JP3723882B2 - Microwell array chip for detection of antigen-specific lymphocytes, detection method and production method of antigen-specific lymphocytes - Google Patents

Description

  The present invention relates to a microwell array chip used for detecting antigen-specific lymphocytes, and a method for detecting and producing antigen-specific lymphocytes.

Conventionally, antigen-specific lymphocytes were detected by adding about 200,000 lymphocytes per well and culturing in the presence of the antigen for 3 days to 1 week using a 96-well plate as shown in FIG. (Non-patent Documents 1 and 2).
The detection method is
1. Cell proliferation (incorporation of ³ H-thymidine, detection of living cells)
2. By measuring antibody / cytokine production.
In this method, it was confirmed that antigen-specific lymphocytes were present in a population of about 200,000 lymphocytes. However, it was not possible to identify individual antigen-specific lymphocytes present in the lymphocyte population.

  On the other hand, in recent years, antigen molecules labeled with a fluorescent dye are mixed with lymphocytes to bind the antigen-specific lymphocytes to the antigen receptor of the antigen-specific lymphocytes, and the lymphocytes bound to the fluorescence-labeled antigens are flowed. A method of detecting by using a cytometer has been developed and used (Non-Patent Document 3). This method makes it possible to identify a single lymphocyte that binds to an antigen. Furthermore, it is possible to sort out one lymphocyte that binds to an antigen.

However, the above-described detection method requires an expensive and complicated device such as a cell sorter for sorting, and also has the following problems.
(1) It is difficult to set the conditions of the instrument for sorting, and skill of instrument operation is required to sort the cells.
(2) Because the background is high, antigen-specific lymphocytes cannot be detected when the frequency of antigen-specific lymphocytes is 0.1% or less.
(3) The efficiency of sorting cells is low.
(4) It takes time to sort out infrequent cells.
(5) Although it can be confirmed that the antigen is bound, it is difficult to analyze the reaction of the lymphocyte bound with the antigen.

As another antigen-specific lymphocyte detection method, magnetic beads-bound antigen is bound to the antigen receptor of antigen-specific lymphocytes by mixing antigen molecules bound to magnetic beads with lymphocytes, and the antigen is then detected using a magnet. A method for sorting specific lymphocytes has also been developed (Non-patent Document 4).
In this method, a complicated apparatus is not required, the cell sorting time is short, and it can be confirmed that the antigen is bound. However, it was not possible to analyze whether the lymphocytes to which the antigen was bound responded to the antigen (intracellular signal transduction, RNA synthesis, protein synthesis, etc.). In addition, when the frequency of antigen-specific lymphocytes was 0.1% or less, antigen-specific lymphocytes could not be detected.
"Lymphocyte function search method", Junichi Yano, Michio Fujiwara, Chugai Medical Co., Ltd. (1994) "Immune experiment manipulation methods I and II" Shunsuke Ueda, Susumu Hamada, Motosu, Toshiyuki Tsujioka, Nanedo (1995) Altman JD, Moss PA, Goulder PJ, Barouch DH, McHeyzer-Williams MG, Bell JI, McMichael AJ, Davis MM. Phenotypic analysis of antigen-specific T lymphocytes, Science, 274: 94-96, 1996 Abts H, Emmerich M, Miltenyi S, Radbruch A, Tesch H, CD20 positive human B lymphocytes separated with the magnetic sorter (MACS) can be induced to proliferation and antibody secretion in vitro.Journal of Immunological Methods 125: 19-28, 1989 .

Therefore, the present invention does not require a complicated device, the cell sorting time is short, it can be confirmed that the antigen is bound, and the low-frequency antigen-specific lymphocyte (0.001% or more) can also be detected. Another object of the present invention is to provide an antigen-specific lymphocyte detection method that can analyze whether lymphocytes bound with an antigen react with the antigen, and that can collect antigen-specific lymphocytes.
A further object of the present invention is to provide a microwell array chip for detecting antigen-specific lymphocytes used in the detection method, and a method for producing antigen-specific lymphocytes using the detection method.

