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WO2000079260A1 - Procede d'electrophorese capillaire destine au criblage de ligands d'affinite utilisant un ligand competitif detectable - Google Patents

Procede d'electrophorese capillaire destine au criblage de ligands d'affinite utilisant un ligand competitif detectable Download PDF

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Publication number
WO2000079260A1
WO2000079260A1 PCT/US2000/017490 US0017490W WO0079260A1 WO 2000079260 A1 WO2000079260 A1 WO 2000079260A1 US 0017490 W US0017490 W US 0017490W WO 0079260 A1 WO0079260 A1 WO 0079260A1
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Prior art keywords
target
plug
ligand
competitive ligand
capillary electrophoresis
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PCT/US2000/017490
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English (en)
Inventor
Yuriy M. Dunayevskiy
Dallas E. Hughes
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Cetek Corporation
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Publication date
Application filed by Cetek Corporation filed Critical Cetek Corporation
Priority to EP00944872A priority Critical patent/EP1188049A1/fr
Priority to JP2001505179A priority patent/JP2003502665A/ja
Priority to CA002375798A priority patent/CA2375798A1/fr
Priority to US10/018,233 priority patent/US6837977B1/en
Publication of WO2000079260A1 publication Critical patent/WO2000079260A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
    • G01N27/44726—Arrangements for investigating the separated zones, e.g. localising zones by optical means using specific dyes, markers or binding molecules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis

Definitions

  • This invention relates to the field of capillary electrophoresis-based screening of materials for unidentified compounds that can bind to a target molecule of interest.
  • complex biological materials any material that may have an effect in a biological system.
  • complex material include, but are not limited to: naturally occurring complex biological materials, such as natural products or extracts; various biological preparations; chemical mixtures; libraries of pure compounds; and synthetic compounds such as combinatorial libraries.
  • major screening problems include: detecting candidate hit compounds that bind to a target molecule of interest, especially those present at low concentrations in screened samples; accounting for unknown components that can interfere with screening agents; and determining the relative value of screened samples for further investigative efforts.
  • the invention provides a means of better identifying ligands having a relative binding strength at or higher than a desired threshold as set by use of a known, preferably detectable, competitive ligand having a selected binding strength or dissociation constant (e.g., K d ⁇ 100 ⁇ M, preferably K d ⁇ 10 ⁇ M) . Identifying and ranking those ligand- containing samples that form the most stable complexes with the selected target, saves time and resources spent on further isolation and characterization of hit compounds. The most stable ligands are potentially more effective and valuable as therapeutic, regulatory and/or diagnostic compounds and drugs .
  • a known, preferably detectable, competitive ligand having a selected binding strength or dissociation constant e.g., K d ⁇ 100 ⁇ M, preferably K d ⁇ 10 ⁇ M
  • the present invention provides a way of identifying new, biologically active compounds when screening various materials, such as naturally occurring complex biological materials, chemical mixtures, synthetic compounds and other materials, for candidate affinity ligands that bind to target molecules of interest (e.g., molecules involved in a disease) .
  • target molecules of interest e.g., molecules involved in a disease
  • candidate ligands have potential regulatory, pharmaceutical, therapeutic, and/or diagnostic applications.
  • the methods of the invention combine competitive binding conditions with capillary electrophoresis (CE) to detect, in a screened sample, an unidentified candidate ligand that binds to the target at or above a selected binding strength, and to characterize that candidate ligand' s relative binding strength.
  • CE capillary electrophoresis
  • the method is particularly useful for detecting moderate-to-tight-binding ligands (e.g., with a dissociation constant of preferably about K d ⁇ 100 ⁇ M, preferably ⁇ lO ⁇ M or even ⁇ lOnM) .
  • moderate-to-tight-binding ligands e.g., with a dissociation constant of preferably about K d ⁇ 100 ⁇ M, preferably ⁇ lO ⁇ M or even ⁇ lOnM.
  • the method involves using a selected target molecule (TG) and a known, preferably moderate-to-tight binding, competitive ligand (CL) that binds to the target (TG) , to screen a sample of, e.g., a complex biological material, potentially containing a candidate ligand (LG) that competes with the CL for binding to the TG.
  • a plug of a mixture of the TG and the sample, and a separate plug of the CL are subjected to capillary electrophoresis under conditions optimized to detect the migration of a chosen molecule in unbound state and/or when bound in a complex.
