WO2007055226A1 - Method of antigen-specific lymphocytes and method of preparing the same - Google Patents

Abstract

[PROBLEMS] To provide a method whereby antigen-specific lymphocytes can be detected by using a microwell array chip and scanning cells over a wide scope on the chip even in the case of using a scanner having the conventional scanning speed. [MEANS FOR SOLVING PROBLEMS] A method of detecting antigen-specific B lymphocytes. This method comprises: preparing a fluorescent labeled antigen as an antigen; preparing a nonspecific substance of the same type as the fluorescent labeled antigen; preparing a microwell array chip having a B lymphocyte in each well; bringing the B lymphocytes on the microwell array chip into contact with the fluorescent labeled nonspecific substance; detecting B lymphocytes emitting fluorescence; bringing the B lymphocytes on the microwell array chip into contact with the fluorescent labeled antigen; detecting B lymphocytes emitting fluorescence; and selecting B lymphocytes that emit fluorescence not after the contact with the nonspecific substance but after the contact with the antigen. A method wherein the antigen-specific B lymphocytes are substituted by antigen-specific T lymphocytes and a fluorescent labeled MHC/peptide complex tetramer is employed as an antigen.

Description

 Specification

 Method for detecting and preparing antigen-specific lymphocytes

 Technical field

 [0001] The present invention relates to a method for detecting and preparing an antigen-specific lymphocyte.

 Background art

 [0002] As a method for detecting antigen-specific lymphocytes (particularly B lymphocytes), there is a method using a flow cytometer. Since antigen-specific B lymphocytes express antigen-specific antibodies on the cell surface, the fluorescent-labeled antigen is bound to the antigen-specific antibodies on the cell surface by incubating the fluorescently labeled antigen with B lymphocytes. The cells are stained with a fluorescently labeled antigen, and B lymphocytes bound with the fluorescently labeled antigen can be detected and separated with a flow cytometer.

 [0003] When antigen-specific B lymphocytes are detected using a fluorescently labeled antigen, a phenomenon occurs in which the fluorescently labeled antigen binds to cells non-specifically. This occurs because fluorescent dyes or antigens bind nonspecifically to the surface of B lymphocytes. Since the 1% strength of B lymphocytes stained in a non-antigen-specific manner is about 0.1%, the antigen-specific B lymphocytes are antigen-specific when the percentage of antigen-specific B lymphocytes is high (about 10%). B cells can be detected without problems using a flow cytometer. However, if the frequency of antigen-specific B lymphocytes is about 1% to 0.1%, that is, if the frequency is about the same as that of cells that are non-specifically stained, the flow cytometer will be antigen-specific. It becomes difficult to distinguish cells stained with fluorescent dyes from cells that are stained nonspecifically (Figure 1). This is due to the fact that the same cell cannot be measured twice in the detection using a flow cytometer because lymphocytes flow through the buffer at a very fast speed at the fluorescence detection site of the device.

[0004] The present inventors have so far used a chip (microweller chip) that has a size and shape of 45,000 wells that can contain exactly one lymphocyte, and 230,000 ur regularly arranged. We have developed a method that can detect antigen-specific B lymphocytes. In this case, B lymphocytes loaded with Fluo-4, a fluorescent substance that binds to Ca ²⁺ and enhances fluorescence, are added to the microwell array chip, and one B lymphoid is added to each well. Place the sphere Stimulate B lymphocytes with antigen on the chip, increase the intracellular Ca concentration of the B lymphocytes stimulated with the antigen, and increase the fluorescence of Fluo-4. It was possible to detect. (Japanese Unexamined Patent Application Publication No. 2004-173681 (Reference 1), Japanese Unexamined Patent Application Publication No. 2004-187676 (Reference 2)).

 Disclosure of the invention

 Problems to be solved by the invention

[0005] However, the increase in intracellular Ca ²⁺ concentration of B lymphocytes stimulated with antigen occurs at a peak of about 30 seconds to 1 minute after stimulation, and then gradually decreases. The fluorescence of cells on the chip can be detected using a fluorescence scanner, but in order to detect antigen-specific B lymphocytes by changing intracellular Ca ²⁺ concentration, it takes 30 seconds to 2 minutes after stimulation. There was a need to scan the chip in between. For this reason, it was difficult to read signals from a wide range of cells. In addition, it was necessary to develop a scanner with a high scanning speed in order to read signals from a wide range of cells.

 [0006] Therefore, an object of the present invention is a method for detecting antigen-specific lymphocytes using a microwell array chip, which scans a wide range of cells on the chip even with a conventional scanning speed scanner. Another object is to provide a method capable of detecting antigen-specific lymphocytes.

 Means for solving the problem

[0007] The present invention is as follows.

 [1] A method for detecting B lymphocytes that specifically bind to an antigen, comprising preparing a fluorescently labeled antigen as the antigen,

 Preparing a non-specific substance similar to the antigen labeled with the fluorescence,

 Prepare a microwell array chip, one for each well of the B lymphocyte force microwell array chip,

 The B lymphocytes on the microwell array chip are brought into contact with the non-specific substance labeled with the fluorescence, and after the contact, B lymphocytes that emit fluorescence are detected,

Next, the B lymphocytes on the microwell array chip are brought into contact with a fluorescently labeled antigen, and after contact, fluorescent B lymphocytes that detect fluorescence are detected, Select B lymphocytes that do not fluoresce after contact with non-specific substances, but fluoresce after contact with antigen

Said method. (First embodiment of the present invention)

 [2] When the antigen is a protein, the non-specific substance is also a protein,

If the antigen is a peptide, the non-specific substance is also a peptide,

When the antigen is a sugar chain, the non-specific substance is also a sugar chain,

When the antigen is a lipid, the non-specific substance is also a lipid.

