JPWO2007024014A1 - Method for measuring intermolecular interaction and measuring apparatus using this method - Google Patents

Abstract

The present invention provides a method and apparatus for measuring an interaction between molecules such as proteins. The present invention prepares a solution containing one of the molecules desired for intermolecular interaction with a fluorescent dye and the other molecule not labeled with the fluorescent dye. Thereafter, a solution containing unlabeled molecules is immobilized on a region of the bottom surface of the well, and a solution containing labeled molecules is reacted with an immobilized solution containing non-labeled molecules, and light is irradiated. As a result, a difference occurs in the fluorescence concentration between one region of the well bottom and another region, and the intermolecular interaction is measured by measuring the difference in fluorescence concentration.

Description

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

In recent years, in order to elucidate life activity, it has been desired to detect intermolecular interactions such as between proteins in living cells. As one of the detection methods, conventionally, there is a method in which an antigenic 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, a molecule serving as a ligand is fluorescently labeled, and a binding state to an antigenic substance serving as a receptor is detected by fluorescent color development (the ligand and the receptor may be reversed, but here the ligand is fluorescently labeled. As described). In order to detect the binding between the receptor and the ligand, it is necessary to detect the fluorescent coloration of the ligand bound to the receptor, and it is necessary to exclude the coloration due to 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. The smaller the dissociation constant, the greater the relationship between the receptor and the ligand. It is understood that the binding is strong (the intermolecular interaction is strong). In the conventional method, the conjugate concentration itself of each sample can be compared and verified by the fluorescence intensity. There is a problem that it is difficult to verify and compare with the ligand concentration, and it is difficult to absolutely verify the binding strength between the receptor and the ligand depending on the dissociation constant.
In addition, in order to solve such a problem, there is a method for measuring an intermolecular interaction using surface plasmon resonance. This measurement method is a method of immobilizing a ligand molecule and reacting a receptor molecule that interacts with the immobilized ligand. That is, between the ligand and the receptor, that is, the specificity of the molecular interaction and the strength of the affinity. It is advantageous in that information such as the speed of binding / dissociation and the concentration of the conjugate can be obtained. However, this measurement method has problems such as the necessity of dedicated equipment and facilities, high measurement costs, and difficulty in noise removal.
For example, “JP 2000-283984 A”, “Experimental Medicine Separate Volume, Close-up Experimental Method Summary”, Yodosha (2002), detection of low affinity interaction using Biacore, Koichi Inagawa, p143-150 and “Experimental Medicine” Separate volume, Experimental Lecture 3 in the Post-Genome Era GFP and Bioimaging ”Yodosha (2000), Fluorescence Correlation Spectroscopy Masataka Kaneshiro Naoto Yoshida, Yasutomo Nomura, p215-222.
[Problems to be solved by the invention]
In view of the above circumstances, the present invention has been provided, and it is possible to easily verify the bond strength between molecules, that is, the intermolecular interaction, from one measurement result, and to greatly reduce the measurement cost. It is an object of the present invention to provide an intermolecular interaction measurement method capable of achieving the above and a measurement apparatus using the measurement method.

According to the intermolecular interaction measurement method of the present invention, one of the molecules whose interaction is desired to be measured is labeled with a fluorescent dye, a solution containing at least the same is prepared, and another molecule is further prepared with the fluorescent dye. A solution preparation step of preparing a solution containing this in an unlabeled state, and an immobilization step of immobilizing the solution containing an unlabeled molecule prepared by the preparation step in a region of the bottom surface of the well opened upward A reaction step in which the solution containing the labeled molecule prepared in the preparation step is stored in the well and reacted with the solution immobilized in the immobilization step, and the solution reacted in the reaction step. An irradiation step of irradiating light, a fluorescence concentration in a region of the bottom surface of the well on which a solution containing a molecule not labeled in the immobilization step is immobilized, and the labeled amount It has a fluorescence measurement step of comparing measured and fluorescence concentration, and in the region other than the one region the solution stored including. The immobilization step includes a step of immobilizing a predetermined molecule in a region of the bottom surface of the well, and a solution containing an unlabeled molecule includes a molecule having an affinity for the predetermined molecule. Preferably, the method includes a step of dropping a solution containing an unlabeled molecule in a region of the bottom of the well, and a step of dropping a solution containing the unlabeled molecule in a region of the bottom of the well And a step of drying the dropped solution may be used. Furthermore, a plurality of solutions prepared in the above solution preparation step are set under a predetermined concentration condition, and the fluorescence concentration corresponding to each solution of the set concentration condition is measured to measure between the molecules desired to be measured. An appropriate concentration condition / dissociation constant (Kd) of the interaction may be calculated.
In addition, according to the intermolecular interaction measuring apparatus of the present invention, the well has a recess that can store a solution, and the bottom surface of the well is one of the molecules for which intermolecular interaction is desired to be measured. A region for immobilizing a solution containing a molecule that is not fluorescently labeled, and another molecule that is desired to measure an intermolecular interaction with the one molecule and that is fluorescently labeled It consists of other areas for receiving the containing solution. The one region is formed as a solid layer on which a solution containing a molecule that is not fluorescently labeled is immobilized on the bottom surface of the well, and the other region is hollow upward from the bottom surface of the well. The solid layer is preferably formed of a layer containing molecules immobilized directly on the bottom surface of the well, and a solution layer containing molecules that are not fluorescently dropped and dropped onto the layer. Alternatively, it may be formed by drying a solution containing non-fluorescent molecules on the bottom of the well. In addition, it is preferable that a plurality of wells are arranged in the vertical direction and the horizontal direction. Furthermore, the solid layer in the one region is a molecule containing a fluorescently labeled molecule that is desired to measure an interaction with the non-fluorescently labeled molecule included in the solid layer. When stored in a region, the fluorescently labeled molecules are not allowed to enter, but the non-fluorescently labeled molecules included in the solid layer are not allowed to enter the other regions. Preferably it is.

