiety; in another, the detection group includes a catalytic moiety capable of catalyzing a detectable reaction.
  • One general embodiment of the method is adapted for determining the extent of interaction of a first hybrid protein having a DNA-binding domain that binds to the selected promoter, and a first interaction domain; and a second hybrid protein having a transcriptional activation domain and a second interaction domain that is to be tested for interaction with the first interaction domain.
  • the promoter is capable of activation by a polypeptide having a transcriptional activation domain when the transcriptional activation domain is in sufficient proximity to the gene.
  • the cells contain the first and second hybrid proteins, and the determining step includes determining the extent of interaction of the two hybrid proteins by the level of e-tag reporter determined.
  • the method may further include adding the test compound to the cells and determining the amount of e-tag reporter produced, and from that, the extent to which the test compound has an effect on binding between the two hybrid proteins.
  • the promoter is a repressible promoter
  • the cells may further contain a repressor gene construct comprising an inducible promoter operatively linked to the coding sequence for a protein capable of binding to and repressing said repressible promoter.
  • the method may be used to determine the extent of binding of a hybrid protein to a designated DNA sequence, where the hybrid protein has a transcriptional activation domain fused to a DNA-binding domain.
  • the designated DNA sequence is operatively linked to the selected promoter, where (i) the promoter is capable of activation by a polypeptide having the transcriptional activation domain when the transcriptional activation domain is in sufficient proximity to the promoter, (ii) the cells contain the hybrid protein, and (iii) said determining includes determining the extent of binding of the hybrid protein to the designated DNA sequence by the level of e-tag reporter determined.
  • the method further includes adding the test compound to the cells after the mixing step, and comparing the determined amount of each separated reporter to the amount determined from cells that were not exposed to the test compound, thereby determining the effect of the compound on interaction between any of the hybrid protein and the designated DNA sequence.
  • the detection moiety D is a fluorescent moiety; in another, the detection moiety includes a catalytic moiety capable of catalyzing a detectable reaction.
  • the probes may have one of the forms given above.
  • Figures 1 A-C illustrate one possible sequence of changes in the structure of a probe during the course of performing the method of the invention.
  • Figure 1 A illustrates one embodiment of the structure of a probe introduced to a cell culture for transport into the cells. Once the probe is taken up by the cell, intracellular deacetylases will remove the acetyl groups, as shown in Figure 1 B, resulting in a probe structure that is too hydrophilic to efficiently pass across the cell membrane.
  • Figure 1C shows a reporter after recognition and action on the deacetylated probe by a reporter gene product.
  • Figures 2A and 2B illustrate an embodiment of the methods of the invention for monitoring the effects of a stimulus on transcription from a defined promoter.
  • an external cellular stimulus causes expression of a reporter enzyme, resulting in action on a probe to release a reporter.
  • Figures 3A and 3B depict hypothetical electropherograms of the separation of probe and reporter molecules resulting from the cellular samples of Figures 2A and B.
  • Figure 3A shows a single peak corresponding to the unmodified probe.
  • Figure 3B shows a second peak arising as a result of enzyme action on the probe to yield a reporter, catalyzed by expression of the reporter enzyme.
  • Figure 6A illustrates an exemplary fluorescent lactam probe for use in the invention. Enzyme products of the probe resulting from successive esterase and lactamase cleavages are shown in Figures 6B and 6C.
  • Figure 7 illustrates another exemplary fluorescent lactam probe for use in the invention.
  • Figure 8A illustrates an exemplary fluorescent lactam probe according to the general structure shown in Figure 7, in which the undeaved probe is capable of exhibiting FRET.
  • Enzyme products of the probe resulting from successive esterase and lactamase cleavages are shown in Figures 8B and 8C.
  • Figures 9A and 9B illustrate an embodiment of the methods of the invention for a multiplexed screen to monitor the effects of a stimulus on transcription from multiple promoters.
  • Figure 9A illustrates individual construction of separate cell lines prior to mixing to conduct the screen.
  • Figure 9B shows the effect of an external cellular stimulus on a mixture of three separate cell lines. Stimulation of transcription causes expression of a reporter enzyme in only one of the cell lines, resulting in cleavage of a probe specifically in the responsive cell line.
  • Figures 10A and 10B depict hypothetical electropherograms of the separation of molecules resulting from the cellular samples of Figures 9A and B.
  • Figure 10A shows three peaks corresponding to the three unmodified probes contained in the mixture of cells in the untreated state of Figure 9A.
  • Figure 10B shows a second peak arising as a result of modification of the probe by the reporter enzyme in one of the three cell lines of the mixture.
  • Figures 11 A and 11 B illustrate an embodiment of the methods of the invention for studying protein-protein interactions based on the forward yeast two-hybrid approach.
  • an external cellular stimulus leads to disruption of the interaction between two peptides, causing a loss in transcription of the reporter gene.
  • Figures 12A and 12B depict hypothetical electropherograms of the separation of probe and reporter molecules resulting from the cellular samples of Figures 11 A and B.
  • Figure 12A shows two peaks corresponding to the unmodified probe and the released reporter.
  • Figure 12B shows loss of the reporter peak due to disrupted expression of the reporter gene.
  • Figures 13A-C illustrate assembly of transcriptional initiation complexes on copies of the same indicator gene.
  • the complexes have common DNA- binding and RNA polymerase-binding domains, but each has different protein- protein interaction domains.
  • Figures 14A and 14B illustrate an embodiment of the methods of the invention in which an external cellular stimulus on a mixture of three separate cell lines causes disruption of the expression of a reporter enzyme in only one of the cell lines, resulting in loss of action on a probe specifically in the responsive cell line.
