JP2003502665A - Capillary electrophoresis for affinity ligand screening using competing detectable ligands - Google Patents

Abstract

  (57) [Summary] The present invention relates to capillary electrophoresis-based methods for screening composites for unspecified affinity ligands that bind to a target of interest. The method combines a plug that is a mixture of target and composite material sample and a plug of a known tightly binding competing ligand under conditions that are optimized such that the two plugs mix during the capillary electrophoresis run. Apply to capillary electrophoresis. Preferably, the movement of the competing ligand is tracked.

Description

Detailed Description of the Invention

CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority from United States Patent Application No. 60 / 140,710, filed June 24, 1999, all of which is incorporated herein by reference. is there.

[0002]   Federal support for research and development None

BACKGROUND OF THE INVENTION The present invention relates to the field of capillary electrophoresis-based substance screening for unspecified compounds that can bind to a target molecule of interest.

Developing screening methods to identify new, biologically active compounds has been
Complex substances, especially “complex biological substances”: can be a particularly specific and difficult endeavor when screening substances that have an effect on biological systems. Examples of complex substances include naturally occurring biological substances, natural products or extracts; nuclide biological regulators; chemical mixtures; libraries of pure compounds; and synthetic compounds such as combinatorial libraries. However, the present invention is not limited to these. A major screening issue is the detection of candidates that find compounds that bind to the target molecule of interest, especially the low concentration of the candidate in the screening sample; the potential for interference with screening reagents. With regard to certain unknown compounds; and to determine if the screening sample is worth further study. In addition, if one or more weakly competing binders are present in high concentration, the signals from low-concentration or strongly binding affinity ligands present in the same sample may be masked.

[0005]   Thus, novel bioactive compounds and potential regulatory compounds, especially of important metabolic pathways,
Rapid, suitable for discovering compounds that bind to essential or disease-related molecules
And there is still a need for economically effective screening tools. Furthermore,
Are identified as containing potential candidate ligands and / or candidate ligands are detected.
A prioritized method for further characterization and testing of samples of material
There is also a demand for it. The present invention is a novel bioactive compound that can be a candidate.
This demand is met by detecting known or unspecified ligands. Specifically, the present invention
Is the binding strength or dissociation constant (eg K_d≦ 100 μM, preferably K _d A known, preferably detectable, competing ligand with ≤10 μM)
And better identify ligands with relative avidity at or above the desired threshold
Provide the means. Identifying and grading samples containing these ligands
To further isolate and characterize the resulting compound.
Saves time and resources. Most stable ligands are therapeutic, regulatory and / or diagnostic
It is more effective and valuable as a compound and drug.

SUMMARY OF THE INVENTION The present invention provides candidate affinity ligands (eg, disease-related molecules) that bind to a target molecule of interest, such as naturally occurring complex biological materials, chemical mixtures, synthetic compounds and other substances. A method of identifying new biologically active compounds for Such candidate ligands have potential for regulatory, pharmaceutical, therapeutic and / or diagnostic applications. The method of the present invention combines competitive binding candidates with capillary electrophoresis (CE) to detect unidentified candidate ligands that bind the target in the screening sample with a certain selective strength or greater, and Characterize the relative binding strengths of the ligands. The method is particularly suitable for ligands that bind moderately to strongly (eg preferably dissociation constant K _d ≦ 100 μM, preferably ≦ 10 μM or even ≦ 10
nM).

The method uses a target molecule (TG) of choice and a known, preferably a moderate to strong binding ligand (CL) that binds the target (TG) and competes with CL for binding to TG. Including a sample potentially containing a candidate ligand (LG), eg, complex biomaterial. In general, the plug of the mixture of TG and sample and the other plug of CL are in unbound conditions and / or under optimized conditions for detecting migration of selected molecules that have bound to form a complex. The bottom is subjected to capillary electrophoresis. Preferably, the molecule to be detected or traced is a known CL and must be detectable in CE, eg by fluorescence or absorption. Tracking the CL at the detection point in the CE device provides a capillary electrophoresis migration pattern or profile of the CL. The migration pattern includes at least one member from the group consisting of a peak indicating unbound competing ligand and a peak indicating a complex of competing ligand bound to the target. Preferably both peaks are detectable and do not contain a candidate ligand.

