JP2010281595A - Method for detecting ligand molecule - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simple method which can detect the ligand molecules specifically bonded to the receptor molecules present on the surfaces of cells with high sensitivity and high precision. <P>SOLUTION: The method for detecting the ligand molecules specifically bonded to the receptor molecules present on the surfaces of the cells in a sample to be inspected includes: a step (a) for bringing the sample to be inspected into contact with the cells having the receptor molecules present on the surfaces thereof and labelled by fluorescence using a first fluorescent substance; a step (b) for bringing detecting molecules labelled by a second fluorescent substance into contact with the fluorescence labelled cells simultaneously with the sample to be inspected after the step (a) or in the step (a); and a step (c) for measuring the fluorescence resonance energy transfer produced between the first fluorescent substance and the second fluorescent substance after the step (b). The fluorescence labelled cells are the molecules present in cell membranes, cytoplasms or cell membranes other than the receptor molecule and labelled by the first fluorescent substance and the detecting molecules are the molecules specifically bonded to the ligand molecules. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for detecting a ligand molecule that specifically binds to a receptor molecule present on the cell surface with high sensitivity and ease, using the cell itself as a detection reagent.

  Various proteins, sugar chains, lipids, and the like exist on the surface of the cells constituting the living body. For example, a receptor, which is a typical protein present on the cell surface, plays an important role in transmitting a signal into the cell by binding to a ligand from outside the cell. In addition, antigens composed of cell surface glycoproteins are important as signals for self-non-self recognition and activation of the exclusion mechanism as foreign substances. For this reason, detecting substances that bind to such cell surface molecules is not only academic but also clinically significant.

In particular, it is clinically useful to test for the presence of antibodies that bind to cell surface molecules.
For example, in cancer patients, some proteins and sugar chains present on the surface of cancer cells may be recognized as foreign substances and produce autoantibodies using these as antigens. Since such autoantibodies against cancer are mainly present in blood, they are expected as screening methods for cancer by blood tests.

Specifically, for example, Patent Document 1 reports a method for detecting cancer by detecting the presence of autoantibodies against hormone receptors whose expression on cell membranes is enhanced by canceration.
Patent Document 2 discloses a method for improving cancer detection sensitivity by detecting a combination of autoantibodies against a plurality of antigens present on the surface of cancer cells. In this method, a combination of a plurality of autoantibodies against proteins well known to be highly expressed in cancer cells such as MUC1 and ErbB2 (Her2 / neu) is used in combination with the patient's serum. This is a highly sensitive detection method.

  In addition, detection of antibodies against erythrocyte surface antigens is performed as a clinical test. For example, when another person's blood cells are mixed in the body, such as when pregnant or transfused, if there is an antigen that the other person's blood cell does not have, an antibody against that antigen is produced. May be. These are called irregular antibodies, and more than 20 types of antigens have now been identified. If a blood transfusion is performed between a person with an irregular antibody and a person with an antigen against the irregular antibody, serious symptoms such as blood aggregation and hemolysis will occur. Is important to.

  As described above, research on cancer autoantibodies has been conducted for a long time, and although many antigens have been identified, antibodies that use biomolecules present on the cell surface as antigens are simple enough to be applicable to clinical tests. It is very difficult to detect with high sensitivity and specificity. In order to detect cancer with high sensitivity, it is preferable to measure autoantibodies against membrane surface molecules that are always displayed on the surface, rather than autoantibodies that use intracellular molecules as antigens. None of the kits are used for testing. For example, as an autoantibody detection kit approved as a cancer detection method, a detection kit for autoantibodies against mutant p53 protein by ELISA method in which a mutant p53 recombinant protein is immobilized [MESACUP® anti-p53 test, MBL However, the mutant p53 protein is a nuclear protein that is excreted when cancer cells die, and is not a biomolecule that is always present on the cell surface.

  A common difficulty in detecting antibodies that use biomolecules present on the surface of cancer cells or blood cells as antigens is that it is difficult to prepare an appropriate antigen for use in detection. This is because surface molecules are often subjected to complex sugar chain modifications, and it is difficult to supply a recombinant protein that retains antigenicity. Although there is a method of directly isolating and purifying a surface molecule from a cell, since the surface molecule is often transmembrane, it is often very difficult to isolate and purify from the cell. In addition, as described in Patent Document 2, it is more effective to detect not only an antibody against one type of surface molecule but also each antibody against a plurality of surface molecules at the same time on one biological sample. Although the sensitivity can be improved and is clinically significant in many cases, it is more difficult to prepare a plurality of artificially prepared antigens because it takes more time and cost.

  For example, as a method for identifying and screening antigens of autoantibodies against cancer cells, recombinant proteins expressed by cloning and expressing genes expressed in cancer cells are prepared and reacted with cancer patient sera. The SEREX method for identifying the antigens of these autoantibodies has been practiced for a long time. Although many autoantibodies have been found by this method, since artificially expressed recombinant proteins are used as antigens, even if this method is used for clinical tests for screening autoantibodies in biological samples Sensitivity and reproducibility cannot be maintained, and it is difficult to become a clinically meaningful test. This is because there is a difference in the structure such as modification between the recombinant protein and the protein present on the cell surface, and even if it is an antibody against the same antigen, the antibody was produced using any part of the antigen as an epitope between individuals. This is because they are different.

  Such problems relating to the preparation of an appropriate antigen for detection can be solved by using the cell itself as the antigen. For example, in the method described in Patent Document 1, autoantibodies are detected by ELISA using a part of the synthetic sequence of the hormone receptor as an antigen, but the sensitivity of cancer detection is further improved by using the cells themselves as antigens. There is a possibility that. In addition, since many surface molecules exist on the cell surface, a plurality of types of antibodies can be detected by a single cell. That is, unlike the case of artificially preparing a recombinant protein, it is not necessary to prepare an antigen for each type of antibody.

  In fact, in the inspection of irregular antibodies, it is difficult to provide multiple antigens as recombinant proteins because all of the antigens have complex sugar chain modifications. For this reason, the erythrocytes themselves presenting the antigen are prepared as antigens. The test is conducted by confirming the antigen-antibody reaction. The most common method for this test method is the indirect globulin method (Coombs method). In this method, three types of red blood cells and serum to be tested are mixed, reacted at 37 ° C. for 10 to 30 minutes, and then an antibody other than an antibody bound to red blood cells is obtained by performing centrifugation and exchanging physiological saline three times. After exclusion, anti-globulin antibodies are mixed, and the presence of irregular antibodies is determined by visually confirming the aggregation of the erythrocytes bound with the irregular antibodies and the anti-globulin antibodies.

  When the indirect globulin method is performed as a test using a normal test tube, centrifugation and washing must be repeated, and since it is a visual judgment, it is not quantitative and is more accurate depending on the ability of the laboratory technician. Will be different. Thus, several methods for improving the indirect globulin method have been disclosed. For example, automation by a gel column method such as an ID-microtyping system (manufactured by Olympus) is performed. This method is a method of separating aggregates of erythrocyte-binding antibodies on a molecular weight column, and does not require washing, and the number of steps can be reduced. Further, for example, by using a microplate such as the method described in Patent Document 3 (MMPH-A method) or a commercially available irregular antibody detection reagent CAPTURE-R (manufactured by Imcore), throughput can be increased. An improved method is also disclosed. In the MMPH-A method, erythrocytes are immobilized on a U-bottom microplate container, reacted with antibodies in the serum to be examined on the surface of the immobilized erythrocytes, washed, and encapsulated with anti-human globulin antibody-sensitized magnetic material. Irregular antibodies are detected by confirming the binding between the particles and the antibodies. In addition, in Patent Document 4, a cell suspension prepared by diluting cells to a predetermined concentration with a cell diluent having a constant osmotic pressure is solidified in a container, and then the cell suspension is used to remove the surplus cells. In addition, a method is disclosed in which cells such as erythrocytes are uniformly formed on the inner wall of a container by washing with a washing liquid adjusted to be hypertonic.