The present invention provides a method for measuring an intermolecular interaction and a measuring apparatus used for a method for measuring an intermolecular interaction. According to this measuring method and measuring apparatus, interacting receptor molecules are fixed and arranged in only one region of the well bottom surface, and ligand molecules exist in other regions in the well (receptor molecule). And the ligand molecule may be reversed, that is, the ligand molecule may be immobilized in one region (hereinafter, this case is referred to as “other case”). The ligand molecule (in other cases, the receptor molecule) is labeled with a fluorescent dye. Therefore, when the receptor-ligand binds to each other, in one immobilized region, a receptor molecule-ligand molecule conjugate and a free receptor molecule (in other cases, a receptor molecule-ligand molecule conjugate). And free ligand molecules), and in other regions of the well there will be free ligand molecules (in other cases free receptor molecules). In this state, when light is irradiated to excite the fluorescent dye, it is the receptor / ligand conjugate that produces fluorescent coloration in one immobilized region, and in the other region free ligand molecules (other cases). In the case of 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 / antigen binding reaction) can be clearly measured at the visual level in the same well, and the surface plasmon resonance method is used. There is no need to use specialized / expensive equipment, and measurement costs can be greatly reduced. Furthermore, in principle, the measurement method and measurement apparatus of the present invention can detect an intermolecular interaction even in a microwell space (1 mm or less). Therefore, it can be utilized in each well of a microplate having a large number of holes, and it becomes possible to easily and quickly detect an appropriate condition of the intermolecular interaction in combination with the visibility of the detection result (described above).
In the above description of the effect of the present invention, the intermolecular interaction between the two regions in the well has been explained by the interaction between the receptor molecule and the ligand molecule. Can be used in general. In other words, it is possible to measure not only the interaction between molecules that are expected to be bound in advance, such as receptor molecules and ligand molecules, but also whether or not they interact between molecules whose presence or absence of interaction is unknown, For example, the presence or absence of interaction between different proteins, between nucleic acids, between proteins and nucleic acids can be measured. In particular, the method and apparatus of the present invention has the effect that it is possible to detect intermolecular interaction even in a minute well space (1 mm or less), and it can be used in each well of a microplate having a large number of holes. In combination with the visibility of detection results (described above), it is possible to easily and quickly detect screening of molecules that interact with each other from many types of molecules (for example, proteins).

