WO2007024014A1

WO2007024014A1 - Method of measuring interaction between molecules and measurement apparatus using the method

Info

Publication number: WO2007024014A1
Application number: PCT/JP2006/317151
Authority: WO
Inventors: Kiichi Fukui; Susumu Uchiyama; Shigeki Kawakami; Satoshi Nishikawa
Original assignee: Osaka University; Kansai Technology Licensing Organization Co., Ltd.
Priority date: 2005-08-24
Filing date: 2006-08-24
Publication date: 2007-03-01
Also published as: JPWO2007024014A1

Abstract

It is intended to provide a method of measuring an interaction between molecules such as proteins and a measurement apparatus therefor. In this method, a solution containing one of molecules, the interaction between which is to be measured, having been labeled with a fluorescent dye and another solution containing the other molecule not labeled with a fluorescent dye are prepared. Next, the solution containing the unlabeled molecule is immobilized to an area in the bottom face of a well. Then, the solution containing the labeled molecule is reacted with the immobilized solution containing the unlabeled molecule followed by photo irradiation. As a result, there arises a difference in fluorescent intensity between the above-described area and the other area in the bottom face of the well. By measuring the difference between the fluorescent intensities, the interaction between the molecules is measured.

Description

m Itoda Intermolecular interaction measurement method and measurement device using this method

The present invention relates to a method for measuring an intermolecular interaction such as between proteins and a measurement apparatus using the method. Background art

In recent years, in order to elucidate life activities, it has been desired to detect intermolecular interactions such as proteins in living cells. As one of the detection methods, there is a conventional method in which a ^ ϊί ^ substance such as an enzyme or a structural protein is bound to a fluorescently labeled antibody and detected under a fluorescent microscope, which is generally called a fluorescent antibody method. . In this method, the ligand molecule is fluorescently labeled, and the binding state to the active substance that becomes the receptor is detected by fluorescent color development (the ligand and the receptor may be reversed, but here the ligand is fluorescently labeled. To explain). In order to detect the binding of the receptor to the ligand, it is necessary to detect the fluorescence of the ligand bound to the receptor, and it is necessary to exclude the coloration of the unbound free ligand. The binding action between the receptor and the ligand is determined by the correlation between the receptor-ligand conjugate concentration, the total ligand concentration, the total receptor concentration, and the dissociation constant. In the conventional method, the conjugate concentration itself of each sample can be compared and verified by the fluorescence intensity. However, the receptor and ligand can be compared with each other. There is a problem that it is difficult to verify and compare the degree of binding of the ligand and the concentration of free ligand, and it is difficult to verify the binding strength between the receptor and the ligand depending on the dissociation constant. In addition, there is a method for measuring intermolecular interactions using surface plasmon resonance to solve such problems. This measurement method is a method in which a ligand molecule is immobilized and a receptor molecule that interacts with the immobilized ligand reacts, and the specificity between the ligand and the receptor, that is, the molecular interaction specificity and affinity. This is the point that we can obtain information such as strength, speed of bond-dissociation, and bond strength. However, this measurement method has problems such as the need for dedicated equipment and facilities, high measurement costs, and difficulty in removing noise. For example, “Japanese Laid-Open Patent Publication No. 2 00 0-2 8 3 9 8 4”, “Experimental Medicine Separate Volume, Close-up Experiment Method”, Yodosha (2002), Biacore-based low affinity interaction Detection, Satoshi Inagawa, l43-150, "Experimental Medicine Separate Volume: Experimental Course in the Age of Genome 3 GFP and Bioimaging" Yodosha (2000), Fluorescence Correlation Spectroscopy Masataka Kaneshiro Naoto Yoshida, Yasutomo Nomura, p2-222 It is shown. Invention s try to solve

The present invention has been provided in view of the above circumstances, and it is possible to easily verify the bond strength between molecules, that is, the intermolecular interaction, from one measurement result, and greatly reduce the measurement cost. It is an object of the present invention to provide an intermolecular interaction measurement method and a measurement apparatus using this measurement method. Disclosure of the invention