  • Figures 15A and 15B depict hypothetical electropherograms of the separation of probe and reporter molecules resulting from the cellular samples of Figures 14A and 14B.
  • Figure 15A shows three peaks corresponding to the three unmodified probes and three peaks corresponding to released reporters contained in the mixture of cells in the untreated state of Figure 14A.
  • Figure 15B shows the loss of one of the reporter peaks due to disruption of expression of the reporter enzyme in one of the three cell lines of the mixture.
  • FIGS 16A and 16B illustrate an embodiment of the invention utilizing a reverse two-hybrid method.
  • an external cellular stimulus disrupts a protein-protein interaction, resulting in loss of expression of a repressor protein, and induction of expression of a reporter enzyme.
  • Figures 17A and 17B depict hypothetical electropherograms of the separation of probe and reporter molecules resulting from the cellular samples of Figures 16A and B.
  • Figure 17A shows one peak corresponding to the probe.
  • Figure 17B shows a second peak arising as a result of action on the probe by the reporter enzyme, to yield a reporter molecule.
  • probe it is useful to consider the functional components of the probe, as used in practicing the methods of the invention.
  • the basic components of a probe which also may be termed groups or moieties, include (1) a detection group, D, (2) a separation modifier, M, (3) a substrate, S, and optionally (4) one or more transport moieties, T.
  • the function of a probe in the invention is to serve as a substrate for a designated enzymatic activity, wherein action of the designated enzyme on the probe results in modification of the probe to generate a corresponding reporter molecule.
  • modifications generally comprise removal of a portion of the probe, or modification in the structure of the probe.
  • the resulting reporter molecule minimally comprises the detection group and the separation modifier.
  • a “reporter,” or “reporter molecule” of the invention is the molecule generated by action of a designated enzyme on a probe that contains the detection group and separation modifier of the probe.
  • a “detection group,” abbreviated “D,” refers to a chemical group or moiety that is capable of being detected by a suitable detection system, or alternatively a means for generating a detection group.
  • Means for generating a detection group may include either incorporation of a reactive group to form a bond with a detectable moiety, or the detection group may be a catalytic moiety capable of catalyzing synthesis of a detection group in an electrophoretic system.
  • One preferred detection group is a fluorescent moiety or other chromogenic moiety.
  • the detection group will typically be common among a set or subset of different probes, but may also differ among probe subsets.
  • the "separation modifier,” abbreviated “M,” is a moiety that confers upon the probe or reporter molecule containing it, a “separation characteristic” that allows separation of each probe or reporter molecule from all other probes and reporters of a designated set.
  • the type of separation characteristic used will typically be determined by the separation platform being employed for analysis of an assay.
  • a set of probes may be made up of a first subset having a group of separation modifiers that impart unique electrophoretic mobilities to the subset when in combination with a first detection group having one defined charge and/or mass, and a second subset having the same group of mobility modifiers in combination with a second detection group with a different charge and/or mass, thus imparting electrophoretic mobilities that are unique among the combination of both subsets.
  • the separation characteristic will be a unique mass.
  • the unique mass of a probe or reporter will serve as the separation characteristic, and the separation modifiers within a set of probes will be any set of structures that vary in mass without significantly impacting the efficiency of detection of a given structure within a set.
  • substrate abbreviated "S,” when used in the context of a probe or probe composition, refers to the part of the structure that is recognized by the designated reporter enzyme of a given assay, and is acted upon by the enzyme to make a product having a separation characteristic that is different from that of the unreacted probe.
  • the action of the enzyme will most preferably be cleavage of the substrate, however modifications comprising additions, conversions, or other types of modification are also contemplated.
  • a “set”, “group”, or “library” of probes refers to a plurality of probes numbering typically at least five, and more typically 10-100 or more probes, wherein the plurality of probes have common substrate moieties and different separation characteristics.
  • probe set refers to a set of probes for use in detecting the level of reporter enzyme activity present in each or any of a plurality of known, selected cell lines.
  • multiplex as applied to methods, assays, detection, analyses, and other processes, means that a given process is conducted for multiple samples, enzymes, targets, and/or molecules simultaneously in a single mixture. Various steps in the methods of the invention may be conducted individually or in a multiplexed fashion.
  • a “multiplexed assay” means the activity of a common reporter enzyme in a plurality of different cell lines is measured simultaneously in a single sample, and/or the detection step conducted to analyze the results of an assay is carried out for multiple assays in a single separation.
  • Transfected cells refers to cultured cells, typically mammalian cells, that have been transfected by an engineered gene construct comprising a selected promoter and the coding sequence for a reporter enzyme, typically an exogenous enzyme.
  • the intracellular activity of the reporter enzyme can be assayed by its ability to act on a probe, thereby releasing a detectable reporter having a unique and defined characteristic that allows it to be separated from other reporters, e.g., by electrophoretic mobility, mass, or other separation properties.
  • the present invention discloses methods for multiplexed cellular assays.
  • the methods can be used for (i) simultaneous detection of transcriptional response of a given cell to multiple different regulatory stimuli, e.g., test compounds, or to different concentration of one or more test compounds, (ii) simultaneous detection of the transcriptional response of multiple cell lines to one or more regulatory stimuli, (iii) simultaneous quantitation of transcription levels from multiple promoters, or (iv) for monitoring effects on multiple protein-protein interactions.