  • the detected or tracked molecule is the known, CL, which must be detectable during CE, for instance, by fluorescence or absorbance.
  • CL the known, CL
  • a capillary electrophoretic migration pattern or profile of the CL is generated.
  • the migration pattern comprises at least one member from the group consisting of a peak representing unbound competitive ligand and a peak representing a complex of the competitive ligand bound to the target.
  • both peaks are detectable, absent any candidate ligand.
  • the invention encompasses a method of screening a complex material for an unidentified candidate ligand that binds to a pre-selected target of interest
  • the method comprises the following steps:
  • step (e) determining whether a migration pattern resulting from step (d) differs from a reference standard, thereby indicating the presence of a candidate ligand in the complex material.
  • Figure 1 is an overlay of several electropherograms resulting from using the competitive-binding, CE method of the invention to screen samples containing various concentrations of a test candidate ligand, hirulog-1, which binds tightly to a target molecule, thrombin; and
  • Figure 2 is an overlay of several electropherograms resulting from using the competitive-binding, CE method of the invention to screen several samples each containing two test candidate thrombin-binding ligands, weak-binding hirugen and tight-binding hirulog-1, at various concentrations .
  • the present invention allows detection of ligands that bind to a selected target, especially moderate-to-tight-binding ones, in mixtures that also contain higher concentrations of competing, weaker-binding ligands.
  • the method does not require knowledge of a candidate ligand' s particular structure or concentration within the screened sample in order for it to be detected.
  • the method also enables screening assays using targets that cannot be detected directly during capillary electrophoresis .
  • the method of the invention works as follows.
  • a predetermined concentration of a target of interest (TG) is first mixed with a sample of complex material to be screened, which may or may not contain one or more potential or candidate ligand (s) (LG) that can bind to the target.
  • the target/sample mixture should be incubated for enough time to allow formation of a complex of the target and any candidate ligand (sometimes referred to as "the TG/LG complex”): e.g., 0.5-30 minutes, preferably 1-5 minutes.
  • a known competitive ligand that is detectable during capillary electrophoresis (e.g., fluorescently labelled CL) - i.e., a compound known to bind to the target, preferably moderately to tightly (e.g., having a dissociation constant K d ⁇ 100 ⁇ M, preferably about ⁇ lO ⁇ M) .
  • CL competitive ligand
  • the CL serves to compete with any candidate LG in the screened sample material in binding to target during the capillary electrophoresis (CE) step, described below.
  • a plug of each is injected sequentially into the capillary of a capillary electrophoresis instrument.
  • the order of injection depends on the relative CE migration rates of the CL and the TG under the selected CE conditions.
  • CE running buffer that will confer a charge on both the CL and the TG.
  • An optional injection of running buffer may be applied between injections to separate the TG/sample and CL plugs.
  • the greater the running buffer plug between the injections the higher the candidate ligand' s affinity must be in order to be detected.
  • relative affinities of candidate ligands can be determined by repeating the screening protocol with each ligand sample and varying the amount of running buffer injected in between the first and second plugs (i.e., varying the distance traveled by the second plug and hence the time interval before the two plugs mingle) .
  • CE conditions are chosen such that detectable analytes from the second-injected plug migrate faster than detectable analytes from the first-injected plug toward a fluorescence detector attached to the CE instrument, during the CE run, so that the detectable analytes from the second plug can migrate through and mingle with detectable analytes from the first plug, prior to the detectable analytes from either of the plugs reaching a detection point in the capillary.
  • detectable analytes refers primarily to the competitive ligand (CL) and the target (TG) , as well as to any candidate ligand from the sample (LG) .
  • CL competitive ligand
  • TG target
  • LG candidate ligand from the sample
  • the CL is detected directly by the detector, while the TG is detected indirectly when it binds to CL to give a shifted CE migration peak representing the CL/TG complex. Any LG present is indirectly detected via its interruption of CL/TG complex formation, as discussed elsewhere in this disclosure.
  • the detectable analytes from the second-injected plug must have a higher capillary electrophoretic mobility in the direction of the detector, than the detectable analytes from the first plug.
  • TG-containing first plug may be a TG/sample plug (as in a screening protocol) , or may be a plug of target alone (as when running a control or reference standard) .
  • the TG analyte has a faster or higher capillary electrophoretic mobility than the CL analyte, so that TG analyte from the second, TG- containing, plug will move through and interact with analyte from the first, CL-containing plug.