The present invention for solving the above problems is as follows.
(1) A microwell array chip having a plurality of microwells, storing one subject lymphocyte in each microwell, and detecting antigen-specific lymphocytes one by one. The microwell is a microwell array chip having a shape and size in which only one lymphocyte is stored in one microwell.
(2) The microwell array chip according to (1), wherein the microwell has a cylindrical shape, a rectangular parallelepiped shape, an inverted conical shape, an inverted pyramid shape, or a combination of two or more thereof.
(3) The diameter of the largest circle inscribed in the planar shape of the microwell is in the range of 1 to 2 times the diameter of the lymphocyte to be stored in the microwell, and the depth of the microwell is stored in the microwell. The microwell array chip according to (1) or (2), which is in the range of 1 to 2 times the diameter of the lymphocyte to be prepared.
(4) A microwell array chip for detecting antigen-specific lymphocytes having a plurality of microwells containing one subject lymphocyte.
(5) The microwell array chip according to (4), wherein the microwell has a diameter of 5 to 100 μm and a depth of 5 to 100 μm.
(6) The microwell array chip according to (4) or (5), wherein the subject lymphocyte is stored in a microwell together with a culture solution.
(7) The microwell array chip according to any one of (4) to (6), wherein the subject lymphocyte is derived from blood.
(8) The microwell array chip according to any one of (4) to (7), wherein the subject lymphocyte is a B lymphocyte or a T lymphocyte.
(9) The antigen is added to each microwell of the microwell array chip according to any one of (4) to (8), the subject lymphocyte is stimulated, and the subject lymphocyte that reacts with the antigen is detected. A method for detecting antigen-specific lymphocytes.
(10) The method according to (9), wherein the cells that react with the antigen are detected using a Ca ion-dependent fluorescent dye.
(11) The method according to (9), wherein detection of a cell that reacts with an antigen is performed using an activation marker protein expressed on the surface of a subject lymphocyte cell stimulated and activated by the antigen as an index.
(12) The method according to (9), wherein the cells that react with the antigen are detected using the degree of polarization of fluorescence emitted from the fluorescent substance in the lymphocyte of the subject lymphocyte as an index.
(13) The method according to (9), wherein the detection of cells that react with the antigen is carried out using proliferation of the subject lymphocyte cells or antibody production as an index.
(14) The method according to any one of (9) to (13), wherein the antigen is a protein, peptide, DNA, RNA, lipid, sugar chain, or organic polymer compound.
(15) The method according to any one of (9) to (13), wherein the antigen is a bacterium, a virus, a self antigen, a cancer antigen, or an allergen.
(16) A method for producing antigen-specific lymphocytes, which comprises recovering, from a microwell, a subject lymphocyte that reacts with the detected antigen by the method according to any one of (9) to (15).

  In the present invention, one lymphocyte in blood is added so as to enter one microwell, and these lymphocytes are stimulated with an antigen. The antigen here refers to pathogens such as bacteria and viruses in infectious diseases. Each lymphocyte in the blood responds to a different antigen. By using the microwell array chip of the present invention, for example, B lymphocytes that react with an antigen can be detected. Furthermore, the detected antigen-specific B lymphocytes can be collected. When antigen-specific B lymphocytes can be recovered, antibody genes that react with antigens (pathogens) can be amplified by PCR or the like in a tube other than microwells. Antibody genes that react with antigens (pathogens) can also be amplified in microwells that have detected antigen-specific B lymphocytes.

  The method for detecting antigen-specific lymphocytes of the present invention is performed using a microwell array chip. Therefore, since the detected antigen-specific lymphocytes are in the microwells, the cells can be easily processed (cell sorting / DNA / RNA preparation). In addition, the response of the cell to the antigen can be detected. It is possible to analyze not only the antigen but also the cellular response to various stimuli and the influence of various reagents on the cellular response.

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

  There is no particular limitation on the shape and dimensions of the microwell, but the shape of the microwell can be, for example, a cylindrical shape. Besides a cylindrical shape, a rectangular parallelepiped, an inverted cone, an inverted pyramid (an inverted triangular pyramid, An inverted quadrangular pyramid, an inverted pentagonal pyramid, an inverted hexagonal pyramid, an inverted polygonal pyramid with a heptagon or more can also be used, and a combination of two or more of these shapes can also be used. For example, some can be cylindrical and the rest can be inverted cones. In the case of an inverted cone shape or inverted pyramid shape, the bottom surface is the opening of the microwell, but a shape obtained by cutting a part from the top of the inverted cone shape or inverted pyramid shape (in this case, the bottom of the microwell is flat) Can also be). In the cylindrical and rectangular parallelepiped, the bottom of the microwell is usually flat, but it can be curved (convex or concave). The bottom of the microwell can be curved as in the case of a shape obtained by cutting a part from the top of an inverted cone or inverted pyramid.

