KR102497904B1 - Method for detecting immune cells using cell centrifugation and enrichment, and Chip for detecting immune cells - Google Patents

Abstract

The present invention relates to a method for detecting immune cells using cell centrifugation and concentration and a chip for detecting immune cells. By detecting specific immune cells after cell enrichment through lysis and cell centrifugation, specific immune cells can be easily detected and counted with the naked eye without using an expensive fluorescence-based flow cytometer.

Description

Method for detecting immune cells using cell centrifugation and enrichment, and chip for detecting immune cells {Method for detecting immune cells using cell centrifugation and enrichment, and Chip for detecting immune cells}

The present invention relates to a method for detecting immune cells using cell centrifugation and concentration and a chip for detecting immune cells.

The CD4 ⁺ T cell count is an important test in the management of human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS), determining when to start antiretroviral therapy and monitoring the effectiveness of treatment. It is widely used to

CD4 ⁺ T cell counts are usually expressed as the absolute number of CD4 ⁺ T lymphocytes per microliter of blood. In addition, the ratio of CD4 ⁺ T cells to the total number of lymphocytes (CD4 percentage) and the ratio of CD4 ⁺ T cells to CD8 ⁺ T cells (CD4/CD8) are particularly useful for monitoring the course of an infection and provide an overall picture of the body's immune strength. can be used to evaluate. The CD4/CD8 ratio is also particularly useful in HIV-infected infants, as CD8 ⁺ T cells are markedly increased, whereas the reduction in CD4 ⁺ T cells due to HIV infection is not evident in childhood.

The current gold standard for CD4 ⁺ T cell enumeration is flow cytometry, a reliable and accurate method, several types of flow cytometers are available with the BD FACSCount, FACSCalibur (BD Biosciences) and EPICS XL (Beckman Coulter) . An intermediate flow cytometer dedicated to CD4 ⁺ T cell enumeration is the FACSCount, and in 2017 another compact intermediate flow cytometer, the Muse Auto CD4/CD4 % System, was developed. However, state-of-the-art CD4 ⁺ T cell counting methods based on flow cytometry are not suitable for resource-constrained settings due to high operation/maintenance costs and skilled manpower requirements.

Currently, the most used IVD flow cytometers are sophisticated, large, heavy, and very expensive instruments operated by dedicated, highly skilled personnel, typically used in large hospitals, semi-automated or fully automated, and with high sample throughput.

Accordingly, less expensive devices dedicated to counting CD4 ⁺ T cells have been developed, resulting in several portable point-of-care (POC) devices on the market that use disposable cartridges for counting CD4 ⁺ T cells. These devices are small, portable, rugged, reasonably priced, battery operated, and can be operated by a healthcare professional close to the patient using a finger prick or venous blood. Two of the cartridge-based POC CD4 devices pre-validated by WHO are the PIMA CD4 and FACSPresto.

However, most first-generation POC CD4 ⁺ T cell counting methods rely on microfluidic adaptations of flow cytometry, such as microfluidic image cell counting, which analyzes images of fluorescently labeled CD4 ⁺ T cells to be counted, or uses manual handling and finger sticks. may still require a greater blood volume than a finger stick.

Therefore, it is necessary to study a method for counting immune cells such as CD4 ⁺ T cells easily, quickly and with high accuracy without advanced optical equipment for fluorescence detection, overcoming the problems of the conventional immune cell counting method as described above. am.

Sci Transl Med. 2013 Dec 4;5(214):214ra170.

The present inventors detect specific immune cells by treating blood with antibodies coupled to magnetic beads and concentrating cells through red blood cell lysis and cell centrifugation, thereby easily detecting immune cells with the naked eye without using fluorescence and without complex and precise optical instruments. The present invention was completed based on the development of an immune cell detection chip capable of detecting immune cells and a method of detecting immune cells using the same.

Accordingly, an object of the present invention is to provide a method for detecting immune cells using cell centrifugation and concentration and a chip for detecting immune cells.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

In order to achieve the above object, the present invention provides a method for detecting immune cells using cell centrifugation and concentration, comprising the following steps:

(a) preparing a bead-antibody complex by conjugating microbeads and an antibody;

(b) reacting the bead-antibody complex with blood separated from the subject;

(c) after the reaction, treating the reactant with red blood cell lysis buffer to dissolve red blood cells and removing the supernatant; and

(d) concentrating the cells by centrifuging the cells after removing the supernatant.

As one embodiment of the present invention, after removing the supernatant in step (c), the step of suspending the cell pellet in polyethylene glycol 8000 (PEG 8000) may be further included, but is not limited thereto.

As another embodiment of the present invention, the antibody may be an antibody against a cluster of designation (CD), but is not limited thereto.

As another embodiment of the present invention, the antibody is at least one selected from the group consisting of anti-CD4, anti-CD19, anti-CD3, anti-CD8, anti-CD14, anti-CD16, anti-CD25, anti-CD45, anti-CD56, and anti-CD127. It may, but is not limited thereto.

As another embodiment of the present invention, the concentration of the antibody in step (a) may be 0.01 mg/mL to 3 mg/mL, but is not limited thereto.

As another embodiment of the present invention, the volume ratio of microbeads:antibody in step (a) may be 1 to 6:1, but is not limited thereto.