FIG. 1 is a diagram showing a microplate used in the intermolecular interaction measurement apparatus of the present invention.
FIG. 2 is an enlarged cross-sectional view of a well disposed on the microplate 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 Scatchard 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 an analysis image of an experimental result in another embodiment.
FIG. 8 is a Scatchard plot diagram showing the experimental results of FIG.
FIG. 9 is an analysis image of an experimental result in still another embodiment.
FIG. 10 is a Scatchard plot diagram showing the experimental results of FIG.

このモデル式からも明らかなようにレセプターとリガンドとの結合体濃度は、総リガンド濃度、総レセプター濃度、解離定数により与えられるものである。従って、結合体濃度（［Ｒ・Ｌ］）は、レセプター濃度が増加するとこれに従って増加し、逆に遊離リガンド（［Ｌ］）は、レセプター濃度が増加すると減少する性質を有することがわかっている。ここで、レセプターとリガンドとが結合し易い場合、すなわち分子間相互作用が強い場合とは解離定数が小さい状態である。また、解離定数はレセプターと対応するリガンドにより決定される固定値である。このことから鑑みてレセプターとリガンドとの両者の結合がし易い場合には、結合体濃度（［Ｒ・Ｌ］）に比して遊離リガンド濃度（［Ｌ］）が小さい状態であることが判る。従って、レセプターとリガンドとの結合のし易さ、すなわち分子間相互作用の強度を検証するには、レセプターとリガンドの結合体濃度と遊離リガンド濃度とを一の反応において同時に比較することが必要とされることが理解される。
次に、レセプターとリガンドの結合状態を測定するための基本原理について説明する。従来より知られている方法としてリガンドを蛍光標識する方法が存在する（レセプターを蛍光標識する場合も存在する）。とりわけ、代表的にレセプターに相当する試料中の抗原に対して蛍光標識した抗体を結合させ、蛍光顕微鏡で検出する方法が存在する。この方法では、抗体をフルオレセインイソチオシアネート（ＦＩＴＣ）やテトラメチルローダミンイソチオシアネート（ＴＲＩＴＣ）等の蛍光色素により標識し、蛍光検出する方法が一般的である。また、検出感度を増大させるため非標識抗体（一次抗体と称する）を抗原と反応させ、蛍光標識した抗体（二次抗体と称する）を、抗原と一次抗体との結合体に結合させ抗原を追跡する方法も存在し、マウス抗体と抗マウス抗体との結合等が揚げられる（後述するが本実施形態では、これを活用している）。
しかしながら、従来の方法のみでは既に分子間相互作用が判明している場合、例えば、特定のレセプター抗原の存在を試料内で検証するために対応するリガンド抗体を付与する場合には有利であるが、レセプターとリガンドの結合体濃度と遊離リガンド濃度との比較検証が困難であるため新たにレセプターとリガンドとの相互作用を検証するには不十分である。
これに対して本発明の測定方法および測定装置では、レセプターとリガンドの結合体濃度と遊離リガンド濃度との比較検証が容易に可能な構成を提供している。再びマイクロプレート１の説明に戻れば、図２ではウェル２内の底面２ｂ近傍の拡大断面図が示されている。この図２は、図１（ａ）の断面図と同一視である。また、図３は図２の平面図を略示しており、図１（ｂ）と同一視である。これらの図に示すようにウェル２内の底面はその一部の領域２ｄ、ここでは中心近傍に所望のレセプター分子を固体化させている。本実施形態においてはレセプター分子としてマウス抗体、リガンド分子として抗マウス抗体を用いており、マイクロプレート１のウェル底面２ｂの一領域２ｄにマウス抗体を所望濃度含む溶液を所定量（１μｌ）滴下し、固体化している。また、ウェル底面２ｂの他の領域２ｃは原始的にはブランクである。ここで、レセプター分子の固定化について言及すれば、ウェル底面２ｄに滴下された溶液を乾燥させて直接的に固定化する方法や、ウェル底面２ｄに所定分子を固定化させた後に該所定分子と親和性が高い分子を含む溶液をマウス抗体を含む溶液と混合させてウェル底面２ｄ上に滴下する方法等が存在する。とりわけ後者の方法は、乾燥によるタンパク質の変質を抑制することができる点で有利である。例えば、ウェル底面２ｄにグルタチオンやニッケルを固定した後に、マウス抗体を含む溶液にＧＳＴタンパク質や金属・ポリヒステジンペプチドを含ませたものをウェル底面２ｄ上に滴下する方法が提供される。この方法の原理は、滴下溶液内のＧＳＴタンパク質等とウェル底面２ｄ上のグルタチオン等との親和性により滴下溶液をウェル底面２ｄに固定化させるものである。
次に、このような状態で形成されたウェル２にリガンド分子を含む溶液を滴下し、ウェル２内に貯留する。このときウェル２内に貯留された溶液（及びウェル底面２ｄに固定化されたレセプター分子を含む溶液）を密閉することが好ましい。前述するように溶液が乾燥すると内部のタンパク質分子（ここではリガンド分子たる抗マウス抗体およびレセプター分子たるマウス抗体）が変質せしめるおそれがあり、これを抑制することができるからである。例えば、液層表面にミネラルオイルやセチルアルコールを滴下して表面被覆する方法やウェル２を物理的に密閉する方法が提供される。ここでは、リガンド分子として前述するようにマウス抗体に対応する抗マウス抗体を用いており、この抗マウス抗体をＦＩＴＣ等の蛍光色素で標識したものを含む所望濃度の溶液を準備し、ウェル２内に滴下して貯留させる。従って、ウェル内２ａでは、その底面の一領域２ｄではレセプターとしてのマウス抗体の一部にリガンドとしての抗マウス抗体が結合し他の部分ではマウス抗体のみ遊離された状態を形成し、他の領域２ｃでは遊離された抗マウス抗体のみが包摂されていることとなる（前述のモデル式参照）。このような状態で、ウェル底面２ｂを上方又は下方から所定強度の光照射すれば、抗マウス抗体に標識した蛍光色素が励起され発色する。その場合、蛍光強度はリガンド濃度に比例し、具体的には領域２ｄではマウス抗体と抗マウス抗体との結合体濃度（前述のモデル式における［Ｒ・Ｌ］）に比例し、領域２ｃでは遊離された抗マウス抗体濃度（前述のモデル式における［Ｌ］）に比例することとなる。従って、それぞれの濃度により領域２ｄ、２ｃとでの蛍光濃度に差が生じることとなり、領域２ｄの蛍光濃度が領域２ｃの蛍光濃度よりも大きければ大きいほどレセプターとしてのマウス抗体とリガンドとしての抗マウス抗体との結合強度が大きい（解離定数が小さい）ことが判別できる。従って、２つの分子間の相互作用が強いか否かを各領域２ｄ、２ｃの蛍光濃度差を一見するだけで測定することが可能となり、レセプターとリガンドの結合濃度［Ｒ・Ｌ］、解離定数Ｋが不明な場合であっても、総レセプター濃度、総リガンド濃度（それぞれ溶夜を準備する段階で既知である）が決定されれば、この条件に対応するレセプターとリガンドの結合強度を容易に理解することができる。