 The method according to [1].

 [3] The method according to [1] or [2], wherein the non-specific substance is substantially the same substance as the antigen except for the antigen epitope.

 [4] The method according to any one of [1] to [3], wherein the fluorescent label for the antigen and the fluorescent label for a non-specific substance are the same.

 [5] The method according to [4], wherein the fluorescently labeled nonspecific substance is removed, and then the fluorescently labeled antigen is contacted.

 [6] The method according to any one of [1] to [5], which is a fluorescent label that can be detected by a fluorescent labeling force scanner.

 [7] A method for preparing an antigen-specific B lymphocyte, comprising collecting the B lymphocyte selected by the method according to any one of [1] to [6].

 [8] A method for detecting T lymphocytes that specifically bind to an antigen, comprising preparing a fluorescently labeled MHC / peptide complex tetramer as the antigen,

Preparing a nonspecific substance similar to the fluorescently labeled antigen,

Prepare a microwell array chip, one for each well of the T lymphocyte force microwell array chip,

The T-lymphocytes on the microwell array chip are brought into contact with the non-specific substance labeled with the fluorescence, and after the contact, the T lymphocytes that emit fluorescence are detected,

Next, the fluorescently labeled MHC / peptide complex tetramer is brought into contact with the T lymphocytes on the microwell array chip, and after contact, fluorescent T lymphocytes are detected, and after contact with a non-specific substance. Fluorescence did not occur and MHC / peptide complex tetramer was contacted Select fluorescent T lymphocytes later

 Said method. (Second embodiment of the present invention)

 [9] The method according to [8], wherein the non-specific substance has the same antigen and MHC, and the peptide is different from the peptide of the antigen.

 [10] The method according to [8], wherein the non-specific substance has the same antigen and peptide and MHC is different from the MHC of the antigen.

 [11] The method according to any one of [8] to [10], wherein the non-specific substance is substantially the same substance as the antigen except for the epitope of the antigen.

 [12] The method according to any one of [8] to [11], wherein the fluorescent label for the antigen and the fluorescent label for a non-specific substance are the same.

 [13] The method according to [12], wherein the fluorescently labeled nonspecific substance is removed, and then the fluorescently labeled antigen is contacted.

 [14] The method according to any one of [8] to [13], which is a fluorescent label that can be detected with a fluorescent labeling force scanner.

 [15] A method for preparing antigen-specific vaginal lymphocytes, comprising collecting vaginal lymphocytes selected by the method according to any one of [8] to [14].

[0008] In the first aspect of the present invention, an antigen obtained by fluorescently labeling an antigen-specific mouse lymphocyte is used to detect the binding of a fluorescently labeled antigen as an index. At that time, the lymphocytes including antigen-specific lymphocytes are seeded on a microwell array chip to fix their addresses (positions). In the present invention, the addressed fixed lymphocytes are first brought into contact with a fluorescently labeled non-specific substance, and the detected lymphocytes bound to the fluorescently labeled non-specific substance are detected. That is, non-specifically stained sputum lymphocytes are detected as noise cells. Next, the fluorescently labeled antigen is brought into contact with the lymphocytes, and the lymphocytes bound with the fluorescently labeled antigen are detected. Because the address on the chip is fixed on the chip, the non-specific substance that is fluorescently labeled does not bind, and the host lymphocyte that is bound by the fluorescently labeled antigen can be identified, and the non-specific signal (Noise) can be removed and specific signals can be detected more accurately.

[0009] In the second aspect of the present invention, antigen-specific sputum lymphocytes are used instead of antigen-specific sputum lymphocytes. Use a fluorescently labeled MHC / peptide complex tetramer as an antigen. The invention's effect

 [0010] The address (position) of the lymphocyte is fixed on the chip. This makes it possible to repeatedly measure the fluorescence signal of the same lymphocyte. In the present invention, lymphocytes with fixed addresses are first brought into contact with a fluorescently labeled nonspecific substance, and lymphocytes to which the fluorescently labeled nonspecific substance is bound are detected. That is, non-specifically stained lymphocytes are detected as noise cells. Next, the fluorescently labeled antigen is contacted with lymphocytes, and the lymphocytes bound with the fluorescently labeled antigen are detected. Since lymphocytes have fixed addresses on the chip, non-specific substances that are fluorescently labeled do not bind, and lymphocytes that have bound fluorescently labeled antigens can be identified, and nonspecific signals (noise) This makes it possible to detect specific signals with higher accuracy.

 BEST MODE FOR CARRYING OUT THE INVENTION

 [0011] [Method for detecting antigen-specific B lymphocytes] (first embodiment of the present invention)

 The method of the present invention is a method for detecting B lymphocytes that specifically bind to a certain antigen (antigen-specific B lymphocytes). In the method of the present invention,

 (1) a fluorescently labeled antigen as an antigen,

 (2) a non-specific substance similar to the above-mentioned antigen, which is a fluorescent-labeled non-specific substance, and

 (3) B lymphocyte force One microwell array chip placed on each well of the microwell array chip,

 Is used.

 [0012] The antigen can be, for example, a protein, peptide, DNA, RNA, lipid, sugar chain, organic polymer compound (for example, environmental hormone) or the like without particular limitation. Alternatively, it can be a bacterium, virus, autoantigen, cancer antigen or allergen.

[0013] The fluorescent substance used for the fluorescent labeling of the antigen is not particularly limited, and can be, for example, FITC (fluorescence isothiocyanate), PE (phycoerythrin), or the like. However, the fluorescent label is a fluorescent label that can be detected by a scanner (having an excitation wavelength excited by the wavelength of the laser mounted on the scanner, and the wavelength detected by the detector mounted on the scanner. It is preferable that the fluorescent dye emits fluorescence of

 [0014] When a plurality of different antigens are labeled with different fluorescent dyes, a plurality of antigen-specific B lymphocytes against a plurality of antigens can be detected.

