JP2006078210A - Screening method of substance to be examined - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a screening method which enables the simple screening of the substance to be examined, which interacts with a ligand and high in both of a bonding speed and a dissociation speed, from a large number of substances to be examined. <P>SOLUTION: In the screening method for sorting a specific substance to be examined from the substances to be examined of one group based on the interaction of the ligand with the substance to be examined, the ligand is set to a solid phase, and a unit for setting the ligand to a solid phase state and a unit for measuring the interaction of the ligand set to the solid phase state and the substance to be examined are separated. An arbitrary dissociation speed constant (kd) of 1×10<SP>-2</SP>(S<SP>-1</SP>) or more and an arbitrary bonding speed constant (ka) of 1×10<SP>5</SP>(M<SP>-1</SP>S<SP>-1</SP>) are set as a sorting standard value to sort the substance to be examined wherein both of the measured dissociation speed constant and the bonding speed constant are reference values or above. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a screening method for a test substance that interacts with a ligand.

  Drugs often interact and function with proteins in vivo. For example, an inhibitor binds to an enzyme and inhibits the enzyme reaction, thereby changing a biochemical reaction in the living body and expressing a pharmacological action. For this reason, it is important to know the strength of binding between a protein and a drug candidate compound in drug search.

In recent years, the surface plasmon resonance method has attracted attention as a method of interaction between proteins and chemical substances. In this method, protein is solid-phased on a metal surface such as gold, and the interaction is quantified using a change in resonance when a chemical substance is donated here. The analyte molecule, which is a chemical substance, adsorbs over time to molecules that interact with the analyte molecule, such as proteins immobilized on the metal surface. The amount of adsorption, that is, the change of the SPR signal at this time changes according to the following equation.
dR / dt = ka ・ Cs ・ (Rmax-R)-kd ・ R Equation (1)
(Where R is the SPR signal, Rmax is the maximum SPR signal, ka is the binding rate constant, kd is the dissociation rate constant, and Cs is the concentration of the analyte molecule near the metal surface.)

  Here, Cs is constant under ideal conditions in which the metal surface is constantly replaced with fresh liquid, and ka and kd can be obtained from the measurement results by solving a simple differential equation. The association constant (KD) indicates the ratio of association to dissociation and is expressed in kd / ka.

  In searching for low molecular weight drugs, screening of at least 10,000 low molecular weight compounds is usually performed for one target protein. The hit compound group obtained as a result is subjected to further detailed evaluation and narrowed down to one lead compound.

  The measurement of the interaction between the protein and the test compound by the surface plasmon resonance method is used in the drug search process as described in Non-Patent Document 1, but the number of measurements per day is about 100 samples. Because of its low processing capacity, it was not used for large-scale screening as described above.

  ELISA and RI labeling methods are mainly used for large-scale screening, but these methods measure the amount of compound binding through a washing operation with a buffer after reacting the protein with the compound for a certain period of time. Therefore, it is difficult to select a compound that binds but has a high dissociation rate. On the other hand, as a pharmaceutical compound, from the viewpoint of side effects, there may be a case where a compound that binds rapidly under a certain compound concentration and rapidly dissociates when taking is stopped. Therefore, there has been a demand for a method for selecting a compound having a large binding rate and dissociation rate in large-scale screening.

BUNSEKI KAGAKU vol.51, No.6, pp.461-468 (2002)

  The present invention has been made to solve the above-described problems of the prior art. That is, the present invention solves the problem of providing a screening method for a test substance that interacts with a ligand, which can easily screen a substance having both a high binding rate and a high dissociation rate from a large number of test substances. It was a problem to be solved.

As a result of intensive studies to solve the above problems, the present inventors have determined that an arbitrary dissociation rate constant (kd) of ¹ × 10 ⁻² (S ⁻¹ ) or higher and an arbitrary dissociation rate constant of ¹ × 10 ⁵ (M ⁻¹ S ⁻¹ ) or higher The binding rate constant (ka) of the above was used as a reference value for selection, and it was found that the above problem can be solved by selecting a test substance in which both the measured dissociation rate constant and the binding rate constant are equal to or higher than the reference value, The present invention has been completed.