さらに、図１〜３に示す本実施形態のように多数のウェル２を配設するマイクロプレート１を用いれば、それぞれのウェルごとに異なる濃度のレセプター、リガンドを準備することで、両者を反応させた場合に種々の濃度条件における分子間相互作用を一見して理解することができる。このようなウェルごとの濃度条件の設定として、図１のマイクロプレート１で例示すれば、縦方向では行ごと（Ａ〜Ｈ行ごと）にリガンド濃度が大きく又は小さくなるように設定（溶液準備）し、横方向では列ごと（１〜１２列ごと）にレセプター濃度が大きく又は小さくなるように整列すれば、測定結果の理解に好適である。
なお、上述するようにレセプターとして領域２ｄにはマウス抗体を含む溶液を固定化させて、領域２ｃでは抗マウス抗体（ＦＩＴＣ標識有り）を含む溶液を貯留させて検証しており、その結果の一部を抜粋したものが図４に示している（実際には９６穴で検証したが発明の理解を助ける趣旨のもと一部のみを示すこととする）。この実験結果において、マウス抗体濃度は１０μｇ／ｍｌで統一されており、抗マウス抗体濃度は、左上方から下方に従って０．２５、０．４、０．５、０．７５、１、１．２５μｇ／ｍｌ、右上方から下方に従って１．５、１．７５、２、３、４、５μｇ／ｍｌとなっている。この図から考察するに蛍光濃度は、マウス抗体濃度１０μｇ／ｍｌ、抗マウス抗体濃度０．４μｇ／ｍｌの条件の場合に大きく差が出ており、マウス抗体と抗マウス抗体の分子間相互作用が活発であることが理解される。
また、他の実験結果を用いた解離定数（Ｋｄ）の算出について説明する。図５を参照すれば、レセプター分子（ここではマウス抗体）とリガンド分子（ここでは抗マウス抗体）との結合を示したスカッチャード（Ｓｃａｔｃｈａｒｄ）プロットが提供されており、各ウェル２ごとのデータをそれぞれプロットし、これに基づく直線グラフが得られている。この縦軸［Ａ・Ｂ］／［Ａ］は、リガンド・レセプター結合分子と遊離リガンド分子との比値（［Ｒ／Ｌ］／［Ｒ］）を表示し、横軸［Ａ・Ｂ］は、リガンド・レセプター結合分子の濃度（［Ｒ・Ｌ］：ｍｏｌ／ｌ）を表示している。ここでスカッチャードプロットの特性に言及すれば、得られた直線の傾きは解離定数に反比例している。具体的には、傾き＝−１／Ｋｄ となる。従って、図５から明らかなようにマウス抗体と抗マウス抗体との相互作用において得られた直線の傾きは、−２×１０^８（＝ｘの係数）であるため、Ｋｄ＝０．５×１０^−８と算出される。このようにして、各ウェル内の分子濃度を測定すれば解離定数Ｋｄを算出することができ、具体的な分子間相互作用を測定することができる。
次に、さらに詳細な他の追加実験結果について言及しておく。図６に示す参照番号は図２および図３に示す参照番号要素と同一を意味する。ここでは、ＣｏｎｃａｎａｖａｌｉｎＡ（以下「Ｃｏｎ Ａ」と称する）と糖タンパク質（ＲＮａｓｅＢ ｇｌｙｃｏｐｒｏｔｅｉｎ：以下、「ＲＮａｓｅＢ」と称する）との相互作用を検出している。
まず、図６（ａ）に示すように１００μｇ／ｍｌの濃度のＣｏｎ Ａをウェル２の中心の領域２ｄに１μｌ滴下した後に、風乾させて固定した。次に、図６（ｂ）に示すようにタンパク質回収率を向上させるためにブロッキング処理を行う。具体的には、ブロッキング溶液（Ｂｌｏｃｋｉｎｇ ｂｕｆｆｅｒ）として１０ｍｇ／ｍｌ濃度のＢＳＡ（牛血清アルブミン）を６０μｌ程、ウェル２内の前記領域２ｄに加え、２０分程室温で培養（インキュベート）した。その後、図６（ｃ）に示すように洗浄液（Ｗａｓｈｉｎｇ ｂｕｆｆｅｒ）を１００μｌ加え、１度目は加えられた洗浄液を直ちに捨て、さらに洗浄液を加えた後（２度目）、１０分程室温で培養した。次に、蛍光標識（ＦＩＴＣラベル）したＲＮａｓｅＢを０．５，１，１．５，２，２．５，３，３．５，４，４．５，５μｇ／ｍｌの濃度に振り分けて、それぞれ別途のウェル２に加えたあと室温で培養した。そして、２４時間程経過した後に測定および画像解析を行い上述と同様にスカッチャードプロット（Ｓｃａｔｃｈａｒｄ ｐｌｏｔ）を作製した。その解析画像およびスカッチャードプロットがそれぞれ図７および図８に示されている。このようにして計測した結果、Ｃｏｎ ＡとＲＮａｓｅＢの解離定数（Ｋｄ）は２．６×１０^−６と算出され、文献値（１．６４×１０^−６）と略一致することが理解されよう。
なお、図７の解析画像は、上から順に番号が付されており、それぞれＲＮａｓｅＢを０．５，１，１．５，２，２．５，３，３．５，４，４．５，５μｇ／ｍｌのものを示している。また、図８のスカッチャードプロットについては、各ウェル２ごとのデータをそれぞれプロットし、縦軸［Ａ・Ｂ］／［Ａ］は、リガンド・レセプター結合分子と遊離リガンド分子との比値（［Ｒ／Ｌ］／［Ｒ］）を表示し、横軸［Ａ・Ｂ］は、リガンド・レセプター結合分子の濃度（［Ｒ・Ｌ］）を表示しており、得られた直線の傾きが解離定数に反比例していることは既に言及したとおりである（このことは後述する図９および図１０も同様）。
次に図９に示す実験ではＡｕｒｏｒａキナーゼ抗体（以下、「Ａｕｒｏｒａ」と称する）とｈｉｓｔｏｎｅＨ３抗体（以下、「Ｈｉｓｔｏｎｅ」と称する）との相互作用を検出している。
まず、図６（ａ）に示すように１０μｇ／ｍｌの濃度のＨｉｓｔｏｎｅをウェル２の中心の領域２ｄに１μｌ滴下した後に、風乾させて固定した。次に、図６（ｂ）に示すようにブロッキング溶液として１０ｍｇ／ｍｌ濃度のＢＳＡを６０μｌ程、ウェル２内の前記領域２ｄに加え、２０分程室温でインキュベートした。その後、図６（ｃ）に示すように洗浄液を１００μｌ加え、１度目は加えられた洗浄液を直ちに捨て、さらに洗浄液を加えた後（２度目）、１０分程室温でインキュベートした。次に、蛍光標識（ＦＩＴＣラベル）したＡｕｒｏｒａを１，２，３，４，５，６μｇ／ｍｌの濃度に振り分けて、それぞれ別途のウェル２に加えたあと室温で培養した。そして、２４時間程経過した後に測定および画像解析を行いスカッチャードプロットを作製した。その解析画像およびスカッチャードプロットがそれぞれ図９および図１０に示されている。このようにして計測した結果、ＡｕｒｏｒａとＨｉｓｔｏｎｅとの解離定数（Ｋｄ）は１．５×１０^−７と算出され、この解離定数値を示す公知文献は存在しないため、この結果が新規発見とされる。なお、図７の解析画像は、上から順に番号が付されており、それぞれＡｕｒｏｒａを１，２，３，４，５，６μｇ／ｍｌに振り分けたものを示しており、図１０のスカッチャードプロットについては前述のとおりである。