Means for deciding a task

According to the intermolecular interaction measurement method of the present invention, one of the molecules for which the interaction is desired to be measured is labeled with a fluorescent dye, and at least the slumber containing this is ditched, and another molecule is added. A solution containing this solution without labeling with a fluorescent dye, and a step containing a non-labeled molecule that has been trapped by a self-groove step in one region of the bottom surface of the well that is opened upward. An immobilization process for immobilizing the night, and a reaction process in which a solution containing the labeled molecule β in the self-preparation process is stored in the well and reacted with the solution immobilized in the self-immobilization process. And an irradiation step of irradiating the solution reacted in the reaction step with light, and the bottom of the i! Well where the intense night containing the label, label, and molecule is immobilized in the immobilization step. "" Fluorescence density in the IS region, ΙΪΓΪΒί It has a fluorescence measurement step of comparing measured and fluorescence concentration, in the identification the region other than the U gamut is reservoir night force 宁留 containing molecules are, the. In addition, the immobilization step has a step of immobilizing a predetermined molecule in a region of the bottom of the well, and has an affinity for the predetermined molecule immobilized in a solution containing the molecule that is labeled. It is preferable to include a step of including a molecule and a step of dripping a frame containing a molecule that is not labeled in the region of the bottom of the well. Further, the fixing may include a step of dripping the night containing the le, nare and molecules, and a step of drying the drenched night. Furthermore, in the night preparation process described above, two or more nights are set with predetermined concentration conditions. By measuring the fluorescence concentration corresponding to each solution, an appropriate concentration condition / dissociation constant (Kd) of the interaction between molecules desired to be measured may be calculated.

In addition, according to the intermolecular interaction measuring apparatus of the present invention, the apparatus has a well that forms a recess capable of storing a solution, and the bottom force in the well is one of the molecules desired to measure the intermolecular interaction. "I region for immobilizing a solution containing molecules that are not fluorescently labeled" and other molecules that are desired to measure intermolecular interactions with the one molecule and are fluorescently labeled It consists of other regions for receiving molecules-containing solutions The ^ B region is a solid layer on which the solution containing molecules that are not fluorescently labeled is immobilized on the bottom of the well. The other region formed is hollow upward from the bottom surface of the well, and the solid layer is dripped onto the layer containing molecules directly immobilized on the bottom surface of the tool. Includes molecules that are not fluorescently labeled? Is preferred, but it may also be formed by drying a solution containing non-fluorescent molecules on the bottom of the tiff itself, and there are several wells in the fc ^ and lateral directions, Furthermore, the self-solid layer in the ^ B region is labeled with a self-fluorescent material included in the solid layer, and is desired to measure interactions with molecules. When the solution containing the fluorescently labeled molecule is stored in the other region, the infiltration of the tiff self fluorescently labeled molecule is not refused, but the Sift self fluorescent label included in the solid layer is not rejected. It is preferred that the structure is designed so that the molecular Sift itself does not penetrate into other areas.