That is, according to the present invention, in the screening method for selecting a specific test substance from a group of test substances based on the interaction between the ligand and the test substance, the ligand is immobilized, and the ligand is immobilized. Is separated from the unit that measures the interaction between the immobilized ligand and the test substance, and any dissociation rate constant (kd) of ^{1x10 -2} (S ^-1 ) or higher and ^{1x10 5 Using} any binding rate constant (ka) of (M ^-1 S ^-1 ) or higher as the reference value for selection, select the test substances for which both the measured dissociation rate constant and the binding rate constant are higher than the above reference values. The above-described screening method is provided.

According to another aspect of the present invention, in a screening method for selecting a specific compound from a group of test compounds based on the interaction between a ligand and a test substance, the ligand is immobilized and immobilized. The dissociation rate constant (kd) and binding rate constant (ka) between the ligand and the test substance are determined from the amount of signal that changes when the test substance solution is brought into contact with the ligand, and ¹ × 10 ⁻² (S ⁻¹ ) Arbitrary dissociation rate constants (kd) above and 1x10 ⁵ (M ^-1 S ^-1 ) or above arbitrary binding rate constants (ka) as the reference values for selection, The screening method described above, wherein both test substances that are equal to or higher than the reference value is selected. In the method, preferably, a unit for immobilizing a ligand is separated from a unit for measuring an interaction between the immobilized ligand and a test substance.

Preferably, the test substance that falls within the range of the binding rate constant and the dissociation rate constant input to the apparatus in advance is automatically selected.
Preferably, the association rate constant and the dissociation rate constant are determined by a surface plasmon resonance apparatus.
Preferably, the selected test substance is visually distinguished on the display device.

  According to the present invention, it is possible to easily screen a substance having both a high binding rate and a high dissociation rate from among a large number of test substances.

The screening method of the present invention is a screening method for selecting a specific test substance from a group of test substances based on the interaction between a ligand and the test substance, and any dissociation rate constant of ¹ × 10 ⁻² (S ⁻¹ ) or more. (Kd) and any binding rate constant (ka) of ¹ × 10 ⁵ (M ⁻¹ S ⁻¹ ) or higher as a reference value for selection, both the measured dissociation rate constant and the binding rate constant are greater than the above reference values. The test substance which is is characterized by selecting.

  In the present invention, the dissociation rate constant (kd) and the binding rate constant (ka) are preferably determined by a surface plasmon resonance measuring apparatus using a solid phase sensor chip. In the surface plasmon resonance method, an interaction such as binding and dissociation can be measured in real time while both the ligand and the test substance are unlabeled. Therefore, even if the test substance has an extremely fast dissociation rate, the binding can be identified.

In the present invention, an arbitrary dissociation rate constant (kd) of ¹ × 10 ⁻² (S ⁻¹ ) or higher and an arbitrary binding rate constant (ka) of ¹ × 10 ⁵ (M ⁻¹ S ⁻¹ ) or higher are selected as reference values. As described above, test substances having both measured dissociation rate constants and binding rate constants equal to or higher than the reference values are selected. More preferably, an arbitrary dissociation rate constant (kd) of 1x10 ^-1 (S ^-1 ) or more and an arbitrary binding rate constant (ka) of 1x10 ⁶ (M ^-1 S ^-1 ) or more are used as selection reference values. Then, a test substance having both the measured dissociation rate constant and the binding rate constant equal to or higher than those reference values is selected.

  In the present invention, it is preferable that the measurement of interaction is performed for all compounds at the same time. The time for both the binding reaction and the dissociation reaction is preferably less than 10 minutes, more preferably less than 3 minutes, and even more preferably less than 1 minute.

  In the present invention, it is preferable to determine the dissociation rate constant and the binding rate constant between the ligand and the test substance from the amount of signal that changes when the test substance solution is brought into contact with the immobilized ligand.

  In the present invention, it is preferable that the molecular weights of all test substances to be screened are previously input to the screening apparatus. The binding amount of each test substance is preferably normalized and displayed with each molecular weight as expressed by the binding amount / molecular weight.