More specifically, the invention includes methods of screening composite materials for unidentified candidate ligands that bind to a given target of interest (eg, those involved in a disease or disorder). The method comprises the following steps: (a) providing a mixture of the composite material and a target at a predetermined concentration, and further at a predetermined concentration of a known detectable competing ligand that binds to the target (preferably K _d ≦ 100 μM, More preferably, a known ligand having a dissociation constant of K _d ≦ 10 μM) is separately provided; and (b) in a capillary electrophoresis apparatus having a detector (preferably a fluorescence detector),
A step of sequentially performing a first plug of the analyte and a second plug of the analyte, the first and second plugs of the analyte comprising a combination selected from the group consisting of: (i) a target / A combination of the first plug of the analyte which is the sample mixture and the one plug of the analyte which is the competitive ligand; and (ii) the first plug of the analyte which is the competitive ligand and the analyte which is the target / sample mixture. A combination of second plugs; (c) the first and second plugs are optimized for detecting at least one member selected from a plug containing uncompetited ligand and a competing ligand complex bound to the target. Under conditions, the detectable analyte from the second plug migrates toward the detector faster than the detectable analyte from the first plug, resulting in a cap A step of subjecting the second plug to capillary electrophoresis optimized to pass the first plug before a detectable analyte reaches the detector during reelectrophoresis; (d) competition at the detector Tracking the ligand and creating a capillary electrophoretic migration pattern; (e) determining whether the migration pattern obtained in step (d) differs from a reference standard, thereby the presence of a candidate ligand in the composite material. Steps to instruct.

[0009]   Other embodiments and details of the present invention are described below.

DETAILED DESCRIPTION OF THE INVENTION Advantageously, the present invention is directed to the detection of ligands that bind to a selected target, particularly medium to strong ligands, in a mixture that also contains high concentrations of competing weakly binding ligands. To enable. Moreover, the method does not require knowledge of the sample to be screened for the specific structure or concentration of the candidate ligands therein. The method also allows for screening assays that use targets that cannot be directly detected during capillary electrophoresis.

Generally, the method of the present invention is carried out as follows. Target of interest (TG
) Is a complex sample to be screened, and is mixed with a complex sample containing or not one or more potential or candidate ligands capable of binding to the target. Allow the target / sample mixture for a sufficient time; eg 0.5-30 minutes,
Incubation is preferably for 1-5 minutes to allow target and any candidate ligand (
Sometimes referred to as the "TG / LG complex"). At the same time binding to a known concentration of a known competing ligand (CL) -ie target, which can be detected during capillary electrophoresis (eg fluorescently labeled CL), preferably to moderate to strong binding (eg K _d ≦ 100 μM, more preferably Ligands with known dissociation constants of about ≦ 10 μM) are prepared separately. CL is the following capillary electrophoresis (CE)
Candidate LG in screening sample material around binding to target during step
Compete with.

After preparation of the target / sample material mixture and the detectable competing ligand (CL) solution, each plug is continuously injected into the capillary of the capillary electrophoresis apparatus. The order of injection depends on the relative CE migration rates of CL and TG under the selected CE conditions. Even when the method is carried out, capillary electrophoresis that imparts charges to both CL and TG (
CE) Use running buffer. A running buffer is optionally provided between the plugs to separate the TG / sample and CL plugs. Generally, the higher the affinity of the candidate ligand that has to be detected, the larger the injection amount of the running buffer between the plugs. That is, the relative affinity of the candidate ligand was determined by repeating the screening protocol using each ligand sample and changing the amount of running buffer injected between the first and second plugs (ie, the second plug was moved). Change the distance, that is, the time interval until the two plugs mix)
Can be determined by.

After injecting the TG / sample and CL plugs, the TG and CL are directed toward the detector and a suitable amount of voltage is applied to induce movement through. In order to perform this screening method, the detectable analyte from the second injected plug during CE is faster than the detectable analyte from the first injected plug.
The detectable analyte from the second plug is moved to the fluorescence detector attached to the device, so that the detectable analyte from the second plug comes first before reaching the detection point in the capillary. It is important to choose CE conditions to overtake and mix the detectable analytes from the plug.

The term “detectable analyte” refers primarily to competing ligands (CL) and targets (TG).
Not only is it meant the candidate ligand (LG) derived from the sample. CL is detected directly by the detector, while TG is detected indirectly by its binding to CL, giving a shift of the moving peak of CE, which represents the CL / TG complex. The presence of LG is detected indirectly via interference with CL / TG complex formation, as discussed elsewhere in this disclosure. Those skilled in the field of capillary electrophoresis will recognize that the detectable analyte from the second plug must have a higher capillary electrophoretic mobility towards the detector as compared to the detectable analyte from the first plug. right.

For example, when the CL analyte plug is injected as the second plug after the first plug containing the target (TG), unbound CL migrates faster than unbound TG, resulting in later injection. It is important that the CL analyte molecule from the second plug overtakes the TG-containing plug and that free or unbound TG molecule and CL can form a complex during CE. The TG-containing first plug could be the TG / sample plug (for screening protocols) or the target alone plug (for measuring control or reference standards).

If the CL plug is first injected before the second plug containing TG, the TG analyte has a higher capillary electrophoretic mobility than the CL analyte, and consequently contains TG. It is important that the TG analyte from the second plug overtakes and interacts with the analyte from the first plug containing CL.