[0015] A method for producing an antigen labeled with a fluorescent dye will be described below.

[0016] FITC labeling method for proteins

 Dialyze the protein solution against 0.1M NaHCO 3 (pH 9.5) to make a lmg / mL solution. So

 Three

 Add 1 mg FITC on Celite 10% (Sigma F1628) to the protein solution (equivalent to 1 mg) and incubate at room temperature for 30 minutes. Then remove Celite by centrifuging for 1 min. The protein solution can then be dialyzed against PBS to produce FITC-labeled protein.

[0017] PR labeling needles for protein sputum

 First, SATA (N-succinimidy 卜 S-acetylthioacetate) is bound to PE. Next, GMB¾ (y-maleimiaobutyric acid N-hydroxysuccinimiae ester) is bound to the protein. Finally, PE and protein are bound via SATA and GMBS.

 (References) Current Protocols in Immunology / edited by John E. Coligan, Ada M Kru isbeek, David H Margulies, Ethan M Shevach, Warren Strober. Published by Greene Publishing Associated and Wiley— Interscience, New York

[0018] A non-specific substance similar to an antigen does not have the specificity of an antigen, but has a material force similar to that of an antigen. Such a non-specific substance can be substantially the same substance as the antigen except, for example, an epitope of the antigen. When the antigen is a protein, the non-specific substance is also a protein. When the antigen is a peptide, the non-specific substance is also a peptide. When the antigen is a lipid, the non-specific substance is also a lipid. Is possible.

 [0019] As the fluorescent label for the non-specific substance, the same fluorescent label for the antigen can be used. In particular, it is preferable that the fluorescent label for the antigen and the fluorescent label for the non-specific substance are the same in view of eliminating non-specific binding by the fluorescent label.

[0020] A non-specific substance labeled with a fluorescent dye can be fluorescently labeled using a method similar to that for an antigen. [0021] The microwell array chip in which one B lymphocyte is arranged in each well of the microwell array chip is, for example, a microwell array chip prepared by the method described in the above-mentioned documents 1 and 2. Can do.

 [0022] [Microwell array chip]

 The microwell array chip of the present invention has a plurality of microwells, stores one subject lymphocyte in each microwell, and is used to detect antigen-specific lymphocytes in units of one. In the well array chip, the microwell has a shape and a size in which only one lymphocyte is stored in one microwell.

 [0023] There are no particular restrictions on the shape and dimensions of the microwell, but the shape of the microwell can be, for example, a cylindrical shape. Besides the cylindrical shape, a rectangular parallelepiped, an inverted cone, an inverted pyramid (inverted) (Triangular pyramid, inverted quadrangular pyramid, inverted pentagonal pyramid, inverted hexagonal pyramid, inverted polygonal pyramid with a heptagon or more), or a combination of two or more of these shapes. it can. For example, some can be cylindrical and the rest can be inverted cones. In addition, in the case of an inverted cone or inverted pyramid, the force is such that the bottom surface becomes the opening of the microwell. The shape of the inverted cone or inverted pyramid is cut from the top (in this case, the bottom of the microwell is flat. Can also be). Cylindrical and rectangular parallelepipeds are usually flat at the bottom of the microwell, but can also be curved (convex or concave). The bottom of the microwell can be curved, as is the case when the top force of the inverted cone or inverted pyramid is cut out.

 [0024] The shape and dimensions of the microwells are such that one lymphocyte can be stored in one microwall, taking into account the type of lymphocytes to be stored in the microwell (such as the shape and dimensions of the lymphocytes). It is determined as appropriate. In order to store one lymphocyte in one microwell, for example, the maximum circle diameter force inscribed in the planar shape of the microwell is 1 to 2 of the diameter of the lymphocyte to be stored in the micro-well. It is appropriate that the range is double, preferably 1.1 to 1.9 times, more preferably 1.2 to 1.8 times. The depth of the microwell should be in the range of 1 to 2 times the diameter of the lymphocytes to be stored in the microwall, preferably in the range of 1.1 to 1.9 times, more preferably in the range of 1.2 to 1.8 times. Is appropriate.

[0025] If the microwell is cylindrical, its dimensions can be, for example, 5-100 μm in diameter, and if the lymphocyte is a B lymphocyte, preferably the diameter is 5-15 μm . Also deep The depth can be, for example, 5 to 100 / ζ πι, and when the lymphocyte is a lymphocyte, preferably the depth can be 5 to 40 / ζ πι. However, the size of the microwell is appropriately determined in consideration of a suitable ratio of the size of the microwell to the diameter of the lymphocyte to be stored in the microwell as described above.

[0026] The number of microwells included in one microwell array chip is not particularly limited, but it is about 500 when the frequency of antigen-specific lymphocytes is higher than 1 in 10 ^5. From, for example, it can range from 2,000 to 1,000,000 per cm ² .

 [0027] The antigen-specific lymphocyte detection microwell array chip used in the present invention has a plurality of microwells, and each microwell contains one specimen lymphocyte. As the microwell array chip, the above microwell array chip can be used as it is.

 [0028] The microwell array chip for detecting antigen-specific lymphocytes can identify antigen-specific lymphocytes at the level of one cell by each microwell containing one sample lymphocyte. It becomes possible. That is, in the method for detecting antigen-specific lymphocytes using this microwell array chip, since the sample lymphocyte contained in the microwell is one, the sample lymphocyte that reacts with the antigen is regarded as one cell. Can be identified.