  In the present invention, it is preferable that a binding rate constant and a dissociation rate constant in a range to be selected are input in advance to a screening apparatus, and a test substance that falls within the range is automatically selected. The selected test substance is preferably displayed on the display device so as to be visually distinguished. The visual distinction method is not particularly limited, and examples thereof include a method of character size, color, character blinking, character color, underline, and framed.

  In the present invention, it is preferable that a ligand (protein or the like) is immobilized. As a solid phase immobilization method, a known method is used. Further, it is preferable that the unit for immobilizing the ligand is separated from the unit for measuring the interaction between the immobilized ligand and the test substance. An example of the apparatus configuration is shown in FIG. By separating the unit for immobilizing the ligand from the unit for measuring the interaction, the measurement processing capability is greatly improved.

  In the present invention, the number of test substances in a group is preferably 1000 or more, more preferably 10,000 or more, and further preferably 100,000 or more.

  The test substance may be a single substance or a mixture of a plurality of substances. The test substance used in the present invention is not particularly limited as long as it is a substance that interacts with a physiologically active substance (ligand) as described below in the present specification, but a low molecular organic compound, a high molecular compound, DNA, etc. And nucleic acids, proteins (including antibodies), peptides or fragments thereof, sugars, amino acids and the like.

  In the present invention, the dissociation rate constant (kd) and binding rate constant (ka) between the ligand and the test substance are preferably measured by an unlabeled detection method. Non-labeling detection methods include surface plasmon resonance (SPR) measurement technology, quartz crystal microbalance (QCM) measurement technology, measurement technology using functionalized surfaces from gold colloidal particles to ultrafine particles, among others. It is particularly preferable to perform surface plasmon resonance measurement using a surface plasmon resonance apparatus.

  Next, a biosensor used in the surface plasmon resonance apparatus will be described. The biosensor substrate is preferably a metal surface or a metal film. The metal constituting the metal surface or the metal film is not particularly limited as long as surface plasmon resonance can occur. Preferred examples include free electron metals such as gold, silver, copper, aluminum, and platinum, with gold being particularly preferred. These metals can be used alone or in combination. In consideration of adhesion to the substrate, an intervening layer made of chromium or the like may be provided between the substrate and the layer made of metal.

  Although the thickness of the metal film is arbitrary, for example, when considering the use for a surface plasmon resonance biosensor, it is preferably 1 angstrom or more and 5000 angstrom or less, and particularly preferably 10 angstrom or more and 2000 angstrom or less. If it exceeds 5000 angstroms, the surface plasmon phenomenon of the medium cannot be sufficiently detected. When an intervening layer made of chromium or the like is provided, the thickness of the intervening layer is preferably 1 angstrom or more and 100 angstrom or less.

  The metal film may be formed by a conventional method, for example, sputtering, vapor deposition, ion plating, electroplating, electroless plating, or the like.

  The metal film is preferably disposed on the substrate. Here, “arranged on the substrate” means that the metal film is arranged so as to be in direct contact with the substrate, and that the metal film is not directly in contact with the substrate, but through other layers. This also includes the case where they are arranged. As a substrate that can be used in the present invention, for example, when considering use for a surface plasmon resonance biosensor, generally, optical glass such as BK7, or synthetic resin, specifically, polymethyl methacrylate, polyethylene terephthalate, polycarbonate A material made of a material transparent to laser light such as a cycloolefin polymer can be used. Such a substrate is preferably made of a material that does not exhibit anisotropy with respect to polarized light and has excellent processability.

  The substrate preferably has a functional group capable of immobilizing a physiologically active substance on the outermost surface of the substrate. The “outermost surface of the substrate” here means “the side farthest from the substrate”.

Preferred functional groups include —OH, —SH, —COOH, —NR ¹ R ² (wherein R ¹ and R ² independently represent a hydrogen atom or a lower alkyl group), —CHO, —NR ³ NR ^1. R ² (wherein R ¹ , R ² and R ³ each independently represents a hydrogen atom or a lower alkyl group), —NCO, —NCS, an epoxy group, or a vinyl group can be mentioned. Here, the number of carbon atoms in the lower alkyl group is not particularly limited, but is generally about C1 to C10, preferably C1 to C6.