The present invention provides a method for measuring an intermolecular interaction and a measuring apparatus used for the method for measuring an intermolecular interaction. According to this measuring method and measuring apparatus, interacting receptor molecules are fixed and arranged only in one region of the bottom of the well, and ligand molecules are formed in the other region of the well (receptor molecule). And the ligand molecule may be reversed, that is, the ligand molecule may be immobilized in the U region (hereinafter, this ^ is referred to as “another case”). The ligand molecule (in other cases ^ is a receptor molecule) is labeled with a fluorescent dye. Therefore, when the receptor 'ligands are bound to each other as a result, in the immobilized ^ S region, the receptor molecule-ligand molecule conjugate and state receptor molecule (in other cases, the receptor molecule and ligand molecule) And other states within the ue / re are ¾ | 隹 state ligand molecules (in other cases, free state receptor molecules). Will exist. In this state, excite the fluorescent dye. When irradiated to cause light, it is the receptor 'ligand conjugate that forms a fluorescent color in the immobilized region, and in other regions it is a ligand molecule in the t-state (in other cases, a free receptor molecule). ). Therefore, when the intermolecular interaction is active, the fluorescence concentration on one immobilized region becomes stronger than the fluorescence concentration on the other region, and the difference in concentration can be viewed at a glance. Become. In other words, if the present invention is used, the state of intermolecular interaction (for example, antibody binding reaction) can be clearly measured at a visual level in the same well, and a special technique such as surface plasmon resonance method can be used. ■ It is not necessary to use expensive equipment, and the measurement cost can be greatly reduced. Furthermore, in principle, the measurement method and measurement apparatus of the present invention can detect intermolecular interactions even in a microwell space (l rmi or less). Therefore, it can also be used for each microplate with a large number of holes! ^, And together with the visibility of detection results (described above), the appropriate conditions for intermolecular interactions can be easily and quickly detected. Is possible.

In the above description of the effect of the present invention, the force that has been explained by the interaction between a receptor molecule and a ligand molecule in relation to the intermolecular interaction between two regions in the well. It can be widely used for general intermolecular interactions. That is, not only measuring the interaction between molecules that are expected to be bound in advance, such as receptor molecules and ligand molecules, but also measuring the force of interaction between molecules with unknown interaction. For example, it is possible to measure the presence or absence of interactions such as between different proteins, between nucleic acids, and between protein and nucleic acids. In particular, the method and apparatus of the present invention has the effect that it is possible to detect intermolecular interactions even in a microscopic space (1 mm or less), and it is also used in each micro plate having a large number of holes. In addition to the visual solubility of the detection results (described above), it is possible to detect many types of molecules (for example, proteins) and other molecules that interact with each other easily and quickly. Is possible. Brief Description of Drawings

FIG. 1 is a view showing a microplate used in the intermolecular interaction measuring apparatus of the present invention.

FIG. 2 is an enlarged cross-sectional view of a well disposed on the microphone opening plate of FIG.

FIG. 3 is a plan view of FIG.

FIG. 4 is a plan view showing one experimental result in the present embodiment. FIG. 5 is a Scatehard plot diagram showing another experimental result in the present embodiment.

FIG. 6 is a schematic diagram showing an experimental process of another embodiment.

FIG. 7 is a square-corner image of an experimental result in another embodiment.

Fig. 8 is a Scatehard plot showing the experimental results of Fig. 7.

FIG. 9 is an angle Zhe image of an experimental result in still another embodiment.

FIG. 10 is a Scatehard plot showing the experimental results of FIG. BEST MODE FOR CARRYING OUT THE INVENTION

Referring to FIG. 1, an example of a microplate 1 used as an intermolecular interaction measurement apparatus of the present invention is shown (hereinafter, an embodiment of the intermolecular interaction measurement apparatus of the present invention will be described as a microphone port plate 1). 1) An example is shown. Specifically, Fig. 1 (a) shows a side view of a conventional microphone plate and Fig. 1 (b) shows a plan view of a conventional microplate. Here, the microplate is a container that is widely used for clinical tests, tests and tests such as DNA analysis and amplification, and the outer shape of the microplate is integrally attached to the base 3 and the base 3. And a cylindrical-shaped well 2 such as a cylindrical shape. A large number of wels 2 are arranged in a matrix in the row or column direction with respect to the base 3.

Furthermore, as shown in FIG. 1 (b), the Uenore 2 is a hollow container having a concave portion 2a having a cylindrical shape or the like in which the upper part is opened at the base 3 and the ite is closed, like a normal test tube. Four rims such as a circle projecting from the base 3 are formed at a portion opening to the base 3. In this way, the well 2 is configured so that a sample can be stored for testing and inspection.