  In addition, an antinuclear antibody test is performed as a screening method for autoantibodies, which is performed as a clinical test. This is a method of detecting antibodies bound to cells on a slide glass using a fluorescently labeled anti-IgG antibody after applying a blood sample to be examined to a slide glass on which cultured pharyngeal cancer cell Hep-2 is fixed. is there. In this method, autoantibodies using intracellular molecules as antigens are detected. Although the detection target is not a cell surface protein, in order to easily detect autoantibodies against multiple biomolecules, the cells themselves are used as antigens. Yes.

  On the other hand, a method using fluorescence energy transfer (FRET) is known as a method for detecting the binding between molecules. FRET refers to the wavelength and amount of fluorescence emitted from a fluorescent material as a result of energy transfer between the two fluorescent materials (donor fluorescent dye and acceptor fluorescent dye) that are close to each other when they are close to each other. Changes from when the two fluorescent substances are separated from each other, and the two types of molecules are labeled with a fluorescent substance having a relationship between a donor fluorescent dye and an acceptor fluorescent dye, and the resulting FRET is detected. Thus, the distance and bond between molecules can be measured. One of the methods using this method is time-resolved FRET (Time-Resolved Fluorescence Resonance Energy Transfer Assay) using a rare earth dye that emits fluorescence for a long time as a donor fluorescent dye. In this method, delayed fluorescence is measured after excitation. In order to measure the fluorescence after the fluorescence of normal fluorescent dyes fades, in a measurement system using a sample having autofluorescence such as a biological sample, it is possible to perform highly sensitive molecular binding measurement with reduced background. .

  For example, Non-Patent Document 1 reports that it is possible to detect FRET between a fluorescently labeled antibody that binds to IL-2Rα on the surface by applying TR-FRET and fluorescently labeling the whole cell. In this method, the amino group on the entire cell surface is biotinylated with NHS esterification-biotin reagent, and then Eu3 + fluorescent dye (FRET donor fluorescent dye) to which streptavidin is further bound, to thereby bind Eu3 + fluorescence. A cell is produced in which the dye is entirely labeled. On the other hand, an acceptor fluorescent dye-binding antibody is prepared by labeling a monoclonal antibody against IL-2Rα to be detected with Cy5 (FRET acceptor fluorescent dye). When the prepared donor fluorescent dye-labeled cells and the acceptor fluorescent dye-conjugated antibody are mixed, the distance between the acceptor fluorescent dye (Cy5) of the acceptor fluorescent dye-conjugated antibody bound to IL-2Rα on the cell membrane surface and Eu3 + on the cell membrane surface is FRET occurs when the distance at which FRET occurs (for example, 10 nm or less) is reached. Therefore, the presence of IL-2Rα on the cell surface can be detected by measuring the amount of fluorescence at the wavelength at which FRET has occurred.

Japanese Patent No. 3995316 US Pat. No. 7,402,403 JP-A-2-124464 Japanese Patent No. 2821040

Lundin, 3 others, Analytical Biochemistry, 2001, 299, 92-97.

  Of the autoantibody detection methods that use the cells themselves as antigens described above, the antinuclear antibody test is generally performed by immobilizing Hep-2 cells and applying a blood sample to the immobilized cells. Various steps such as antibody reaction, washing operation, secondary antibody binding reaction, washing operation, detection with a fluorescence microscope are required. That is, there is a problem that a plurality of processes that are complicated and difficult to automate are included. Further, since the container for immobilizing the cells is a slide glass, there is a problem that the throughput is poor.

  On the other hand, the method described in Patent Document 2, which is an improved method of the indirect globulin method, does not require washing and is a method that can be automated, but requires centrifugation and is therefore better than antinuclear antibody testing. There is a problem that the throughput is not sufficient. In addition, since the MMPH-A method described in Patent Document 3 uses erythrocytes solid-phased in a U-bottom microplate container, it is a method that can process a large number of specimens at a time, but erythrocytes are monolayered. However, there is a problem that the process of solid-phase formation is complicated and it is very difficult to form a uniform monolayer.

  The method described in Non-Patent Document 1 is a method for detecting a specific antigen present on the cell surface. The cell surface is fluorescently labeled by NHS esterification of an amino group on the cell surface. All the free amino groups above are fluorescently modified. For this reason, depending on the amino acid sequence of the cell surface antigen, the antigenicity is lost due to the fluorescent label, and accurate measurement may not be possible.

  The present invention relates to a simple method capable of detecting a ligand molecule that specifically binds to a receptor molecule present on the cell surface with high sensitivity and high accuracy.

  As a result of diligent research to solve the above problems, the present inventors have used a ligand molecule that specifically binds to a receptor molecule present on the cell surface as a detection reagent using the cell itself on which the receptor molecule is present on the cell surface. And can be detected by detecting FRET between a fluorescently labeled cell and a molecule that specifically binds to a ligand molecule labeled with a fluorescent substance different from the cell, and the cell membrane, cytoplasm, and It has been found that by labeling one or more selected from the group consisting of molecules existing in the cell membrane other than the receptor molecule with a fluorescent substance, the cells can be fluorescently labeled without impairing the three-dimensional structure of the receptor molecule, The present invention has been completed.

That is, the present invention
(1) A method for detecting a ligand molecule that specifically binds to a receptor molecule present on the cell surface in a test sample, wherein (a) the receptor molecule is present on the cell surface and the first fluorescence A step of bringing a test sample into contact with a fluorescently labeled cell labeled with a substance; and (b) after the step (a) or simultaneously with the test sample in the step (a), A step of contacting a detection molecule labeled with a second fluorescent substance, and (c) a fluorescence resonance energy generated between the first fluorescent substance and the second fluorescent substance after the step (b). Measuring one or more metastases, wherein one or more selected from the group consisting of molecules existing in a cell membrane, cytoplasm, and a cell membrane other than the receptor molecule are labeled with a first fluorescent substance. Cell The detection molecule is a molecule that specifically binds to the ligand molecule, and a method for detecting a ligand molecule,
(2) The step (a) is a step of adding the test sample to a cell solution containing the fluorescently labeled cells, and the step (b) is directly added to the cell solution in the step (a). In the step of adding the detection molecule, the measurement of fluorescence resonance energy transfer in the step (c) uses the cell solution itself to which the detection molecule is added in the step (b) as a measurement sample. A method for detecting a ligand molecule according to (1),
(3) The ligand molecule according to (1) or (2), wherein the fluorescently labeled cell is a cell in which a portion having high lipid affinity in the first fluorescent substance is embedded in a cell membrane Detection method,
(4) The method for detecting a ligand molecule according to any one of (1) to (3), wherein the measurement of the fluorescence resonance energy transfer is performed by a time-resolved fluorescence energy transfer method,
(5) The method for detecting a ligand molecule according to any one of (1) to (4), wherein the ligand molecule is an antibody,
(6) The ligand according to any one of (1) to (5), wherein the detection molecule is at least one selected from the group consisting of an anti-globulin antibody, protein A, and protein G. Molecule detection method,
(7) The method for detecting a ligand molecule according to any one of (1) to (6), wherein the fluorescence resonance energy transfer is measured by detecting light having a wavelength of 650 nm or more,
(8) The method for detecting a ligand molecule according to any one of (1) to (7), wherein the test sample is one selected from the group consisting of blood, serum, and plasma,
(9) The method for detecting a ligand molecule according to any one of (1) to (8), wherein the fluorescently labeled cells are one type selected from the group consisting of normal cells or cancer cells,
(10) The method for detecting a ligand molecule according to any one of (1) to (9), wherein the fluorescently labeled cells are blood cells.
(11) A method for detecting an antibody against a cancer cell surface antigen in a biological sample, wherein (a′1) a biological sample to be examined is brought into contact with a cancer cell labeled with a first fluorescent substance. Process,
(B′1) After the step (a′1) or simultaneously with the test sample in the step (a′1), the cancer cell is contacted with a detection molecule labeled with a second fluorescent substance. A process of
(C′1) after the step (b′1), measuring a fluorescence resonance energy transfer that occurs between the first fluorescent substance and the second fluorescent substance, and the cancer cell comprises: One or more cells selected from the group consisting of a cell membrane, a cytoplasm, and a molecule present in a cell membrane other than the cancer cell surface antigen are cells labeled with a first fluorescent substance, and the detection molecule is the antibody A method for detecting an antibody, which is a molecule that specifically binds to
(12) A method for detecting an antibody against a red blood cell surface antigen in a blood sample, comprising: (a′2) contacting a blood sample to be examined with a red blood cell labeled with a first fluorescent substance; ,
(B′2) After the step (a′2) or simultaneously with the test sample in the step (a′2), the red blood cells are contacted with a detection molecule labeled with a second fluorescent substance. And (c′2) measuring the fluorescence resonance energy transfer that occurs between the first fluorescent substance and the second fluorescent substance after the step (b′2), and the red blood cells Is a cell in which one or more selected from the group consisting of a cell membrane, a cytoplasm, and a molecule existing in a cell membrane other than the erythrocyte surface antigen is labeled with a first fluorescent substance, A method for detecting an antibody against an erythrocyte surface antigen, which is a molecule that specifically binds to an antibody;
(13) A kit for detecting a ligand molecule that specifically binds to a receptor molecule present on a cell surface in a test sample, the first fluorescent substance for labeling the cell, and the second fluorescent substance And the first fluorescent substance and the second fluorescent substance are those in which a fluorescence resonance energy transfer occurs between the first fluorescent substance and the second fluorescent substance in proximity to each other. A ligand molecule detection kit,
(14) The first fluorescent substance includes a fluorescent substance having a part having high lipophilicity, a fluorescent substance that can be directly taken into a cell by passive transport or cell endocytosis, and a fluorescent nanoparticle. The ligand molecule according to (13), wherein the ligand molecule is one or more selected, and the detection molecule is one or more selected from the group consisting of an antiglobulin antibody, protein A, and protein G. Detection kit,
Is provided.