As a method for introducing these functional groups on the outermost surface, a polymer containing a precursor of those functional groups is coated on a metal surface or a metal film, and then the precursor is located on the outermost surface by chemical treatment. The method of producing | generating the functional group of is mentioned. For example, after polymethylmethacrylate containing —COOCH ₃ groups is coated on a metal film, the surface is brought into contact with an aqueous NaOH solution (1N) at 40 ° C. for 16 hours to form —COOH groups on the outermost surface.

  In the biosensor surface obtained as described above, the physiologically active substance can be immobilized on the metal surface or metal film by covalently binding the physiologically active substance (ligand) via the functional group. .

  The physiologically active substance (ligand) immobilized on the biosensor surface is not particularly limited as long as it interacts with the measurement target. For example, immune proteins, enzymes, microorganisms, nucleic acids, low molecular organic compounds, non-molecular compounds, Examples include immune proteins, immunoglobulin-binding proteins, sugar-binding proteins, sugar chains that recognize sugars, fatty acids or fatty acid esters, or polypeptides or oligopeptides having ligand binding ability.

  Examples of immunity proteins include antibodies and haptens that use the measurement target as an antigen. As the antibody, various immunoglobulins, that is, IgG, IgM, IgA, IgE, IgD can be used. Specifically, when the measurement target is human serum albumin, an anti-human serum albumin antibody can be used as the antibody. In addition, when using pesticides, insecticides, methicillin-resistant Staphylococcus aureus, antibiotics, narcotics, cocaine, heroin, cracks, etc. as antigens, for example, anti-atrazine antibodies, anti-kanamycin antibodies, anti-methamphetamine antibodies, or pathogenic E. coli Among them, antibodies against O antigens 26, 86, 55, 111, 157 and the like can be used.

  The enzyme is not particularly limited as long as it shows activity against the measurement object or a substance metabolized from the measurement object, and various enzymes such as oxidoreductase, hydrolase, isomerase , A desorbing enzyme, a synthesizing enzyme, etc. Specifically, if the measurement object is glucose, glucose oxidase can be used, and if the measurement object is cholesterol, cholesterol oxidase can be used. In addition, when pesticides, insecticides, methicillin-resistant Staphylococcus aureus, antibiotics, narcotics, cocaine, heroin, cracks, etc. are used as measurement objects, they exhibit specific reactions with substances metabolized from them, such as acetylcholinesterase. Enzymes such as catecholamine esterase, noradrenaline esterase and dopamine esterase can be used.

The microorganism is not particularly limited, and various microorganisms including Escherichia coli can be used.
As the nucleic acid, one that hybridizes complementarily with the nucleic acid to be measured can be used. As the nucleic acid, either DNA (including cDNA) or RNA can be used. The type of DNA is not particularly limited, and may be any of naturally derived DNA, recombinant DNA prepared by gene recombination technology, or chemically synthesized DNA.
Examples of the low-molecular organic compound include any compound that can be synthesized by an ordinary organic chemical synthesis method.

The non-immune protein is not particularly limited, and for example, avidin (streptavidin), biotin or a receptor can be used.
As the immunoglobulin-binding protein, for example, protein A or protein G, rheumatoid factor (RF) and the like can be used.
Examples of sugar-binding proteins include lectins.
Examples of the fatty acid or fatty acid ester include stearic acid, arachidic acid, behenic acid, ethyl stearate, ethyl arachidate, and ethyl behenate.

  When the physiologically active substance is a protein or nucleic acid such as an antibody or an enzyme, the immobilization can be performed by covalently bonding to a functional group on the metal surface using the amino group, thiol group or the like of the physiologically active substance. .

  The biosensor on which a physiologically active substance is immobilized as described above can be used for detection and / or measurement of a substance that interacts with the physiologically active substance.

  The surface plasmon resonance biosensor is a biosensor used in the surface plasmon resonance biosensor, and includes a part that transmits and reflects light emitted from the sensor, and a part that fixes a physiologically active substance. And may be fixed to the main body of the sensor or may be removable.