The shape and dimensions of the microwell are appropriately determined so that one lymphocyte can be stored in one microwell in consideration of the type of lymphocyte to be stored in the microwell (such as the shape and size of the lymphocyte). It is determined.
In order to store one lymphocyte in one microwell, for example, the diameter of the largest circle inscribed in the planar shape of the microwell is 1 to -1 of the diameter of the lymphocyte to be stored in the microwell. It is suitable that it is in the range of 2 times, preferably in the range of 1.1 to 1.9 times, more preferably in the range of 1.2 to 1.8 times.
The depth of the microwell may be in the range of 1 to 2 times the diameter of the lymphocyte to be stored in the microwell, 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.

  When the microwell is cylindrical, its dimensions can be, for example, 5-100 μm in diameter, and preferably when the lymphocyte is a B lymphocyte, the diameter is 5-15 μm. The depth can be, for example, 5 to 100 μm. When the lymphocyte is a B lymphocyte, the depth can be preferably 5 to 40 μm. 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.

The number of microwells that one microwell array chip has is not particularly limited, but is 1 cm ² from the viewpoint that when the frequency of antigen-specific lymphocytes is from 1 to 10 ⁵ , it is about 500. For example, it can range from 2,000 to 1,000,000.

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

  The microwell array chip for detecting antigen-specific lymphocytes of the present invention can identify antigen-specific lymphocytes at the level of one cell at a time, because each microwell contains one specimen lymphocyte. become. That is, in the method for detecting antigen-specific lymphocytes using the microwell array chip of the present invention, since one sample lymphocyte is contained in the microwell, one sample lymphocyte that reacts with the antigen is one. It can be identified as a cell.

  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. By administering this antibody to patients with infectious diseases, it is considered that they can be used for the treatment and prevention of infectious diseases.

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.
In the microwell, the subject lymphocyte is stored together with the culture solution, for example. Examples of the culture solution include any of the following.
1.137 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl ₂ , 1 mM MgCl ₂ , 1 mg / ml glucose, 1 mg / ml BSA, 20 mM HEPES (pH 7.4)
2. RPMI1640 medium containing 10% FCS (fetal calf serum)
3. RPMI1640 medium containing 1mg / ml BSA
4. Dulbecco's MEM medium containing 10% FCS (fetal calf serum)
5. Dulbecco's MEM medium containing 1 mg / ml BSA The subject lymphocytes can be blood-derived, for example, B lymphocytes or T lymphocytes. In addition, tonsillar gland (lymph node), lymphoid tissue-derived lymphocytes such as spleen, lesion site infiltrating lymphocytes such as cancer infiltrating lymphocytes and the like can be mentioned.

[Method for detecting antigen-specific lymphocytes]
The method for detecting antigen-specific lymphocytes of the present invention includes adding an antigen to each microwell of the microwell array chip of the present invention, stimulating lymphocytes, and detecting lymphocytes that react with the antigen.

Antigen can be added to each microwell as follows.
1. Use a pipette to add the antigen solution to cover the entire surface of the microwell array chip.
2. Add the antigen solution one well at a time using an automatic spotter.

  The antigen detected by the antigen-specific lymphocyte detection method of the present invention is not particularly limited, and examples thereof include proteins, peptides, DNA, RNA, lipids, sugar chains, or organic polymer compounds (for example, environmental hormones). ) Etc. Alternatively, it can be a bacterium, virus, autoantigen, cancer antigen or allergen.

The cells can be cultured, for example, by suspending lymphocytes in a culture solution, dispensing the cells into microwells, and then culturing in air or in a CO ₂ incubator at room temperature or 37 ° C.