  In the method for detecting a ligand molecule of the present invention, since the cell itself is used as a detection reagent, the ligand molecule can be more easily and accurately obtained than when an artificially prepared receptor molecule such as a recombinant protein is used. Can be detected. In general, a measurement system using cells has a solid phase treatment or immobilization treatment of cells, a plurality of washing operations, centrifugation operations, etc., and the process is complicated. Since the method for detecting a ligand molecule uses FRET, it can be carried out without requiring such treatment, and is very simple. In addition, when preparing fluorescently labeled cells, the cell membrane is fluorescently labeled, so there is almost no effect on the three-dimensional structure of the receptor molecule, and there is no change in the binding (antigenicity) with the ligand molecule. Ligand molecules can be detected accurately.

It is the figure which compared the conceptual diagram (B) of the detection method of the ligand molecule of this invention at the time of using the fluorescence labeled cell which labeled the cell membrane with the conceptual diagram (A) of the method described in the nonpatent literature 1. In Example 1, it is the figure which showed the measurement result of the fluorescence intensity of 665 nm of each test sample. In the comparative example 1, it is the figure which showed the measurement result of the fluorescence intensity of 665 nm of each test sample. In the comparative example 2, it is the figure which showed the measurement result of the fluorescence intensity of 665 nm of each test sample. In Example 2, it is the figure which showed the measurement result of the fluorescence intensity of 730 nm of each test sample. In the comparative example 3, it is the figure which showed the dispersion | distribution degree (relative value) of the magnetic particle of each test sample.

  In the present invention, the receptor molecule means a molecule that is a biomolecule presented on the cell surface and specifically binds to a ligand molecule to be detected. The receptor molecule is not particularly limited as long as it is presented on the cell surface, and may be a protein, a sugar chain, or a lipid. There may be. Further, a part of the molecule may be embedded in the cell membrane, or a molecule that penetrates the cell membrane. In the present invention, cancer cell surface antigens and erythrocyte surface antigens are particularly preferable.

  In the present invention, the ligand molecule is not particularly limited as long as it is a molecule that specifically binds to the portion of the receptor molecule presented on the cell surface, and may be a protein or a low molecular weight compound. Also good. In the present invention, it is preferably a biomolecule contained in a biological sample, and more preferably an antibody or a biomolecule that functions as an information transmitting substance. Examples of biomolecules that function as information transmission substances include hormones and vitamins.

  More specifically, when the ligand molecule is an antibody, the receptor molecule is an antigen such as a cancer cell surface antigen or an erythrocyte surface antigen, and when the ligand molecule is an information transmitter such as a hormone, the receptor molecule Is a receptor for the information transmitting substance.

  In the present invention, the detection molecule means a molecule that specifically binds to a ligand molecule. The detection molecule used in the present invention is not particularly limited as long as it is a molecule that can bind without inhibiting the binding between the ligand molecule bound to the receptor molecule present on the cell surface and the receptor molecule. Instead, it may be a biomolecule such as a protein or a synthetic compound. Specifically, when the ligand molecule is an antibody, a molecule that binds to a portion other than the antigen recognition site (variable region) of the antibody, such as an antiglobulin antibody (anti-IgG antibody, anti-IgM antibody), proteinA, proteinG. Etc. Further, when the ligand molecule is a protein such as a hormone (including a peptide), an antibody specific for the ligand molecule, a protein capable of binding to the ligand molecule, and the like can be mentioned.

  In the method for detecting a ligand molecule of the present invention, a cell in which a receptor molecule is present on the cell surface is labeled with a first fluorescent substance, a test sample is brought into contact therewith, and then the ligand molecule labeled with a second fluorescent substance. A detection molecule that specifically binds to is added. When a ligand molecule is present in the test sample, the ligand molecule is bound to a receptor molecule on the cell surface, and a detection molecule is further bound to the ligand molecule. And a second fluorescent material labeled with the detection molecule. That is, the ligand molecule in the test sample can be detected by detecting FRET generated between the first fluorescent substance and the second fluorescent substance.

  The first fluorescent substance for labeling the cells and the second fluorescent substance for labeling the detection molecules are not particularly limited as long as FRET is generated between the two in close proximity to each other, and the reaction using FRET Any combination of fluorescent materials commonly used in the system may be used. Further, either the first fluorescent substance or the second fluorescent substance may be a donor fluorescent dye, and any of them may be an acceptor fluorescent dye. Further, the acceptor fluorescent dye may be a fluorescent substance, or a quenching substance (quencher) that converts fluorescent energy received from the donor fluorescent dye into thermal energy.

  In the present invention, the donor fluorescent dye used as the first fluorescent substance or the second fluorescent substance is preferably a fluorescent substance that enables TR-FRET. Specifically, it is preferable to use a rare earth complex (such as europium or terbium) that enables long-lived fluorescence as the donor fluorescent dye. Many cells have autofluorescence, but by using TR-FRET for detection, the background during measurement by autofluorescence can be reduced.

  In addition, when using cells that easily absorb light of a specific wavelength, such as erythrocytes, as the cells labeled with the first fluorescent substance, the donor fluorescent dye used as the first fluorescent substance or the second fluorescent substance and As the acceptor fluorescent dye, the excitation wavelength of the donor fluorescent dye, the excitation wavelength of the acceptor fluorescent dye (the wavelength of light emitted from the donor fluorescent dye and received by the acceptor fluorescent dye by energy transfer), and the measurement wavelength after energy transfer ( It is preferable to use a combination of fluorescent dyes in which the fluorescence wavelength emitted from the acceptor fluorescent dye after energy transfer (hereinafter sometimes referred to as “FRET light wavelength”) avoids the absorption wavelength band of the cell. .