  The phenomenon of surface plasmon resonance is due to the fact that the intensity of monochromatic light reflected from the boundary between an optically transparent substance such as glass and the metal thin film layer depends on the refractive index of the sample on the metal exit side. Yes, so the sample can be analyzed by measuring the intensity of the reflected monochromatic light.

  As a surface plasmon measuring device for analyzing the characteristics of a substance to be measured using a phenomenon in which surface plasmons are excited by light waves, an apparatus using a system called Kretschmann arrangement can be cited (for example, Japanese Patent Laid-Open No. 6-167443). See the official gazette). A surface plasmon measuring apparatus using the above system basically includes a dielectric block formed in a prism shape, for example, and a metal film formed on one surface of the dielectric block and brought into contact with a substance to be measured such as a sample liquid. A light source that generates a light beam; an optical system that causes the light beam to enter the dielectric block at various angles so that a total reflection condition is obtained at an interface between the dielectric block and the metal film; and It comprises light detecting means for detecting the surface plasmon resonance state, that is, the state of total reflection attenuation by measuring the intensity of the light beam totally reflected at the interface.

  In order to obtain various incident angles as described above, a relatively thin light beam may be incident on the interface by changing the incident angle, or a component incident on the light beam at various angles is included. As described above, a relatively thick light beam may be incident on the interface in a convergent light state or a divergent light state. In the former case, a light beam whose reflection angle changes according to the change in the incident angle of the incident light beam is detected by a small photodetector that moves in synchronization with the change in the reflection angle, or the direction in which the reflection angle changes Can be detected by an area sensor extending along the line. On the other hand, in the latter case, it can be detected by an area sensor extending in a direction in which each light beam reflected at various reflection angles can be received.

  In the surface plasmon measuring apparatus having the above configuration, when a light beam is incident on a metal film at a specific incident angle that is greater than the total reflection angle, an evanescent wave having an electric field distribution is generated in the substance to be measured in contact with the metal film. The evanescent wave excites surface plasmons at the interface between the metal film and the substance to be measured. When the wave number vector of the evanescent light is equal to the wave number of the surface plasmon and the wave number matching is established, both are in a resonance state, and the light energy is transferred to the surface plasmon. The intensity of the reflected light decreases sharply. This decrease in light intensity is generally detected as a dark line by the light detection means. The resonance described above occurs only when the incident beam is p-polarized light. Therefore, it is necessary to set in advance so that the light beam is incident as p-polarized light.

  If the wave number of the surface plasmon is known from the incident angle at which this total reflection attenuation (ATR) occurs, that is, the total reflection attenuation angle (θSP), the dielectric constant of the substance to be measured can be obtained. In this type of surface plasmon measurement apparatus, as shown in Japanese Patent Application Laid-Open No. 11-326194, in order to measure the total reflection attenuation angle (θSP) with high accuracy and a large dynamic range, It is considered to use detection means. This light detection means is provided with a plurality of light receiving elements arranged in a predetermined direction, and arranged so that different light receiving elements receive light beam components totally reflected at various reflection angles at the interface. Is.

  In that case, there is provided differential means for differentiating the light detection signals output from the light receiving elements of the arrayed light detection means with respect to the arrangement direction of the light receiving elements, and based on the differential value output by the differential means. In many cases, the total reflection attenuation angle (θSP) is specified to obtain a characteristic related to the refractive index of the substance to be measured.

  Moreover, as a similar measuring device using total reflection attenuation (ATR), for example, “Spectroscopic Research” Vol. 47, No. 1, (1998), pages 21 to 23 and pages 26 to 27 are described. Devices are also known. This leakage mode measuring device is basically a dielectric block formed in a prism shape, for example, a clad layer formed on one surface of the dielectric block, and formed on the clad layer to be in contact with the sample liquid. Optical waveguide layer to be generated, a light source for generating a light beam, and the light beam to the dielectric block at various angles so that a total reflection condition is obtained at the interface between the dielectric block and the cladding layer. The optical system includes an incident optical system and light detection means for detecting the excitation state of the waveguide mode, that is, the total reflection attenuation state by measuring the intensity of the light beam totally reflected at the interface.