  For example, (1) a Ca ion-dependent fluorescent dye is used to detect cells that react with an antigen. (2) An activation marker protein expressed on the surface of a subject lymphocyte cell stimulated and activated by an antigen is used. The measurement can be performed using (3) the degree of polarization of fluorescence emitted from the fluorescent substance in the subject lymphocyte as an index, or (4) the proliferation or antibody production of the sample lymphocyte.

  More specifically, for example, when an antigen binds to an antigen receptor (immunoglobulin) of B lymphocytes, intracellular signal transduction occurs first, followed by cell proliferation and antibody production. Accordingly, by detecting intracellular signal transduction, cell proliferation, and antibody production by various methods, cells that react with the antigen can be detected. Alternatively, for example, when an antigen binds to an antigen receptor of T lymphocytes, intracellular signal transduction occurs first, followed by cell proliferation and cytokine production. Therefore, cells that react with antigens can be detected by detecting intracellular signal transduction, cell proliferation, and cytokine production by various methods.

Detection of a cell that reacts with an antigen by detecting intracellular signal transduction can be performed, for example, by changing the concentration of intracellular Ca ions by using a Ca ion-dependent fluorescent dye.
For intracellular Ca ion concentration change, Fura-2, Fluo-3 or Fluo-4 is used as a fluorescent dye, and a fluorescence microscope or a microarray scanner is used as a detection device.

  Specifically, as shown in FIG. 1, Fura-2 or Fluo-3, which is a Ca ion-dependent fluorescent dye, is introduced into B lymphocytes. Next, when B lymphocytes are stimulated with an antigen, the Ca ion concentration in the B lymphocytes increases. As a result, Ca ions bind to the Ca ion-dependent fluorescent dye, and the fluorescence intensity is enhanced. A low Ca ion concentration indicates a bluish color, and a high Ca ion concentration indicates a reddish color. In this method, B lymphocytes (antigen-specific) in which intracellular Ca ions are increased by stimulation with an antigen can be detected using a microwell array chip.

Detection of cells that react with an antigen by detecting cell proliferation can also be performed, for example, by measuring the number of cells using a living cell-specific fluorescent dye. Specifically, in this method, cells are proliferated when B lymphocytes are stimulated with an antigen and cultured in a CO ₂ incubator at 37 ° C. for 3 days. After cells grow, add fluorescein diacetate (FDA) or carboxyfluorescein diacetate, succinimidyl ester (CFSE) solution to the culture medium . These reagents permeate the membranes of living cells and are degraded by esterases in the cells to produce membrane-impermeable fluorescent dyes. Since the emission of this fluorescent dye is proportional to the number of cells, the number of living cells can be measured by measuring the sum of the fluorescence intensities emitted by the living cells in the well using a fluorescence microscope or a microarray scanner.

It is also possible to detect cells that react with the antigen by measuring antibody production. Antibody production can be detected by measuring the antibody immunochemically.
Specifically, when B lymphocytes are stimulated with an antigen and cultured for 1 week at 37 ° C. in a CO ₂ incubator, the antibody is secreted into the culture medium. Antigen-specific antibodies secreted into the culture medium are detected by ELISA (enzyme-labeled immunosorbent method).
Alternatively, signal transduction, cell proliferation, and antibody production can be detected using mitogens, lectins, antibodies, cytokines, PMA, and Ca ionophores.

In the following, description will be made based on FIG. 2 from the dispensing of cells to the microwell array chip, the antigen stimulation, and the removal in the method using a fluorescent dye.
(1) Dispensing cells Put cells into each microwell.
The cells to be placed in the microwell can be obtained, for example, by separating a lymphocyte fraction from peripheral blood and further separating and purifying the B lymphocyte fraction.
Next, the cells are suspended in the Fluo3 / AM (2 μM) solution, placed at room temperature for 30 minutes, and further washed with a buffer solution to remove the dye not loaded in the cells.
Dispense the cells from which the dye has been removed into microwells.
A sticker is put on both sides of the microwell array chip, a cover glass is placed on the chip, and the buffer solution is filled to prevent drying.
(2) Fluorescence measurement First, the fluorescence of unstimulated cells is measured. The fluorescence intensity (A) at that time is measured.
Next, the antigen solution is poured into the gap between the slide glass and the cover glass to replace the buffer solution, and the fluorescence of the cells stimulated with the antigen is measured. Measure fluorescence intensity (B) 1-2 minutes after stimulation. Select cells in wells with high fluorescence intensity ratio (B / A) before and after stimulation.
(3) Removal (recovery) of cells in response to antigen stimulation
If air is introduced into the gap between the slide glass and the cover glass, the cover glass is easily peeled off. Collecting antigen-specific lymphocytes by selecting and removing cells that have reacted by antigen stimulation according to the ratio (B / A) of fluorescence intensity of unstimulated cells to that of cells stimulated by antigen Can do.