As another embodiment of the present invention, the blood may be at least one selected from the group consisting of whole blood and peripheral blood mononuclear cells (PBMC), but is not limited thereto.

As another embodiment of the present invention, the microbead may have a diameter of 0.1 μm to 4 μm, but is not limited thereto.

As another embodiment of the present invention, the concentration of PEG 8000 may be 5% (v/v) to 25% (v/v), but is not limited thereto.

As another embodiment of the present invention, the step of centrifuging and concentrating the cells in step (d) may use a cytospin, but is not limited thereto.

As another embodiment of the present invention, the cell centrifugation using the cytospin may be performed at a speed of 5000 rpm to 9000 rpm, but is not limited thereto.

In addition, the present invention comprises a chamber 1 (11, 21) in which the reaction between the bead-antibody complex and the blood separated from the subject is performed;

Chamber 2 (12, 22) where the red blood cell lysis reaction takes place;

Chamber 3 (13, 23) containing a solution used for the immune cell detection reaction including the red blood cell lysis solution;

Chamber 4 (14, 24) in which cell centrifugation and concentration are performed;

filter papers 16 and 25 made of paper that absorbs water;

Slides 17 and 26 made of a transparent material for observation of microscopic images and closely attached to the filter paper; and

As an immune cell detection chip (10, 20) composed of barcodes (18, 27),

The bead-antibody complex provides a chip for detecting immune cells, characterized in that it is prepared by conjugating microbeads and antibodies.

As an embodiment of the present invention, the immune cell detection chip (10, 20) further includes a chamber 5 (15), and in the chamber 5, a cell pellet suspension by treatment with polyethylene glycol 8000 (PEG 8000) is performed. this happens, or

A reaction in which the cell pellet is suspended by the polyethylene glycol 8000 treatment may occur in the chamber 4 (24), but is not limited thereto.

As another embodiment of the present invention, the material of the slides 17 and 26 may be transparent glass or plastic, but is not limited thereto.

As another embodiment of the present invention, the immune cell detection chips 10 and 20 may have a length of 7 cm to 13 cm, a height of 5 cm to 10 cm, and a width of 0.5 cm to 5 cm, but are limited thereto. It doesn't work.

The immune cell detection method of the present invention treats blood with antibodies bound to microbeads through an immune cell detection chip, and detects specific immune cells after cell concentration through red blood cell lysis and cell centrifugation, without going through a staining process. immune cells can be observed with the naked eye, and specific immune cells can be easily detected and counted without using an expensive fluorescence-based flow cytometer.

1 is a schematic diagram showing a process of an immune cell detection method according to an embodiment of the present invention.
2A is a front view of chip 1 (10) for detecting immune cells according to an embodiment of the present invention.
2B is a side view of chip 1 (10) for detecting immune cells according to an embodiment of the present invention.
2C is a side view of chip 2 (20) for detecting immune cells according to an embodiment of the present invention.
3a to 3d are diagrams showing the results of detecting B cells using an anti-CD19 antibody through an immune cell detection method according to an embodiment of the present invention.
4 is a diagram showing CD4 ⁺ T cell detection results according to the particle size of microbeads using the immune cell detection method according to an embodiment of the present invention.
5 is a diagram showing CD4 ⁺ T cell detection results according to the amount of anti-CD4 antibody using the immune cell detection method according to one embodiment of the present invention.
6a to 6c are views showing CD4 ⁺ T cell detection results according to PEG8000 concentration using the immune cell detection method according to an embodiment of the present invention.
7a and 7b are diagrams showing CD4 ⁺ T cell detection results according to FACS buffer treatment using the immune cell detection method according to one embodiment of the present invention.
8 is a view showing the detection results of CD4 ⁺ T cells according to the rotation speed of cell centrifugation using the immune cell detection method according to one embodiment of the present invention.
9 is a view showing the result of methylene blue staining using the immune cell detection method according to one embodiment of the present invention.
10 is a view showing the results of detecting CD8 ⁺ T cells using the method for detecting immune cells according to an embodiment of the present invention.

The present invention provides a method for detecting immune cells using cell centrifugation and concentration, comprising the following steps:

(a) preparing a bead-antibody complex by conjugating microbeads and an antibody;

(b) reacting the bead-antibody complex with blood separated from the subject;

(c) after the reaction, treating the reactant with red blood cell lysis buffer to dissolve red blood cells and removing the supernatant; and

(d) concentrating the cells by centrifuging the cells after removing the supernatant.

In the present invention, “immune cells” means bone marrow progenitor cells (generated from bone marrow cells such as bone marrow-derived suppressor cells, monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes) and lymphoid progenitor cells (T cells, B cells and It refers to any cell of the immune system of hematopoietic stem cell (eg, in the bone marrow) origin, arising from two main lineages: natural killer (NK) cells, which arise from cells of the lymphatic system. The immune cells include, for example, B cells, CD4 ⁺ T cells, CD8 ⁺ T cells, CD3 ⁺ T cells, CD14 ⁺ monocytes, CD16 ⁺ NK cells, CD25 ⁺ T cells, CD45 ⁺ leukocytes, CD56 ⁺ NK cells, CD127 ⁺ T cells, CD4 ^- CD8 ^- double negative T cells, γδ T cells, regulatory T cells, antigen presenting cells (APCs), natural killer cells, and dendritic cells.