The rim 4 protrudes slightly from the upper surface of the base 3, which is used to prevent sample evaporation and sample mixing (cross contamination) between each well 2 during the test. . Specifically, a cap is attached to the rim 4 or an adhesive film is attached on one side to prevent sample mixing. By providing the rim 4, the adhesion to the cap or tt adherence film is improved. be able to.

Here, in the microplate 1 as shown in FIG. 1, the distance between the centers of the adjacent wells 2 in the row and column directions is standardized, and the number of the wells 2 in one microplate 1 is 8 rows XI 2 columns. (9 6 pieces), 3 rows x 8 columns (2 4 pieces) etc. The lower end of the wel 2 has a flat (or substantially spherical) bottom surface 2 b as shown in the side view of FIG. Furthermore, the base 3 of the microplate 1 is flat as shown in FIG. 1 and extends downward from the peripheral edge of the flat plate. Some have a side wall (so-called skirt shape). Further, the microplate 1 is made of a transparent synthetic resin, glass or the like, and is preferably made of a low fluorescent material. The features of the microplate 1 described so far are also included in the conventional microplate.

Here, as a premise for explaining the characteristics of the present embodiment, the basic principle of the intermolecular interaction measurement method of the present invention will be described based on specific experimental examples in the present embodiment. First, an overview of the intermolecular interactions between receptors and ligands. Receptors are proteins in the cell membrane, cytoplasm, or nucleus that bind ligands (specific substances), that is, neurotransmitters, hormones, cell growth factors, and other substances to initiate cellular responses. have. This receptor and ligand bind to the state according to the law of mass action. This is shown in modeno below.

(Formula)

R + L R L

If the dissociation constant is ¾,

[3 [L] Ichi (ER) _T- [RL]) [L]

K ^A one [RL] - R]

Where [R] _T is the total concentration of R ([R] _T = [R] + [R'L]). As is clear from this model equation, the concentration of the receptor and ligand conjugate is It is given by the total ligand concentration, the total receptor concentration, and the dissociation constant. Therefore, the conjugate concentration ([R-L]) increases as the receptor concentration increases, and conversely

([L]) has been shown to have the property of decreasing with increasing receptor concentration. Here, the receptor and ligand can be easily bonded to each other with a bond: ^, that is, a strong intermolecular interaction ¾ ^ is a state with a small dissociation constant. Dissociation is a fixed value determined by the ligand corresponding to the receptor. From this, it can be seen that the free ligand concentration ([L]) is small compared to the conjugate concentration ([R'L]) when both the receptor and the ligand are easily bound. . Therefore, in order to verify the ease of binding between the receptor and the ligand, that is, the strength of the intermolecular interaction, it is necessary to simultaneously compare the receptor and ligand conjugate concentration and the ¾ϋ ligand concentration in It is tempted to be done.

Next, the basic principle for measuring the binding state between the receptor and the ligand will be described. As a conventionally known method, S has a method for fluorescently labeling a ligand. There is also a case of fluorescent labeling). In particular, there is a method in which a fluorescently labeled antibody is bound to an antigen in a sample typically corresponding to a receptor and detected by fluorescence microscopy. In this method, a method is generally used in which the antibody is labeled with a fluorescent dye such as fluororesin sothiocyanate (FITC) or tetramethinororhodamine sothiocyanate (TRITC), and the fluorescence is detected. In addition, in order to increase the detection sensitivity, an unlabeled antibody (referred to as a primary antibody) is reacted with, and a fluorescently labeled antibody (referred to as a secondary antibody) is bound to a conjugate with the 17-fire antibody. There is also a method of tracing, and the binding between the mouse antibody and the anti-mouse antibody is raised (this is used in the present embodiment as described later).

However, when the intermolecular interaction is already known only by the conventional method, for example, it is ^ iJ to give the corresponding ligand antibody to verify the presence of a specific receptor antigen in the sample, Comparison of receptor-ligand binding strength and reciprocal ligand concentration Since verification is difficult, it is not sufficient to newly verify the interaction between receptor and ligand.