  In the leakage mode measuring apparatus having the above-described configuration, when a light beam is incident on the cladding layer through the dielectric block at an incident angle greater than the total reflection angle, the light waveguide layer transmits a specific wave number after passing through the cladding layer. Only light having a specific incident angle having a wave length propagates in the waveguide mode. When the waveguide mode is excited in this way, most of the incident light is taken into the optical waveguide layer, resulting in total reflection attenuation in which the intensity of light totally reflected at the interface is sharply reduced. Since the wave number of guided light depends on the refractive index of the substance to be measured on the optical waveguide layer, knowing the specific incident angle at which total reflection attenuation occurs, the refractive index of the substance to be measured and the measurement object related thereto The properties of the substance can be analyzed.

  In this leakage mode measuring apparatus, the above-mentioned array-shaped light detecting means can be used to detect the position of the dark line generated in the reflected light due to the total reflection attenuation, and the above-described differentiating means is applied in conjunction therewith. Often done.

  In addition, the surface plasmon measurement device and the leakage mode measurement device described above may be used for random screening to find a specific substance that binds to a desired sensing substance in the field of drug discovery research. In this case, the thin film layer A sensing substance is fixed on the sensing substance on the sensing substance (a metal film in the case of a surface plasmon measuring apparatus, a clad layer and an optical waveguide layer in the case of a leakage mode measuring apparatus), and various analytes are placed on the sensing substance. A sample solution dissolved in a solvent is added, and the total reflection attenuation angle (θSP) is measured every time a predetermined time elapses.

  If the analyte in the sample liquid binds to the sensing substance, the refractive index of the sensing substance changes with time due to this binding. Therefore, by measuring the total reflection attenuation angle (θSP) every predetermined time and measuring whether or not the total reflection attenuation angle (θSP) has changed, the binding state of the analyte and the sensing substance is determined. It is possible to determine whether or not the analyte is a specific substance that binds to the sensing substance based on the result. Examples of the combination of the specific substance and the sensing substance include an antigen and an antibody, or an antibody and an antibody. Specifically, a rabbit anti-human IgG antibody can be immobilized on the surface of the thin film layer as a sensing substance, and a human IgG antibody can be used as the specific substance.

  Note that, in order to measure the binding state between the subject and the sensing substance, it is not always necessary to detect the angle of the total reflection attenuation angle (θSP) itself. For example, a sample solution can be added to the sensing substance, and the amount of change in the total reflection attenuation angle (θSP) thereafter can be measured, and the binding state can be measured based on the magnitude of the amount of change in angle. If the above-described arrayed light detecting means and differentiating means are applied to a measuring apparatus using total reflection attenuation, the change amount of the differential value reflects the angle change amount of the total reflection attenuation angle (θSP). Therefore, the binding state between the sensing substance and the analyte can be measured based on the amount of change in the differential value (see Japanese Patent Application No. 2000-398309 by the present applicant). In such a measurement method and apparatus using total reflection attenuation, a sample liquid consisting of a solvent and an analyte is placed on a cup-shaped or petri-shaped measuring chip in which a sensing substance is fixed on a thin film layer formed in advance on the bottom surface. The amount of change in angle of the total reflection attenuation angle (θSP) described above is measured.

  Furthermore, a measuring apparatus using total reflection attenuation capable of measuring a large number of samples in a short time by sequentially measuring a plurality of measuring chips mounted on a turntable or the like is disclosed in JP-A-2001-2001. No. 330560.

  In one embodiment of the present invention, after the liquid in the flow path system of the surface plasmon resonance measuring apparatus is exchanged from a solution not containing the test substance to a solution containing the test substance, the surface of the surface is stopped in a state where the liquid flow is stopped. Changes in plasmon resonance can be measured. By such measurement, it is possible to suppress the noise width of the signal change of the reference cell and the baseline fluctuation within the measurement time, and thereby it is possible to acquire highly reliable combined detection data. The time for stopping the flow of the liquid is not particularly limited, but is, for example, 1 second to 30 minutes, preferably 10 seconds to 20 minutes, and more preferably about 1 minute to 20 minutes.

  In the present invention, preferably, a reference cell that does not bind a substance that interacts with a test substance and a detection cell that binds a substance that interacts with the test substance are connected in series and installed in the flow path system, A change in surface plasmon resonance can be measured by flowing a liquid through the reference cell and the detection cell.