  • regulatory stimuli e.g., test compounds
  • concentration of one or more test compounds e.g., test compounds
  • simultaneous detection of the transcriptional response of multiple cell lines to one or more regulatory stimuli e.g., simultaneous detection of the transcriptional response of multiple cell lines to one or more regulatory stimuli
  • simultaneous quantitation of transcription levels from multiple promoters e.g., simultaneous quantitation of transcription levels from multiple promoters
  • a preferred embodiment of these methods makes use of one or more reporter gene constructs that couple a single reporter enzyme coding sequence with one or more of a plurality of different promoter regions, and a set of tagged probes, each having a unique separation characteristic, that are subject to modification by the reporter enzyme, producing a set of tagged reporters that can be separated from each other by differences between their separation characteristics, e.g. electrophoretic mobility, mass, shape, or other physical property.
  • the transfected cells are exposed to the regulatory stimuli, e.g., test compound(s), in individual assay wells.
  • the cells in each well typically transfected with the same reporter construct, are exposed to one of a plurality of different probes, to allow uptake of the probe into the cells in each well.
  • the different wells are exposed to one or more of a group of different regulatory stimuli, or differing concentrations of the same regulatory stimulus, being tested for the ability to regulate gene expression in the gene controlled by the construct promoter.
  • the regulatory stimuli are test compounds, but other stimuli, such as heat, pH environment, concentration of external cations, or the presence of cells capable of interacting with the transfected cells may also be tested.
  • the cells are then incubated under conditions that allow differential gene expression in response to the added stimulus.
  • the released tag reporters are obtained from the cells, either before or after combining the cells from the different wells, and the combined tag reporters are separated.
  • the level of transcriptional response of cells in each well can then be determined from the mobilities and amounts of the detected tag reporters, which are each uniquely associated with a given, known probe.
  • multiplexing is carried out at the detection step, but promoter constructs are assayed individually.
  • multiplexed assays are achieved by combining a set of cell populations, where each population contains a distinct probe and different cell type that has the potential for different response to one or more regulatory stimuli, e.g., test compounds.
  • the different cell lines may be formed by transfecting different cell lines with the same promoter-enzyme coding sequence construct, or by transfecting the same (or different) cell line with each of a plurality of different constructs, each containing a different selected promoter (corresponding to the promoter of a gene of interest) and operably linked to the coding sequence of a desired assay enzyme, as discussed below.
  • Cellular treatments that affect transcription from specific promoters will cause expression or repression of the gene for the reporter enzyme in that particular cell population, causing changes in the amount of degradation of the corresponding probe.
  • the generation of specific reporters is determined by separation of the products of the cellular assay, and is correlated with an effect of the treatment on transcription from a defined reporter gene construct.
  • multiplexing occurs both at the cell-response step in the method, and at the detection step.
  • Another embodiment of the invention employs reporter gene constructs coupling one of a plurality of reporter enzyme coding sequences with one of a plurality of promoter regions.
  • each probe of the plurality comprises a substrate moiety for one of the plurality of reporter enzymes, and has a unique separation characteristic subject to modification by one of the plurality of reporter enzymes, thereby producing one of a plurality of tagged reporters that can be separated from each other by differences between their separation characteristics, as described above.
  • a single cell line is transfected with a plurality of reporter gene constructs, each having a unique reporter enzyme, enabling a multiplexed determination of the effects of a cellular treatment on different genetic constructs in the same cell.
  • This embodiment of the invention will require that the conditions of the assay be such that each and all of the reporter enzymes employed will be capable of showing enzymatic activity.
  • reporter enzymes for use in combination include, e.g., ?-lactamase, ⁇ -galactosidase, luciferase, Agueorin, Green Fluorescent Protein (GFP), esterases, proteases, kinases, phosphatases, and nucleases.
  • N Green Fluorescent Protein
  • the level of multiplexing X that is obtained using a mixture of singly-transfected cell lines, each containing a distinct probe (as described in the preceding paragraph), can be increased to N(X),
  • the multiple lines can also be combined for a multiplexed assay.
  • conditions for the assay must allow each reporter enzyme employed to be active.
  • the present invention provides methods for multiplexed cellular assays. Any of the embodiments disclosed above can be used for a simultaneous quantitation of transcription levels from multiple c/s-acting regulatory sequences (herein referred to generally as "Promoters"), or for monitoring effects on multiple protein-protein interactions that control transcription of multiple reporter genes. These methods make use of reporter gene constructs that couple a single reporter enzyme coding sequence with different promoter regions, and a set of probes that are subject to modification by the reporter enzyme, producing a set of reporters that can be separated from each other by differences in their separation characteristics. These probes are derivatives of probes containing transport moieties, such as ester modifications, that provide for membrane permeabilization.
  • cellular enzymes remove these transport moieties, thereby trapping the probe within the cell.
  • Multiplexed assays are achieved by generating independent cell lines that contain a distinct promoter region controlling expression of a common reporter gene, and a distinct probe, then combining a set of cell populations prior to cellular treatment. Those treatments that affect transcription from specific promoters will cause expression or repression of the gene for the reporter enzyme in that particular cell population, within the mixture of cell populations, causing changes in modification of the corresponding probe to produce the corresponding reporter.
  • the generation of specific reporters is determined after separation of the reporter products of the reaction, and is correlated with an effect of the treatment on transcription from a defined promoter.
  • FIGS 1A-1C illustrate a probe 10 and its transformations through the course of the method of the invention.
  • the probe generally includes a substrate 20 that is linked to a tag that includes a detection group 21, such as a fluorescent reporter, and a separation modifier Mj, indicated at 22, that imparts to the tag a selected separation characteristic, such as, e.g., charge-to-mass ratio, and thus a selected electrophoretic mobility.