In the present invention, "detection" means both measuring and confirming the presence or absence of immune cells, or measuring and confirming a change in the level (number of cells) of immune cells. In the same context, in the present invention, measuring the level of the immune cells means measuring the presence or absence (ie, measuring the presence or absence) or measuring the level of qualitative or quantitative change of the immune cells. . The measurement can be performed without limitation including both qualitative methods (analysis) and quantitative methods. Types of qualitative and quantitative methods for measuring the presence or absence of immune cells are well known in the art, and the experimental methods described herein are included. A specific immune cell level comparison method for each method is well known in the art.

In the present invention, in the step (a), “microbead” is a particle used as a marker to visually identify immune cells without fluorescence, and the diameter of the particle may be 0.1 μm to 5 μm, preferably. 0.1 μm to 4 μm, 0.1 μm to 3 μm, 0.1 μm to 2 μm, 0.5 μm to 5 μm, 0.5 μm to 4 μm, 0.5 μm to 3 μm, 0.5 μm to 2 μm, 1 μm to 5 μm, 1 μm to 4 μm, 1 μm to 3 μm, 1 μm to 2.5 μm, 1 μm to 2 μm, or 1 μm, but is not limited thereto.

In the present invention, the type of microbead is not limited as long as it falls within the above particle diameter range, for example, magnetic bead, polystyrene particle, silicate particle, or metal (gold, silver) , copper, mercury, or cadmium) particles. According to one embodiment of the present invention, the microbead may be a magnetic bead that has a brown color and can be observed with a microscope so as to be visible to the naked eye without dye, but is not limited thereto.

According to one embodiment of the present invention, the microbead may be coated with streptavidin, but is not limited thereto.

In the present invention, in step (a), “antibody” refers to a polypeptide containing a framework region derived from an immunoglobulin gene or a fragment thereof that specifically binds to and recognizes an antigen. Recognized immunoglobulin genes include a myriad of immunoglobulin variable region genes, including kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes. Light chains were classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and consequently define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. Typically, the antigen-binding region of an antibody becomes most critical for binding specificity and affinity. In some embodiments, antibodies or fragments of antibodies may be from different organisms, including humans, mice, rats, hamsters, camels, and the like. Antibodies of the present invention are antibodies that have been modified or mutated at one or more amino acid positions to improve or modulate a desired function of the antibody (e.g., glycosylation, expression, antigen recognition, effector function, antigen binding, specificity, etc.) may also include

Exemplary immunoglobulin (antibody) structural units include tetramers. Each tetramer consists of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The N terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively. The Fc (i.e., fragment determinable region) is the "base" or "tail" of an immunoglobulin and typically consists of two heavy chains contributing to two or three constant domains, depending on the class of antibody. By binding to specific proteins, the Fc region ensures that each antibody generates an appropriate immune response against a given antigen. The Fc region also binds various cellular receptors, such as Fc receptors, and other immune molecules, such as complement proteins.

According to one embodiment of the present invention, the antibody may be biotin attached, but is not limited thereto.

In the present invention, the antibody may be an antibody against a cluster of designation (CD), for example, the antibody is anti-CD4, anti-CD19, anti-CD3, anti-CD8, anti-CD14, anti-CD16, anti-CD16 It may be one or more selected from the group consisting of CD25, anti-CD45, anti-CD56, and anti-CD127, but is not limited thereto.

At this time, the concentration of the antibody in step (a) is 0.01 mg/mL to 3 mg/mL, 0.01 mg/mL to 2 mg/mL, 0.01 mg/mL to 1 mg/mL, 0.01 mg/mL to 0.5 mg/mL. mL, 0.1 mg/mL to 3 mg/mL, 0.1 mg/mL to 2 mg/mL, 0.1 mg/mL to 1 mg/mL, 0.1 mg/mL to 0.5 mg/mL, or 0.2 mg/mL; , but not limited thereto.

In addition, the volume ratio of microbeads to antibody used in the preparation of the bead-antibody complex in step (a) is 1 to 6:1, 1 to 4:1, 1 to 3:1, 1 to 2.5:1, 1.5 to 6:1. 6:1, 1.5 to 4:1, 1.5 to 3:1, 2 to 6:1, 2 to 4:1, or 2:1, but not limited thereto.

In the present invention, in the step (b), the blood is separated from the subject and may be at least one selected from the group consisting of whole blood and peripheral blood mononuclear cells (PBMC), but is not limited thereto. . "Whole blood" means blood including all blood components such as red blood cells, white blood cells, platelets, plasma, etc. without any component being removed by purification. The whole blood or peripheral blood mononuclear cells can be collected from any animal, such as humans, rabbits, rats, dogs, cats and other mammals.

In the present invention, after removing the supernatant in step (c), the step of suspending the cell pellet in polyethylene glycol 8000 (PEG 8000) may be further included, but is not limited thereto.

At this time, the PEG 8000 has a concentration of 5% (v / v) to 25% (v / v), 5% (v / v) to 20% (v / v), 5% (v / v) to 15% (v/v), 5% (v/v) to 10% (v/v), 7% (v/v) to 20% (v/v), 7% (v/v) to 15% (v/v) /v), 7% (v/v) to 10% (v/v), 7.5% (v/v) to 15% (v/v), 7.5% (v/v) to 10% (v/v) ), or 7.5% (v/v), but is not limited thereto.