以上、本発明の分子間相互作用測定装置としてマイクロプレート１を用いた実施形態を一例として説明してきたが、本発明はこれに限定されるものではなく、少なくとも１つのウェルの底面（平面でなくとも良い）の一部領域と他の領域とで包含する分子が異なっているように構成された他の装置であっても良く、また、上述の抗体での実例反応で検証したが本発明の思想に鑑みれば相互作用を所望する２つ以上の分子の測定全般に活用することができることが理解できよう。また、本実施形態では２つの分子間の相互作用の場合のみを例示したが、他の固体化領域以外のウェル内領域に貯留させる溶液に他のリガンド分子（非標識）を包含させて競合反応させる方法にも応用することができ、又反応感度を高めるために標識しない一次抗体を抗原（レセプター）に結合させ、所望する抗体（リガンド）を二次抗体として標識し抗原と一次抗体との結合体に結合させて反応させる方法に応用しても良く、いわゆる蛍光測定法（相互作用させる一方の分子を標識し、発する蛍光で測定する方法全般）に応用することができる。さらに、蛍光色素としてはＦＩＴＣに特に限定されるものでなく、他の蛍光色素、例えば、ｃｙ５のごときでも差し支えない。なお、本明細書においてレセプターとリガンドとの表現を用いてきたが、これは質量作用の法則（モデル式参照）における結合作用（平衡作用）に従って説明する便宜上用いたものであり、レセプターとリガンドとを逆に当てはめても良く、相互作用の有無が不明な分子間において相互作用するか否かを測定することであっても（例えば、異なるタンパク質相互間、核酸相互間、タンパク質・核酸相互間等の相互作用の有無を測定する）本発明の特徴（異なる分子が包含される領域を形成して反応させる構成）およびその思想を逸脱するものではないことが当業者には容易に理解できるであろう。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 microplate 1). An example of 1 is shown. Specifically, FIG. 1A shows a side view of a conventional microplate, and FIG. 1B shows a plan view of the conventional microplate. Here, the microplate is a container widely used for tests and tests such as clinical tests, DNA analysis and amplification, and a base 3 having a substantially rectangular outer shape and a cylindrical shape integrally attached to the base 3. It has a cylindrical well 2 such as. A number of wells 2 are arranged in a matrix in the row or column direction with respect to the base 3.
Furthermore, as shown by the broken line in FIG. 1B, the well 2 is a hollow container having a concave portion 2a such as a cylindrical shape whose upper part is opened at the base 3 and the other is closed, like a normal test tube, A rim 4 having a circular shape or the like protruding from the base 3 is formed at a portion opening in the base 3. In this way, the well 2 is configured to be able to store a sample when performing a test or inspection.
The rim 4 slightly protrudes from the upper surface of the base 3, and this is used to prevent sample evaporation and sample mixing (cross contamination) between the wells 2 during the test. Specifically, a cap is attached to the rim 4 or an adhesive film is attached to one side to prevent mixing of the sample. By providing the rim 4, the adhesion with the cap or the adhesive film is improved. Can do.
Here, in the microplate 1 as shown in FIG. 1, the distance between the centers of adjacent wells 2 in the row and column directions is standardized, and the number of wells 2 in one microplate 1 is 8 rows × 12 columns ( 96), 3 rows × 8 columns (24), and the like. The lower end of the well 2 has a flat (or substantially spherical) bottom surface 2b as shown in the side view of FIG. Further, the base portion 3 of the microplate 1 has a flat plate shape as shown in FIG. 1 or a shape having a side wall extending downward from the peripheral edge portion of the flat plate (so-called skirt shape). The microplate 1 is made of a transparent synthetic resin, glass or the like, and is preferably made of a low fluorescent material. The characteristics 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, the intermolecular interaction between the receptor (receptor) and the ligand will be outlined. A receptor is a protein in the cell membrane, cytoplasm, or nucleus that binds to a ligand (specific substance), that is, a neurotransmitter, hormone, cell growth factor, or other substance, and initiates a cell reaction. Have. The receptor and ligand bind to an equilibrium state according to the law of mass action. A model expression of this is shown below.