Preferably, the known detectable competing ligand is captured by the detector during CE,
The movement is tracked to create a capillary electrophoresis movement pattern or profile. The CE migration pattern contains at least one if not both of the following: peaks for unbound (free) CL and peaks for CL / TG complex. For example, CL can be fluorescently labeled so that CL and CL / TG peaks can be monitored by laser-induced fluorescence detection. Unbound CL and bound CL / in CE
While it would be beneficial to utilize CE conditions that allow the observation of both TG complexes, only one or the other peak in the CE profile need be observed for the performance of this screening method. In most screens performed according to the invention, two capillary electrophoresis peaks will be observed; one for CL / TG.
Corresponding to the complex and the other to either unbound CL.

The concentration of CL and TG in each plug is usually optimized to provide a detectable percentage of CL bound to TG in the absence of other target binding ligands in CE, as discussed in detail below. Has been done. Those skilled in the art will appreciate that T without other ligands
It will be readily understood what TG concentration ratio to CL is used to obtain the desired capillary electrophoresis profile or migration pattern that corresponds to the desired amount of CL complexed with G. For example, if the target has a single CL binding site and is a detectably tightly binding competing ligand, it is desirable that at least 90-95% of the CL bind to TG in the absence of the other ligand. If you do
It is preferred to use equimolar concentrations for each.

In general, in order to practice the method of the invention with the aim of screening complex materials for novel affinity ligands, known competing ligands are detectable in CE and the target of choice. Stably binds to form an LC / TG complex,
The resulting migration pattern of CE must be different from the CE profile of unbound CL alone. The formation of the CL / TG complex must result in at least one detectable decrease in the area of the unbound or free CL peak and can occur in another, but not absolute, CL / TG peak. preferable. Although not absolute, preferably the CL and target concentrations and CE run time are sufficient for the CL plugs to mix with the target-containing plugs during CE, and either or both plugs have CE.
It is preferable that it is the time to reach the detection point in the device. At least C during CE
The concentration and time at which L and the target can interact are sufficient to form a target / CL complex in which all the target and CL have a detectable migration peak that is distinguishable from the migration peak of unbound CL. There must be. In other words, a given target concentration, a given competing ligand concentration, and capillary electrophoresis conditions are such that a measurable change in the capillary electrophoretic migration pattern produced by the method steps in the absence of other target binding ligands will result. To be selected. The measurable change is at least 10% in the peak region of at least one peak selected from the group comprising a peak representing an unbound competitive ligand and a peak representing a complex of a competitive ligand bound to a target,
Preferably it comprises at least 50%, conveniently at least 75%.

Candidate affinity ligands (LGs), particularly moderate to tightly bound LGs (eg K _d ≦ 100 μM, preferably ≦ 10 μM), are detectable when present in the sample of complex material. The peaks of free or unbound CL will increase (compared to migration patterns obtained from control CE measurements of TG and CL plugs alone in the absence of other target binding ligands). Therefore, the detectable CL / TG complex peak will have a reduced area compared to the migration pattern from the reference or control CE measurements. This is because LG in CE maintains binding to TG, CL cannot bind to TG, and the CL plug moves through the plug of the mixture of samples containing TG and LG. Although it is beneficial to detect both peaks, it is required to observe only one of the peaks in CE, preferably the peak of unbound or free CL.

When screening samples containing candidate LG, the reduction of CL / TG complex peaks depends on both the affinity of LG for TG and the concentration of LG in the biological sample. Thus, CE conditions, including the time that allows interaction between the TG and CL plugs, can be adjusted to facilitate detection of the presence of LG that binds to TG with more than the desired strength or affinity.

In order to detect weakly binding ligands, or low concentrations of candidate ligands, CE conditions are controlled to limit the time of interaction between the tightly bound CL plug and the TG containing plug, Assay sensitivity will be adjusted so that at least a portion of the TG / weakly bound IG complex formed can be detected. Reducing assay sensitivity, and thereby limiting the detection of only tightly bound targets, is achieved by increasing the time it takes for the CL to contact the target (TG / sample plug and CL plug injection). By further increasing the spacing of or by focusing on the charge-to-mass ratio corresponding to TG and CE and selecting the appropriate CE conditions). Such an increase in time would give the weakly binding LG / TG complex more time to dissociate before interaction of TG and CL during CE. That is, the target is free to bind to the competing ligand. When following CL, this would result in a larger CL / TG complex peak and / or a reduction of unbound CL peak in CE (indicating the absence of tight binders in the screening sample).

By following the migration of detectable CL during capillary electrophoresis, the present invention allows for convenient screening of candidate ligands for targets that are not themselves detectable during capillary electrophoresis. Such undetectable targets are, for example, membrane-bound protein receptors that are not easily purified or are relatively insoluble.

The reference standard for this method was to use the same injection sequence and capillary electrophoresis conditions as the protocol for screening a sample of composite material, and to continuously inject target plugs and CL plugs containing no composite material. Including the migration pattern of CL generated by capillary electrophoresis.

Detection methods are known to those of skill in the art of capillary electrophoresis and include, but are not limited to, fluorescence, UV absorption and the like. A convenient and convenient detection means is CE
The use of fluorescent labels on the molecules that are tracked in.