In the present invention, the step of centrifuging the cells in step (d) may use a cytospin (cytospin), but is not limited thereto.

In the context of the present invention, “centrifugation” means a process involving the use of centrifugal force to separate a mixture, for example, separation of a solid from a mixture. Increasing the effective gravity force on the test tube results in more rapid and complete collection of sediment ("pellets") at the bottom of the vesicle. The solution ("supernatant") can then be rapidly decanted from the vesicle without disturbing the precipitate. The speed of centrifugation is specified by the acceleration applied to the sample, typically measured in revolutions per minute (rpm). In centrifugation, the sedimentation velocity of a particle is a function of the size and shape of the particle, the centrifugal acceleration, the volume ratio of solids present, the density difference between the particle and the liquid, and the viscosity. In the present invention, cells may be concentrated through the centrifugation.

In the present invention, “cytospin” refers to a method of immunostaining after putting a certain number of cells on a slide for immunostaining, spinning (centrifugation), and adhering to the slide, and using centrifugal force to Since it serves to spread cells well on a slide and gather them in one place, it has the advantage of being able to clearly see the shape and characteristics of cells after staining, and is a useful method mainly for surface marker staining of cells cultured in suspension.

In the present invention, the cell centrifugation using the cytospin is 5000 rpm to 9000 rpm, 5000 rpm to 8500 rpm, 5000 rpm to 8000 rpm, 5000 rpm to 7500 rpm, 6000 rpm to 9000 rpm, 6000 rpm to 8000 rpm, 6000 rpm to 7500 rpm, 6500 rpm to 9000 rpm, 6500 rpm to 8000 rpm, 6500 rpm to 7500 rpm, or 7000 rpm for 1 minute to 10 minutes, 1 minute to 8 minutes, 1 minute to 6 minutes, 3 minutes to 10 minutes, 3 to 8 minutes, 3 to 6 minutes, or 5 minutes, but is not limited thereto.

As an immune cell detection method according to the present invention, a process of preparing a microbead-antibody complex, reacting with blood, and centrifuging and concentrating cells using cytospin may be referred to as “Immunospin”.

Through the immune cell detection method according to the present invention, immune cells can be observed with the naked eye without a separate staining process, and according to one embodiment of the present invention, the staining process is additionally performed for cell identification, It can be performed to create slides after spin, or without cytospin, and to identify cells in several situations.

The immune cell detection method according to the present invention shortens the test time to within 35 minutes, preferably within 30 minutes, compared to the test time of the existing immunochemical staining method and the flow cytometry method, which are 1 hour or more and 40 minutes or less, respectively. Compared to staining and flow cytometry, immune cells can be detected without washing and without using a secondary antibody. In addition, since the cell shape and frequency can be measured simply enough without cell staining and fluorescence when detecting immune cells, there is an advantage in that expensive optical devices are not required.

In addition, the present invention comprises a chamber 1 (11, 21) in which the reaction between the bead-antibody complex and the blood separated from the subject is performed;

Chamber 2 (12, 22) where the red blood cell lysis reaction takes place;

Chamber 3 (13, 23) containing a solution used for the immune cell detection reaction including the red blood cell lysis solution;

Chamber 4 (14, 24) in which cell centrifugation and concentration are performed;

filter papers 16 and 25 made of paper that absorbs water;

Slides 17 and 26 made of a transparent material for observation of microscopic images and closely attached to the filter paper; and

As an immune cell detection chip (10, 20) composed of barcodes (18, 27),

The bead-antibody complex provides a chip for detecting immune cells, characterized in that it is prepared by conjugating microbeads and antibodies.

In the present invention, the chip for detecting immune cells (10, 20) further includes a chamber 5 (15), and in the chamber 5, a reaction in which cell pellet suspension by treatment with polyethylene glycol 8000 (PEG 8000) occurs or ,

A reaction in which the cell pellet is suspended by the polyethylene glycol 8000 treatment may occur in the chamber 4 (24), but is not limited thereto.

In the present invention, the immune cell detection chips 10 and 20 include immune cell detection chip 1 (10) and immune cell detection chip 2 (20).

In the present invention, the immune cell detection method may be performed using the immune cell detection chip, but is not limited thereto.

In the present invention, "precision" is a criterion indicating how close the results are to each other by measuring or calculating several times. It indicates the homogeneity of the observation, and the smaller the deviation of the observed value, the more precise it is.

In the present invention, “accuracy” indicates the degree of proximity of a measured value to a known true value or standard value, and includes recovery rate and the like.

In the present invention, "linearity" means the degree to which the corresponding test shows a response value linearly according to the concentration in the quantitative measurement range.

In one experimental example of the present invention, precision, accuracy, and linearity when detecting immune cells by the method according to the present invention were confirmed. Precision, accuracy, linearity, etc. are indicators of important basic performance among the analytical performance indicators that clinical examinations must have. Precision is expressed as CV%, and accuracy is expressed by comparing with known values. As a result of checking the precision, accuracy, and linearity when detecting , the precision (CV%) was 2.4% ~ 8.2%, the accuracy was -1.0% ~ -4.6%, the bias was -1.0% ~ -4.6%, and the linearity was R ² = 0.986 (y = 0.9738x + 0.1429), which was confirmed to be very good (see Experimental Example 1).