(Formula)

As is clear from this model formula, the concentration of the conjugate between the receptor and the ligand is given by the total ligand concentration, the total receptor concentration, and the dissociation constant. Thus, it is known that the conjugate concentration ([R · L]) increases with increasing receptor concentration, and conversely, free ligand ([L]) has the property of decreasing with increasing receptor concentration. . Here, when the receptor and the ligand are easily bound, that is, when the intermolecular interaction is strong, the dissociation constant is small. The dissociation constant is a fixed value determined by the receptor and the corresponding ligand. In view of this, when the binding between the receptor and the ligand is easy, it can be seen that the free ligand concentration ([L]) is smaller than the conjugate concentration ([R · L]). . 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 free ligand concentration in one reaction. It is understood that
Next, the basic principle for measuring the binding state between the receptor and the ligand will be described. As a conventionally known method, there is a method of fluorescently labeling a ligand (there is also a case where a receptor is fluorescently labeled). 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 with a fluorescence microscope. In this method, an antibody is generally labeled with a fluorescent dye such as fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (TRITC), and fluorescence detection is generally performed. In addition, in order to increase detection sensitivity, an unlabeled antibody (referred to as a primary antibody) is reacted with an antigen, and a fluorescently labeled antibody (referred to as a secondary antibody) is bound to a conjugate of the antigen and the primary antibody to track the antigen. There is also a method to do this, and the binding between the mouse antibody and the anti-mouse antibody is raised (which will be described later, but this is utilized in this embodiment).
However, it is advantageous when the intermolecular interaction is already known only by the conventional method, for example, when a corresponding ligand antibody is given to verify the presence of a specific receptor antigen in a sample, Since it is difficult to verify and compare the concentration of the receptor-ligand conjugate and the concentration of free ligand, it is insufficient to verify the interaction between the receptor and the ligand.
In contrast, the measurement method and measurement apparatus of the present invention provide a configuration that allows easy comparison and verification between the receptor-ligand conjugate concentration and the free ligand concentration. Returning to the description of the microplate 1 again, FIG. 2 shows an enlarged cross-sectional view of the vicinity of the bottom surface 2 b in the well 2. FIG. 2 is identical to the cross-sectional view of FIG. FIG. 3 schematically shows the plan view of FIG. 2 and is the same view as FIG. As shown in these drawings, a desired receptor molecule is solidified in the bottom surface of the well 2 in a partial 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. A predetermined amount (1 μl) of a solution containing a mouse antibody at a desired concentration is dropped on one region 2d of the well bottom 2b of the microplate 1, It is solidified. The other region 2c of the well bottom 2b is originally blank. Here, regarding the immobilization of the receptor molecule, a method of drying and directly immobilizing the solution dropped on the well bottom surface 2d, or fixing the predetermined molecule on the well bottom surface 2d and There is a method in which a solution containing a molecule having high affinity is mixed with a solution containing a mouse antibody and dropped onto the well bottom surface 2d. In particular, the latter method is advantageous in that it can suppress protein alteration due to drying. For example, a method is provided in which glutathione or nickel is immobilized on the well bottom surface 2d, and then a solution containing a mouse antibody containing a GST protein or a metal / polyhistidine peptide is dropped onto the well bottom surface 2d. The principle of this method is to immobilize the dropping solution on the well bottom surface 2d by the affinity between the GST protein or the like in the dropping solution and glutathione etc. on the well bottom surface 2d.
Next, a solution containing a ligand molecule is dropped into the well 2 formed in such a state and stored in the well 2. At this time, the solution stored in the well 2 (and the solution containing the receptor molecule immobilized on the well bottom surface 2d) is preferably sealed. As described above, when the solution is dried, the internal protein molecules (here, the anti-mouse antibody as a ligand molecule and the mouse antibody as a receptor molecule) may be altered, and this can be suppressed. For example, a method of dripping mineral oil or cetyl alcohol onto the surface of the liquid layer to coat the surface and a method of physically sealing the well 2 are provided. Here, as described above, an anti-mouse antibody corresponding to a mouse antibody is used as a ligand molecule, and a solution having a desired concentration containing the anti-mouse antibody labeled with a fluorescent dye such as FITC is prepared. It is dripped and stored. Accordingly, in the well 2a, a region 2d on the bottom thereof forms a state where an anti-mouse antibody as a ligand binds to a part of the mouse antibody as a receptor and only the mouse antibody is released in the other part. In 2c, only the released anti-mouse antibody is included (see the above model equation). In this state, if the well bottom surface 2b is irradiated with light of a predetermined intensity from above or below, the fluorescent dye labeled with the anti-mouse antibody is excited and develops color. In that case, 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), and in the region 2c, the fluorescence intensity is free. It is proportional to the anti-mouse antibody concentration ([L] in the above-mentioned model formula). Accordingly, a difference occurs in the fluorescence concentration in the regions 2d and 2c depending on the respective concentrations. The larger the fluorescence concentration in the region 2d is, the larger the fluorescence concentration in the region 2c, and the anti-mouse as the ligand and the mouse antibody as the receptor. It can be determined that the binding strength with the antibody is large (the dissociation constant is small). Therefore, it is possible to determine whether the interaction between the two molecules is strong or not by simply looking at the difference in fluorescence concentration between the regions 2d and 2c, the receptor-ligand binding concentration [R · L], the dissociation constant. Even if K is unknown, once the total receptor concentration and total ligand concentration (each known at the stage of preparation of lysis) are determined, the binding strength between the receptor and ligand corresponding to this condition can be easily determined. I can understand.