 As a result, the detected antigen-specific lymphocyte can be taken out and the antigen-specific antibody gene or T cell receptor gene can be cloned. For example, if an antigen-specific antibody gene can be cloned, it can be used to produce human monoclonal antibodies in large quantities. It is considered that this antibody can be used for the treatment and prevention of infectious diseases by administering it to patients with infectious diseases.

 [0030] However, the same microwell may contain cells other than lymphocytes together with the subject lymphocytes. This is because cells other than lymphocytes do not react with the antigen and are not detected.

 [0031] In the microwell, the subject lymphocyte is stored together with, for example, a culture solution. Examples of the culture solution include any of the following.

 1.137 mM NaCl, 2.7 mM KC1, 1.8 mM CaCl, 1 mM MgCl, lmg / ml glucose, lmg /

 twenty two

ml BSA, 20mM HEPES (pH7.4) 2. RPMI1640 medium containing 10% FCS (fetal calf serum)

 3. RPMI1640 medium containing lmg / ml BSA

 4. Dulbecco's MEM medium containing 10% FCS (fetal calf serum)

 5. Dulbecco's MEM medium containing lmg / ml BSA

 [0032] In the method of the present invention, first, a B-lymphocyte on the microwell array chip is contacted with a non-specific substance labeled with a fluorescence, and after the contact, B-lymphocytes that emit fluorescence are detected. . Next, a fluorescently labeled antigen is brought into contact with the B lymphocytes on the microwell array chip, and after the contact, fluorescent B lymphocytes are detected. Fluorescence can be detected with a scanner. Fluorescence does not fluoresce after contact with non-specific substances, but fluoresces after contact with antigen Antigen-specific B lymphocytes. By selecting such B lymphocytes, antigen-specific B Lymphocytes are obtained.

 [0033] For example, the addition of the non-specific substance and the antigen to each microwell can be performed as follows.

 1. Use a pipette to add non-specific substance solution or antigen solution to cover the entire surface of the microwell array chip.

 2. Add nonspecific substance solution or antigen solution one by one using an automatic spotter.

FIG. 2 shows a method for detecting antigen-specific B lymphocytes using a cell chip using a fluorescently labeled antigen. A cell expressing an antigen-specific antibody is identified by binding the fluorescently labeled antigen to an antigen-specific antibody on the cell surface and detecting the fluorescence. In this case, since the addresses of the B lymphocytes stored in the micro-well are determined, it is possible to perform the staining operation twice or more, that is, the binding operation with a fluorescently labeled antigen or the like.

[0035] More specifically, it is as follows.

Seed B lymphocytes in a microwell array chip and place one B lymphocyte on each well. Next, a non-specific substance similar to the fluorescently labeled antigen is added on the chip. For example, if the antigen used is a protein, a protein unrelated to the fluorescently labeled antigen is added to the chip. At this point, non-specifically stained B lymphocytes are detected using a scanner. Next, the fluorescently labeled antigen is added to the chip, the antigen-specific antibody is bound to the B lymphocytes expressing the antigen-specific antibody on the cell surface, and the antigen-specific B re-ligation is performed with a scanner. The signal of the lymphocyte is detected. When using a microwell array chip, the B lymphocytes in the microwell array basically do not move due to staining, washing, etc., and the cell address is fixed, so the same cell is nonspecific. It is possible to compare simple staining with antigen-specific staining. In other words, non-specifically stained B lymphocytes are not stained by non-specific staining as opposed to antigen-specific B lymphocytes, and B lymphocytes stained with a fluorescent dye only when antigen is used are antigen-specific. Can be identified as B lymphocytes

[0036] When antigen-specific B lymphocytes are detected using a fluorescently labeled antigen, the intensity of the fluorescent dye bound to the B lymphocytes hardly changes over time. Therefore, when detecting fluorescence using a scanner, scan It may take 5 minutes or 10 minutes (the time that the buffer is not dried and the state of the cells is not deteriorated). In addition, when observing changes in intracellular Ca ²⁺ concentration with changes in Fluo-4 fluorescence, the fluorescence of antigen-specific stimulated B lymphocytes varies depending on the scan time, ranging from low fluorescence intensity to strong fluorescence intensity. Fluorescent cells are observed. That is, the fluorescence intensity is dispersed. However, when antigen-specific B lymphocytes are detected using a fluorescently labeled antigen, there is no variation or change in fluorescence intensity depending on the scanning time, so a uniform signal can be obtained. This makes it easy to distinguish between negative and positive signals.

 [0037] The present invention includes a method for preparing antigen-specific B lymphocytes, which comprises recovering B lymphocytes selected by the above detection method. The selected B lymphocytes can be collected, for example, as follows. Collect B lymphocytes detected using a micromanipulator under a microscope or fluorescence microscope. Alternatively, B lymphocytes detected using an automatic cell recovery device (Sugino Machine Co., Ltd.) can be recovered.

[0038] The collected antigen-specific B lymphocytes are placed in a reaction solution containing reverse transcriptase, and reverse transcription is performed to convert antibody mRNA into cDNA, followed by PCR or 5'-RACE. Amplify the antibody cDNA using. An antigen-specific antibody can be prepared by introducing the amplified antibody cDNA into a protein expression vector and introducing it into an animal cell. Alternatively, the recovered antigen-specific B lymphocytes can be grown using cytoforce-in and CD40 antibodies, etc., and antigen-specific antibody-producing hybridomas can be created using the expanded antigen-specific B lymphocytes. Thus, an antigen-specific antibody can be prepared.

 [0039] [Method for detecting antigen-specific T lymphocytes] (second embodiment of the present invention)

 The method of the present invention detects antigen-specific T lymphocytes by staining the antigen-specific T lymphocytes with a fluorescently labeled MHC / peptide complex tetramer that only detects antigen-specific B lymphocytes. Can also be used.