The methods of the invention can be used to easily and rapidly detect, identify and characterize tightly bound ligands or compounds of interest from among those in a sample of complex biological material (eg, natural Mid-autumn or synthetic compound mixture). This method successfully and selectively detects low concentrations of the compound of interest that bind tightly to the desired target of choice, even when the screening sample contains high concentrations of weakly binding candidate ligands or entities. It is especially useful to do.

Prior to carrying out the screening according to the invention, all CE conditions have to be optimized together to detect the ligand of interest with an affinity in the desired range, as known by the person skilled in the art of capillary electrophoresis. I won't. Criteria must be established to determine what is the weakly binding compound of interest (WB) and what is the ligand with a strong binding affinity. The cut-offs that determine the relative binding strengths of different ligands or compounds of interest are mainly capillary length, injection site to detector length, voltage and temperature during CE, buffer composition (eg pH and / or salt). Concentration). Even strong binders can be found by performing CE at elevated temperature (eg> 25 ° C).

The methods of the invention are particularly useful for identifying candidate compounds of interest in a screening sample that have binding strengths above a selected threshold and determining their relative binding strengths. "Medium to tightly bound" and "weakly bound" ligands have faster off-rates ( _Koff ) and higher dissociation constants (KD), and have little or no association in capillary electrophoresis. It forms a non-retained target / ligand complex, a target / ligand complex that is unstable and rapidly dissociates before contacting CL and before reaching the detector. In contrast, more strongly or tightly bound ligands have lower dissociation constants and slower off-rates, and they generally bind even when they come into contact with CL during capillary electrophoresis and pass through the detector. Form a target / ligand complex that maintains Typical ligands with each binding strength have the properties shown in Table 1.

[0029]

[Table 1]

After the capillary electrophoresis conditions are selected, the screening itself is performed with a ligand / target complex and a strong ligand / target complex with a low dissociation rate of the candidate ligand / target complex.
Take advantage of the fact that it differs from the target. That is, optimizing CE conditions completely or fully dissociates the weak candidate ligand / target complex formed during pre-CE incubation of the sample with the target, and the target molecule in CE for binding to a known competing ligand. Can be sufficiently free and detectable. At the same time, this same optimized CE condition keeps the strong candidate ligand / target complex formed during the pre-CE incubation bound, reducing the availability of free target and thereby the TG / CL complex. Formation can be reduced. As a result, the changes that occur in the CE migration patterns of known competing ligands indirectly indicate the presence of tightly bound candidates.

One of the method embodiments has a weakly binding ligand (eg, K _d > 100 μM).
Which has a dissociation constant of about 100 μM), and strongly bound ligands (for example, having a dissociation constant of about K _d ≦ 100 μM, preferably ≦ 10 μM).
The target and material samples are first mixed and incubated together to allow binding of the target to the candidate ligand (LG) present in the sample. Then, (1) plug of target / sample mixture (including unbound TG, unbound LG, and LG./TG complex); and (2) continuous injection of plug containing CL into the capillary. Be seen. The order of injection will be determined by the known relative CE mobilities of CL and TG. In some examples, CL plugs are injected first; in others, target / sample plugs are injected more and more. In some examples, a CE running buffer plug is injected between the TG / sample plug and CL plug.

An exemplary screen according to the invention that tracks CE migration of competing ligands uses the following steps with the following results: A mixed plug of target and sample composite material is injected into the capillary electrophoresis apparatus. In this example, TG is negatively charged and has a relatively slow CE mobility. 2. Then, the CL plug is injected. In this example, CL is negatively charged and TG
Has a higher CE mobility than 3. Target and CL are directed towards a detector mounted on the CE device, whether unbound (TG) or bound (LG / TG complex) to a candidate ligand in the sample when a voltage is applied. Moving. The detector is a fluorescence detector. 4. Since CL has high mobility, it starts catching up with TG which is moving slower during CE traveling. 5. TGs dissociated from the TG / LG complex during CE running are free to associate with CL as the CL plug overtakes the TG-containing plug. TG that maintains the binding with LG does not participate in the binding with CL. 6. CL concentration and CE conditions (including relative time until CL and TG / sample plugs are mixed) are those in which the LG / TG complex is moderately or weakly bound (eg, dissociation constant K _d ≧ 100 μM, preferably ≧ 10 μM). If CL is TG
It can be set up to dissociate most of the weak LG / TG complex before catching up with. Free or unbound TG binds to CL and is large, detectable CL / TG
Peaks and / or reduced free or unbound CL peaks are provided. 7. If the LG / TG complex was moderately to tightly bound (eg K _d ≦ 1 μM), the complex would be united during CE transit and even less free TG could bind to CL. That is, the CL / TG peak will be lower and the free CL peak will be larger in the absence of LG compared to the control. 8. That is, the decrease in the CL / TG complex peak and the increase in the free CL peak indicate that there is at least one strongly binding candidate ligand (LG) bound to the target, which interferes with CL binding. It is shown that. Higher CL / TG signals and / or lower free CL peaks indicate the presence of weakly binding ligands.