  For example, when erythrocytes are used as fluorescently labeled cells, it is preferable to use a fluorescent dye having a wavelength that avoids absorption of hemoglobin contained in the erythrocytes. That is, the excitation wavelength of the donor fluorescent dye is 400 nm or less, more preferably 320 nm or less, the excitation wavelength of the acceptor fluorescent dye is 600 nm or more, more preferably 650 nm or more, and the wavelength of FRET light is 650 nm or more, more preferably 700 nm. The above is preferable. Examples of useful acceptor fluorescent dyes include APC, Cy5 (manufactured by GE Healthcare Bioscience), Alexa Fluor (registered trademark) series (manufactured by Invitrogen), and Atto dye series (manufactured by Atto tec). .

  In the method for detecting a ligand molecule of the present invention, instead of a recombinant protein of a receptor molecule, a cell in which the receptor molecule is present on the cell surface, the cell membrane, the cytoplasm, and a molecule present in a cell membrane other than the receptor molecule A fluorescently labeled cell in which at least one selected from the group consisting of the above is labeled with a first fluorescent substance is used for detection of a ligand molecule. Easy detection of the presence of ligand molecules that bind to receptor molecules on cell membranes, which are difficult to artificially isolate and express, because the cells themselves that have receptor molecules on the cell surface are used for ligand molecule detection. Can do. In particular, when detecting a plurality of ligand molecules, preparing a recombinant protein to be used as a detection reagent for each ligand molecule requires a great deal of effort. Since it is sufficient to appropriately use cells expressing the receptor molecule, it is very simple. For example, by using one type of cell expressing a plurality of types of receptor molecules, a plurality of types of ligand molecules can be detected by a single detection operation.

  In addition, antibodies that are specifically detected in patients with a specific disease such as cancer, irregular antibodies in blood, and the like are often identified, but antigens are often not identified. In the present invention, since the cell itself is used for detection, it is possible to detect a ligand molecule whose receptor molecule has not been identified. For example, even when an antibody is used as a cancer marker and its antigen is unidentified, the antibody can be detected by using cells collected from a cancer patient.

  The cell used as the fluorescently labeled cell is not particularly limited as long as the receptor molecule is present on the cell surface, and may be a cell directly taken out from a living body or a cultured cell. Good. Further, the cell may be a cell in which the receptor molecule originally exists on the cell surface, or a cell in which the receptor molecule is expressed later by gene transfer or the like. Examples of the cells taken out from the living body include blood cells and cells constituting various organs. Examples of blood cells include erythrocytes, platelets, leukocytes, lymphocytes and the like. Further, it may be a normal cell or a cell causing some kind of disease such as a cancer cell or a cell at an inflammatory site.

  A cell used as a fluorescence-labeled cell may be in a state where a receptor molecule on the cell surface retains the ability to bind to a ligand molecule (sometimes referred to as “antigenic” in the present specification). It may be a living cell or a fixed cell. In the present invention, the ligand-labeled cell is preferably a living cell because the ligand molecule can be detected under conditions closer to the state of functioning in vivo.

  In the fluorescently labeled cells used in the method for detecting a ligand molecule of the present invention, one or more selected from the group consisting of molecules present in cell membranes other than the cell membrane, cytoplasm, and receptor molecule are labeled with a fluorescent substance. In the present invention, since the receptor molecule itself is not labeled, the binding ability of the receptor molecule to the ligand molecule is not impaired, and the whole cell is labeled as in the method described in Non-Patent Document 1. Thus, the sensitivity and specificity of the ligand molecule detection can be improved as compared with the method.

  FIG. 1 is a conceptual diagram (B) of the method for detecting a ligand molecule of the present invention when using fluorescently labeled cells labeled with a cell membrane, compared with the conceptual diagram (A) of the method described in Non-Patent Document 1. FIG. In the method described in Non-Patent Document 1, cell surface antigen 1 is targeted for detection. However, since free amino groups on the cell surface are uniformly labeled with fluorescent substance 2, release in cell surface antigen 1 is performed. The amino group is also labeled with the fluorescent substance 2. Then, when the antibody 4 against the cell surface antigen 1 labeled with the fluorescent substance 3 is added, when the other molecules in the vicinity thereof are labeled with the fluorescent substance 2 instead of the cell surface antigen 1, the antibody 4 is It can bind to cell surface antigen 1 and FRET occurs. On the other hand, when the cell surface antigen 1 itself is labeled with the fluorescent substance 2, the antibody 4 cannot bind to the cell surface antigen 1 and FRET does not occur. That is, the method described in Non-Patent Document 1 has a problem that the detection result varies depending on the amino acid sequence of the cell surface antigen to be detected, the fluorescent labeling method, and the like.

  In contrast, in the method for detecting a ligand molecule of the present invention, since the cell membrane itself is labeled with the fluorescent substance 5, the antigenicity of the receptor molecule 6 on the cell surface does not change, and the ligand molecule 7 to be detected is a receptor molecule. 6, and the detection molecule 9 labeled with the fluorescent substance 8 is bound to the ligand molecule 7, whereby FRET occurs, and the ligand molecule in the test sample is stably detected. be able to.

  As described above, in the present invention, a ligand molecule is detected by detecting FRET generated between a first fluorescent substance labeled with a cell and a second fluorescent substance labeled with a detection molecule. For this reason, it is necessary that the distance between the second fluorescent substance for labeling the detection molecule further bound to the ligand molecule bound to the receptor molecule and the first fluorescent substance for labeling the cell is a distance at which FRET occurs. . In the present invention, a complex composed of a receptor molecule, a ligand molecule and a detection molecule (hereinafter referred to as “three-party complex”) is obtained by fluorescently labeling molecules present in cell membranes other than the cell membrane, cytoplasm, and receptor molecule. In some cases, the distance between the first fluorescent material and the second fluorescent material can be sufficiently shortened.

  The method of fluorescently labeling molecules present in cell membranes other than the cell membrane, cytoplasm, and receptor molecule is not particularly limited as long as it does not affect the binding between the receptor molecule and the ligand molecule present on the cell surface. Alternatively, any method used in the technical field may be used.

  The fluorescent labeling of the cell membrane may be performed by binding a fluorescent substance directly to the cell membrane, but it is preferable to label using the affinity between the cell membrane and the fluorescent substance. For example, the cell membrane itself can be fluorescently labeled by staining it with a fluorescent substance having at least a part having high lipophilicity. Examples of the fluorescent substance having a portion having high lipophilicity include a fluorescent substance having a fatty chain or a fatty acid. The cell membrane is a lipid bilayer composed of phospholipids, and the cell membrane is fluorescently labeled by embedding high lipid affinity sites (lipid tails) such as fatty chains and fatty acids in the fluorescent substance in the lipid bilayer. . Since this method does not cause any chemical changes other than embedding a fluorescent substance in the cell membrane, it is a labeling method with low cytotoxicity. In addition to being able to stain cells alive, expression of receptor molecules by fluorescent labeling is possible. Etc. are preferred without change.

  Examples of the fluorescent substance having a high lipophilic site include CellVue (registered trademark) Kits (manufactured by Polysciences, US Pat. No. 5,665,328), which is a fluorescent substance having a long fatty acid, and the fluorescent substance carbocyanine. Etc. The carbocyanine dye DiI, which is one of the commonly used carbocyanines, is a perchlorate (1,1 ′) of 1,1′-dioctadecyl-3,3,3 ′, 3′-tetramethylindocarbocyanine. -Dioctadedecyl-3,3,3 ', 3'-tetramethyllindocarbocyine perchlorate), which has a structure in which two (di) octadecyl groups are bonded to indocarbocyanine, and has high affinity for the acyl groups of lipid bilayers Hydrophobic octadecyl groups (lipid tails) are embedded in the cell membrane and can label whole cells. In particular, since carbocyanine DiD and CellVue (registered trademark) series are fluorescent materials having fluorescence characteristics in which the excitation wavelength and the wavelength of FRET light are out of the absorption wavelength band of hemoglobin, red blood cells are used as fluorescently labeled cells. Particularly preferred.