[Example]
Hereinafter, the present invention will be described in detail with reference to examples.
Example 1
1. Separation of B lymphocytes The lymphocyte fraction was separated from peripheral blood using Ficoll-Paque (Pharmacia, Uppsala, Sweden), and further B was separated from lymphocyte fraction using AutoMACS (Miltenyi Biotec, Bergisch Gladbach, Germany). The lymphocyte fraction was further separated and purified.

2. Introduction of Fluo3 into cells (see Fig. 1)
2 × 10 ⁶ B lymphocytes are suspended in 2 μM Fluo3 / AM (Dojin, Kumamoto) / RPMI1640 / 10% FCS solution and incubated at room temperature for 30 minutes. Cells were washed with RPMI1640 / 10% FCS solution to remove Fluo3 / AM that was not introduced into the cells. Thereafter, the cells were suspended in RPMI1640 / 10% FCS solution.

3. Microwell array chip (see Fig. 2)
The microwell array chip is manufactured using poly (dimethylsiloxane) (PDMS), and microwells with a diameter of 10 μm and a depth of 32 μm are arranged on a 2 cm x 2 cm chip at intervals of 30 μm vertically and horizontally (center of the microwell). The distance from the center to the center is 40 μm). A sticker having a thickness of 1 mm, a width of about 1 mm, and a length of 2 cm was attached to both sides of the microwell array chip.

4. Microarray Scanner This device basically uses a microarray scanner (CRBIO IIe-FITC) from Hitachi Software Engineering Co., Ltd. (Yokohama), with the following changes.
1. One of the mounted lasers (for Cy3, 532 nm; for Cy5, 635 nm) is replaced with a 473 nm laser.
2. The depth of focus is ± 25 μm in the conventional one, but this apparatus is changed to ± 50 μm.

5. Detection of activated B lymphocytes using a microwell array chip (see Figure 2)
The cell suspension was added to the microwell array chip and allowed to stand for 5 minutes. Cells that did not enter the microwells were washed away using RPMI 1640/10% FCS solution. The diameter of the lymphocyte is about 8 μm (8 μm ± 1 μm), and since the diameter of the microwell used is 10 μm, one lymphocyte is contained in one microwell. A cover glass was placed on the seal and RPMI 1640/10% FCS solution was filled between the chip and the cover glass. The microwell array chip was inserted into a microarray scanner, scanned at a resolution of 10 μm, and data was stored (fluorescence data before antigen stimulation: A).
Next, the RPMI1640 / 10% FCS solution between the chip and the cover glass was removed, and the antigen (10 μg / mL) dissolved in the RPMI1640 / 10% FCS solution was added thereto. One minute after adding the antigen, the microwell array chip was inserted into a microarray scanner, scanned at a resolution of 10 μm, and the data was saved (fluorescence data after antigen stimulation: B).
The ratio of fluorescence intensity before and after stimulation (B / A) was calculated, and wells with a high ratio were identified. There were antigen-specific B lymphocytes in this well.

Example 2
The efficiency of introduction of cells into microwells was examined using a fluorescence microscope or a microarray scanner. Mouse lymphocytes were fluorescently labeled using CellTracker Orange (Molecular Probe).
Mouse lymphocytes were obtained as follows.
The spleen was removed from the mouse and transferred to a plastic petri dish containing PBS. The spleen is sandwiched between two meshes, and lymphocytes are removed from the spleen by grinding. The extracted lymphocytes were suspended in RPMI 1640, 10% FCS solution, and the number of lymphocytes was counted.