On the other hand, the measurement method and measurement apparatus of the present invention share a configuration that allows easy comparison and verification of the receptor-ligand conjugate concentration and the soot-ligand concentration. Returning to the description of the microplate 1 again, FIG. 2 shows an enlarged sectional view of the vicinity of the bottom surface 2 b in the well 2. This FIG. 2 is identical to the cross-sectional view of FIG. 3 schematically shows the plan view of FIG. 2, which is identical to FIG. 1 (b). As shown in these figures, the bottom surface of the well 2 is solidified with a desired receptor molecule in a part of the region 2d, here, in the vicinity of the center. In this embodiment, a mouse antibody is used as a receptor molecule and an anti-mouse antibody is used as a ligand molecule, and a predetermined amount of a solution containing the mouse antibody at a desired concentration in a region 2 d of the bottom surface 2 b of the microplate 1 is used. (1 μ 1) Dropped and solidified. In addition, the other area 2 c of the bottom surface 2 b is originally blank. Here, with regard to the immobilization of the receptor molecule, a method in which the solution dropped on the bottom surface 2 d of the well is dried and directly immobilized, a predetermined molecule is immobilized on the bottom surface 2 d of the well, and then the predetermined molecule is immobilized. A method in which a liquid containing a molecule having a high affinity is mixed with a solution containing a mouse antibody and dropped on the bottom 2d of the well. In particular, the latter method is advantageous in that protein degradation due to drying can be suppressed. For example, after fixing dartathione or nickel on the bottom 2d of the well, a method of dropping GST protein or metal / polyhistidine peptide on the bottom 2d of the well in the night including mouse antibody is used. . The principle of this method is that the GST protein, etc. The dripping solution is immobilized on the dwell bottom 2d by affinity with gnolethione etc. on the surface 2d.

Next, a dwell containing ligand molecules is dropped on the well 2 formed in this state and stored in the well 2. At this time, it is preferable to seal the night (the solution containing the receptor molecule immobilized on the bottom surface 2d) stored in the well 2. This is because if the solution is dried as described above, the internal protein molecules (here, the anti-mouse antibody as a ligand molecule and the mouse antibody as a single receptor molecule) may be altered, and this can be suppressed. For example, dripping mineral ino-cetyl alcohol onto the surface of the liquid layer to coat the surface of the liquid layer is a method of physically sealing the well 2. Here, as described above, an anti-mouse antibody corresponding to a mouse antibody is used as a ligand molecule, and a night of a desired concentration including the anti-mouse antibody labeled with a fluorescent dye such as FITC is ditched, Drop it into the Uenore 2 and store it. Therefore, in the 2a in the well, the U region 2d on the bottom surface of the mouse antibody as a receptor binds to a part of the mouse antibody as a receptor, and in the other part, only the mouse antibody is formed, In the other region 2c, only the released anti-mouse antibody is included (see the model equation above). In such a state, when the illuminant bottom surface 2b is irradiated with light of a predetermined intensity I from above or below, the fluorescent dye labeled with the anti-mouse antibody is excited and develops color. The fluorescence intensity is proportional to the ligand concentration. Specifically, in the region 2d, the fluorescence intensity is proportional to the conjugate concentration of the mouse antibody and the anti-mouse antibody ([R ■ L] in the above model equation). Then, it is proportional to the concentration of the released anti-mouse antibody ([L] in Modeno ^: mentioned above). Therefore, there is a difference in the fluorescence concentration between the regions 2d and 2c depending on the respective concentrations. The greater the fluorescence concentration of the region 2d, It can be discriminated that the binding strength between the mouse antibody and the anti-mouse antibody as a ligand is large (dissociation is small). Therefore, it is possible to measure whether the interaction between two molecules is strong or not by simply looking at the difference in fluorescence concentration between the regions 2d and 2c. The receptor and ligand binding concentrations [R ■ L] Even if the dissociation constant K is unknown, if the total receptor concentration and total ligand concentration (known at the time of ¾ Pf solution for each solution) are determined, the receptor corresponding to this condition The binding strength of the ligand can be easily reduced.