  A fluorescent substance having a site with high lipophilicity can be embedded in a cell membrane and labeled with a cell simply by mixing with a cell. For example, the cell membrane can be fluorescently labeled by exchanging the cell culture solution with a fluorescent substance solution and incubating for a predetermined time. Depending on the type of fluorescent substance, the fluorescent substance may be added to the culture solution as it is. Alternatively, the method for detecting a ligand molecule of the present invention may be performed using fluorescently labeled cells in a state mixed with a fluorescent substance solution, and the fluorescently labeled solution is used after removing the fluorescent substance solution and eliminating excess fluorescent substance. You may go. Furthermore, it is also preferable to use fluorescently labeled cells that have been washed with a culture solution or an appropriate buffer after removing the fluorescent substance solution. For example, in the case of CellVue (registered trademark) Kits or carbocyanine, after removing the culture solution from the cells, the fluorescent substance solution is mixed and incubated at room temperature for 2 to 5 minutes, and FCS (Fetal calf Serum) is added. After stopping the reaction, fluorescence-labeled cells can be produced simply by washing with a medium.

  The replacement of the culture solution and the fluorescent substance solution, the washing operation of the fluorescently labeled cells, and the like can be performed in the same manner as usual medium exchange. For example, when the cells labeled with the first fluorescent substance are adhesive cells, the supernatant is removed from the culture vessel using an aspirator or the like, and then a fluorescent substance solution or the like may be added. In addition, when the cells labeled with the first fluorescent substance are floating cells, the mixed solution of the fluorescent substance solution and the cells is allowed to stand for a predetermined time or precipitated by centrifugation, The liquid may be removed, and the fluorescent substance solution or the like may be removed from the cells by filter filtration or the like.

  In addition, when a ternary complex is formed by fluorescently labeling the cytoplasm, the distance between the first fluorescent substance and the second fluorescent substance labeled with the cytoplasm immediately below the cell membrane is the same as when the cell membrane itself is stained. Can be short enough. Cytoplasmic fluorescent labeling can be performed, for example, by incorporating a fluorescent substance into cells. As a fluorescent substance to be incorporated into cells, a fluorescent substance having preferable fluorescence characteristics can be appropriately selected and used from fluorescent substances usually used for labeling cells. In addition, cells with fluorescent labeling of cytoplasm can also be produced by incorporating fluorescent nanoparticles, such as NEO-STEM (made by Bititals), in which a desired fluorescent substance is encapsulated in a silica shell. Can do.

  Such fluorescent substances and fluorescent nanoparticles are taken into cells by passive transport or cell endocytosis. For this reason, similarly to the fluorescent substance having a portion having a high lipophilicity as described above, the fluorescent nanoparticle or the like can be taken into the cell and the cytoplasm can be fluorescently labeled simply by mixing with the cell. For example, fluorescent nanoparticles are taken up into cells by incubating the cells for 2-3 hours in a solution containing the particles.

  In addition, when a molecule other than the receptor molecule is fluorescently labeled, the first fluorescent substance and the second fluorescent substance are formed when a ternary complex is formed as in the case of labeling the cell membrane or cytoplasm. Can be sufficiently shortened. The molecule present on the cell membrane may be a molecule present on the cell surface or a molecule present on the cell membrane inside the cell. For example, using an intracellular expression system using a known gene recombination technique, a fusion protein with a fluorescent substance of a protein localized in the cell membrane or a protein (including a partial protein such as a domain) located directly under the cell membrane When expressed in, the expressed fluorescent substance fusion protein can be localized in the cell membrane. Examples of the protein localized in the cell membrane include a domain that binds to lipids and an actin protein that is a main constituent molecule of the cytoskeletal system that lines the cell membrane.

  In particular, by using a labeling method that uses a fluorescent substance having a highly lipophilic site or a labeling method that incorporates fluorescent nanoparticles into the cell, cells with fluorescent labeling of the cell membrane or cytoplasm can be produced very easily. be able to. These labeling methods can mix cells and fluorescent substances, and can label cells with a fast reaction time of about 5 minutes. Exclusion of free excess fluorescent substances can be achieved by eliminating fluorescence from cells. This is a very simple method that requires only the work of removing the substance solution. Therefore, by using the labeling method, it becomes possible to produce fluorescently labeled cells for each examination in a clinical laboratory. For example, not only cultured cells and commercially available red blood cells but also disease cells taken from the living body, cells taken from patients in cross-compatibility tests of blood transfusion tests, etc. are used to produce fluorescently labeled cells on the spot It is possible to easily cope with cases that must be done. In addition, since it is a label for a universal cell membrane lipid, any cell can be used in common, and there is no need to change the reagent for each test object.

  The detection molecule can be labeled with the second fluorescent substance by a conventional method. For example, when the detection molecule is a protein containing an antibody, it can be labeled by binding a maleimidated or NHS esterified fluorescent substance. In addition, after binding Biotin to the detection molecule, a fluorescent substance bound to streptavidin can be indirectly bound. In this case, it is necessary to consider the labeling method so that the fluorescent substance does not bind to the recognition site of the ligand molecule. For example, when a ligand molecule recognition site contains many cysteines having a thiol group that reacts with maleimide, the fluorescent substance is labeled by a method other than maleimidation.

  In addition, when the detection molecule is a relatively stable molecule such as an anti-globulin antibody, protein A, or protein G, it can be stored for a long time after the labeling. Therefore, the detection molecule labeled with the second fluorescent substance is used as a test reagent. Can be provided as is. For this reason, for example, the fluorescent substance having a high lipophilic site, the fluorescent substance that can be directly taken into the cell by passive transport or cell endocytosis, and cells such as fluorescent nanoparticles are used for the first time. By making a reagent for labeling with a fluorescent substance and a molecule for detection labeled with a second fluorescent substance into a kit and using the kit, the method for detecting a ligand molecule of the present invention can be carried out more easily.

  The test sample used in the method for detecting a ligand molecule of the present invention is not particularly limited as long as the sample is expected to contain a ligand molecule to be detected. Preferably there is. Examples of such biological samples include blood, serum, plasma, bone marrow fluid, lymph fluid, saliva, ascites, exudate, sputum, semen, urine, bile, pancreatic fluid, amniotic fluid, feces, peritoneal lavage fluid, intestinal lavage fluid, lung Examples include lavage fluid, bronchial lavage fluid, and bladder lavage fluid. In the present invention, blood, serum, plasma, bone marrow fluid, lymph fluid and the like are particularly preferable, and serum or plasma is more preferable.

The biological sample used as the test sample may be a sample immediately after collection or may be stored for a certain period after collection. A biological sample can be stored by a conventional method.
In addition, the biological sample may be a sample collected from a living organism such as a human or a prepared sample. The preparation method is not particularly limited as long as it does not impair the ligand molecules contained in the sample, and the preparation method usually used for biological samples and the like can be performed. . Examples of the preparation method include washing and removing mucus, dilution using physiological saline, buffer solution, and the like, separation or concentration of cellular components.

Specifically, the method for detecting a ligand molecule of the present invention includes the following steps (a) to (c).
(A) A step of bringing a test sample into contact with a fluorescently labeled cell in which a receptor molecule is present on the cell surface and labeled with the first fluorescent substance.
(B) A step of contacting the fluorescently labeled cells with a detection molecule labeled with a second fluorescent substance after the step (a) or simultaneously with the test sample in the step (a).
(C) A step of measuring fluorescence resonance energy transfer (FRET) generated between the first fluorescent material and the second fluorescent material after the step (b).
Hereinafter, it demonstrates for every process.