  • a detection group 21 such as a fluorescent reporter
  • Mj separation modifier
  • the detection group and separation modifier are show in parentheses to indicate that they may be represented in the structure in either order, or their functionalities may be embodied by a single moiety.
  • All of the detection groups of a probe set may be the same, or multiple detection groups that can be simultaneously detected may be employed to further enhance the multiplexing capacity of the assay. Exemplary probes for a probe set useful in the invention will be described below.
  • transport moieties 23 generally ester groups, preferably acetyl groups, that facilitate transport of the probe across a cell membrane by rendering the probe nonpolar.
  • the transport moiety is joined to the probe, normally through either the detection group or separation modifier, by a linkage, such as an ester linkage, that is cleavable by an intracellular enzyme, removing the transport moiety to generate the probe of the assay.
  • Probe indicated at 11 in Figure 2B, is generated within the cell by removal of the transport moieties from the probe, rendering the resulting probe less hydrophobic, thereby inhibiting the rate of loss of the probe by transport out of the cell, and inhibiting any lost probe from being taken up by a second cell.
  • the substrate or bulk of the substrate is released, as shown Figure 1 C, yielding an reporter 12 composed of the detection group 21 , the separation modifier 22 and any residue S' of the cleaved substrate, indicated as 24 in Figure 1C.
  • This reporter by virtue of its unique separation modifier, can be electrophoretically separated from all other reporters derived from the set of probes in the multiplexed assay.
  • the set of probes constitute one aspect of the invention, as exemplified by probe 10 in Figure 1A, wherein each probe in the set comprises a different separation modifier.
  • the reporter 12 will also have an electrophoretic mobility that is different from that of the probe itself 11.
  • reporter enzyme that is expressed, one that does not normally occur in the cells, or occurs only at low levels, or can be suppressed to low levels under selected growth conditions.
  • the transfected cells are mixed with a probe from the above probe set, such as probe 10 in Figure 1A, which is taken up by the cells, and sequestered within the cells by cleavage of the transport moiety, thereby generating a probe, indicated as 11 in Figure 2A.
  • a probe from the above probe set, such as probe 10 in Figure 1A
  • the cells are exposed to a stimulus 34 indicated as S, which has the potential to impact the level of expression of the reporter gene, by acting at the level of promoter 32.
  • the control may be either induction or repression, and the control exerted by the stimulus may be either indirect, e.g., by inhibiting or promoting the level of protein kinase action on an upstream control element, or direct, e.g., by inhibiting or promoting the binding of a regulatory factor, e.g., transcription factor, to the promoter.
  • a regulatory factor e.g., transcription factor
  • the stimulus is a test compound whose ability to affect regulation of the selected gene is being assayed.
  • the stimulus applied is effective to induce transcription of the reporter gene under the control of the selected promoter.
  • This leads to increased levels of the reporter enzyme (RE), indicated at 36 in Figure 2B.
  • the enzyme interacts with probe 11 , cleaving it into a reporter 12 and a substrate fragment.
  • the level of expression of the gene under the control of the selected promoter can be determined.
  • the effect of the stimulus on the transcriptional regulation of the reporter gene of interest can be determined.
  • the reporter is isolated from the cells by cell lysis, and collection of a soluble cell fraction.
  • Figures 3A and 3B show electropherograms of detectable molecules isolated from cells before and after a stimulus that is effective to promote expression of the reporter enzyme. Before cellular treatment, and in the absence of expressed reporter enzyme, only intact probe will be detected, as shown in Figure 3A. In cells in which a stimulus has been effective to promote gene expression, and thus expression of the reporter enzyme, the observed signal includes both intact probe and cleaved reporter molecules, as seen in Figure 3B. By comparing levels of electrophoretic probes in the two figures, the ability of the stimulus to promote gene expression can be quantitated or semi- quantitated.
  • the probes employed in the method are derivatives of probes used as reagents for the assay.
  • the probes of the invention preferably comprise at least one transport moiety, such as one or more acetyl groups, that is attached to the probe through a bond that can be cleaved intracellularly, e.g., by a de- acetylase.
  • the transport moiety facilitates entry of the probes into the assay cells. Removal of the transport moiety likewise inhibits passage of the probe back out of the cells, or re-uptake of any lost probe by a second cell.
  • the derivative probes of the invention have three important features. First, they will act as substrates for the reporter enzyme produced by the reporter gene construct.
  • each reporter released from its corresponding probe has a unique separation characteristic, e.g. charge-to-mass ratio, relative to that of other reporters from a set of probes (preferably combined with a relatively low mass, e.g., less than 1-5 kDal), that allows a plurality, e.g., 10 to 100 or more, different reporters, to be readily separated and uniquely identified according to each reporter's known separation characteristic, e.g., electrophoretic mobility.
  • the resulting reporter will carry a detection group or means for generating a detection group.
  • the detection group may be either a fluorescent or other chromogenic moiety, or a catalytic moiety capable of catalyzing synthesis of a detection group in an electrophoretic system.
  • the probes in a set of n probes have the form T x - (D, Mj) - S, where T x represents one or more transport moieties.
  • T x represents one or more transport moieties.
  • S is the substrate moiety recognized by the reporter enzyme of the assay, wherein the action of the enzyme on the probe produces a reporter of the form (D, M) - S', where S' is the residue of S remaining with the reporter after reaction of S with the enzyme.
  • D and M are shown in parentheses to indicate that there is no preferred order of these components in the probe or probe structure. Both T and S may be linked to the probe through either D or M.