Furthermore, if a microplate 1 having a large number of wells 2 is used as in the present embodiment shown in FIGS. 1 to 3, a different concentration of receptor and ligand can be β for each well. At first glance, understand the interaction between molecules under various concentration conditions. can do. As an example of setting the concentration condition for each well, in the case of microplate 1 in Fig. 1, the concentration is set so that the ligand concentration becomes larger or smaller for each row (each row A to H) in the vertical direction. It is suitable for understanding the measurement results if the receptor concentration is arranged so that the receptor concentration is increased or decreased in each row (every 1 to 12 rows) in the horizontal direction.

As described above, as a receptor, a solution containing a mouse antibody is immobilized in region 2d, and in region 2c, a night of night containing anti-mouse antibody (with FITC label) is stored for verification. An excerpt of the result is shown in Fig. 4 (actually, it was verified with 96 holes, but only a part is shown for the purpose of helping to summarize the invention). In this fruit, the mouse antibody concentration is unified at 10 g / ml, and the anti-mouse antibody concentration is 0.25, 0.4, 0.5, 0. 75, 1, 1.25 / ig / ml, 1.5, 1.75, 2, 3, 4, 5 / ig / ml from upper right to lower. Considering this figure, the fluorescence concentration is greatly different between the mouse antibody concentration lO zg / ral and the anti-mouse antibody concentration 0.4 _g / _ra l. The interaction is active.

We also explain the calculation of dissociation constant (Kd) using other experimental results. Referring to Figure 5, a Scatchard plot showing the binding of a receptor molecule (here mouse antibody) and a ligand molecule (here anti-mouse antibody) is shown. A line graph based on this is obtained. This vertical axis

[A-B] Z [A] displays the ratio ([R / L] / [R]) between the ligand 'receptor binding molecule and the escape ligand molecule, and the horizontal axis [Α B] is the ligand 'Receptor binding molecule concentration ([R · L]: mol / 1) is displayed. If we refer to the characteristics of the scatter plot here, the slope of the ia line obtained will be inversely proportional to the dissociation. Specifically, the slope = — 1 ZKd. Therefore, as is clear from FIG. 5, since the slope of the straight line obtained in the interaction between the mouse antibody and the anti-mouse antibody is 1 2 X 1 0 ⁸ (= coefficient of x), Kd = 0. is calculated as 5X 1 0 one ^8. In this way, the dissociation Kd can be calculated by measuring the molecular concentration in each well, and a specific intermolecular interaction can be measured.

Next, I will mention other more detailed results. Reference numbers shown in Figure 6 are figures

Means the same as the reference number element shown in 2 and FIG. Here, the interaction between ConcanavalinA (hereinafter referred to as Γϋοη AJ) and glycoprotein (RNaseB glycoprotein: hereinafter referred to as “RNaseBj”) is detected. First, as shown in Fig. 6 (a), 1 μ の of Con A having a concentration of 100 ig / ml was dropped onto the central region 2 d of the well 2 and then air-dried and fixed. Next, as shown in Fig. 6 (b), blocking treatment is performed to improve the protein recovery rate. Specifically, add 10 mg / ml BSA (bovine serum albumin) as a blocking buffer to the region 2d in the well 2 and incubate (incubate) for 20 minutes at 1¾ temperature. did. Then, as shown in Fig. 6 (c), wash the washing buffer (100 μm), discard the added washing solution for the first time, and add the washing solution (second time) for 10 minutes. ¾ Cultivated at temperature. Next, RNaseB labeled with a fluorescent label (FITC label) was distributed to concentrations of 0.5, 1, 1. 5, 2, 2.5, 3, 3.5, 4, 4.5, 5 μg / ml. Then, after cultivating each in separate wel 2, it was incubated at room temperature. Then, after 24 hours of injury, perform measurement and image cornering, and perform a scatter plot as described above.