  • the known, detectable competitive ligand is tracked at a detector during CE, so that its migration is tracked, thereby generating a capillary electrophoretic migration pattern or profile.
  • the CE migration pattern comprises at least one of the following, if not both: an unbound (free) CL peak and a CL/TG complex peak.
  • the CL can be fluorescently labeled, allowing monitoring of the CL and/or CL/TG peaks by laser-induced fluorescence detection.
  • it is advantageous to use CE conditions allowing observation of both an unbound CL and an bound CL/TG complex during CE one need observe only one or the other peak in the CE profile for the present screening method to work. Two capillary electrophoretic peaks will be observed in most screenings performed according to the invention: one corresponding to the CL/TG complex and the other corresponding to any unbound CL.
  • concentrations of the CL and the TG in their respective plugs are optimized to provide, during CE, a detectable proportion of the CL bound to the TG in the absence of any other target-binding ligand (LG) , as discussed further below.
  • LG target-binding ligand
  • the known, competitive ligand in order to practice the method of the invention for screening complex material for new affinity ligands, the known, competitive ligand must be detectable during CE and bind to the chosen target stably to form a CL/TG complex, so that the resulting CE migration pattern differs from the CE profile of unbound CL alone.
  • Formation of a CL/TG complex should produce at least an observable decrease in the area of the unbound or free CL peak, and preferably but not necessarily, a separate CL/TG peak.
  • the CL and target concentrations and the CE run time should be used in amounts sufficient to allow the CL plug to mingle with a target- containing plug during CE, by the time either or both of the plugs reach the detection point in the CE instrument.
  • the CL and target concentrations and the time allowed for their interaction during CE should be sufficient to allow enough of the total target and CL available to form a target/CL complex having a detectably different migration peak from that of the unbound CL.
  • the predetermined target concentration, predetermined competitive ligand concentration, and capillary electrophoresis conditions are pre-selected to produce, absent any other target-binding ligand, a measurable change in the capillary electrophoretic migration pattern generated by the steps of the method.
  • the measurable change comprises a change of at least 10%, preferably at least 50%, advantageously at least 75%, in the peak area of at least one peak selected from the group consisting of a peak representing unbound competitive ligand and a peak representing a complex of the competitive ligand bound to the target.
  • a detectable change is seen in both the unbound CL peak area and the bound CL/TG peak area.
  • LG candidate affinity ligand
  • a moderate-to-tight-binding LG e.g., K d ⁇ 100 ⁇ M, preferably ⁇ lO ⁇ M
  • any detectable CL/TG complex peak will decrease in area, as compared to the migration pattern from the reference or control CE run. This is because the LG remains bound to the TG and does not allow the CL to bind to the TG during the CE run, as the CL plug migrates through the plug of the mixture of TG and LG-containing sample.
  • the sensitivity of the assay for detecting weak-binding ligands or low concentrations of candidate ligands may be adjusted by regulating CE conditions to limit the time of interaction between the plug of tight-binding CL and the TG- containing plug, so that at least some portion of any TG/weak-binding LG complex formed may be detected. Decreasing the assay's sensitivity and thus limiting detection to only tight-binding targets, can be achieved by increasing the time before the CL contacts the target (by spacing further apart the TG/sample plug and the CL plug injections, or by selecting appropriate CE conditions in light of the TG's and CE ' s respective charge-to-mass ratios) .
  • the present invention advantageously allows one to screen for candidate ligands to a target that is not itself detectable during capillary electrophoresis.
  • undetectable targets may be, for instance, membrane-bound proteins receptors that are not easily purified or are relatively insoluble.
  • a reference standard for this method comprises the migration pattern of the CL resulting from capillary electrophoresis of sequential injections of a plug of the target without the complex material and a plug of the CL, using the same order of injection and same capillary electrophoresis conditions as in the protocol for screening a sample of complex material.
  • Methods of detection are known to one of ordinary skill in the art of capillary electrophoresis, and include but are not limited to fluorescence, ultraviolet absorbance, and the like.
  • An advantageous and simple detection means is the use of a fluorescent label on the molecule tracked during CE .
  • the method of the invention can be used to detect, identify and characterize, both easily and rapidly, moderate-to-tight binding ligands or hit compounds present in samples of complex biological materials (e.g., natural extracts or synthetic compound mixtures) .