[0040] The method for detecting an antigen-specific T lymphocyte of the present invention is a method for detecting a T lymphocyte (antigen-specific T lymphocyte) that specifically binds to a certain antigen,

 (1) As an antigen, a fluorescently labeled MHC / peptide complex tetramer,

 (2) a non-specific substance similar to the antigen labeled with fluorescence, and

 (3) A microwell array chip in which one T lymphocyte is placed in each well of the microwell array chip.

 Is used.

 [0041] MHC class I molecule / peptide complex tetramer is a cDNA of the extracellular part (solubilized part) of Major Histocompatibility Complex (MHC) class I molecule, introduced into an expression vector, Create a protein (no steric structure formed at this point). The prepared protein is incubated in the presence of j8 2-microglobulin and an antigenic peptide to produce a solubilized MHC classl molecule in which the peptide binds and forms an accurate three-dimensional structure. Next, piotin is bound to this molecule. MHC class I molecule / peptide complex tetramers can be made by incubating Piotiny MHC class I molecule / peptide complexes with PE-labeled streptavidin. (Reference) Altman JD, Moss PA, Goulder PJ, Barouch DH, McHeyzer— Williams MG, Bell JI, McMichael AJ, Davis MM. P henotypic analysis of antigen-specific T lymphocytes. Science. 274: 94-9b, 1996 (Correction: Davis MM. Science, Science 280: 1821, 1998)

 [0042] Preparation of MHC classll molecule Z peptide complex tetramer is as follows.

MHC class II molecule Alpha chain and j8 chain molecule The extracellular sequence of the cDNA of the extracellular portion is linked to the 3 'side of the cDNA sequence of the leucine zipper motif and the gene sequence of the pyotin 匕 site, and expressed in insect cells. Solubilized MHC class II molecule A molecule in which α-chain and j8-chain molecules are associated via a leucine zipper is formed, and it binds to the piotin 匕 site. And pep After binding the tide, it can be incubated with streptavidin to form a tetramer. (Reference: Novak EJ, Liu AW, Nepom GT, Kwok WW, MHC class II t etramers identify peptide—specific human CD4 + T cells proliferating in response to influenza A antigen. J. Clin. Invest. 104: R63— R67 ( 1999))

 [0043] The fluorescent label for the antigen is the same as that for B lymphocytes. When multiple antigens (MHC / peptide complex tetramers) are labeled with different fluorescent dyes, antigen-specific T lymphocytes against multiple antigens can be detected.

 [0044] The non-specific substance similar to the antigen can be substantially the same substance as the antigen except for the antigen epitope. Specifically, when the antigen is an MHC / peptide complex tetramer, the non-specific substance is the same as the antigen and MHC, and the peptide is different from the peptide of the antigen, or the non-specific substance. This means that the antigen and peptide are the same, and the MHC is different from the MHC of the antigen.

 [0045] The fluorescent label for the antigen and the fluorescent label for the non-specific substance are preferably the same as in the case of B lymphocytes.

 [0046] In the method of the present invention, first, a T-lymphocyte on a microwell array chip is brought into contact with a non-specific substance that is fluorescently labeled, and after the contact, T lymphocytes that emit fluorescence are detected. Next, the fluorescently labeled MHC / peptide complex tetramer is brought into contact with the T lymphocytes on the microwell array chip, and after the contact, fluorescent T lymphocytes are detected. Then, since no fluorescence is emitted after contact with a non-specific substance, and the T lymphocytes that fluoresce after contacting the MHC / peptide complex tetramer are antigen-specific T lymphocytes, they are selected.

 [0047] The present invention includes a method for preparing antigen-specific T lymphocytes, which comprises recovering T lymphocytes selected by the above detection method. The selected T lymphocyte can be collected, for example, as follows. Collect T lymphocytes detected using a micromanipulator under a microscope or fluorescence microscope. Alternatively, T lymphocytes detected using an automatic cell recovery device (Sugino Machine Co., Ltd.) can be recovered.

[0048] The collected antigen-specific T lymphocytes are placed in a reaction solution containing reverse transcriptase, and reverse transcription is performed to convert the antibody mRNA into cDNA, followed by PCR or 5'-RACE. Is used to amplify the T cell receptor (TCR) cDNA. Amplify TCR cDNA with protein expression vector Antigen-specific T cells can be generated by introducing the antibody into other T lymphocytes and expressing the antigen-specific TCR on the cell surface.

 Example

 [0049] Hereinafter, the present invention will be described in more detail by way of examples.

[0050] Comparative Example 1

 Figure 1 shows the detection of antigen-specific B lymphocytes using a fluorescently labeled antigen using a flow cytometer.