In the present invention, the amount of liquid exchange per measurement (Ve) with respect to the volume (Vs ml) of the cell used for measurement (the total volume of those cells when the reference cell and the detection cell are used). ml) (Ve / Vs) can be, for example, 1 or more and 100 or less. Ve / Vs is more preferably 1 or more and 50 or less, and particularly preferably 1 or more and 20 or less. The volume (Vs ml) of the cell used for the measurement is not particularly limited, but is preferably about 1 × 10 ^{−6 to} 1.0 ml, particularly preferably about 1 × 10 ⁻⁵ to 1 × 10 ⁻¹ ml. Further, the time required for exchanging the liquid is preferably 0.01 seconds or more and 100 seconds or less, and more preferably 0.1 seconds or more and 10 seconds or less.

  The following examples further illustrate the present invention, but the scope of the present invention is not limited to these examples.

Example 1
Using the screening SPR system shown in FIG. 1, ligand fixation and compound binding measurement were performed.
(1) Immobilization of Ligand Using the protein immobilizer shown in FIG. 1 and the following sensor chip, a ligand was immobilized on the sensor chip under the following immobilization conditions.

  Sensor chip: 1 cm (1 cm cover glass with gold deposited to a thickness of 500 angstroms) treated with Model-208 UV-ozone cleaning system (TECHNOVISION INC.) For 30 minutes, then spin coater (MODEL ASS-303, manufactured by ABLE) and rotated at 1000 rpm, 50 μl of a methyl ethyl ketone solution (2 mg / ml) of polymethyl methacrylate (PMMA) was dropped into the center of the gold-deposited cover glass, and the rotation was stopped after 2 minutes. When the film thickness was measured by a measurement method (In-Situ Ellipsometer MAUS-101, manufactured by Five Lab), the thickness of the polymethyl methacrylate film was 200 angstrom.This sample is called a PMMA surface chip.This PMMA surface The chip was immersed in an aqueous NaOH solution (1N) at 40 ° C. for 16 hours and then washed with water three times.This sample is called a PMMA / COOH surface chip. After immersing in 2 ml of a mixture of -2,3-dimethylaminopropylcarbodiimide (400 mM) and N-hydroxysuccinimide (100 mM) for 60 minutes, an aqueous solution of α-amino-polyethyleneoxy-ω-carboxylic acid (average molecular weight 5000) ( (10 mM) was immersed in 2 ml for 16 hours, and finally washed 5 times with water.

The ligand is immobilized by the following procedure.
Preparation of ligand solution: Dissolve carbonic acid dehydrogenase in acetate buffer (pH 5.0) to 0.1 mg / ml.
Activation of sensor chip surface: NHS (0.1M) / EDC (0.4M) = 1/1, 25 ° C, 30 minutes Immobilization of ligand: The above ligand solution, 25 ° C, 30 minutes Blocking: 1M ethanolamine solution (pH8) .5), 25 ° C, 30 minutes

(2) Compound binding measurement Measurement compound: Indapamide
Temperature: 25 ℃
Running buffer: Phosphate buffer (pH7.4, DMSO 5% included)
Compound concentration: 0.5 μM
Measurement time: 30 seconds

(3) Calculation of ka, kd, KD From the measurement results of the signal change of surface plasmon resonance, the following formulas (1), (2) and (3) are used to determine the adsorption rate coefficient (ka) and the separation rate coefficient (kd ) And the dissociation constant (KD) was calculated from the determined adsorption rate coefficient (ka) and desorption rate coefficient (kd) by the equation represented by KD = kd / ka.

_{dθ / dt = ka × c s} × (1-θ) -kd × θ (1)
(In the formula, θ represents the adsorption rate (= adsorption amount / saturated adsorption amount), ka represents the adsorption rate coefficient, kd represents the desorption rate coefficient, and c _s represents the concentration of the molecule to be analyzed in the vicinity of the metal surface.)
^{∂c / ∂t = D × ∂ 2} c / ∂x 2 (2)
(Distance from wherein, x is a metal surface, D is the diffusion coefficient of the analyte molecule, c is represents the concentration of the analyte molecule, and c = c _s when x = 0.)
θ = R / Rmax (3)
(In the formula, θ represents an adsorption rate (= adsorption amount / saturated adsorption amount), R represents a surface plasmon signal, and Rmax represents a signal when the molecule to be analyzed is saturated adsorbed.)