  • This method is particularly useful to detect, successfully and selectively, low concentrations of tight-binding hit compounds to a selected target of interest, even if the screened sample contains high concentrations of weak-binding candidate ligands or hits.
  • all CE conditions have to be optimized to detect hit ligand (s) with a desired range of affinities, as will be appreciated by one of ordinary skill in capillary electrophoresis.
  • WB weak-binding hit compound
  • a strong- binding affinity ligand One must define criteria for determining what are a weak-binding hit compound (WB) and a strong- binding affinity ligand.
  • the cut-off for determining the relative binding strength of different ligands or hit compounds is determined primarily by factors such as capillary length, injection-to-detector length, voltage and temperature during CE, buffer composition (e.g., pH and/or salt concentration) .
  • buffer composition e.g., pH and/or salt concentration
  • tight binders can be found by conducting the CE at higher temperatures (e.g. > 25°C) .
  • the method of the invention is particularly advantageous in identifying, in a screened sample, candidate hit compound (s) having a binding strength higher than a selected threshold, and for determining their relative binding strength.
  • “Moderate-to-strong binding” ligands and “weak-binding” ligands have faster off-rates (Koff) and higher dissociaton constants (KD) , and form target/ligand complexes that hold together for little or none of a capillary electrophoretic run, i.e., target/ligand complexes that are unstable and fall apart quickly before coming into contact with CL and before reaching detector.
  • ligands of a particular binding strength have the respective characteristics shown in Table 1.
  • the screening itself uses the fact that the rate of candidate ligand/target complex dissociation is different between weak ligand/target and strong ligand/target complexes. That is, CE conditions can be optimized to achieve complete or sufficient dissociation of any weak candidate ligand/target complex formed during the pre-CE incubation of sample and target, to free up enough target molecules during the CE for binding to the known competitive ligand, detectably. At the same time, those same optimized CE conditions should allow any moderate-to-tight candidate ligand/target complex that forms during the pre-CE incubation, to remain bound, thus reducing the availability of free target and thereby reducing formation of the TG/CL complex. As a result, the presence of the moderate-to- tight-binding candidate is indicated indirectly, by the change it produces in the CE migration pattern of the known competitive ligand.
  • One embodiment of the method detects a moderate-to- tight-binding ligand (e.g., having a dissociation constant of about K d ⁇ 100 ⁇ M, preferably ⁇ lO ⁇ M) , in preference to a weak-binding ligand (e.g., having a dissociation constant of about K d >100 ⁇ M) .
  • the target and a sample of material are first mixed and incubated together to allow binding of the target to any candidate ligand (LG) within the sample. Subsequently, sequential injections of: (1) a plug of the target/sample mixture (containing unbound TG, unbound LG, and any LG/TG complex) ; and (2) a plug containing CL, are injected into the capillary.
  • the order of the plug injections will be determined by the known, relative CE mobilities of the CL and TG. In some cases, the CL plug is injected first; in other cases, the target/sample plug is injected first. In some cases, a plug of CE running buffer may be injected between the TG/sample and CL plugs.
  • An exemplary screening according to the invention in which the CE migration of the competitive ligand is tracked, uses the following steps, with the following results:
  • a plug of a mixture of target and sample complex material is injected into a capillary electrophoresis instrument.
  • the TG is negatively charged and has a relatively slow CE mobility.
  • the detector may be a fluorescence detector.
  • TG that has dissociated from any TG/LG complex during the CE run will be free to bind to CL as the CL plug passes through the TG-containing plug. Any TG remaining bound to LG will not be available to bind to CL. 6.
  • the CL concentrations and CE conditions can be set so that, if the LG/TG complex is moderate-to-weak (e.g., having a dissociation constant K lOO ⁇ M, preferably ⁇ lO ⁇ M) , most of the weak LG/TG complex will have dissociated before the CL catches up to the TG.
  • the free or unbound TG will bind to the CL and give a large, detectable CL/TG peak, and/or a reduced free or unbound CL peak.
  • the LG/TG complex is moderate-to-strong (e.g., K d ⁇ l ⁇ M) , the complex will hold together during the CE run and there will be less free TG available to bind to the CL. Thus, the CL/TG peak will be reduced compared to the control without LG present, and the free CL peak will be large.