[0051] The spleen cells of transgenic mice (HyHELlO-tg mice) carrying the antibody specific to Hen egg lysozyme (HEL) (HyHELlO-tg mice) and spleen cells of normal mice are mixed appropriately, and Hy HEL10-tg mice The cell suspension was prepared so that the total number of spleen cells was 10% (10HEL), 1% (1HEL), 0.1% (0.1HEL), and 0% (0HEL). The samples were stained with piotinylated H EL and PE labeled streptavidin and analyzed with a flow cytometer. The vertical axis shows the fluorescence intensity of the cells stained with PE-HEL, and the horizontal axis shows the fluorescence intensity of the cells stained with the FITC-B220 antibody. Cells are divided into four fractions with PE and FITC fluorescence intensities. The cells in the lower left fraction indicate cells that did not bind to either PE-HEL or FITC-B220 antibody, and the cells in the lower right fraction bound only to the FITC-B220 antibody. Cells that did not bind are shown. That is, B lymphocytes derived from normal mice. The upper right is a cell to which both FITC-B220 antibody and PE-HEL are bound, and is a cell population of HEL-specific B lymphocytes. The percentage of HEL-specific B lymphocytes in all cells is shown. Since the proportion of B lymphocytes in the spleen cells of HyHEL10-tg mice is approximately 50%, in the sample containing 10% HyHEL10-tg mouse spleen cells, approximately 5.6% of the cells are HEL-specific B lymphocytes. Detected as At 1%, the expected proportion of HEL-specific B lymphocytes is 0.5%, but in reality 1.6% are detected. In addition, in the case of 0.1% and 0%, 0.8% and 0.7% of cells are detected in the HEL-specific B lymphocyte fraction. That is, in the analysis using a flow cytometer, PE-HEL-bound cells were present at a ratio of about 0.7 to 0.8% even in the 0% cell group of the negative control. This is thought to be due to non-specific binding of fluorescently labeled proteins, and causes background noise in the analysis. Example 1

1. Almost all B lymphocytes of spleen cells of HyHELlO-tg mice express HEL-specific antibodies on the cell surface. The HyHEL10-tg mouse spleen cells were prepared splenocytes wild-type mice, respectively were combined, total 2xl0 ⁶ to give a cell, HyHELlO- tg 100% splenocytes of whole mouse, 10%, 1 Cell suspensions that accounted for%, 0.1%, 0.01%, and 0% were prepared.

2. Centrifuge the cells for 1,500 rpm for 5 minutes, discard the supernatant, and then add 1 μM Fluo4 (Molecular Probes, Oregon, USA), 0.2% (v / v) Fl 27 (Molecular Probes, Oregon, USA) ) Loadi ng buffer (20mM HEPES pH7.6, 137mM NaCl, 2.7mM KC1, 1.8mM CaCl, O.lmM M

 2 gCl 2, l.lmM Glucose, 0.2% BSA) 1 ml was added and suspended, and then shaken at room temperature for 30 minutes.

 2

 3. About 10 mL of loading buffer was added to the cells, centrifuged at 1,500 rpm for 5 minutes, and the supernatant was removed.

4. The cells were suspended in 10% FCS / RPMI and centrifuged at 1,500 rpm for 5 minutes.

 5. To the cell precipitate, 2 μg of anti-1¾γR antibody (2.4G2, BD Pharmingen, USA) was added, stirred, and allowed to stand at room temperature for 15 minutes. The cells were then suspended in 50 μL or 200 μL of 10% FCS / RPMI.

 6. Add 50 μL of cell suspension to 45,000 or 200 μL to a 230,000 microwell array chip, leave it for 2 to 3 minutes, and stir by pipetting about 3 times.

 7. Remove the buffer on the tip using a pipette, add 50 L (45,000 wells, the same applies below) or 200 L (the same applies below for 230,000 tooltips), and mix gently with a pipette. After the buffer was removed. This was repeated twice more to remove cells that did not enter the well. Finally, 50 L or 200 L of buffer was added, and the cells were covered with a cover glass. Thereafter, the buffer was changed about three more times.

 8. Insert the cell array chip into the scanner (Hitachi Software Engineering Co., Ltd., CRBI 0 Ile-FITC), excitation wavelength 473nm, fluorescence wavelength 535nm, excitation wavelength 532nm, fluorescence wavelength 585nm, resolution 2.5 μm The fluorescence of each cell was measured (Scan 1).

9. After removing the chip from the scanner, remove the buffer while covering the cover glass, add 2 μg / ml of piotinylated BSA to the 50 μL or 200 μL chip, and shield from light for 5 minutes at room temperature. Left to stand. 10. Remove the buffer with the tip, wash the buffer with 50 L or 200 L, wash once, and add 50 μL of PE-labeled streptavidin (Sav-PE, BD Pharmingen, USA) diluted 1: 500 to the tip. Alternatively, 200 μL was added and the mixture was allowed to stand at room temperature for 3 minutes.

 11. After washing the cells three times with buffer one, insert the chip into a scanner (Hitachi Software Engineering Co., Ltd., CRBIO Ile-FITC), excitation wavelength 473 nm, fluorescence wavelength 535 nm, excitation wavelength 532 nm, fluorescence wavelength The fluorescence of each cell was measured at 585 nm and a resolution of 2.5 m (Scan 2).

 12. Remove the chip from the scanner, remove the buffer while covering the cover glass, add 2 g / ml pyotinylated HEL to the 50 μL or 200 μL chip, and leave it in the dark for 5 minutes.

13. After the cells were washed once with buffer 1, 50 μL to 200 μL of Sav-PE diluted 1500-fold with buffer was added to the chip, and left for 3 minutes in the dark.

 14. After washing the cells on the chip three times with a buffer, insert the chip into a scanner (Hitachi Software Engineering Co., Ltd., CRBIO Ile-FITC), excitation wavelength 473nm, fluorescence wavelength 535ηm, excitation wavelength 532nm, fluorescence The fluorescence of each cell was measured at a wavelength of 585 nm and a resolution of 2.5 m (Scan 3).

 15. Measure the fluorescence intensity of each cell using the software supplied with the scanner (Hitachi Software Engineering Co., Ltd., DNASIS). Cells stained with pyotinylated BSA / PE-sav are non-antigen-specific. Cells stained with piotiny HEL / PE-sav without staining with pyotinylated BSA / PE-sav were detected as antigen (HEL) -specific cells (FIG. 3).

 [0053] Fig. 3 shows the results of detection of antigen (HEL) -specific B lymphocytes by a cell chip using fluorescently labeled antigen (PE-HEL).