Mouse lymphocytes were fluorescently labeled as follows.
2 x 10 ⁶ B lymphocytes in 1 μM CellTracker Orange (Molecular Probes, USA), 0.02% Pluronic F-127 (Molecular Probes, USA) Loading Buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 1.8 The suspension was suspended in mM CaCl ₂ , 1 mM MgCl ₂ , 1 mg / ml Glucose, 1 mg / ml BSA) and incubated at room temperature for 30 minutes with shaking. Cells were washed with RPMI1640 / 10% FCS solution to remove CellTracker Orange that was not introduced into the cells. Thereafter, the cells were suspended in RPMI1640 / 10% FCS solution.
A cell suspension (100 μL) of fluorescently labeled mouse lymphocytes (10 ⁵ cells / μL) was added so as to cover the microwell array, and the cells were seeded.

The shape and dimensions of the microwell array used here are as follows.
Microwells with a diameter of 10 μm and a depth of 12 μm are arranged on a 2 cm × 2.5 cm chip at 15 μm vertical and horizontal intervals (the distance from the center of the microwell to the center is 25 μm). A sticker having a thickness of 1 mm, a width of about 1 mm, and a length of 2 cm was attached to both sides of the microwell array chip.

  After waiting for the cells to sink into the wells, the cells outside the wells were washed away by pipetting. By repeating this series of cell seeding and washing operations several times, cells can be placed in more wells. Finally, the cells were washed with a buffer (RPMI1640 / 10% FCS solution) so that no cells remained outside the wells. A cover glass was applied to prevent drying, and at the same time, the liquid level was made uniform to improve reading accuracy. This was observed under a fluorescence microscope (BX-URA2 / BX51W, Olympus Optical Industry, Japan), and inserted into a microarray scanner (CRBIO IIe-FITC, Hitachi Software Engineering, Japan) to read the fluorescence. The results are shown in FIG.

The diameter of the lymphocyte is about 8 μm, and the diameter of the used microwell is 10 μm. Therefore, one lymphocyte enters one microwell. FIG. 4 shows one cluster (30 × 30) which is a part of the chip.
The left figure shows the result of observation with a fluorescence microscope. It was confirmed that cells were contained in about 85% of the wells. The right figure shows the result of observing another sample with a microarray scanner, and it was confirmed that cells were contained in about 99% of the wells.

Example 3
[Detection of antigen-specific B lymphocytes by microarray scanner]
Healthy individuals were inoculated with hepatitis B virus vaccine, and B lymphocytes were prepared from peripheral blood on the 4th or 6th day by conventional methods. Human B lymphocytes containing calcium fluorescent indicator Fluo-4 / AM (Molecular Probes) 4 μM, CellTracker Orange (Molecular Probes) 1 μM, Pluronic F-127 (Molecular Probes) 0.02% buffer (Loading Buffer (20 mM HEPES, Fluo-4 and CellTracker Orange were introduced into the cytoplasm by suspending in 137 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl ₂ 2H ₂ O, 1 mM MgCl ₂ , 1 mg / mL glucose, 1 mg / mL BSA). B lymphocytes loaded with Fluo-4 and CellTracker Orange were seeded in the same manner as in Example 2 on the same microwell array chip as in Example 2.

Then, the B lymphocytes loaded with Fluo-4 and CellTracker Orange seeded on the microwell array chip were stimulated with the hepatitis B virus antigen HBs protein, and the fluorescence of the B lymphocytes before and after the stimulation was measured with the microwell array canner. Observed. The results are shown in FIG.
Stimulation with hepatitis B virus antigen HBs protein was given as follows.

  Remove the RPMI1640 / 10% FCS solution from the microwell array chip taken out from the microarray scanner with the chip, and add hepatitis B virus antigen HBs protein diluted to 100 μg / mL in RPMI1640 / 10% FCS solution instead. Then, it was inserted again into the microarray scanner and scanned with a resolution of 2.5 μm after about 1 minute.

The left figure of FIG. 5 is a fluorescence image before stimulating lymphocytes. The lymphocytes into which Fluo-4 had been introduced emitted a slight fluorescence, so the image showed a pale light blue color, but the fluorescence intensity was quite low.
On the other hand, the right figure is a fluorescence image after stimulating lymphocytes with an antigen (hepatitis B virus antigen HBs protein 100 μg / mL). Most lymphocytes have no change in fluorescence intensity. However, the fluorescence of antigen-specific B lymphocytes (arrow part) increases, and the image shows red color.
Furthermore, by observing the fluorescence of CellTracker Orange, it was confirmed that there was one cell in each well before and after stimulation, and there was no movement between the wells before and after stimulation (no data).