  • moderate-to-strong e.g., K d ⁇ l ⁇ M
  • Exemplary complex materials that can be screened by the present method include, but are not limited to, naturally occurring complex biological materials, chemical mixtures, and synthetic compounds such as combinatorial libraries.
  • Naturally occurring complex biological materials include natural extracts, complex biological material is selected from the group consisting of combinatorial chemical libraries, extracts of terrestrial plants, extracts of marine plants, cells from higher animals including humans, eubacteria, actinomycetes, bacteria, extracts from non- recombinant or recombinant microorganisms, microbial fermentation broths, fungi, protozoa, algae, archaebacteria, worms, insects, marine organisms, sponges, corals, crustaceans, viruses, phages, tissues, organs, blood, soil, sea water, water from a fresh-water body, humus, detritus, manure, mud, and sewage or partially purified fractions thereof.
  • Complex material samples may be diluted and/or fractionated prior to screening by the present methods .
  • targets examples include, but are not limited to, enzymes, receptors, proteins, polypeptides, nucleic acids, polynucleotides, carbohydrates, and chemically, enzymatically, or recombinantly modified forms thereof, the modified forms having been modified for improved electrophoretic properties.
  • the target molecules need not be in purified form, and can be presented as part of, e.g., a cell extract or sonicate, as long as the target itself is accessible for binding to the known competitive ligand.
  • the target may be a G-protein coupled receptor (GPCR) embedded in cell membrane.
  • GPCR G-protein coupled receptor
  • CL include, but are not limited to, naturally occurring compounds, synthetic compounds, antibodies, proteins, peptides, oligonucleotides and other drugs known to bind to the target of interest.
  • the competitive ligand may be modified as needed to confer a desired charge to the molecule.
  • the CL is moderate-to-tight-binding, with a dissociation constant of about K ⁇ ⁇ 10 ⁇ M and an off- rate of about K 0 ff ⁇ 1.0 (s ⁇ l) .
  • one may use a CL having a K,-j ⁇ lOnM and a K 0 ff ⁇ 0.01 (s * ⁇ l) .
  • a weaker- binding CL may be used (e.g., having a K within about lO ⁇ M- lOO ⁇ M, and an off-rate within about 1.0 (s _1 ) ⁇ K off ⁇ 1.0 (s -1 ) ) , if used at a higher concentration than used for a moderate-to-tight-binding CL, so as to compensate for the weaker affinity to target.
  • the methods of the invention use conventional capillary electrophoresis instruments as known in the art. These may use a capillary or a microchip having a plurality of conduits, in each of which CE can be performed.
  • the CE capillary has a length within a range of about 0.5-1000 cm, and a diameter within a range of about 10 ⁇ m-250 ⁇ m.
  • Typical microchip dimensions are in a range of about 0.5-20 cm, and have conduit diameters in a range of about 10 ⁇ m-250 ⁇ m.
  • a variety of buffer conditions and other factors can be adjusted to achieve a desired sensitivity in the methods of the invention - for instance, to favor detection of tighter-binding candidate ligands having a K d ⁇ 100 ⁇ M, preferably K d ⁇ 10 ⁇ M, even more selectively K d 10nM.
  • Factors that can be varied include, but are not limited to: capillary length; the distance between the CE starting point and the detector; CE run time; voltage and temperature during CE; and buffer composition, such as its pH and/or salt concentration (i.e., ionic strength).
  • Buffer pH values may be within a range of about pH3-10, preferably pH 5-8.
  • Capillary electrophoresis voltages may be within a range of about 5-30kV.
  • CE running buffer e.g., NaCl or KC1
  • a range of about 0-500mM advantageously within 10-100 mM, particularly for proteinaceous targets.
  • a CE run time may be within a range of about 2.5-10.0 minutes.
  • a distance between a capillary electrophoresis start point and the detector may be within a range of about 0.5-1000 cm.
  • the temperature at which CE is performed can be varied, generally within a range of about 0-60 °C, advantageously 5-37°C. Higher temperatures (e.g., about 25-60°C) can be used to limit the sensitivity of the present screening assay to more tightly binding candidate ligands. Tight-binding ligand/target complexes are generally more stable at higher temperatures, thereby dissociating to a lesser degree than weak ligand/target complexes at the same temperatures.
  • the present invention can be used in tandem with, or compared against, other capillary electrophoretic methods for screening complex biological materials, such as those found in Hughes et al . , U.S. Patent No. 5,783,397, wholly incorporated herein by reference.