[0054] Similar to FIG. 1 1 "^ 1" ¾ to 10 ratio of the spleen cells of spleen cells and normal Mausu of _§ Mausu 10:90, 1

: 99, 0.1: 99.9 A mixed cell suspension was prepared. After seeding the cells on the chip, the cells are first stained on the chip with piotin-labeled BSA (b—BSA) and PE-labeled streptavidin (Sav-PE), and the signal is read with a scanner. The cells were stained on the chip using b-HEL) and Sav-PE, and the signal was read with a scanner. In the figure, the horizontal axis shows the fluorescence intensity (A) stained with b-BSA / Sav-PE, and the vertical axis shows the fluorescence intensity (B) stained with bHEL / Sav-PE. The percentage of cells with a B / A ratio of 10 times or more The result is shown. As in Fig. 1, assuming that the proportion of B lymphocytes in the spleen cells of HyHEL10-tg mice is about 50%, 1% is 0.56% cell strength 0.1% 0.049% cells are detected. It is thought that antigen-specific B lymphocytes are detected more accurately with a lower frequency than the meter.

 [0055] 16. Thereafter, the chip was taken out of the scanner, the cover was removed, and the cover glass was removed. After adding buffer on the chip to prevent the cells from drying, the detected antigen-specific B lymphocytes were collected using a micromanipulator (TransferMan NK2, Eppendori) under a fluorescence microscope (BX51WI, Olympus), and RT solution (GSP mix 1 ^ 1, 5x 1st strand buffer 3 ^ 1, dNTP 1.5 ^ 1, lmg / ml BSA 1.5 ^ 1, NP40 1.5 ^ 1, RNaseOUT 0.2 μ 1, rev erse transcriptase 0.2 μ 1, and DEPC- H 0 It was transferred to a PCR tube containing 6.1 ^ 1).

 2

 Then, RT reaction (55 degreeC, 60 minutes) was performed, and cDNA was produced.

With 17. to prepare cDNA, it was amplified cDNA by PCR using primers to amplify the ^{HEL cDNA (1 st PCR 94 °} C 3min; 94 ° C 20sec, 60 ° C 20sec, 72 ° C 1.5min 30cycle; 72 ° C 2 min; cDNA 1.5 ^ 1, 10X Taq Buffer 2 μ \, dNTP 1.6 ^ 1, 1 ^st primerl (H chain TTTGA AGAAA GGGGTTGTAG (SEQ ID NO: 1), L chain CCACACAAAC TCAGGGAAAG (SEQ ID NO: 2) 0.6 μ 1, 1 ^st Primer2 (H chain TGCAGAGACA GTGACCAGAG (SEQ ID NO: 3), L chain CAGCCCGTTT TATTTCCAG (SEQ ID NO: 4)) 0.6 μ 1, ExTaq 0.1 1, ddH 0 13.61

^{2 μ 1, 2 nd PCR 94} ° C 3min; 94 ° C 20sec, 60 ° C 20sec, 72 ° C 1.5min 30cycle; 72 ° C 2min; 100X 1 st PCR product 2 μ \, 10X Taq Buffer 2 μ \, dNTP 1.6 ^ 1, nest primerl (H chain AGCCTAAAAGATGATGGTGT T (SEQ ID NO: 5), L chain CAGTAATCACCGTCCCAG TT (SEQ ID NO: 6)) 0.6 1, nest Primer2 (H chain CAGTAATCACCGTCCCAGTT (SEQ ID NO: 7), L chain CCAAAATCTTCAGTCTCCACA (SEQ ID NO: 8 >> 0.6 μ 1, ExTaq 0.1 1, dd H 0 13.1 l) The amplified cDNA was separated by agarose gel electrophoresis (cDNA 10 μ 1, 1%

2

 agarose gel / EtBr 20 μg / lxTAE, 100V), and it was checked whether the target cDNA was amplified (Fig. 4).

[0056] FIG. 4 shows the detection result of the HEL-specific antibody (HyHELLlO) gene. The PCR results of H chain of HEL specific antibody (HyHELLlO) are shown. CDNA is prepared by RT reaction from one cell collected by microarray method, and PCR reaction is performed using HyHELLlO antibody gene specific primer. The cDNA was amplified by As a result, HyHELLlO cDNA (about 400 bp) was confirmed even in samples diluted from 10% to 0.01%. P is shows results using 100% HEL- tg mouse splenocytes ¹⁰⁶ from RNA extracted at positives Control, Ru.

 [0057] Comparative Example 2 and Example 2

 Using a system using Piotin® HEL and PE-labeled streptavidin, we examined how low the frequency of antigen-specific B lymphocytes was actually detected. In the same manner as in Example 1 above, the HEL-specific antibody (HyHELlO) gene transgenic mice (HyHELlO-1 g mice) were replaced with normal mouse splenocytes by 10% of HEL-tg mouse splenocytes, In the case of a flow cytometer (Comparative Example 2), the cells are stained with Piotin® HEL and PE-labeled streptavidin to obtain a 1%, 0.1%, and 0.001%. Analyze the percentage of cells with positive fluorescence using Tonic Dickinson's FACSCanto, sort the cells with positive fluorescence using Beckman Coulter's EPICS ELITE, and sort the sorted cells on the slide glass. Expanded above, one B lymphocyte was collected in a tube using a micromanipulator under a microscope and examined for the presence of HEL-specific antibody genes by RT-PCR reaction. When 100% HEL-specific B lymphocytes were used, the antibody gene was also amplified with an average of about 50% B lymphocyte power.