(Scatchard plot) was prepared. The angle Zhe image and scutchyard plot are shown in Figure 7 and Figure 8, respectively. As a result of the measurement, the angular separation constant (Kd) of Con A and RNase B is calculated as 2.6 X 10 " ^6, which is almost the same as the literature value (1. 64X 10" ⁶ ). Be.

In addition, the Kakuzhe images in Fig. 7 are numbered in order from the top, and RNaseB is assigned to 0.5, 1, 1. 5, 2, 2. 5, 3, 3. 5, 4, 4. 5 respectively. , 5 μg / ml. In addition, for the scatter plot of Fig. 8, the data for each of the two well plots are plotted, and the vertical axis [A · B] / [A] is the ratio value of the ligand 'receptor binding molecule and the taste ligand molecule ( [R / L] / [R]) is displayed, and the horizontal axis [A ■ B] indicates the concentration of ligand / receptor binding molecule ([R · L]), and the slope of the obtained ¾ line As mentioned earlier, is inversely proportional to dissociation (this is also true for Fig. 9 and Fig. 10 described later).

Next, in the experiment shown in FIG. 9, the interaction between the Aurora kinase antibody (hereinafter referred to as “Aurora”) and the hi stone H3 antibody (hereinafter referred to as “Hi _S tone”) is detected.

First, as shown in FIG. 6 (a), 1 xl of Histone having a concentration of 10 μg / ml was dropped on the central region 2 d of the well 2 and then air-dried and fixed. Next, as shown in Fig. 6 (b), blocking was performed, and as a sputum, BSA at a concentration of 10 mg / ml was added to the knitting area 2 d in the well 2 and incubated for 20 minutes at room temperature. Thereafter, as shown in FIG. 6 (c), the washing solution was 100 μΐ, the first washing solution was immediately discarded, and further the washing solution was added (second time), followed by incubation at a temperature of 10 minutes at 3 ° C. Next, fluorescently labeled (FITC-labeled) Aurora was distributed to concentrations of 1, 2, 3, 4, 5, and 6 μg / ml, and each was placed in a separate well 2 and incubated at room temperature. After about 24 hours, measurements and image analysis were performed, and a scatter plot was created. The horned eyelid image and scutchyard plot are shown in Fig. 9 and Fig. 10, respectively. As a result of measuring this manner, the dissociation constant of the Aurora and Histone (Kd) was calculated to be 1. 5 X 10- ^7, known in the literature indicating the dissociation ¾m value because it does not exist, the result is O look new onset Is done. In addition, the Kakuzhe images in Fig. 7 are numbered sequentially from the top.

Aurora is distributed to 1, 2, 3, 4, 5, 6 g / ml, and the scatchart plot in Fig. 10 is as described above.

As described above, the embodiment using the microplate 1 as the intermolecular interaction measuring apparatus of the present invention has been described as an example. However, the present invention is not limited to this, and the bottom surface of at least one well (not a plane) Other regions configured to have different molecules to be included in some of the regions and other regions, and verified by the above-described example reaction with the antibody. In view of this idea, it can be used for the measurement of two or more molecules for which interaction is desired. In this embodiment, the interaction between two molecules! Although only ^ is illustrated, it can also be applied to a method in which other ligand molecules (unlabeled) are included in the solution stored in the area inside the well other than the solidification area to cause a competitive reaction. In order to increase the sensitivity, a non-labeled seven-antibody is bound to (receptor), and the desired antibody (ligand) is labeled as a secondary antibody and bound to a conjugate of the antigen and the seven-antibody. This method can also be applied to the so-called fluorescence measurement method, which is a method of measuring one of the interacting molecules with the emitted fluorescence (^! 殳). Further, the fluorescent dye is not particularly limited to FITC, and other fluorescent dyes such as cy 5 may be used. In this specification, the expression of receptor and ligand has been used, but this is used for the convenience of explanation according to the binding action (equilibrium action) in the law of mass action (see the model formula). May be applied in reverse, and even measuring the force of interaction between molecules with unknown interaction (for example, between different proteins, between nucleic acids, protein 'nucleic acid It is easy for those skilled in the art not to deviate from the characteristics of the present invention (configuration in which different molecules are included and reacted) and the idea thereof. Will understand.