In another experimental example of the present invention, as a result of observing PBMC after reacting a complex of anti-CD19 antibody and magnetic beads with whole blood, it was confirmed that B cells could be detected with the naked eye without going through another staining process (see Experimental Example 2). ).

In another experimental example of the present invention, as a result of detecting CD4 ⁺ T cells according to the bead particle size with the immune cell detection method according to the present invention, when the magnetic beads exceed a certain size, intercellular aggregation occurs and affects the result, It was confirmed that the range of 1 to 2.5 μm was appropriate (see Experimental Example 3-1), and as a result of detecting CD4 ⁺ T cells according to the amount of anti-CD4 antibody, the reaction did not occur well even if the amount of antibody was too small or too large. As it was found that the antibody was required, it was confirmed that the appropriate amount of the antibody was in the range of 1 to 4 μL (see Experimental Example 3-2).

In addition, as a result of detecting CD4 ⁺ T cells according to the concentration of PEG8000 by the immune cell detection method according to the present invention, it was confirmed that cell morphology was stably maintained for a long time and detection was easy when PEG8000 in the concentration range of 7.5% to 10% was treated. (Refer to Experimental Example 3-3), as a result of CD4 ⁺ T cell detection according to FACS buffer treatment, when using FACS buffer, the cell membrane and cell morphology are clearer than when only PBS is used, and when treated with PEG8000, cells for a long time It was confirmed that the deformation of was less and aggregation did not occur (see Experimental Example 3-4).

In addition, as a result of detecting CD4 ⁺ T cells according to the rotation speed of cell centrifugation with the method for detecting immune cells according to the present invention, it was found that there was a difference in cell type and degree of concentration depending on the rotation speed of cell centrifugation. It was confirmed that the range within rpm to 7500 rpm was appropriate (see Experimental Example 3-5).

In another experimental example of the present invention, it was confirmed that CD8 ⁺ T cells could be detected by the immune cell detection method according to the present invention (see Experimental Example 4).

In the present invention, in the chamber 1 (11, 21), cells positive for one or more specific antigens selected from the group consisting of CD4, CD19, CD3, CD8, CD14, CD16, CD25, CD45, CD56, and CD127 in the blood and A reaction occurs between the bead-antibody complex, and the bead-antibody complex that reacts with the antigen in the blood in the chamber 1 (11, 21) can be used by putting microbeads and antibodies conjugated into chamber 1, It may be included in chamber 1, but is not limited thereto.

In the present invention, in the chamber 2 (12, 22), a red blood cell lysis reaction occurs, and by removing red blood cells leaving only immune cells, many immune cells per unit area can be seen during centrifugation and concentration.

In the present invention, the chamber 3 (13, 23) is a chamber containing a solution that can be used in the immune cell detection process, for example, the solution is a general ammonium chloride solution that dissolves red blood cells or a BD FACS lysis buffer ( BD Biosciences, San Jose, CA, USA), red blood cell lysis solution, or another solution that can be used in the immune cell detection process, and the type of the solution is not limited. According to one embodiment of the present invention, an appropriate amount of the red blood cell lysis solution contained in chamber 3 can be transferred to chamber 2 where the red blood cell lysis reaction takes place. It may be, but is not limited thereto.

In the present invention, in the chamber 4 (14, 24), cells are centrifuged by centrifugal force, and a reaction of concentrating may occur thereby. It can be connected with the slides 17 and 26.

In the present invention, the filter papers 16 and 25 have a diameter of 1 mm to 15 mm, 1 mm to 13 mm, 1 mm to 10 mm, 1 mm to 7 mm, 3 mm to 15 mm, 3 mm to 13 mm, 3 mm to 10 mm, 3 mm to 7 mm, 5 mm to 15 mm, 5 mm to 13 mm, 5 mm to 10 mm, 5 mm to 7 mm, or 6 mm holes The slides 17 and 26 may be in close contact with or coupled to the filter paper, and the cells may be concentrated after centrifugation with the hole size of the filter paper, and immune cells may be observed by observing the cells concentrated on the slide under a microscope. can be detected.

In the present invention, the type of the slides 17 and 26 is not limited as long as it is a transparent material, and may be, for example, transparent glass or plastic.

In the present invention, the filter papers 16 and 25 and the slides 17 and 26 have a length of 0.5 cm to 5 cm, 0.5 cm to 4 cm, 0.5 cm to 3 cm, 1 cm to 5 cm, and 1 cm to 4 cm. cm, 1 cm to 3 cm, 2 cm to 5 cm, 2 cm to 4 cm, 2 cm to 3 cm, or 2.5 cm, and heights 5 cm to 10 cm, 5 cm to 9 cm, 5 cm to 8 cm, It may consist of 6 cm to 10 cm, 6 cm to 9 cm, 6 cm to 8 cm, 7 cm to 10 cm, 7 cm to 9 cm, 7 cm to 8 cm, or 7.5 cm, but is not limited thereto.

In the present invention, the chamber 5 (15) is a chamber in which cell pellets are suspended by processing polyethylene glycol 8000 (PEG 8000) in the chip 1 (10) for detecting immune cells, and in the chip 2 (20) for detecting immune cells In the chamber 4 (24), a reaction in which the cell pellet is suspended may occur by treating the polyethylene glycol 8000.