  The measured values of the fluorescence intensity of 25 (5 × 5) cells surrounded by the frame shown in FIG. 5 are plotted and shown in FIG.

  The fluorescence intensity of antigen-specific B lymphocytes (points with arrows) was approximately 77000 (77454) before stimulation, but was approximately 350,000 (349242) after stimulation, indicating a 4.5-fold increase. In contrast, the 24 lymphocytes that did not respond to stimulation had an average of 52000 (51683.875) before stimulation, an average of 39000 (38720.833) after stimulation, and an average of 0.75 times, and no significant change was observed in fluorescence intensity.

  In FIG. 6, the central straight line (diagonal line) shows the fluorescence intensity when the fluorescence intensity before and after stimulation does not change, and the two straight lines (hatched line) on both sides show the fluorescence intensity after stimulation relative to the fluorescence intensity before stimulation. The fluorescence intensity is shown when the intensity increases 4 times (upper straight line) or 1/4 times (lower straight line). The fluorescence intensity of antigen-specific B lymphocytes (points with arrows) is located slightly above the upper straight line. One microwell contains one lymphocyte, and it can be seen that one antigen-specific B lymphocyte stimulated with hepatitis B virus antigen HBs protein can be detected by this method.

Example 4
[Detection of antigen-specific B lymphocytes using a microwell array chip]
Healthy volunteers (# 1, # 2) who had increased the titer of antibodies against HBs antigen of hepatitis B virus by vaccination with hepatitis B virus vaccine until now Vaccine (HBs antigen 10 μg) was inoculated, peripheral blood before and after inoculation (4 days, 6 days, 8 days and 10 days later) was collected, lymphocytes were separated, and B lymphocytes were further separated and prepared. The B lymphocytes were labeled with Fluo-4 (Molecular Probes) 4 μM and CellTracker Orange (Molecular Probes) 1 μM in the same manner as in Example 3, and then seeded on a microwell array chip, and hepatitis B virus antigen (HBs Stimulated with 100 μg / mL protein. Measure the fluorescence of cells before and after antigen stimulation using a microarray scanner. Cells with weak Fluo-4 fluorescence signal before stimulation and enhanced Fluo-4 fluorescence signal after stimulation (antigen-specific B lymphocytes) ) Was counted. Next, the fluorescence of CellTracker Orange was observed, and the total number of B lymphocytes was counted. The frequency (percentage) obtained by dividing the number of cells with increased Fluo-4 fluorescence in response to the antigen divided by the total number of B lymphocytes was calculated and shown in the graph. The results are shown in FIG.

The number of cells (antigen-specific B lymphocytes) whose fluorescence signal was enhanced after stimulation was measured as follows.
Fluorescent images of Fluo-4 and CellTracker Orange before and after antigen stimulation are obtained using a microarray scanner. Compared with these, CellTracker Orange fluorescence signal shows that the cell position does not move before and after antigen stimulation, Fluo-4 fluorescence signal is weak and displayed in blue before stimulation, Fluo-4 after stimulation Cells with enhanced fluorescence signal and displayed in red were counted as cells with enhanced signal (antigen-specific B lymphocytes). In order to calculate the frequency, cells (antigen-specific B lymphocytes) with an enhanced fluorescence signal of Fluo-4 were counted in 5 arbitrarily selected clusters (4500 wells). In addition, the total number of cells contained in the same 5 clusters (4500 wells) was measured using fluorescence images of CellTracker Orange (Molecular Probe).

From the results shown in FIG.
1. Hepatitis B virus antigen-specific B lymphocytes in the peripheral blood of healthy volunteers whose hepatitis B virus titer of antibodies to HBs antigen was increased by inoculation with hepatitis B virus vaccine Only 1-2% of lymphocytes.
2. It can also be seen that the ratio of antigen-specific B lymphocytes to the total B lymphocytes peaked at 4-6 days after vaccination and then decreased to the ratio before vaccination.
3. Thus, the presence / absence, frequency and change of antigen-specific B lymphocytes in human peripheral blood can be detected by this method.