  First, as a step (a), a test sample is brought into contact with fluorescently labeled cells. Thereby, when a ligand molecule is contained in the test sample, the receptor molecule and the ligand molecule on the surface of the fluorescently labeled cell are bound. Specifically, for example, a test sample may be added to the culture medium of fluorescently labeled cells, and the culture medium of fluorescently labeled cells is added to the test sample or a test sample dilution prepared with an appropriate buffer. It may be exchanged. The exchange to the test sample or its diluted solution can be performed by the same method as the usual medium exchange, similarly to the labeling of the cells with the fluorescent substance having a site having high lipid affinity. The temperature at which the test sample is brought into contact with the receptor molecule and the ligand molecule for the binding reaction is not particularly limited, but the binding between the two molecules is a reaction between biomolecules, so that the temperature is 20 to 37 ° C. preferable.

  By incubating the fluorescently labeled cells in a culture solution containing a test sample, or a test sample or a diluted solution thereof (hereinafter sometimes referred to as “a culture solution containing a test sample”) for a predetermined time, The ligand molecule and the receptor molecule in the test sample can be bound more sufficiently, and a more stable detection result can be obtained. The incubation time and temperature can be appropriately determined depending on the type of test sample, the preparation method, etc. For example, the receptor molecule and the ligand molecule can be bound by incubating at 20 to 37 ° C. for 1 to 15 minutes. Can do.

  Next, as a step (b), the fluorescence-labeled detection molecule further brought into contact with the fluorescence-labeled cell with which the test sample is contacted. As a result, since the ligand molecule and the detection molecule are bound to each other, a triplet complex is formed when the ligand molecule is contained in the test sample. Specifically, the detection molecule may be added directly to the culture solution containing the test sample, or by adding a solution containing the detection molecule to the fluorescently labeled cells from which free ligand molecules have been removed by washing. Alternatively, the fluorescently labeled cells and the detection molecule may be contacted. In addition, both the test sample and the detection molecule may be simultaneously brought into contact with the fluorescently labeled cells by adding them to the culture solution.

  By incubating the fluorescently labeled cells for a predetermined time in the presence of the detection molecule, a three-component complex can be formed more sufficiently. The incubation time and temperature can be appropriately determined depending on the type of test sample, the preparation method, etc. For example, by incubating at 20 to 37 ° C. for 1 to 15 minutes, the receptor molecule and the ligand molecule are bound. Can do.

  When the detection molecule is added directly to the culture solution containing the test sample, or when the test sample and the detection molecule are simultaneously brought into contact with the fluorescently labeled cells, they are contained in the test sample. Considering the abundance of substances other than ligand molecules that can be bound to detection molecules, even if detection molecules bind to substances other than ligand molecules, a three-component complex is formed so that the presence of ligand molecules can be detected. In addition, it is important to add a sufficient amount of detection molecules to the culture solution or the like. On the other hand, if any fluorescent dye is present at a certain concentration or more between two fluorescent dyes that cause FRET, the distance between molecules in the solution may be reduced, and FRET may occur without intermolecular bonding. For this reason, in consideration of the above two points, it is important that the concentration of the fluorescently labeled detection molecule is set to a concentration at which nonspecific FRET does not occur and the ligand molecule bound to the fluorescently labeled cells can be detected.

  In addition, after transferring the fluorescently labeled cells from the reaction vessel in which the fluorescently labeled cells and the test sample are contacted in the step (a) to another reaction vessel, the fluorescently labeled cells and the detection molecules are used as the step (b). However, from the viewpoint of easy operability and suppression of contamination, the fluorescently labeled cells and the detection are performed in the same reaction container as the reaction container in which the fluorescently labeled cells and the test sample are contacted in step (a). It is preferable to contact the molecule for use.

  Next, as a step (c), FRET generated between the first fluorescent material and the second fluorescent material is measured. When FRET is detected, it can be determined that a tripartite complex has been formed and a ligand molecule is contained in the test sample. On the other hand, when FRET is not detected, it can be determined that the ligand molecule is not contained in the test sample. In the method for detecting a ligand molecule of the present invention, since FRET is used, the ligand molecule can be detected with high sensitivity.

  In the present invention, in particular, a time-resolved FRET (TR-FRET) that uses a rare-earth complex that is long-lived fluorescence as a donor fluorescent dye to be excited, and measures the fluorescence intensity of FRET light after a certain delay time after excitation. Is preferably used. In this method, autofluorescence such as a biological sample is short-lived fluorescence, which is quenched, and then only the remaining FRET light is measured to eliminate autofluorescence derived from cells and test samples. Sensitive measurement can be performed.

  Time-resolved is a reaction that excites the donor fluorescent dye by irradiating pulsed light, and measures the fluorescence intensity of the fluorescence wavelength after energy transfer emitted from the acceptor fluorescent dye after a certain time. This process is repeated several times. The average value obtained is usually used as the fluorescence intensity. Furthermore, the fluorescence intensity of the fluorescence wavelength of the donor fluorescent dye itself is also measured, and the influence of absorption and the like derived from the biological sample is eliminated by using the ratio of the fluorescence intensity of the energy transfer and the fluorescence intensity of the donor fluorescent dye itself. It is also possible to make an accurate measurement.

  The measurement of FRET light can be performed using a conventional method. For example, FRET can be measured by measuring the fluorescence intensity at the wavelength of FRET light using a fluorescence measuring instrument such as a fluorescence plate reader. It is more preferable to use a plate reader capable of time-resolved measurement.

  In order to determine whether or not FRET has been detected in step (c), steps (a) to (c) are performed using a negative control such as a buffer solution containing no ligand molecule, instead of the test sample. The measured value obtained can be used as a reference. For example, when the measured value of the fluorescence intensity of the FRET light obtained from the test sample is larger than the measured value obtained from the negative control, FRET is detected, and a ligand molecule is contained in the test sample. Can be determined. In order to more accurately determine the presence or absence of FRET detection, it is preferable to perform the negative control and the test sample in the same step when performing steps (a) to (c).

  In addition, the fluorescence intensity of the FRET light or the fluorescence intensity of the fluorescence wavelength when the acceptor fluorescent dye is present alone is measured over time from the time when the detection molecule is brought into contact with the fluorescently labeled cells in the step (b). It is also possible to determine whether or not FRET has been detected by examining changes in fluorescence intensity. For example, when FRET does not occur, the fluorescence intensity of the FRET light does not change. On the other hand, when FRET occurs, the fluorescence intensity of the FRET light is longer than a point of time when the detection molecules are brought into contact with each other. It becomes larger after elapse.

  Further, in place of the test sample, steps (a) to (c) are performed using a sample solution having a known ligand molecule content, and a measurement value obtained from the test sample based on the obtained measurement value. Thus, the ligand molecules contained in the test sample can be quantitatively measured.

  In the present invention, since the presence of the formed ternary complex is directly measured by measuring FRET light, indirect such as chemiluminescence detection by enzyme label widely used for detection of antigen-antibody reaction. Unlike the method of measuring, there is no time lag with the reaction to form a ternary complex, and the formed ternary complex can be monitored over time. Furthermore, by measuring with a constant reaction time for each step for each measurement sample, FRET light is emitted before the binding reaction between the receptor molecule and the ligand molecule or the binding reaction between the ligand molecule and the detection molecule reaches equilibrium. Even if it is measured, the ligand molecule can be detected with high accuracy. In other words, in the present invention, it is not necessary to wait for the binding reaction between the receptor molecule and the ligand molecule or the binding reaction between the ligand molecule and the detection molecule to reach equilibrium, so the ligand molecule can be detected in a short time. It becomes. Specifically, for example, all the steps (a) to (c) can be performed within 30 minutes.

  Since the FRET light occurs only when the first fluorescent substance and the second fluorescent substance come close to each other, even if a free second fluorescent substance (that is, a free detection molecule) is present, Measurement is possible. Therefore, the measurement of FRET light may be performed using the culture solution or the like to which the detection molecule is added in the step (b) as the measurement sample, and using the fluorescence-labeled cells from which the free detection molecule is removed by washing as the measurement sample. Also good.