 [0058] When using a chip (Example 2), splenocytes prepared as described above are seeded on the chip, and on the chip, first stained with Piotin-BSA and PE-labeled streptavidin, and then scanned. Cells non-specifically stained with na were detected. Next, spleen cells were stained on the chip with piotinylated HEL and PE-labeled streptavidin, and fluorescence-positive cells were detected with a scanner. Cells that were not stained with non-specific staining but were stained specifically with HEL were detected as HEL-specific B lymphocytes, and the cells were collected using a micromanipulator under a fluorescence microscope. The existence of a specific antibody gene was examined. When 100% HE L-specific B lymphocytes were used, the antibody gene was also amplified with an average of about 50% B lymphocyte power. B lymphocytes are present in about 50% of HEL splenocytes.

[0059] As shown in Table 1 below, in the case of a flow cytometer, 0.09% of cells were detected, exceeding the predicted 0.05% with 0.1% HEL, and the predicted 0.005% was greatly increased with 0.01%. Over 0.06 3% of cells are detected, indicating that the noise is high. On the other hand, the chip according to the present invention With the method used, 0.124% of the cells were detected as positive, with 0.0024% of the cells being slightly lower than the expected 0.005% compared to the expected 0.005% when the cell strength of 0.01% was close to the expected 0.05%. It can be seen that the noise is lower in the method. In addition, when checking whether HEL-specific B lymphocytes were actually collected using PCR, in the case of a flow cytometer, the proportion of positive cells decreased from 0.1% to 0.01%. In the case of the chip method, HEL-specific antibody genes are amplified from 44 to 45% of cells, and even when 100% HEL-specific B lymphocytes are used, an average of about 50% of B lymphocyte force is also antibody gene In view of the amplification, it is suggested that even when 0.01%, antigen-specific B lymphocytes are detected with a probability close to 100%.

[0060] [Table 1]

 Industrial applicability

 [0061] The present invention is a method for detecting and preparing an antigen-specific lymphocyte, and is useful in the medical field related to immunity such as antibody therapy.

 Brief Description of Drawings

 [0062] FIG. 1 shows the results of detection of antigen-specific B lymphocytes using a fluorescence-labeled antigen by a flow cytometer.

 [FIG. 2] A method for detecting antigen-specific B lymphocytes with a cell chip using a fluorescently labeled antigen.

 [Fig. 3] Detection results of antigen (HEL) -specific B lymphocytes with a cell chip using fluorescently labeled antigen (PE-HEL).

 [FIG. 4] Detection result of HEL-specific antibody (HyHELlO) gene.

Claims

The scope of the claims  [1] A method for detecting B lymphocytes that specifically bind to an antigen,  As the antigen, preparing a fluorescently labeled antigen,  Preparing a non-specific substance similar to the antigen labeled with the fluorescence,  Prepare a microwell array chip, one for each well of the B lymphocyte force microwell array chip,  The B lymphocytes on the microwell array chip are brought into contact with the non-specific substance labeled with the fluorescence, and after the contact, B lymphocytes that emit fluorescence are detected,  Next, the B lymphocytes on the microwell array chip are brought into contact with a fluorescently labeled antigen, and after contact, fluorescent B lymphocytes are detected,  Select B lymphocytes that do not fluoresce after contact with non-specific substances, but fluoresce after contact with antigen  Said method. [2] If the antigen is a protein, the non-specific substance is also a protein,  If the antigen is a peptide, the non-specific substance is also a peptide,  When the antigen is a sugar chain, the non-specific substance is also a sugar chain,  When the antigen is a lipid, the non-specific substance is also a lipid.  The method of claim 1. [3] The method according to claim 1 or 2, wherein the non-specific substance is substantially the same substance as the antigen except for the epitope of the antigen.  4. The method according to any one of claims 1 to 3, wherein the fluorescent label for the antigen and the fluorescent label for a non-specific substance are the same. [5] The method according to claim 4, wherein the fluorescently labeled non-specific substance is removed, and then the fluorescently labeled antigen is contacted.  6. The method according to any one of claims 1 to 5, wherein the fluorescent label is a fluorescent label that can be detected by a scanner. [7] A method for preparing antigen-specific B lymphocytes, comprising recovering the B lymphocytes selected by the method according to any one of claims 1 to 6. [8] A method for detecting T lymphocytes that specifically bind to an antigen,  As the antigen, a fluorescently labeled MHC / peptide complex tetramer is prepared, a non-specific substance similar to the fluorescently labeled antigen is prepared,  Prepare a microwell array chip, one for each well of the micro-lymphocyte array chip,  The fluorescently labeled non-specific substance is brought into contact with the lymphocytes on the microwell array chip, and after the contact, the fluorescent lymphocytes are detected.  Next, the fluorescently labeled MHC / peptide complex tetramer is brought into contact with the amber lymphocytes on the microwell array chip. Select fluorescent lymphocytes that do not fluoresce but contact MHC / peptide complex tetramers and then fluoresce  Said method.  [9] The method according to claim 8, wherein the non-specific substance has the same antigen and MHC, and the peptide is different from the peptide of the antigen.  [10] The method according to claim 8, wherein the non-specific substance has the same antigen and peptide, and MHC is different from that of the antigen.  [11] The non-specific substance is substantially the same substance as the antigen except for the epitope of the antigen. 0 !, displacement force The method according to item 1. 12. The method according to any one of claims 8 to 11, wherein the fluorescent label for the antigen and the fluorescent label for a non-specific substance are the same. [13] The method according to claim 12, wherein the fluorescently labeled non-specific substance is removed, and then the fluorescently labeled antigen is contacted.  [14] The method according to any one of [8] to [13], wherein the fluorescent label is a fluorescent label that can be detected by a scanner.  [15] A method for preparing antigen-specific vaginal lymphocytes, comprising collecting vaginal lymphocytes selected by the method according to any one of claims 8 to 14.