Exemplary composite materials that can be screened by this method include, but are not limited to, naturally occurring composite biological materials, chemical mixtures and synthetic compounds such as combinatorial libraries. Naturally occurring complex biomaterials include natural extracts, which include combinatorial chemical substance libraries, terrestrial plant extracts, marine plant extracts, cells derived from higher animals including humans, eubacteria, actinomycetes,
Bacteria, extracts derived from non-recombinant or recombinant microorganisms, microbial fermentation broth, fungi, protozoa, algae, archaea, worms, insects, marine organisms, sponges, corals, crustaceans, viruses, phages, tissues, organs, It is selected from the group comprising blood, soil, seawater, freshwater water, humus, organic sediments, organic fertilizers, mud and sewage, or partially purified fragments thereof. The composite material is diluted and / or fragmented before it is subjected to screening by this method.

Examples of targets that can be used in the method of the present invention include enzymes, receptors, proteins, polypeptides, nucleic acids, polynucleotides, carbohydrates and their forms that are chemically, enzymatically or recombinantly modified. In addition, the modified shape is changed and the electrophoretic property is improved, but is not limited thereto. The target molecule need not be in purified form and can be provided, for example, as part of a cell extract or sonicate, as long as the target itself is available for binding known competing ligands. For example, the target may be a G-protein coupled receptor (GPCR) immobilized within the cell membrane.

Examples of known CE-detectable competing ligands (CL) include naturally occurring compounds,
It includes, but is not limited to, synthetic compounds, antibodies, proteins, peptides, oligonucleotides and other drugs known to bind to a target of interest. Competing ligands will be modified as necessary to provide the molecule with the desired charge. Preferably, CL is strongly bound from the inside, its dissociation constant is about K _d ≦ 10 μM, and the off rate is about K _off ≦ 1.0 (s ⁻¹ ). Conveniently, K _d ≦ 10
CL of nM and K _off ≦ 0.01 (s ⁻¹ ) may be used. When used at a concentration higher than that used for CL that strongly binds from the inside for the purpose of compensating for the weak affinity for the target, weak binding CL (for example, K _d of about 10 μm is used).
M-100 μM, and the off speed is about 1.0 (s ⁻¹ ) ≦ K _off ≦ 1.
0 (s ⁻¹ ) range) can be used.

The method of the present invention uses a general capillary electrophoresis apparatus known in the art.
These would use capillaries or microchips with multiple conduits capable of performing CE in each. Preferably, the CE capillaries are about 0.5-10.
It has a length in the range of 00 cm and a diameter in the range of about 10 μm-250 μm.
Typical microchip dimensions are about 0.5-20 cm, and about 10 μm.
It has a conduit diameter in the range of −250 μm.

As mentioned above, it is possible to obtain the desired sensitivity in the method of the invention by adjusting various buffer conditions and other factors-for example K _d ≦ 100 μM, preferably K _d ≦ 10 μM, even more selected. In general, it is possible to obtain favorable sensitivity for the detection of more tightly bound candidate ligands with K _d ≦ 10 nM. Variables include: capillary length; distance between CE start point and detector; CE transit time; voltage and temperature during CE; and its pH and / or buffer such as salt concentration (ie ionic strength). Compositions include, but are not limited to. Some examples are given below, but additional modifications will be apparent to those skilled in the art of capillary electrophoresis in view of the present disclosure.

The buffer pH value will be in the range of about pH 3-10, preferably pH 5-8. The capillary electrophoresis voltage will be in the range of about 5-30 kV. The salt concentration of the CE running buffer (eg, NaCl or KCl) is, for example, about 0-500 mM.
, Conveniently in the range of 10-100 mM, especially with regard to proteinaceous targets. Higher salt concentrations (eg, in the range of about 200 mM-1M) tend to help stabilize the target / ligand complex. Therefore, the use of lower salt concentrations is advantageous when detecting candidate ligands that exhibit stronger or stronger binding than those that exhibit weaker binding.

Increasing the CE transit time before contact between the TG-sample plug and the CL plug is also advantageous for detecting candidate ligands that show tighter binding. For example, CE transit time may be in the range of about 2.5-10.0 minutes. Similarly, the distance between the start of capillary electrophoresis and the detector will be in the range of about 0.5-1000 cm.

The temperature at which CE is carried out can also be varied and is generally in the range of about 0-60 ° C, conveniently 5-37 ° C. Use higher temperatures (eg about 25-60 ° C),
It is also possible to limit the sensitivity of the screening assay to candidate ligands that bind more tightly. Tightly bound ligand / target complexes are generally stable at high temperatures, and therefore at a lower rate they dissociate compared to weaker ligand / target complexes.