  • the detection group is a fluorescent moiety, such as fluorescein.
  • Figure 4 shows a number of suitable reporter structures derived from probe cleavage, each having a fluorescein detection group. These reporters comprise a tag moiety (D - Mj) linked to an oligonucleotide through a phosphodiester linkage. As can be seen, the reporters have different separation modifiers, in addition to fluorescein and a single deoxynucleotide derived from the oligonucleotide probe that binds to target (shown as "dC" or "dT” in the figure). Each of these reporters has a unique charge/mass ratio that allows it to be separated from any of the other reporters on an electrophoretic platform. The ability to resolve each of the reporter structures electrophoretically is shown in Figure 5.
  • the oligonucleotide probes containing the tag moieties shown in Figure 4 would be suitable for use in an assay system in which the reporter enzyme (RE) is a nuclease.
  • the reporter enzyme (RE) is a nuclease.
  • the (D - M j ) groups may also be linked to a peptide substrate that can be cleaved by a selected peptidase, an ester linkage that allows cleavage with a selected esterase, a /?-glucoside linkage that can be cleaved by a ?-glucosidase, a ?-lactam or ⁇ -lactam linkage that can be cleaved by a Mactamase, or any other linkage or group that can be cleaved by a suitable cleaving enzyme, such as a hydrolase or phosphatase.
  • a suitable cleaving enzyme such as a hydrolase or phosphatase.
  • FIG. 6A shows a Mactam structure suitable for use as a probe in the invention, in which cleavage by ⁇ -lactamase either converts a leucodye to a fluorescent molecule, or causes a spectral shift in fluorescent emission.
  • the separation modifier is indicated as M, giving each resulting fluorescent reporter ( Figure 6C) a unique charge/mass ratio, and thus a unique electrophoretic mobility among a set of reporters.
  • the figures show cleavage of the transport moieties, T, of the probe (6A) by an intracellular esterase to produce a probe (compound 6B), which serves as a substrate for a ⁇ -lactamase reporter enzyme.
  • FIG. 7 An exemplary structure of a second class of probes useful in practicing the invention, in which cleavage by ⁇ -lactamase causes elimination of a leaving group, is shown in Figure 7.
  • This structure comprises two fluorophores with overlapping spectra attached to the 7 and 3' positions of a cephalosporin (Zlokamik). The close proximity of the two fluorophores allows them to exhibit fluorescence resonance energy transfer, or FRET (J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum, New York, 1983.).
  • FRET fluorescence resonance energy transfer
  • Figure 7A illustrates one embodiment of a probe in which 7-hydroxycoumarin is attached to the 7 position of cephalosporin, and serves as a donor fluorophor to fluorescein, which is attached to the 3' position of the cephalosporin.
  • Cleavage of the ⁇ -lactam ring of cephalosporin causes spontaneous elimination of the group attached to the 3' position, in this case fluorescein, reestablishing fluorescence emission from the donor by disruption of FRET.
  • the free thiol group remaining on the fluorescein as a leaving group completely quenches its fluorescence.
  • the probe shown in Figure 7A contains several transport moieties, which are removed by intracellular esterases to produce the compound shown in 7B, and preferably includes a separation modifier, M, attached to a non-quenched fluorescent product of ⁇ - lactamase cleavage. Action by a ⁇ -lactamase enzyme produces the two products shown in 7C. Additional embodiments of this class of probe structures include those reversing the position of attachment to cephalosporin of the donor and acceptor fluorophores.
  • Probe structures with a leaving group removed by ⁇ -lactamase that do not make use of FRET also find use.
  • a probe containing a single detectable moiety is shown in Figure 8.
  • This probe design comprises a fluorescent detection group D and separation modifier M attached to the 7 position of cephalosporin, and a non-fluorescent leaving group LG attached to the 3' position of cephalosporin.
  • cleavage of the probe by ⁇ -lactamase will lead to elimination of the leaving group, resulting in a change in the mobility of the resulting fluorescent reporter relative to the undeaved probe.
  • the probes in a probe set may have, as their detection group, a moiety capable of catalyzing a localized detectable reaction, termed a catalytic group.
  • the reporters produced by probe cleavage are separated electrophoretically, then combined with or exposed to reagents necessary for catalytic synthesis of a molecule that generates a detectable signal.
  • the catalytic group can be a photosensitive moiety capable of producing singlet oxygen in the presence of light. Singlet oxygen in turn reacts with a leucodye, oxidizing it to a fluorescent molecule.
  • This embodiment may be accomplished by introducing substrates for the catalytic group into the electrophoretic medium, or by bringing the separated reporters into contact with the development reagents, e.g., in a T-junction within an electrophoretic system.
  • the released reporter molecules can serve as a required subunit in a multisubunit holoenzyme. Construction, structures, and properties of such catalytic electrophoretic tags, and systems for development detectable reactions with separated electrophoretic tag reporters, are described in co-owned U.S. Patent Application for "Methods and Reagents for Catalytic Multiplexed Assays ", Serial No. 09/293,821 , filed 5/26/2001 , incorporated by reference and attached hereto.
  • FIG. 9A illustrates the processes conducted to prepare multiple cell cultures for combining in a multiplex assay.
  • the figure shows three individual cell types, indicated at 44, 46, and 48.
  • the probes with the transport moieties removed are indicated at 56, 58, and 60, and are specific to the cell culture.
  • a multiplexed assay may include a large number of such separately prepared cell cultures, e.g., 10-50 different samples, or more.
  • a stimulus 70 such as a test compound having potential regulatory effect on one or more of the promoters of interest.