In the present invention, the immune cell detection chips 10 and 20 have a length of 7 cm to 13 cm, 7 cm to 12 cm, 7 cm to 11 cm, 8 cm to 13 cm, 8 cm to 12 cm, and 8 cm. to 11 cm, 9 cm to 13 cm, 9 cm to 12 cm, 9 cm to 11 cm, or 10 cm, height 5 cm to 10 cm, 5 cm to 9 cm, 5 cm to 8 cm, 6 cm to 10 cm , 6 cm to 9 cm, 6 cm to 8 cm, 7 cm to 10 cm, 7 cm to 9 cm, 7 cm to 8 cm, or 7.5 cm, and widths 0.5 cm to 5 cm, 0.5 cm to 4 cm, 0.5 It may consist of cm to 3 cm, 1 cm to 5 cm, 1 cm to 4 cm, 1 cm to 3 cm, 2 cm to 5 cm, 2 cm to 4 cm, 2 cm to 3 cm, or 2.5 cm, Not limited to this.

In the present invention, the immune cell detection chip 1 (10) has the configuration of chamber 1 (11), chamber 2 (12), chamber 3 (13), chamber 4 (14), and chamber 5 (15), If the chip 2 (20) for detecting immune cells includes the configuration of chamber 1 (21), chamber 2 (22), chamber 3 (23), and chamber 4 (24), the order is limited in the position of the chambers in the chip. It is not. In the case of chip 1 for detecting immune cells, each chamber can automatically move the solution using an automatic pipette dispenser device (liquid handler) that moves the solution, and in the case of chip 2 for detecting immune cells, a pumping device is used to transfer the sample. Can be injected or moved, but is not limited thereto.

In the present invention, the immune cell detection chips 10 and 20 may be used in a rotatable device. The rotatable device may be a centrifugal separator, and may be used in, for example, a low-speed centrifuge, a high-speed centrifuge, or an ultracentrifuge, but is not limited thereto.

Hereinafter, preferred embodiments and experimental examples are presented to aid understanding of the present invention. However, the following Examples and Experimental Examples are only provided to more easily understand the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

[Example]

Example 1. Material preparation

Experimental materials used in the present invention are as follows.

- Biotinylated anti-CD19 monoclonal antibody (HIB19) (eBioscience, Thermo Fisher Scientific)

- Biotinylated anti-CD3 monoclonal antibody (OKT3) (eBioscience)

- Biotinylated anti-CD4 monoclonal antibody (RPA-T4) (eBioscience)

- Dynabeads MyOne Streptavidin C1 (Magnetic bead, Invitrogen),

- HulaMixer Sample Mixer (ThermoFisher)

- BD FACS lysing buffer (BD Biosciences, San Jose, CA, USA)

- Stain buffer (BD Biosciences, San Jose, CA, USA)

- 5% FBS PBS (phosphate buffered saline),

- Slide glass, cover glass,

- Disposable transfer pipette,

- Methylene blue staining solution (Sigma-Aldrich, St. Louis, MO, USA)

- Wright staining solution (YD Wright Stain, YD Diagnostics, Yongin, Korea)

Example 2. Functionalization of beads with anti-CD4 antibody

Streptavidin-coated magnetic beads (Dynabeads MyOne Streptavidin C1) with an average diameter of 1 μm were conjugated with a biotinylated anti-CD4 antibody (eBioscience). 6 μL of magnetic beads diluted 1:10 were mixed gently with 50 μL FACS buffer containing 1% bovine serum albumin (BSA), followed by 3 μL of monoclonal anti-CD4 antibody (diluted 1:10) 0.2 mg/mL, eBioscience) was added to the magnetic beads. There was no washing process capable of removing unbound antibody, and antibody-bound magnetic beads were blocked using 1% bovine serum albumin in FACS buffer. The antibody functionalized magnetic beads were stored at 4°C for later use.

Example 3. Anti-CD19 antibody and bead reaction for B cell detection

B cells were detected using the immune cell detection method of the present invention as follows.

By the method of Example 2, 0.5 μL (0.25 μg) of biotinylated anti-CD19 antibody at a concentration of 0.5 mg/mL and streptavidin-coated magnetic beads were either undiluted or diluted at a ratio of 1:10, and by concentration (0.5 μg). μL, 1 μL, or 10 μL) for 30 minutes, reacted with peripheral blood mononuclear cells (PBMC) for 15 minutes, and then observed the mononuclear cells (PBMC) to detect B cells.

Example 4. Red blood cell lysis for CD4+ T cell detection

In order to detect CD4+ T cells, the immune cell detection method of the present invention was used and treated according to the "antibody reaction - erythrocyte lysis or non-lysis - PEG8000 treatment - staining or no staining" protocol. Specifically, 100 μL of freshly collected peripheral EDTA (ethylenediamineteraacetic acid)-treated blood was put into a tube, followed by 3 μL of 1:10 diluted anti-CD4 antibody and 6 μL of 1:10 diluted magnetic beads (Dynabeads MyOne Streptavidin C1, Invitrogen) and incubated for 15 minutes at room temperature. Then, red blood cells were lysed with 500 μL BD FACS Lysis Buffer (BD Biosciences, USA) and incubated for 10 to 20 minutes at room temperature. Then, after removing the supernatant, the cell pellet was suspended in 7.5% PEG8000 PBS solution.