The present invention is directed to other capillary electrophoresis methods suitable for screening complex biomaterials, as seen in Hughes et al., US Pat. No. 5,783,397, which is incorporated by reference in its entirety. Can be used in series or compared.

Furthermore, if the concentration of the candidate ligand is known, the present invention can be used to determine the relative binding strength or affinity of the candidate ligand in the screening sample. Relative affinity was determined by varying the time before the CL plug contacted the TG / sample plug with multiple CE runs and mixing, and the relative area of the CE peaks observed in each run (unbound CL and TG). / CL complex) is observed, measured, and compared.

Furthermore, the person skilled in the art will combine the screening method with fragment, purification and / or analytical techniques known in the art (liquid chromatography or mass spectrometry),
It will be appreciated that screening methods can be used to isolate and characterize candidate ligands that are detected in composite material samples.

[0044] EXAMPLES The present invention is further described by the following non-limiting examples, the results of the CE are illustrated in Figures 1 and 2. 1 and 2 show a plurality of electrophoretic images in which a plurality of samples were screened by the method of the present invention, each sample containing a different concentration of a candidate ligand. The X-axis represents the elapsed time from the start of CE travel and the Y-axis represents the relative fluorescence signal (ie amount). However, X axis and Y
The axes are offset in each electrophoretic image to show the difference in height between the unbound CL peak and the CL / TG complex peak observed under various conditions.

Example 1 FIG. 1 shows a target of 50 nM, ie, the anticoagulant protein thrombin (TG), for 5 minutes prior to CE, at various concentrations of test candidate ligand: strong binding synthetic ligand, hirulog-1. -1) (D-Phe-Pro-Arg-Pro- (Gly
) 4-Asn-Gly-Asp-PHe-Glu-Glu-Ile-Pro-G
lu-Glu-Tyr-Leu; Kd ~ 2 x ^10-9 M). Strongly binding competitive ligand that is also fluorescently labeled, single chain (ss) D
NA oligonucleotide 5'-fluorocein-GGTTGGGTGTGGTTTG
G-3 ′ (K _d = 3 × 10 ⁻⁹ M; hereinafter “FCL”) was adjusted. A mixture of target and candidate ligand (TG / LG) was first injected into the CE device for 10 seconds to provide the first plug, followed by a second plug of FCL for 10 seconds.

A voltage of about 20 kV is applied to start electrophoresis, and unbound competing ligand (FCL)
The peak of the bound FCL / TG complex was monitored in CE using laser-induced fluorescence and a fluorescence detector located at the detection point along the capillary.

Referring to the electrophoretic image shown in FIG. 1, hirolog-1 in the target / sample mixture
When (hirolog-1) is not present, a large FCL / TG complex peak is observed in addition to the peak of unbound (free) FCL. Increasing the concentration of the test candidate ligand, hirulog-1, decreased the peak of the FCL / TG complex and showed unbound F.
The amount of CL increases. This is because Hilllog-1 bound to the target FCL binding site during pre-CE incubation and remained bound during CE running, allowing the ECL plug to catch up with the plug in the TG / sample mixture and bind FCL when interacting. This is because the amount of unbound TG has decreased. 50 equal to the concentration of TG in the sample
When nM of Hilllog-1 is present, obviously all TGs are bound to Hilllog-1, resulting in the peak of the FCL / TG complex disappearing completely and the peak of unbound FCL appearing. did.

Example 2 FIG. 2 shows weakly binding test candidate ligands hirugen (hirudin Gly-54-Leu-65 with sulfated tyrosine).
The experiment shows that the test candidate ligand, which binds tightly, hirulog-1, was detected even when the carboxyl terminal degree decapeptide; Kd = 1.5 × 10 ⁻⁷ M) was present at a high concentration. Here, thrombin target (TG) is incubated with CE for 5 minutes with samples containing various concentrations of hirugen and hirulog-1 prior to CE.
Each of these target / sample mixtures is subjected to CE screening as follows. Inject the plug of the target / sample mixture into the CE device for 10 seconds,
Give the first plug. Then inject a second plug of 100 nM FCL for 10 seconds,
Then, a voltage of about 20 kV is applied to start electrophoresis. CE migration of FCL and FCL / TG complexes is monitored by laser induced fluorescence. Also in this case FCL
Shows higher mobility than TG, the peak of unbound FCL is FCL / T
It appears before the peak of the G complex (see Figure 2).

In the absence of hirugen or hirulog-1, FC moves faster during CE
When L overtakes the slowly moving TG and FCL passes the TG, FCL /
A TG complex is formed. This results in an electrophoretic profile similar to that of FIG. 1, top (FIG. 2, top): both unbound FCL and FCL / TG peaks are observed.

Incubation of the thrombin target with 10,000 nM hirugen prior to CE makes no difference in the CE profile of the competing ligand (FCL). This is because hirugen is very weak and most or all of the hirugen / TG complexes formed during precapillary cubation dissociated during CE running before the FCL caught up with the TG and made contact. In other words, under these conditions, the method is not sensitive to the relatively weakly bound ligand, hirugen.