The results are shown below.
ka = 4.0 × 10 ⁵ (M ^-1 s ^-1 )
kd = 2.2 × 10 ^-1 (s ^-1 )
KD = 5.5 × 10 ^-7 (M)

Example 2
An experiment was conducted in the same manner as in Example 1 using a sensor chip produced in the same manner as in Example 1 except that the following compound F-1 was used instead of PMMA. As a result, the same result as in Example 1 was obtained.

Example 3
Using the SPR system for screening shown in FIG. 1 and the sensor chip prepared in Example 2, ligand fixation and screening of a large number of compounds are automatically performed.
(1) Ligand fixation The ligand is immobilized by the following procedure.
Preparation of ligand solution: Dissolve 0.1 mg of carbonic acid dehydrogenase in 1 ml of acetate buffer (pH 5.0).
Activation of sensor chip surface: NHS (0.1M) / EDC (0.4M) = 1/1, 25 ° C, 30 minutes Immobilization of ligand: The above ligand solution, 25 ° C, 30 minutes Blocking: 1M ethanolamine solution (pH8) .5), 25 ° C, 30 minutes

(2) Binding measurement and compound selection Measurement compound: 5000 arbitrary compounds in a group Temperature: 25 ° C
Running buffer: Phosphate buffer (pH7.4, DMSO 5% included)
Compound concentration: 0.1, 1, 10 μM (DMSO 5% PBS buffer)
Measurement time: 30 seconds

Calculation of ka and kd: Calculated by the same method as (3) of Example 1. The binding curve used for the calculation is appropriately selected from the curves obtained at the above three compound concentrations.
Selection of compounds: Compounds having ka of 1.0 × 10 ⁶ (M ⁻¹ s ⁻¹ ) or more and kd of 1.0 × 10 ⁻¹ (s ⁻¹ ) or more are automatically selected.
Display: The number of the compound selected above is displayed blinking on the display device.

FIG. 1 shows an example of an SPR system for performing the screening method of the present invention.

Claims (6)

In a screening method for selecting a specific test substance from a group of test substances based on the interaction between the ligand and the test substance, the ligand is immobilized, and the unit for immobilizing the ligand is immobilized. The unit for measuring the interaction between the ligand and the test substance is separated, and any dissociation rate constant (kd) of ^{1x10 -2} (S ^-1 ) or higher, and 1x10 ⁵ (M ^-1 S ^-1 ) The screening method described above, wherein a test substance having both the measured dissociation rate constant and binding rate constant equal to or higher than the reference value is selected using the above arbitrary binding rate constant (ka) as a reference value for selection. In a screening method for selecting a specific compound from a group of test compounds based on the interaction between a ligand and a test substance, the ligand is immobilized and the solution of the test substance is contacted with the immobilized ligand The dissociation rate constant (kd) and binding rate constant (ka) between the ligand and the test substance are obtained from the signal amount that changes when the signal is applied, and an arbitrary dissociation rate constant (kd) of ¹ × 10 ⁻² (S ⁻¹ ) or more is obtained. ), And any binding rate constant (ka) of ¹ × 10 ⁵ (M ⁻¹ S ⁻¹ ) or more, and the measured dissociation rate constant and binding rate constant are both above the above reference values The screening method described above, wherein a test substance is selected. The screening method according to claim 2, wherein the unit for immobilizing the ligand is separated from the unit for measuring the interaction between the immobilized ligand and the test substance. The screening method according to any one of claims 1 to 3, wherein a test substance that falls within a range of a binding rate constant and a dissociation rate constant input to the apparatus in advance is automatically selected. The screening method according to claim 1, wherein the binding rate constant and the dissociation rate constant are determined by a surface plasmon resonance apparatus. The screening method according to claim 1, wherein the selected test substance is visually distinguished on a display device.

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