Furthermore, if the microplate 1 which arrange | positions many wells 2 like this embodiment shown in FIGS. 1-3 is used, both will be made to react by preparing a receptor and a ligand of a different density | concentration for each well. In this case, the interaction between molecules at various concentration conditions can be understood at a glance. As an example of setting the concentration conditions for each well, in the case of the microplate 1 shown in FIG. 1, the ligand concentration is set so as to increase or decrease for each row (A to H rows) in the vertical direction (solution preparation). In the horizontal direction, it is suitable for understanding the measurement results if the receptor concentration is arranged so as to increase or decrease for each column (every 1 to 12 columns).
As described above, as a receptor, a solution containing a mouse antibody is immobilized in the region 2d, and in the region 2c, a solution containing an anti-mouse antibody (with FITC label) is stored for verification. An excerpt of the part is shown in FIG. 4 (actually, it was verified with 96 holes, but only a part is shown for the purpose of helping understanding of the invention). In this experimental result, the mouse antibody concentration is unified at 10 μg / ml, and the anti-mouse antibody concentration is 0.25, 0.4, 0.5, 0.75, 1, 1.25 μg from the upper left to the lower. / Ml, 1.5, 1.75, 2, 3, 4, 5 μg / ml from upper right to lower. Considering this figure, the fluorescence concentration differs greatly when the mouse antibody concentration is 10 μg / ml and the anti-mouse antibody concentration is 0.4 μg / ml, and the interaction between the mouse antibody and the anti-mouse antibody is intermolecular. It is understood that it is active.
The calculation of the dissociation constant (Kd) using other experimental results will be described. Referring to FIG. 5, a Scatchard plot showing the binding of a receptor molecule (here mouse antibody) and a ligand molecule (here anti-mouse antibody) is provided. Each plot is made, and a straight line graph based on this is obtained. The vertical axis [A · B] / [A] displays the ratio value ([R / L] / [R]) between the ligand / receptor binding molecule and the free ligand molecule, and the horizontal axis [A · B] , The concentration of ligand / receptor binding molecule ([R · L]: mol / l) is displayed. Here, referring to the characteristics of the Scatchard plot, the slope of the obtained straight line is inversely proportional to the dissociation constant. Specifically, the slope = −1 / Kd. Therefore, as apparent from FIG. 5, the slope of the straight line obtained in the interaction between the mouse antibody and the anti-mouse antibody is −2 × 10 ⁸ (= coefficient of x), so Kd = 0.5 × 10 Calculated as ^-8 . Thus, if the molecular concentration in each well is measured, the dissociation constant Kd can be calculated, and a specific intermolecular interaction can be measured.
Next, other additional experimental results in more detail will be mentioned. The reference numbers shown in FIG. 6 mean the same as the reference number elements shown in FIGS. Here, an interaction between Concanavalin A (hereinafter referred to as “Con A”) and a glycoprotein (hereinafter referred to as “RNase B”) is detected.
First, as shown in FIG. 6A, 1 μl of Con A having a concentration of 100 μg / ml was dropped on the center region 2d of the well 2, and then air-dried and fixed. Next, as shown in FIG. 6B, a blocking treatment is performed to improve the protein recovery rate. Specifically, 60 μl of 10 mg / ml BSA (bovine serum albumin) as a blocking buffer was added to the region 2d in the well 2 and incubated (incubated) at room temperature for about 20 minutes. Thereafter, as shown in FIG. 6 (c), 100 μl of a washing buffer was added, and the first added washing solution was immediately discarded. After further addition of the washing solution (second time), the cells were incubated at room temperature for about 10 minutes. Next, RNase B labeled with fluorescence (FITC label) was distributed to concentrations of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, and 5 μg / ml. After adding to a separate well 2, incubation was performed at room temperature. Then, after about 24 hours, measurement and image analysis were performed, and a Scatchard plot was prepared in the same manner as described above. The analysis image and Scatchard plot are shown in FIGS. 7 and 8, respectively. As a result of the measurement, the dissociation constant (Kd) of Con A and RNase B is calculated to be 2.6 × 10 ^−6, and it will be understood that it substantially matches the literature value (1.64 × 10 ⁻⁶ ). .
Note that the analysis images in FIG. 7 are numbered sequentially from the top, and RNase B is 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, respectively. 5 μg / ml is shown. For the Scatchard plot of FIG. 8, the data for each well 2 is plotted, and the vertical axis [A · B] / [A] indicates the ratio value of the ligand / receptor binding molecule and the free ligand molecule ([[ R / L] / [R]) and the horizontal axis [A • B] indicates the concentration of ligand / receptor binding molecule ([R • L]), and the slope of the obtained straight line is dissociated. As described above, it is inversely proportional to the constant (this applies to FIGS. 9 and 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 histone H3 antibody (hereinafter referred to as “Histone”) is detected.