However, the method detects Hilllog-1 which is a tightly bound candidate ligand.
The Hilllog-1 / TG complex is strong enough to maintain the complex during CE until FCL catches up with its target. Due to the stability of the Hilllog-1 / TG complex, less TG can bind to FCL. As a result, the FCL / TG peak decreases. As seen in the electropherograms, the Hilllog-1 signal can be detected even in the presence of up to 1,000-fold molar excess of weakly bound hirugen.

As indicated, the invention provides a method for determining which sample to be screened contains low concentrations of strongly binding compounds in the presence of high concentrations of weakly binding compounds. This method is advantageous when screening complex mixtures, such as natural extracts, which often contain such mixtures of candidate ligands.
The relative affinity of the ligand for the target can also be estimated by adjusting the conditions, repeating the screening method under different CE conditions, and comparing the electrophoretic images obtained. Variable conditions include, but are not limited to, sample concentration or dilution, buffer pH and / or salt concentration, CE voltage, capillary temperature and the time interval or distance between the first and second plugs.

Although the present invention has been described in connection with exemplary embodiments, those skilled in the art can read the above specification to perform the methods described herein without departing from the spirit of the invention as defined by the claims. Various changes or equivalent replacements could be made. Accordingly, the patent protection afforded by the patents issued herein is limited only by the appended claims and equivalents thereof.

[Brief description of drawings]

FIG. 1 shows a plurality of electrophoresis samples obtained by screening samples containing various concentrations of hirulog-1, a test candidate ligand that strongly binds to a target molecule, thrombin, using the competitive binding CE method of the present invention. A stack of images; and

FIG. 2 shows the results obtained by screening a plurality of samples containing various concentrations of two test candidate thrombin-binding ligands, weak-binding hirugen and strong-binding hirulog-1, using the competitive binding CE method of the present invention. It is a superposition of a plurality of electrophoretic images.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) // C12N 15/09 ZNA G01N 27/26 331K C12N 15/00 ZNAA F term (reference) 2G045 AA40 DA36 FB05 GC15 4B024 AA11 CA01 HA11 4B063 QA18 QQ21 QQ42 QQ79 QR56 QS16 QX02

Claims (33)