  That is, in steps (a) to (c), after adding a test sample to a cell solution containing fluorescently labeled cells, a detection molecule is added directly to the cell solution, and the cell solution itself is used as a measurement sample. FRET is measured as a measurement system that does not perform a solution exchange process such as a cleaning operation. The cell solution containing fluorescently labeled cells may be a culture solution or a solution prepared using an appropriate buffer solution. In addition, incubation may be performed as necessary after adding the test sample and after adding the detection molecule.

  As described above, the method for detecting a ligand molecule of the present invention can be carried out in a homogeneous measurement system that does not perform a solution exchange process, that is, a measurement system that does not require processing such as cell centrifugation or cell solid phase. it can. And since there is no need for washing and centrifugation steps, even when floating cells are used as fluorescently labeled cells, the fluorescently labeled cells to be used are used in reaction containers, unlike the MMPH-A method and antinuclear antibody test. There is no need for solidification and no immobilization. That is, although the method for detecting a ligand molecule of the present invention is a method using cells, complicated and difficult pretreatment such as solid phase treatment is unnecessary, and a plurality of specimens such as a multiwell plate are used. Can be used at the same time.

  In general, the cleaning process is likely to cause variation in data. However, in the present invention, by using a homogeneous measurement system, it is possible to obtain a more reliable measurement result with less variation. Furthermore, since the number of processes is small, not only can data be obtained in a shorter time, but also the difference in data and daily difference by the operator can be reduced.

  In addition, after mixing fluorescently labeled cells, test samples, and detection molecules, it is only necessary to measure FRET light, and a normal microplate can be used, and a centrifuge, solution aspiration, and dispensing operations are not required. Therefore, it is possible to easily automate, and it is expected that the apparatus in the case of automation will be a simple and inexpensive apparatus configuration including only a detector. Furthermore, since the steps of centrifugation and washing can be omitted, even when used for clinical examinations, the examination throughput can be made very good.

  Thus, by using the method for detecting a ligand molecule of the present invention, the presence of a ligand molecule that binds to a plurality of receptor molecules on a cell membrane that are difficult to artificially isolate and express can be easily detected. For example, a screening system for autoantibodies against cancer in a biological sample such as blood can be prepared using cancer cells as fluorescently labeled cells. It is also possible to create a screening system that detects the presence of autoantibodies in biological samples against cells of various organs.

  In the method for detecting a ligand molecule of the present invention, unfixed living cells can be used as fluorescently labeled cells. In other words, since the receptor molecule and the ligand molecule can be bound in a state closer to the state of functioning in the living body, the reactivity between the antibody in the biological sample such as blood and the surface antigen on the fluorescently labeled cells is good. In addition, since the presence of autoantibodies against a plurality of factors can be detected, the presence of a disease can be detected with high sensitivity. Furthermore, by using erythrocytes as fluorescently labeled cells, it is possible to easily test for irregular antibodies in blood and to perform cross-compatibility tests.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

[Example 1]
<Method of detecting autoantibodies in serum using breast cancer cells with fluorescently labeled cell membrane>
First, breast cancer cultured cell strain MCF7, which is known to have enhanced estrogen receptor and progesterone receptor, was labeled with a fluorescent substance (lipid tail fluorescent dye) having a site with high lipophilicity. Specifically, carbocyanine DiD (Molecular-probe) (Em644 nm / Ex665 nm) was mixed with breast cancer cultured cells and incubated for 5 minutes, then the supernatant was removed by centrifugation, suspended in PBS and fluorescently labeled. A cell solution of breast cancer cells was prepared.
Next, the obtained cell solution is dispensed onto a microplate so that the number of fluorescently labeled breast cancer cells is 1000 / well, and further, cancer patient serum (5 samples), healthy patient serum (5 samples), or buffer solution And mixed and incubated at 37 ° C. for 10 minutes. Thereafter, an anti-IgG antibody (manufactured by CisBio) labeled with Eu3 + fluorescent dye prepared in advance was further added to each well so as to be 10 nM, mixed, and incubated at 37 ° C. for 10 minutes to react. I let you. The microplate was placed in a Tecan plate reader, excited at 320 nm, and the fluorescence intensities at 620 nm and 665 nm were measured. The measurement result of the fluorescence intensity of 665 nm of each test sample is shown in FIG.

As a result, it was confirmed that the fluorescence intensity of 665 nm was enhanced in all five specimens of cancer patient sera. On the other hand, in the healthy patient serum and the buffer, neither increase in fluorescence intensity was confirmed.
That is, it is clear that by using the method for detecting a ligand molecule of the present invention, an antibody against a cancer cell surface antigen expressed in breast cancer cultured cell MCF7 strain, which is present in the serum of a cancer patient, can be detected. .

[Comparative Example 1]
<Detection method of autoantibodies in serum using breast cancer cells fluorescently labeled by NHS esterification>
Breast cancer cultured cell strain MCF7 was fluorescently labeled with NHS esterified Cy5 (GE-Healthcare) (Em649 nm / Ex670 nm) and suspended in PBS to prepare a cell solution of NHS esterified fluorescently labeled breast cancer cells.
The cancer patient serum (5 samples), healthy patient serum (5 samples) examined in Example 1, and self in buffer solution, except that the prepared NHS esterified fluorescently labeled breast cancer cells were used. Antibody was detected. The measurement result of the fluorescence intensity of 665 nm of each test sample is shown in FIG.
As a result, the enhancement of the fluorescence intensity in the cancer patient serum was weak, and the difference from the healthy patient serum could not be detected in the three samples. There was no enhancement of serum intensity of healthy patients and fluorescence intensity of the buffer.
From this result, it was suggested that the fluorescent labeling by NHS esterification might enter some free amino acids of the antigen on the cell surface and lose its antigenicity, so the presence of antibodies against some antigens could not be detected .

[Comparative Example 2]
<Detection method of autoantibodies in serum using peptide antigen instead of fluorescently labeled cells>
Instead of fluorescently labeled breast cancer cells, a polypeptide composed of a partial sequence of estrogen receptor and a polypeptide composed of a partial sequence of progesterone receptor were respectively labeled with Cy5 (GE-Healthcare) (Em649nm / Ex670nm). Except for the above, in the same manner as in Example 1, cancer patient sera examined in Example 1 (5 samples), healthy patient sera (5 samples), and autoantibodies in the buffer were detected. The measurement result of the fluorescence intensity of 665 nm of each test sample is shown in FIG.
As a result, it was confirmed that three of the five serum samples of cancer patients showed an increase in fluorescence intensity, but the remaining two samples did not show an increase in fluorescence intensity. Based on this result, the autoantibodies in the serum of cancer patients recognize epitopes other than the assumed partial sequence of each receptor, or recognize cancer-specific substances other than each receptor. The possibility is suggested.

  From the results of Example 1 and Comparative Examples 1 and 2, in the system for screening autoantibodies in blood, the case where an artificially produced antigen such as a peptide antigen is used instead of the whole cell as an antigen. It is clear that the detection can be performed without losing antigenicity by using a fluorescent material having a higher detection sensitivity and fluorescently labeling cells with a lipid-affinity site.

[Example 2]
<Detection of irregular antibodies in serum using erythrocytes whose cell membrane is fluorescently labeled>
First, erythrocytes were prepared by fluorescently labeling a cell membrane with a fluorescent substance (lipid tail fluorescent dye) having a high lipophilic site. Specifically, CellVue (registered trademark) Kits (manufactured by Polysciences) was mixed with antigen-presenting type O red blood cells (manufactured by Orthodiagnostics), incubated for 5 minutes, centrifuged, and the supernatant was aspirated, and LISS A cell solution of fluorescently labeled erythrocytes was prepared by suspending in a buffer solution.
Next, the obtained cell solution is dispensed into a microplate in an equal amount in each well, and anti-E serum, anti-C serum, anti-e serum, or negative control buffer containing an irregular antibody is added. Mix and incubate at 37 ° C. for 10 minutes. Thereafter, an anti-IgG antibody (manufactured by CisBio) labeled with Eu3 + fluorescent dye prepared in advance was added to each well, mixed, and incubated at 37 ° C. for 10 minutes for reaction. The microplate was placed in a Tecan plate reader, excited at 320 nm, and the fluorescence intensity at 730 nm was measured. The measurement result of the fluorescence intensity of 730 nm of each test sample is shown in FIG.