  • the present methods can be used to determine the relative binding strength or affinity of a candidate ligand in a screened sample, if the concentration of the candidate ligand is known.
  • the relative affinity can be determined by varying, over several CE runs, the amount of time before the CL plug contacts and mingles with the TG/sample plug, and observing, measuring, and comparing the relative areas of the observed CE peaks (representing unbound CL and TG/CL complex) in each run.
  • the present screening method can be coupled with fractionation, purification, and/or analytical techniques (such as liquid chromatography or mass spectrometry) , as known in the art, in order to isolate and to characterize a candidate ligand detected in a complex material sample by the screening method.
  • analytical techniques such as liquid chromatography or mass spectrometry
  • FIGS. 1 and 2 show overlays of several electropherograms from screening several samples according to the method of the invention, with each sample having a different concentration of a candidate ligand.
  • the X-axis represents the time elapsed from the start of the CE run
  • the Y-axis represents the relative fluorescence signal (i.e., amount) of the CL detected by the detector of the CE instrument.)
  • the X-axis and Y-axis are offset for each electropherogram, in order to visualize the height differences in the unbound CL peak and CL/TG complex peak observed under the various conditions .
  • Figure 1 demonstrates an experiment where 50 nM of a target, the anticoagulant protein, thrombin (TG) , is first incubated, prior to CE, for 5 minutes with different concentrations of a test candidate ligand: the tight-binding synthetic ligand, hirulog-1 (D-Phe-Pro-Arg-Pro- (Gly) 4 -Asn- Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu; K d ⁇ 2 x 10 "9 M) .
  • a test candidate ligand the tight-binding synthetic ligand, hirulog-1 (D-Phe-Pro-Arg-Pro- (Gly) 4 -Asn- Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu; K d ⁇ 2 x 10 "9 M) .
  • the mixture of target and candidate ligand (TG/LG) is first injected into the CE instrument for about ten (10) seconds to give a first plug, followed by a 10-second injection of FCL as the second plug.
  • the thrombin target (TG) is incubated, prior to CE, for five minutes with samples containing hirugen and hirulog-1 at different concentra- tions.
  • Each of these target/sample mixtures is subjected to a CE screening run, as follows.
  • a plug of target/sample mixture is pressure-injected into the CE instrument for 10 seconds to give a first-injected plug.
  • a second injection plug of 100 nM FCL is then injected for 10 seconds, and then voltage of about 20kV is applied to start electrophoresis.
  • the CE migration of the FCL and FCL/TG complex are monitored by way of laser-induced fluorescence. Again, the FCL has a higher electrophoretic mobility than the TG, so that the unbound FCL peak appears before that of the FCL/TG complex (see Figure 2) .
  • the method detects the tight-binding candidate ligand, hirulog.
  • the hirulog-1/TG complex is sufficiently strong to remain together during the CE run until the FCL catches up to the target. Due to the stability of the hirulog-1/TG complex, there is less TG available to bind to the FCL. This results in a decrease in the FCL/TG peak. As seen in the electropherograms, the hirulog-1 signal is detectable even in the presence of up to 1,000-fold molar excess of the weak-binding hirugen.
  • the invention thus provides a method for determining which screened samples contain low concentrations of tight-binding compounds in the presence of high concentrations of weak-binding compounds.
  • This method is advantageous for screening complex mixtures such as natural extracts, which often contain such a mix of candidate ligands.
  • Some conditions that may be varied include, but are not limited to, sample concentration or dilution amount, buffer pH and/or salt concentration, CE voltage, capillary temperature, and the interval of time or distance between the first and second plugs.

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Abstract

L'invention concerne un procédé basé sur l'électrophorèse capillaire permettant de cribler des matières complexes à la recherche de tout ligand d'affinité non identifié liant une cible d'intérêt. Ledit procédé consiste à soumette un tampon constitué d'un mélange de la cible et de l'échantillon de matière complexe, et un tampon séparé constitué d'un ligand compétitif à liaison renforcée connu à une électrophorèse capillaire dans des conditions optimisées visant à permettre le mélange des deux tampons au cours de l'électrophorèse capillaire. De préférence, on surveille la migration de ce ligand compétitif.
