Classifications

• • 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.
• 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 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 .
• 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.
• 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
• 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).
• 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.
• 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 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.

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• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
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• Investigating Or Analysing Biological Materials (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

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• 2000
  • 2000-06-23 CA CA002375798A patent/CA2375798A1/en not_active Abandoned
  • 2000-06-23 JP JP2001505179A patent/JP2003502665A/ja active Pending
  • 2000-06-23 WO PCT/US2000/017490 patent/WO2000079260A1/en active Application Filing
  • 2000-06-23 EP EP00944872A patent/EP1188049A1/de not_active Withdrawn

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