  • the stimulus induces transcription of the RCS under the control of promoter Pi, but not those under the control of promoters P 2 and P 3 , with the result that the reporter enzyme RE is produced in cells of the first cell population only, as shown.
  • the cells are lysed to obtain the probes and reporters from the mixture of cell lines, e.g., by isolating a soluble cell extract. The detectable molecules within the cell extract are then separated electrophoretically.
  • Figures 10A and 10B show electropherograms corresponding to an assay result before exposing the cells to stimulus (10A), and after exposure to a stimulus that induces transcription of the reporter gene under the control of promoter Pi, but not the genes under the control of promoters P 2 and P 3 (10B).
  • the only peaks observed are those corresponding to undeaved probes 56, 58, and 60.
  • a single cleaved reporter 64 is identified. The identity of the reporter is determined from the expected electrophoretic mobility of a reporter derived from the first probe 56, indicating that the stimulus acted only on the gene with promoter i.
  • a second method for screening a plurality of test compounds in a single determination can be employed where it is not advantageous to test multiple compounds in a single assay, such as in the case of an expectation of drug interactions.
  • the cells from multiple independent assays are combined, and the probes and reporters are isolated and separated electrophoretically, as above.
  • the present method is also easily adapted to multiplexed methods for determining the interaction between two proteins, for example, in an assay to screen for a compound capable of inhibiting the interaction between two intracellular proteins.
  • the assay format is a two-hybrid protein system, such as the yeast two-hybrid system, described in detail in for example, by Meng, Topcu, Walhout, and Buckholz.
  • This assay is based on the reconstruction of a transcriptional activator from two hybrid proteins: a first hybrid protein containing a DNA-binding domain that binds to the selected promoter, and a first interaction domain; and a second hybrid protein having a second interaction domain to be tested for interaction with the first interaction domain, fused to a transcriptional activation domain.
  • a functional transcriptional activator is produced, having both a DNA binding domain that binds to the promoter sequence, and an activation domain that is positioned to interact with RNA polymerase II, promoting transcriptional activation of the gene of interest.
  • the cells in the assay are also transfected with a genetic construct 77 of the type described above, having a selected promoter P controlling a reporter gene coding sequence encoding a reporter enzyme RE.
  • the cells are also contacted with a probe of the type described above, from a set of probes, allowing it to be taken up by the cells and modified by intracellular enzymes to produce a probe 78 that is retained within the cell.
  • FIG. 14 Application of this multiplexed two-hybrid system to a multiplexed assay for the screening of a test compound on a set of protein-protein interactions is illustrated in Figure 14.
  • a test compound S indicated at 82, is tested for its ability to block or inhibit the binding of the two interacting moieties that together form a transcriptional activator in particular two-hybrid transcription factors.
  • the figure shows three distinct cell lines, indicated at 100, 101 , 102, generated separately, then combined in a single test mixture.
  • Such an assay may include a large number of such cell lines, e.g., 10-50 different cell lines, or more.
  • each cell line Prior to the screen, the cells in each cell line have been transfected with expression vectors for the two hybrid proteins, as well as a genetic construct 77 that includes a promoter P ⁇ of a selected gene whose transcriptional response is being measured, and the coding sequence RCSi for a selected reporter enzyme.
  • a different selected probe is added to each cell line for transport into cells and conversion to a probe, indicated at 105, 106, and 107, prior to mixing the cell lines for the screen.
  • each probe will include a transport moiety that is cleaved once the probe enters the intracellular compartment, inhibiting its exit from the cell and subsequent re-uptake by a cell from a different line.
  • test agent 82 is then added to the mixed cell lines, and the cells are incubated under conditions, e.g., under growth and time conditions, that allow for detection in differences in transcription regulation. Following this, the cells are lysed and the probes and reporters extracted for analysis.
  • the above embodiment illustrates use of the forward two-hybrid multiplexed system for testing the ability of a binding agent to block specific interacting moieties, by preparing a plurality of cell lines, each cell line having a designated pair of interaction moieties in the hybrid proteins and a designated probe.
  • This embodiment may be used to screen for changes in hybrid protein interactions that are mediated by the test agent either directly, as shown in Figure 14, or indirectly via, e.g., a signal transduction pathway.
  • the different cell lines will have one of a plurality of selected promoter sequences in the promoter/regulated enzyme construct and the first hybrid protein with the corresponding DNA-binding domain, and the interacting domains of the two hybrid proteins will be the same among the different cell lines.
  • the ability of a test agent to disrupt transcription from selected promoter regions can be monitored.
  • a reverse two-hybrid strategy will result in the gain of a signal (rather than loss) as a result of a change in a cell elicited by a cellular treatment.
  • this type of screen is advantageous for applications requiring high sensitivity, and is usually preferred for drug screening.
  • the reverse two-hybrid strategy differs from a forward two-hybrid approach, however, in that the gene 122 induced by interaction of the two hybrid proteins encodes a transcriptional repressor that acts in trans to block transcription of the reporter gene coding sequence RCS.
  • the repressor gene is tetR, encoding the tetracycline gene repressor protein TetR, indicated at 123, which binds to an Operator sequence 124 normally present upstream of the tetracycline resistance gene.
  • a reporter gene 125 is constructed that positions the Tet Operator between a constitutive promoter 126 and a reporter gene coding sequence.
  • a different type of hybrid protein approach called the one-hybrid system allows application of the methods of the invention to screens for DNA binding proteins.
  • a single hybrid protein is made, comprising a DNA binding domain of interest fused to a transcriptional activator domain.