The process of treating the PEG8000 PBS solution is a process of adding a kind of diluent to prevent the cells from being too concentrated and to maintain the shape of the cells.

The above method can be applied not only to CD4 but also to all CD antigens such as CD3, CD8, CD16, and CD56.

Example 5. Cell centrifugation and staining using Cytospin

The cell suspension of Example 4 was loaded into cytospin cuvettes (Shandon, Inc., Pittsburgh, PA) corresponding to chamber 4 of the chip for detecting immune cells according to the present invention, and a glass slide and paper were loaded. After mounting on a card, centrifugation at 800 x g, 7000 rpm for 5 minutes allowed a monolayer of cells to be deposited on the slide. Slides were air-dried and counterstained with Wright staining, and cell types were identified by nuclear morphology and cytoplasmic staining (Wright staining solution, YD Wright Stain, YD Diagnostics, Korea). All cell counts were performed using a light microscope.

[Experimental example]

Experimental Example 1. Performance evaluation

1-1. precision

Intra-run and inter-run inaccuracies of the immune cell detection method according to the present invention were measured for each run and twice a day over 5 consecutive working days (total of 20 repetitions) of Multi-check Normal and Low Process Control (BD Biosciences), respectively. was evaluated for duplicate measurements of

The precision evaluation results were as shown in Table 1 below.

%CD4 T cells Mean% Precision (CV)
repeatability Between-run Within laboratory
(between days) Low CD4 control material (12.7 (8.7~16.7), BM11202L) 12.2% 4.1% 7.1% 8.2% Normal CD4 control material (48.1 (41.6~54.6), BM1120N) 48.0% 1.0% 2.1% 2.4%

As shown in Table 1, the precision (coefficient of variation, CV%) was 2.4% to 8.2%.

1-2. accuracy

Accuracy (bias) is the result of the immune cell detection method CD4/CD4% according to the present invention for the corresponding CD4 count results of Multi-check Normal and Low process controls (BD Biosciences) by flow cytometer BD FACSCanto II (BD Biosciences) Evaluated against established results.

The accuracy evaluation results were as shown in Table 2 below.

%CD4 T cells Mean%
(Method of the present invention) Assigned %CD4 FACS bias Relative bias Low CD4 control material (12.7 (8.7~16.7), BM11202L) 12.2% 12.8% -0.6 -4.6% Normal CD4 control material (48.1 (41.6~54.6), BM1120N) 48.0% 48.5% -0.5 -1.0%

As shown in Table 2 above, the accuracy showed -1.0% to -4.6% bias in the reference test result (assigned %CD4).

1-3. linearity

Linearity was measured using Multi-check Normal control (BD Biosciences) and very low (0.2% lymphocytes) CD4 samples in 6 different ratios (5/0, 4/1, 3/2, 2/3, 1/4, 0/5). ), and the corresponding CD4 count results were obtained in Multi-check Normal process control (BD Biosciences) by flow cytometer BD FACSCanto II (BD Biosciences, San Jose, CA). Analyzes were performed in duplicate for all, and each parameter correlation coefficient (R ² ) was calculated after linear regression.

R ² = 0.986, y = 0.9738x + 0.1429

As a result, R ² > 0.975 showed excellent linearity.

Experimental Example 2. B cell detection result

As a result of observing PBMC after reacting the complex of anti-CD19 antibody and magnetic beads with whole blood by the method of Example 3, as shown in Figure 3a, 1 μL of magnetic beads diluted at a ratio of 1: 10 were reacted It was confirmed that a large amount of beads bound to a large number of B cells. In FIG. 3a, WB-buffy was prepared by taking the buffy layer in which many leukocytes were collected from whole blood and made a slide without lysis of red blood cells, and it was found that red blood cells were observed together. It was found that PBMC was separated from monocytes, and red blood cells were removed. Unlike the method of dissolving red blood cells, PBMC were separated using density and centrifugal force, and only monocytes were purely isolated.

When the magnetic beads were diluted at a ratio of 1:100, aggregation was significantly reduced, but the amount of beads bound to cells and the number of cells were significantly reduced.

FIG. 3B shows the result of detecting B cells by treating the complex with 0.5 μL (0.25 μg) of anti-CD19 antibody at a concentration of 0.5 mg/mL and 0.5 μL of undiluted magnetic beads, magnified at 1000x magnification, in FIG. 3B, magnified 400x. One drawing is shown in Figure 3c.

In addition, when the anti-CD19 antibody and magnetic beads were treated, the detection results in CD19 ⁺ cells were compared with the results in CD19 ^- cells and are shown in FIG. 3D .

As described above, it was confirmed that B cells can be detected with the naked eye using the antibody-bead complex according to the present invention without undergoing another staining process.

Experimental Example 3. Results of CD4+ T cell detection by the immune cell detection method of the present invention

3-1. Detection results according to bead particle size

In order to confirm the detection result of CD4 ⁺ T cells according to the particle size of the streptavidin-coated magnetic beads, in the method of Example 4, the diameter condition of the magnetic beads was set to 1 μm or 4.5 μm, and the results were observed at 200x magnification. .

As a result, as shown in FIG. 4, it was confirmed that cell aggregation occurred when magnetic beads having a diameter of 4.5 μm were reacted.