First, as shown in FIG. 6A, 1 μl of Histone having a concentration of 10 μg / ml was dropped on the center region 2d of the well 2 and then air-dried and fixed. Next, as shown in FIG. 6B, 60 μl of 10 mg / ml BSA as a blocking solution was added to the region 2d in the well 2 and incubated at room temperature for about 20 minutes. Thereafter, as shown in FIG. 6 (c), 100 μl of the washing solution was added, and the added washing solution was immediately discarded, and after further addition of the washing solution (second time), the mixture was incubated at room temperature for about 10 minutes. Next, fluorescence-labeled (FITC label) Aurora was distributed to concentrations of 1, 2, 3, 4, 5, and 6 μg / ml, added to separate wells 2, and then incubated at room temperature. Then, after about 24 hours, measurement and image analysis were performed to prepare a Scatchard plot. The analysis image and Scatchard plot are shown in FIGS. 9 and 10, respectively. As a result of the measurement, the dissociation constant (Kd) between Aurora and Histone was calculated to be 1.5 × 10 ^−7, and there is no known literature showing this dissociation constant value, and this result was newly discovered. The In addition, the analysis image of FIG. 7 is numbered sequentially from the top, and shows aurora distributed to 1, 2, 3, 4, 5, 6 μg / ml, respectively, and the Scatchard plot of FIG. 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 devices configured such that molecules included in some regions and other regions are different, and verified by an example reaction with the above-described antibody. In view of the idea, it can be understood that the present invention can be used in general measurement of two or more molecules for which interaction is desired. In the present embodiment, only the interaction between two molecules has been exemplified, but other ligand molecules (unlabeled) are included in the solution stored in the well region other than the solidified region, thereby causing a competitive reaction. In order to increase the reaction sensitivity, a primary antibody that is not labeled is bound to an antigen (receptor), and a desired antibody (ligand) is labeled as a secondary antibody to bind the antigen to the primary antibody. The present invention may be applied to a method of reacting by binding to a body, and can be applied to a so-called fluorescence measurement method (a general method of measuring with fluorescence emitted by labeling one molecule to be interacted). Further, the fluorescent dye is not particularly limited to FITC, and other fluorescent dyes such as cy5 may be used. In this specification, the expression of the receptor and the ligand has been used, but this is used for convenience of explanation according to the binding action (equilibrium action) in the law of mass action (see the model equation). May be applied in reverse, and even when measuring whether or not there is an interaction between molecules with unknown interaction (for example, between different proteins, between nucleic acids, between proteins and nucleic acids, etc.) Those skilled in the art can easily understand that the present invention does not depart from the characteristics of the present invention (configuration in which different molecules are included and reacted) and the idea thereof. Let's go.

Claims (9)

Prepare a solution containing at least one of the molecules whose interaction is desired to be measured with a fluorescent dye and prepare a solution containing the same without labeling other molecules with the fluorescent dye. Process,
An immobilization step of immobilizing a solution containing unlabeled molecules prepared by the preparation step in a region of the bottom surface of the well opened upward;
A reaction step of storing a solution containing labeled molecules prepared in the preparation step in the well and reacting with the solution immobilized in the immobilization step;
An irradiation step of irradiating light to the solution reacted in the reaction step;
Fluorescence concentration in one region of the bottom surface of the well to which a solution containing a molecule not labeled in the immobilization step is immobilized, and fluorescence in a region other than the one region in which the solution containing the labeled molecule is stored A fluorescence measurement process for comparing and measuring the concentration,
A method for measuring an intermolecular interaction, comprising: The immobilization step includes a step of immobilizing a predetermined molecule on a region of the bottom surface of the well, and a molecule having an affinity for the predetermined molecule immobilized on the solution containing the unlabeled molecule. The method for measuring an intermolecular interaction according to claim 1, further comprising: dropping the solution containing the unlabeled molecule in a region of the bottom surface of the well. 2. The immobilization step includes a step of dropping a solution containing the unlabeled molecule in a region of the bottom surface of the well, and a step of drying the dropped solution. The method for measuring intermolecular interaction as described. A plurality of solutions prepared in the solution preparation step are set under a predetermined concentration condition, and the interaction between molecules desired to be measured is measured by measuring the corresponding fluorescence concentration for each solution under the set concentration condition. The method for measuring an intermolecular interaction according to claim 1, wherein an appropriate concentration condition / dissociation constant is calculated. Having a well forming a recess capable of storing a solution;
The bottom surface in the well has a region for immobilizing a solution containing a molecule that is one of the molecules whose intermolecular interaction is desired to be measured and is not fluorescently labeled, and the one molecule. Composed of other molecules for which a measurement of intermolecular interactions is desired and for receiving solutions containing fluorescently labeled molecules,
The one region is formed on the bottom surface of the well as a solid layer on which the solution containing the molecule not fluorescently labeled is immobilized, and the other region is hollow upward from the bottom surface of the well. A device for measuring intermolecular interactions, characterized in that it is. The solid layer is formed of a layer containing molecules directly immobilized on the bottom surface of the well, and a layer of a solution containing the non-fluorescently labeled molecules dropped onto the layer. The intermolecular interaction measuring apparatus according to claim 5. 6. The intermolecular interaction measuring apparatus according to claim 5, wherein the solid layer is formed by drying a solution containing the non-fluorescent molecules on the bottom surface of the well. The intermolecular interaction measuring apparatus according to any one of claims 5 to 7, wherein a plurality of the wells are arranged in the vertical direction and the horizontal direction, respectively. The solid layer in the one region has a solution containing a molecule labeled with a fluorescent molecule that is desired to measure an interaction with the non-fluorescent labeled molecule included in the solid layer in the other region. When stored, the fluorescently labeled molecules are not allowed to enter, but are configured so that the non-fluorescently labeled molecules contained in the solid layer do not enter the other region. The intermolecular interaction measuring apparatus according to claim 5, wherein

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