As a result, sera containing irregular antibodies increased the fluorescence intensity at 730 nm after FRET, but the buffer solution did not increase the fluorescence intensity.
That is, it is clear that irregular antibodies in serum can be detected by using the method for detecting a ligand molecule of the present invention.

[Comparative Example 3]
<Method for detecting autoantibodies in serum using MMPH-A method>
First, a microplate container in which red blood cells were solid-phased in a monolayer on the container bottom was prepared. Specifically, first, a 10 μg / ml lectin solution was dispensed into a multiwell plate, incubated at 4 ° C. for 12 hours, and then washed 5 times with PBS to prepare a lectin-coated plate. Antigen-presenting type O red blood cells (manufactured by Orthodiagnostics) were dispensed onto this plate, placed at room temperature for 10 minutes, and then washed three times with physiological saline.
Separately, the magnetic particle inclusion body and 10 μg / ml anti-IgG antibody were mixed, placed at 37 ° C. for 60 minutes, and then washed three times with PBS to prepare an anti-IgG-sensitized magnetic particle inclusion body.
Serum containing a LISS buffer and an irregular antibody was dispensed into a microplate container on which the prepared erythrocytes were immobilized, placed at room temperature for 10 minutes, and then washed 6 times with physiological saline. Furthermore, after the anti-IgG-sensitized magnetic particle inclusion body is dispensed and placed at room temperature for 3 minutes, the magnet plate is placed under the microplate container, and the state of dispersion of the magnetic particles in each well is visually confirmed. did. FIG. 6 shows the degree of dispersion (relative value) of the magnetic particles in each test sample.
As a result, in the serum containing irregular antibodies, the dispersion of the magnetic particles was confirmed, but in the buffer solution, the magnetic particles were collected and collected at the bottom of the plate.

  In the MMPH-A method, there are not only complicated pretreatment steps such as preparation of a lectin-coated plate and anti-IgG-sensitized magnetic particles, but also nine washing steps during the reaction. It took a long time to react, and a lot of waste liquid came out. On the other hand, as shown in Examples 1 and 2, in the ligand molecule detection method of the present invention, there is no need for a washing step, and a series of steps from preparation of fluorescently labeled cells to reaction and detection can be performed in 30 minutes. It was possible to carry out the process, and no waste liquid was generated with the cleaning.

  The method for detecting a ligand molecule of the present invention can be easily and highly sensitively detected for a ligand molecule that specifically binds to a receptor molecule present on the cell surface, and thus can be used particularly in fields such as clinical examinations.

  DESCRIPTION OF SYMBOLS 1 ... Cell surface antigen, 2 ... Fluorescent substance, 3 ... Fluorescent substance, 4 ... Antibody, 5 ... Fluorescent substance, 6 ... Receptor molecule, 7 ... Ligand molecule, 8 ... Fluorescent substance, 9 ... Detection molecule

Claims (14)

A method for detecting a ligand molecule that specifically binds to a receptor molecule present on a cell surface in a test sample,
(A) contacting a test sample with a fluorescently labeled cell in which a receptor molecule is present on the cell surface and labeled with a first fluorescent substance;
(B) after the step (a) or simultaneously with the test sample in the step (a), contacting the fluorescently labeled cells with a detection molecule labeled with a second fluorescent substance;
(C) measuring the fluorescence resonance energy transfer that occurs between the first fluorescent material and the second fluorescent material after the step (b);
Have
The fluorescently labeled cells are cells in which at least one selected from the group consisting of a cell membrane, a cytoplasm, and a molecule present in a cell membrane other than the receptor molecule is labeled with a first fluorescent substance;
A method for detecting a ligand molecule, wherein the molecule for detection is a molecule that specifically binds to the ligand molecule. The step (a) is a step of adding the test sample to a cell solution containing the fluorescently labeled cells,
The step (b) is a step of adding the detection molecule directly to the cell solution in the step (a),
2. The detection of a ligand molecule according to claim 1, wherein the measurement of the fluorescence resonance energy transfer in the step (c) uses the cell solution itself to which the detection molecule is added in the step (b) as a measurement sample. Method.   The method for detecting a ligand molecule according to claim 1 or 2, wherein the fluorescently labeled cells are cells in which a portion having a high lipid affinity in the first fluorescent substance is embedded in a cell membrane.   The method for detecting a ligand molecule according to any one of claims 1 to 3, wherein the fluorescence resonance energy transfer is measured by a time-resolved fluorescence energy transfer method.   The method for detecting a ligand molecule according to any one of claims 1 to 4, wherein the ligand molecule is an antibody.   The method for detecting a ligand molecule according to any one of claims 1 to 5, wherein the detection molecule is at least one selected from the group consisting of an anti-globulin antibody, protein A, and protein G.   The method for detecting a ligand molecule according to any one of claims 1 to 6, wherein the fluorescence resonance energy transfer is measured by detecting light having a wavelength of 650 nm or more.   The method for detecting a ligand molecule according to any one of claims 1 to 7, wherein the test sample is one selected from the group consisting of blood, serum, and plasma.   The method for detecting a ligand molecule according to any one of claims 1 to 8, wherein the fluorescently labeled cells are one type selected from the group consisting of normal cells or cancer cells.   The method for detecting a ligand molecule according to any one of claims 1 to 9, wherein the fluorescently labeled cells are blood cells. A method for detecting an antibody against a cancer cell surface antigen in a biological sample, comprising:
(A′1) a step of bringing a biological sample to be examined into contact with a cancer cell labeled with the first fluorescent substance;
(B′1) After the step (a′1) or simultaneously with the test sample in the step (a′1), the cancer cell is contacted with a detection molecule labeled with a second fluorescent substance. A process of
(C′1) After the step (b′1), measuring a fluorescence resonance energy transfer generated between the first fluorescent material and the second fluorescent material;
Have
The cancer cell is a cell in which one or more selected from the group consisting of a cell membrane, a cytoplasm, and a molecule present in a cell membrane other than the cancer cell surface antigen is labeled with a first fluorescent substance;
A method for detecting an antibody, wherein the detection molecule is a molecule that specifically binds to the antibody. A method for detecting an antibody against a red blood cell surface antigen in a blood sample, comprising:
(A′2) a step of bringing a blood sample to be examined into contact with a red blood cell labeled with the first fluorescent substance;
(B′2) After the step (a′2) or simultaneously with the test sample in the step (a′2), the red blood cells are contacted with a detection molecule labeled with a second fluorescent substance. Process,
(C′2) After the step (b′2), measuring fluorescence resonance energy transfer that occurs between the first fluorescent material and the second fluorescent material;
Have
The erythrocytes are cells labeled with a first fluorescent substance, wherein one or more selected from the group consisting of molecules present in cell membranes, cytoplasm, and cell membranes other than the erythrocyte surface antigen,
A method for detecting an antibody against an erythrocyte surface antigen, wherein the detection molecule is a molecule that specifically binds to the antibody. A kit for detecting a ligand molecule that specifically binds to a receptor molecule present on a cell surface in a test sample,
A first fluorescent substance for labeling cells and a detection molecule labeled with a second fluorescent substance,
The kit for detecting a ligand molecule, wherein the first fluorescent material and the second fluorescent material are those in which fluorescence resonance energy transfer occurs between the first fluorescent material and the second fluorescent material. The first fluorescent substance is selected from the group consisting of a fluorescent substance having a high lipophilic site, a fluorescent substance that can be directly taken into cells by passive transport or cell endocytosis, and fluorescent nanoparticles. One or more,
14. The kit for detecting a ligand molecule according to claim 13, wherein the detection molecule is at least one selected from the group consisting of an anti-globulin antibody, protein A, and protein G.

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