Claims

Scope of wording

1. One of the molecules for which interaction is desired to be measured is labeled with a fluorescent dye, and at least a solution containing the same is impregnated, and another molecule is included without being labeled with a fluorescent dye. A solution tree-making process to delight the night,

Immobilization step of immobilizing a solution containing a non-labeled molecule ^ (tagged) by the tiff self ¾1 step in the area of the bottom of the well opened upward,

A reaction step in which the stagnation night containing the labeled molecule prepared in the above step is stored in the frog well, and the stagnation is fixed by the immobilization step, and

An irradiation step of irradiating light to the solution ^ 5 produced by the reaction step;

tiff self-immobilization process labeled ヽ containing molecules ΐ Fluorescence concentration in the region of the bottom of the knitting well where the night was immobilized, and a solution containing the molecules identified in the previous statement were stored l A fluorescence measurement process for comparing and measuring the fluorescence concentration in a region other than its own region,

A method for measuring an intermolecular interaction, comprising:

2. The self-immobilization step includes a step of immobilizing a predetermined molecule in the U region on the bottom surface of the well, and an affinity for the predetermined molecule immobilized on the night containing the unlabeled molecule. The method includes a step of including a molecule having a property, and a step of dripping a night containing a molecule that has been preliminarily identified in the region of the bottom surface of the well. A method for measuring an intermolecular interaction described in 1.

3. Does the immobilization process include molecules that are not S-labeled in the ~ R region of the bottom of the knitting well? The method for measuring an intermolecular interaction according to claim 1, further comprising a step of dripping night and a step of drying the drenched night. -

4. Multiple solutions to be drowned in the knitting solution purification process are set under a predetermined concentration condition, and measurement is desired by measuring the fluorescence concentration corresponding to each solution under the set concentration condition. The method for measuring intermolecular interactions according to (1) to (3), characterized in that the appropriate concentration condition ■ dissociation is calculated among the interactions between molecules.

5. It has a well that forms a recess capable of storing the night, and the bottom of the well is one of the molecules for which intermolecular interaction is desired to be measured and is not fluorescently labeled. Accepts a solution containing a fluorescently labeled molecule, which is the other molecule for which measurement of molecular interaction with the one molecule is desired, and the ^ B region for immobilizing the night And consists of other page areas for

Fujimi U area is not fluorescently labeled on the bottom of Fujimiwell! /, An intermolecular interaction measurement device, characterized in that it is formed as a solid layer in which a molecule-containing solution is fixed, and the SiifS and other regions are hollow upward from the bottom surface of the well.

6. The tiff self-solid layer is formed of a layer containing molecules directly immobilized on the bottom surface of the well and a layer containing a molecule not labeled with self-fluorescence dropped onto the layer. The intermolecular interaction measuring device according to claim 5, wherein

7. The molecule according to claim 5, wherein the tiff self-solid layer is formed by drying the solution containing the fluorescent layer and molecules on the bottom surface of the tfif self-well. Interaction measurement device.

8. The intermolecular interaction measuring device according to any one of claims 5 to 7, characterized in that a plurality of self-wels are arranged in a longitudinal direction and;

9. The braided self-solid layer in the region of the tooth is a reservoir containing a molecule that is desired to measure the interaction with the non-fluorescently labeled molecule contained in the solid layer. Is not allowed to enter the molecule labeled with Fujimi, but the tiff is not labeled with the molecule contained in the solid layer. The intermolecular interaction measuring device according to claim 5, wherein the intermolecular interaction measuring device is configured not to intrude.