PCT/US2000/017490 1999-06-24 2000-06-23 Procede d'electrophorese capillaire destine au criblage de ligands d'affinite utilisant un ligand competitif detectable WO2000079260A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP00944872A EP1188049A1 (fr) 1999-06-24 2000-06-23 Procede d'electrophorese capillaire destine au criblage de ligands d'affinite utilisant un ligand competitif detectable
JP2001505179A JP2003502665A (ja) 1999-06-24 2000-06-23 検出可能な競合リガンドを使用した、親和性リガンドのスクリーニングに適したキャピラリ電気泳動法
CA002375798A CA2375798A1 (fr) 1999-06-24 2000-06-23 Procede d'electrophorese capillaire destine au criblage de ligands d'affinite utilisant un ligand competitif detectable
US10/018,233 US6837977B1 (en) 1999-06-24 2000-06-23 Capillary electrophoresis method for screening for affinity ligands using a detectable competitive ligand

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US14071099P 1999-06-24 1999-06-24
US60/140,710 1999-06-24

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EP1432987A2 (fr) * 2001-09-06 2004-06-30 Beckman Coulter, Inc. Essais homogenes a base de particules par electrophorese capillaire avec detection fluorescence induite par laser
US7074334B2 (en) 2001-05-23 2006-07-11 Klaus Wanner Method for determining the binding behavior of ligands which specifically bind to target molecules
US7672786B2 (en) 2003-07-02 2010-03-02 Sergey Krylov Non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM)—based methods for drug and diagnostic development
GB2481168A (en) * 2009-09-22 2011-12-14 Selcia Ltd Affinity capillary electrophoresis method for assessing a biological interraction a ligand/receptor pair such as G protein coupled receptor and its targets
CN111220676A (zh) * 2019-11-13 2020-06-02 上海药明生物技术有限公司 利用毛细管电泳技术检测含聚乙二醇的蛋白质样品纯度的方法
US12203130B2 (en) 2018-12-20 2025-01-21 Krylov Sergey N Binder selection using capillary electrophoresis

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WO1996033412A1 (fr) * 1995-04-20 1996-10-24 Perseptive Biosystems, Inc. Compositions, procedes et dispositif pour analyse par electroseparation ultrarapide
EP0848251A2 (fr) * 1996-12-16 1998-06-17 Beckman Instruments, Inc. Dosages homogènes en ligne utilisant l'électrophorèse
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US7074334B2 (en) 2001-05-23 2006-07-11 Klaus Wanner Method for determining the binding behavior of ligands which specifically bind to target molecules
EP1432987A2 (fr) * 2001-09-06 2004-06-30 Beckman Coulter, Inc. Essais homogenes a base de particules par electrophorese capillaire avec detection fluorescence induite par laser
EP1432987A4 (fr) * 2001-09-06 2005-04-06 Beckman Coulter Inc Essais homogenes a base de particules par electrophorese capillaire avec detection fluorescence induite par laser
US7179658B2 (en) 2001-09-06 2007-02-20 Beckman Coulter, Inc. Particle based homogeneous assays using capillary electrophoresis with laser-induced fluorescence detection
US7556932B2 (en) 2001-09-06 2009-07-07 Beckman Coulter, Inc. Particle based homogeneous assays using capillary electrophoresis with laser-induced fluorescence detection
US7672786B2 (en) 2003-07-02 2010-03-02 Sergey Krylov Non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM)—based methods for drug and diagnostic development
US8224582B2 (en) 2003-07-02 2012-07-17 Sergery Krylov Non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM)-based methods for drug and diagnostic development
GB2481168A (en) * 2009-09-22 2011-12-14 Selcia Ltd Affinity capillary electrophoresis method for assessing a biological interraction a ligand/receptor pair such as G protein coupled receptor and its targets
GB2481168B (en) * 2009-09-22 2012-06-27 Selcia Ltd Capillary electrophoresis method for assessing the binding of chemical compounds to biological targets
US12203130B2 (en) 2018-12-20 2025-01-21 Krylov Sergey N Binder selection using capillary electrophoresis
CN111220676A (zh) * 2019-11-13 2020-06-02 上海药明生物技术有限公司 利用毛细管电泳技术检测含聚乙二醇的蛋白质样品纯度的方法
CN111220676B (zh) * 2019-11-13 2022-11-29 上海药明生物技术有限公司 利用毛细管电泳技术检测含聚乙二醇的蛋白质样品纯度的方法

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JP2003502665A (ja) 2003-01-21
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