  • This type of screen is a more generalized approach to the methods described above for monitoring control of gene expression, except that interaction of any DNA-binding protein with any binding site is monitored.
  • either DNA binding or the disruption of DNA binding can be monitored.
  • the cells employed in the method are typically mammalian cells that can be grown readily in culture. A variety of cell types, including cells derived from a variety of human normal and cancer tissues are available.
  • the genetic reporter constructs used in the assay each including the coding sequence for a reporter enzyme operatively connected to a selected promoter, is prepared according well-known recombinant methods. Promoter sequences of selected genes are available from a variety of sources, and sequences therefore can be obtained from standard sequence databases, such as GenBank, which can be accessed at www.ncbi.nlm.nih.gov/Genbank/.
  • the probes to be delivered inside cells to serve as substrates for the reporter enzyme may contain transport moieties linked to the probes by a bond that is recognized by an intracellular enzyme, e.g., an intracellular deacetylase, such that the probes are trapped inside the cells following their transport into the cell and removal of the transport moiety.
  • an intracellular enzyme e.g., an intracellular deacetylase
  • typical reporter constructs are exemplified by vectors such as those of the Hybrid HunterTM system from Invitrogen, the MATCHMAKER LexA two-hybrid system from CLONTECH, and the DupLEX-ATM system from OriGene.
  • upstream of the reporter gene coding sequence is a Promoter sequence (P), which functions as a binding site for RNA polymerase and another sequence-specific DNA binding protein.
  • the first hybrid protein fuses the first interacting domain to the DNA-binding domain of a sequence-specific DNA-binding protein, e.g. GAL4 (Clontech) or LexA (Invitrogen, OriGene), wherein the DNA-binding domain is capable of binding to the Promoter upstream of the reporter gene coding sequence.
  • the second hybrid protein fuses the second interacting domain to a transcriptional activation domain, e.g.
  • the LexA DNA-binding domain is fused to a first interacting domain to form a first hybrid protein.
  • a second hybrid protein is formed from fusion of a second interacting domain to the VP16 transcriptional activator domain.
  • the LexA binding site is inserted adjacent to the coding sequence for the tetracycline repressor (TetR), which in turn represses transcription of a designated reporter gene.
  • TetR tetracycline repressor
  • TetR tetracycline repressor
  • Cell lines containing the appropriate vector constructs are generated by transfection.
  • cells may be transiently transfected for immediate screening. Long-term use of transfected cell lines requires that stable cell lines be generated by drug-resistance selection. Reverse hybrid assay systems require use of stable cell lines.
  • individual cell lines typically the same cell line transfected with the same reporter construct
  • test compounds prior to testing the cells in individual wells are exposed to different probes, to allow uptake of a different probe to the cells in each separate well.
  • a different test compound or different concentration of the same compound or for controls, no compound at all
  • the cells are incubated under conditions that allow differential gene-transcription control. Typical incubation conditions involve incubation at 24-37°C for several minutes to several hours, e.g., overnight. Thereafter, the tags released during the assay are obtained, either before or preferably after the cells are combined.
  • the tags may be obtained, for example, by lysing the cells, and obtaining the tags in a soluble cell extract.
  • the combined tags are then separated, and detected, e.g., fluorometrically, to determine the levels of transcription associated with each of the different test compounds or different concentrations of the same compound.
  • the different cells produced by transfecting different cell lines with the same gene construct, or the same cell line with different gene constructs, are first mixed with different probes, to generate different cell samples, each containing a distinct probe.
  • the different cells, and the associated probes are then combined prior to conducting a gene- transcription assay.
  • the mixed cell lines are placed in a common well, e.g., one of the wells in a microtitre plate, in order to test the transcription response of each of a plurality of promoters, or in the two-hybrid example, where the cells have different two-hybrid protein binding regions.
  • the mixed cells are exposed to the assay stimulus, e.g., a test compound or one of a plurality of test compounds, and incubated in the presence of the stimulus for a period sufficient to observe changes in expression level of the reporter gene.
  • the assay stimulus e.g., a test compound or one of a plurality of test compounds
  • treatment with chemical compounds is generally performed overnight.
  • Treatment with protein target candidates in the form of purified proteins or conditioned culture media involves pre-incubation at a certain dose in cell culture. The duration of treatment depends on the nature of the responses to be tested.
  • the cells are washed and lysed to release intracellular probe and reporter compounds. Washing and lysing are carried out by conventional means, e.g., addition of a non-ionic surfactant. If desired the cell extract containing the probes and reporters may be concentrated, e.g., by lyophilization, or via a sample-stacking step during electrophoresis prior to electrophoretic separation.
  • the probe substrate may include one member of a binding pair, such as an antigen, biotin, or the like, which allows the undeaved probe to be captured on a solid-phase substrate coated with the other member of the binding pair, e.g., antibody or avidin, or may be reacted with a soluble form of the other binding pair, to produce a complex that does not compete with the reporters in the electrophoretic system employed.
  • the probe-binding member complex may have a charge opposite to that of the released reporter, at the pH of the electrophoretic medium employed.
  • the released reporters and, optionally, the soluble probes are then separated, e.g., electrophoretically, preferably using a capillary or microchannel electrophoretic system that allows separation and detection of small amounts of reporters.
  • electrophoretic conditions e.g., separation medium, pH, and voltage levels are selected according to convention criteria to optimize separation.
  • the migration positions and relative levels of reporters, and optionally, probes can be determined by analysis of electropherograms such as illustrated above in Figures 10 and 15. From this, the identity of the reporters and probes, i.e., the correspondence between given probes/reporters and given cell samples can be assigned, and the level of change in transcription of the selected gene(s) can be determined.