Explanatory drawing of the method of measuring the density | concentration change of intracellular Ca ion by using fluorescent pigment | dye dependent on Ca ion. Explanatory drawing until the dispensing of the cell to the microwell array chip | tip, antigen stimulation, and taking out in the method using a fluorescent dye. A 96-well plate used for conventional antigen-specific lymphocyte measurement. Test results of introduction efficiency of cells into microwells using a fluorescence microscope or a microarray scanner. Detection results of antigen-specific B lymphocytes by microarray scanner. FIG. 6 is a plot of fluorescence intensity for 25 (5 × 5) cells surrounded by a frame shown in FIG. 5. The frequency of cells (antigen-specific B lymphocytes) with enhanced fluorescence signal after antigen stimulation is shown.

Claims (16)

A microwell array chip having a plurality of microwells having a bottom , wherein the microwell uses a microwell array chip having a shape and a dimension in which only one lymphocyte is stored in one microwell, and the microwell array storing one lymphocyte specimen into each microwell chips, loaded with an antigenic, stimulate lymphocyte specimen, viewed including detecting the analyte lymphocytes that respond to the antigen, and the object After storing the sample lymphocytes in the microwell, cover the microwells with a cover glass to prevent drying of the sample lymphocytes, and stimulate the sample lymphocytes and detect the sample lymphocytes under the cover glass. A method for detecting a stimulating antigen-specific lymphocyte. A microwell array chip having a plurality of microwells having a bottom, wherein the microwell uses a microwell array chip having a shape and a dimension in which only one lymphocyte is stored in one microwell, and the microwell array Storing one subject lymphocyte in each microwell of the chip, adding an antigen, stimulating the subject lymphocyte, and detecting the subject lymphocyte that reacts to the antigen, the subject lymphocyte In the microwell, the liquid containing the sample lymphocytes is added so as to cover the microwells of the microwell array chip, and the cell seeding process waiting for the sample lymphocytes to settle in the microwells, A method for detecting antigen-specific lymphocytes, comprising repeating the washing process of washing out the external subject lymphocytes. The method according to claim 2, wherein the cell seeding process and the washing process are repeated until the subject lymphocytes are stored in at least 85% of the microwells. The method according to claim 1, wherein the subject lymphocyte is stored in a microwell together with a culture solution. The method according to claim 1, wherein the subject lymphocyte is derived from blood. The method according to any one of claims 1 to 5 , wherein the subject lymphocyte is a B lymphocyte or a T lymphocyte. The method according to any one of claims 1 to 6 , wherein a cell that reacts with an antigen is detected using a Ca ion-dependent fluorescent dye. 8. The method according to claim 7 , wherein the cells reacting with the antigen are detected by measuring the fluorescence intensity before and after adding the antigen. The method according to any one of claims 1 to 6 , wherein detection of a cell that reacts with an antigen is performed using an activation marker protein expressed on the surface of a subject lymphocyte cell stimulated and activated by the antigen as an index. The method according to any one of claims 1 to 6 , wherein cells that react with an antigen are detected using the degree of polarization of fluorescence emitted from the fluorescent substance in the lymphocyte of the subject lymphocyte as an index. The method according to any one of claims 1 to 6 , wherein detection of a cell that reacts with an antigen is performed using proliferation of a subject lymphocyte cell or antibody production as an index. The method according to any one of claims 1 to 11 , wherein the antigen is a protein, peptide, DNA, RNA, lipid, sugar chain, or organic polymer compound. The method according to any one of claims 1 to 11 , wherein the antigen is a bacterium, virus, autoantigen, cancer antigen or allergen. The microwells cylindrical, rectangular, inverted cone, or an inverted pyramidal or shape a combination of two or more of these A method according to any one of claims 1 to 13. The diameter of the largest circle inscribed in the planar shape of the microwell is in the range of 1 to 2 times the diameter of the lymphocyte to be stored in the microwell, and the depth of the microwell is to be stored in the microwell. The method according to any one of claims 1 to 13 , which is in the range of 1 to 2 times the diameter of lymphocytes. A method for producing antigen-specific lymphocytes, comprising recovering subject lymphocytes that react with a detected antigen from a microwell by the method according to any one of claims 1 to 15 .