[Claims] 1. A method of screening a composite material for an unspecified candidate ligand that binds to a preselected target, comprising: (a) providing a mixture of a composite material and a target at a predetermined concentration, And (b) continuously injecting a first plug of the analyte and a second plug of the analyte into a capillary electrophoresis apparatus having a detector, which separately provides a known, detectable competing ligand that binds to the target. (I) a combination of the first plug of the analyte that is the target / sample mixture and the first plug of the analyte that is the competing ligand; and (ii) A) a combination of a first plug of the analyte that is a competing ligand and a second plug of the analyte that is the target / sample mixture; (C) under a condition optimized for detecting at least one member selected from a plug containing an unbound competing ligand and a competing ligand complex bound to a target, the second plug The detectable analyte from the first migrates towards the detector faster than the detectable analyte from the first plug, resulting in the second analyte before reaching the detector during capillary electrophoresis. Subjecting the first and second plugs to capillary electrophoresis under conditions where the plugs are optimized to overtake the first plug; (d) tracking the competing ligands with a detector, and capillary electrophoresis. Creating a migration pattern; (e) determining whether the migration pattern obtained in step (d) is different from the reference standard, thereby indicating the presence of a candidate ligand in the composite. And a method including the steps of. 2. The reference standard is: (a) using the same injection sequence and capillary electrophoresis conditions as used in claim 1, a predetermined concentration of target plug and competing ligand free of complex material. 2. A method according to claim 1, which comprises a migration pattern obtained by: (b) tracking the competing ligands in the detector; 3. The first plug is a target / sample mixture, and the second
The method of claim 1, wherein the plug is a competing ligand. 4. The method of claim 3, wherein the competing ligand has a faster capillary electrophoretic mobility in the detector direction as compared to the target. 5. The method of claim 1, wherein the first plug is a competing ligand and the second plug is a target / sample mixture. 6. The method of claim 5, wherein the target has a faster capillary electrophoretic mobility in the detector direction as compared to the competing ligand. 7. The migration pattern comprises at least one member selected from the group comprising a peak representing uncombined competing ligand and a peak representing a complex of competing ligand bound to the target. The method according to 3 or 5. 8. The target comprises a member selected from the group comprising enzymes, receptors, proteins, polypeptides, nucleic acids, polynucleotides, carbohydrates and their chemically, enzymatically or recombinantly modified forms. The method of claim 1, 3 or 5, wherein the modified shape is modified with respect to improving electrophoretic properties. 9. The complex material is a combinatorial chemical substance library, terrestrial plant extract, marine plant extract, cells derived from higher animals including humans, eubacteria,
Actinomycetes, bacteria, non-recombinant or recombinant microbial extracts, microbial fermentation broth, fungi, protozoa, algae, archaea, helminths, insects, marine organisms, sponges, corals, crustaceans, viruses, phages, tissues, 6. An organ, blood, soil, seawater, freshwater water, humus, organic sediment, compost, mud and sewage, or a group comprising partially purified fragments thereof, claim 1, 3 or 5 The method described. 10. The method of claim 1, 3 or 5 wherein said known competing ligand is selected from the group consisting of naturally occurring compounds, synthetic compounds, antibodies, proteins, peptides and oligonucleotides known to bind to a target. The method described in. 11. The method according to claim 1, 3 or 5, wherein the known competing ligand is detectable by a fluorescence detector. 12. A dissociation constant (K _d ) in which the competing ligand is in the range of about 10 μM-100 μM, and an off rate (K _d ) in the range of about 1.0 (s ⁻¹ ) -10 (s ⁻¹ ). The method of claim 1, 3 or 5 having a _Koff ). 13. The predetermined concentration of the competing ligand is at least about 5.0 μM.
13. The method of claim 12, wherein 14. The known competing ligand has a dissociation constant (K _d ) in the range of about 10 nM-10 μM and a range of about 0.010 (s ⁻¹ ) -1.0 (s ⁻¹ ). Method according to claim 1, 3 or 5, having a certain off-rate (K _off ). 15. The known competing ligand has a dissociation constant of about K _d ≦ 10 nM,
And having an off rate of about K _off ≦ 0.01 (s ⁻¹ ).
The method described in. 16. The predetermined target concentration, predetermined competitive ligand concentration, and capillary electrophoresis conditions are preselected to produce a measurable change in the capillary electrophoresis migration pattern in the absence of other target binding ligands. Claim 1
The method according to 3 or 5. 17. The peak area of at least one peak selected from the group comprising a peak representing an unbound competing ligand and a peak representing a mixture of competing ligands bound to a target. 17. The method of claim 16, comprising at least 10% change. 18. The peak area of at least one peak selected from the group comprising a peak representing an unbound competing ligand and a peak representing a mixture of competing ligands bound to a target. 17. The method of claim 16, comprising at least 50% change. 19. The peak area of at least one peak selected from the group comprising a peak representing an unbound competing ligand and a peak representing a mixture of competing ligands bound to a target. 17. The method of claim 16, comprising at least 75% change. 20. The measurable change is at least 10% change in peak area of a peak representing unbound competing ligand, and the peak area of a peak representing a mixture of competing ligands bound to a target. 17. The method of claim 16 including a change of at least 10%. 21. The capillary electrophoresis condition is about 10 μM-100 μM.
Optimum for detection of candidate ligands with dissociation constants (K _d ) in the range of, and off rates (K _off ) in the range of about 1.0 (s ⁻¹ ) -10 (s ⁻¹ ). The method according to claim 1, 3 or 5, which has been modified. 22. The capillary electrophoresis condition is a dissociation constant (K _d ) in the range of about 10 nM-10 μM, and about 0.01 (s ⁻¹ ) -1.0 (s ⁻¹ ).
6. The method of claim 1, 3 or 5, wherein candidate ligands having off-rates ( _Koff ) in the range are optimized for detection. 23. The capillary electrophoresis conditions are optimized to detect candidate ligands having a dissociation constant of about K _d ≦ 10 nM and an off rate of about K _off ≦ 0.01 (s ⁻¹ ). The method according to claim 1, 3 or 5, wherein 24. The method of claim 1, 3 or 5, further comprising injecting a plug of capillary electrophoresis running buffer between the first and second plugs. 25. The method according to claim 1, 3 or 5, wherein the capillary electrophoresis is carried out with a running buffer having a pH value in the range of about pH 3 to pH 10. 26. The capillary electrophoresis is performed with a running buffer having a pH value in the range of about pH5-pH8.
The method described in. 27. The method of claim 1, 3 or 5, wherein capillary electrophoresis is performed with a running buffer having a salt concentration in the range of about 0-500 mM. 28. The method of claim 1, 3 or 5, wherein capillary electrophoresis is performed at a temperature in the range of about 0-60 ° C. 29. The method of claim 1, 3 or 5, wherein the capillary electrophoresis is performed at a temperature in the range of about 5-37 ° C. 30. The method of claim 1, 3 or 5, wherein capillary electrophoresis is performed for a transit time in the range of about 0.5-60 minutes. 31. The method of claim 1, 3 or 5, wherein the distance between the starting point of the capillary electrophoresis and the detector is in the range of about 0.5-1000 cm. 32. The method of claim 1, 3 or 5, wherein the capillary electrophoresis is performed in a capillary having a length in the range of about 0.5-1000 cm. 33. The method according to claim 1, 3 or 5, wherein capillary electrophoresis is performed in the conduit of the microchip.