  • the method allows a large number of test compounds, and/or different concentrations of a single test compound, to be detected in a simple, multiplexed format that is both reliable and quantitative.
  • levels of transcription in response to a large number of test compounds or test-compound concentrations can be identified and compared readily using simple electrophoretic separation and detection.
  • the method allows a large number of cellular responses to be quantitated using both a single cell- incubation format and a single detection format.
  • the simultaneous screening and detection features simplify and speed up a screen, and provides for internal controls or standards for the measured levels of expression.
  • the construction and structural features of the probes themselves, discussed below, permits electrophoretic separation of a large number of reporters in a single electrophoretic format, e.g., capillary electrophoresis, microfluidic device, conventional gel electrophoresis, HPLC, or mass spectrometry, thus permitting sample multiplexing of many samples, e.g., 10-100 samples, or more, at a time.

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Abstract

L'invention concerne des méthodes de criblage cellulaire multiplexé. On peut mettre en application ces méthodes afin de quantifier simultanément les niveaux de transcription de promoteurs ou de médicaments multiples ou de contrôler les effets exercés sur des interactions multiples protéine-protéine. Ces méthodes utilisent des produits de recombinaison de gènes rapporteurs couplant une seule séquence codante d'enzyme rapporteur à une ou plusieurs régions du promoteur, ainsi qu'un ensemble de sondes soumises à une dégradation par l'enzyme rapporteur, ce qui produit un ensemble de rapporteurs pouvant être séparés les uns des autres par leurs différences de mobilité. On exécute des essais multiplexés par combinaison d'un ensemble de population cellulaire, chaque population contenant un produit de recombinaison distinct de gène rapporteur et une sonde distincte. On exécute ensuite différents traitements sur le mélange de cellules. Les traitements induisant la transcription de promoteurs spécifiques provoqueront la synthèse de l'enzyme rapporteur dans une population cellulaire déterminée, ce qui conduit à la dégradation de la sonde correspondante. On détermine la génération des rapporteurs spécifiques par électrophorèse ou d'autres moyens de mesure de la mobilité et on la met en corrélation avec un effet du traitement sur la transcription d'un promoteur défini.
PCT/US2002/021339 2001-07-09 2002-07-05 Methode et composition de criblage cellulaire WO2003006947A2 (fr)

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WO2004087887A2 (fr) 2003-04-01 2004-10-14 Monogram Biosciences, Inc. Complexes intracellulaires utilises comme biomarqueurs
WO2013138585A1 (fr) * 2012-03-16 2013-09-19 The Broad Institute, Inc. Procédés multiplex pour analyser simultanément des populations de cellules mélangées
WO2014053479A1 (fr) 2012-10-02 2014-04-10 Roche Diagnostics Gmbh Procédés permettant de libérer spécifiquement un sous-groupe d'objets
US9081019B2 (en) 2008-12-01 2015-07-14 Laboratory Corporation Of America Holdings Methods and assays for measuring p95 and/or p95 complexes in a sample and antibodies specific for p95
US9766242B2 (en) 2009-01-15 2017-09-19 Laboratory Corporation Of America Holdings Methods of determining patient response by measurement of HER-3 and P95
US10416162B2 (en) 2007-12-20 2019-09-17 Monogram Biosciences, Inc. Her2 diagnostic methods
US10451614B2 (en) 2016-03-15 2019-10-22 Laboratory Corporation Of America Holdings Methods of assessing protein interactions between cells

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US20040229294A1 (en) 2002-05-21 2004-11-18 Po-Ying Chan-Hui ErbB surface receptor complexes as biomarkers

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US5658736A (en) * 1996-01-16 1997-08-19 Genetics Institute, Inc. Oligonucleotide population preparation

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004087887A2 (fr) 2003-04-01 2004-10-14 Monogram Biosciences, Inc. Complexes intracellulaires utilises comme biomarqueurs
US10416162B2 (en) 2007-12-20 2019-09-17 Monogram Biosciences, Inc. Her2 diagnostic methods
US9081019B2 (en) 2008-12-01 2015-07-14 Laboratory Corporation Of America Holdings Methods and assays for measuring p95 and/or p95 complexes in a sample and antibodies specific for p95
US10273308B2 (en) 2008-12-01 2019-04-30 Laboratory Corporation Of America Holdings Methods of producing antibodies specific for p95
US9766242B2 (en) 2009-01-15 2017-09-19 Laboratory Corporation Of America Holdings Methods of determining patient response by measurement of HER-3 and P95
US10775382B2 (en) 2009-01-15 2020-09-15 Laboratory Corporation Of America Holdings Methods of determining patient response by measurement of HER-3
WO2013138585A1 (fr) * 2012-03-16 2013-09-19 The Broad Institute, Inc. Procédés multiplex pour analyser simultanément des populations de cellules mélangées
US10724099B2 (en) 2012-03-16 2020-07-28 The Broad Institute, Inc. Multiplex methods to assay mixed cell populations simultaneously
WO2014053479A1 (fr) 2012-10-02 2014-04-10 Roche Diagnostics Gmbh Procédés permettant de libérer spécifiquement un sous-groupe d'objets
US9506918B2 (en) 2012-10-02 2016-11-29 Roche Diagnostics Operations, Inc. Methods of specifically releasing a sub-group of objects
US10451614B2 (en) 2016-03-15 2019-10-22 Laboratory Corporation Of America Holdings Methods of assessing protein interactions between cells

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