As such, it was found that when the magnetic beads exceed a certain size, intercellular aggregation occurs, which affects the result. was used.

3-2. Detection results according to the amount of anti-CD4 antibody

In order to confirm the detection result of CD4 ⁺ T cells according to the amount of the anti-CD4 antibody, the conditions of the anti-CD4 antibody in the method of Example 4 were diluted 1:10 at a concentration of 0.2 mg/mL, 0.5 μL of the anti-CD4 antibody, Results were observed with 1 µL or 5 µL.

As a result, as shown in FIG. 5, the detection result was weak when reacting with 0.5 μL of antibody, the detection result was good when reacting with 5 μL of antibody, and the detection result when reacting with 1 μL of antibody. was confirmed to appear properly.

As such, it was found that an appropriate amount of antibody was required because the reaction did not occur well even if the amount of antibody was too small or too large, and it was confirmed that the amount of antibody was in the range of 1 to 4 μL, and 3 μL of antibody was used.

3-3. Detection result according to PEG8000 concentration

In order to confirm the detection result of CD4 ⁺ T cells according to the PEG8000 concentration, in the method of Example 4, the PEG8000 concentration was set to 5%, 10%, or 25%, and the result was observed at 400x magnification.

As a result, as shown in FIG. 6a, it was confirmed that the shape deformation severely affected the results when the PEG8000 concentration was as high as 25% or more.

In addition, using Ficoll70 instead of PEG8000, 10% Ficoll70 and 10% PEG8000 were treated for 3 hours and observed at 200x and 400x magnification, as shown in FIG. 6b, there was no special effect when 10% Ficoll70 was treated , In the case of treatment with 10% PEG8000, it was confirmed that the staining was maintained stably over time.

In particular, as shown in FIG. 6c , it was confirmed that the treatment with 10% PEG8000 was helpful in reading the results by maintaining a stable cell shape for a long time when observed at 400x magnification compared to the case without treatment with PEG8000.

Accordingly, when detecting CD4 ⁺ T cells, 7.5% PEG8000 was used since treatment with PEG8000 in the concentration range of 7.5% to 10% stabilized the cell morphology for a long time and facilitated detection.

3-4. Detection result by FACS buffer treatment

In order to confirm the detection result of CD4 ⁺ T cells according to the FACS buffer treatment, the case where PBS containing 1% bovine serum albumin (BSA) was treated as a FACS buffer together with PEG8000 according to the method described in Example 4 above, and PBS alone The treated cells were observed.

As a result, as shown in FIG. 7a, it was confirmed that a more vivid and distinct staining image was observed when FACS buffer was used.

In addition, as shown in Figure 7b, when FACS buffer was used, the cell membrane and cell morphology were clearer than when only PBS was used, and when treated with PEG8000, cell deformation was less and aggregation did not occur even after 24 hours. confirmed that

3-5. Detection result according to cell centrifugation rotation speed

In order to confirm the detection result of CD4 ⁺ T cells according to the rotation speed of the cell centrifugation, in the method of Example 5, the rotation speed of the cell centrifugation was set to 2000 rpm or 7500 rpm, and the results were observed.

As a result, as shown in FIG. 8, it was found that cell morphology and degree of concentration differed depending on the rotational speed of cell centrifugation, and it was confirmed that the range within 5000 rpm to 7500 rpm was appropriate, and centrifuged.

3-6. Methylene blue staining result

Whole blood was reacted with anti-CD4 antibody and magnetic beads in the same manner as in Example 4, and red blood cells were incubated with 500 μL BD FACS lysis buffer (BD Biosciences, USA) at room temperature for 10 to 20 minutes. Then, after cytospin or without cytospin, when slides were made and stained with methylene blue for 2 minutes, 3 minutes, or 4 minutes to identify cells, as shown in FIG. 9, clear cell staining results were confirmed. Bar, it was found that optimal dyeing results can be obtained when dyeing is performed for about 2 minutes.

Experimental Example 4. Detection of CD8+ T cells

In addition to CD4 ⁺ T cells, other immune cells could be detected by the method of Example 4. Among them, when whole blood was treated with anti-CD8 antibody and magnetic beads, reacted for 30 minutes, cells were centrifuged, and cells were observed at magnifications of 400x (left figure) and 200x (right figure), as shown in FIG. 10, CD8 ⁺ It was confirmed that T cells were detected.

The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

10: chip 1 for detecting immune cells
20: chip 2 for detecting immune cells
11, 21: chamber 1
12, 22: chamber 2
13, 23: chamber 3
14, 24: chamber 4
15: chamber 5
16, 25: filter paper
17, 26: slide
18, 27: barcode

Claims (15)

A method for detecting immune cells using cell centrifugation and concentration, comprising the following steps:
(a) preparing a bead-antibody complex by conjugating microbeads and an antibody;
(b) reacting the bead-antibody complex with blood separated from the subject;
(c) after the reaction, treating the reactant with red blood cell lysis buffer to dissolve red blood cells and then removing the supernatant; and
After removing the supernatant, suspending the cell pellet in polyethylene glycol 8000 (PEG 8000); and
(d) concentrating the cells by centrifuging the cells after removing the supernatant.
delete According to claim 1,
Characterized in that the antibody is an antibody against a cell surface antigen cluster (cluster of designation, CD), the method.
According to claim 3,
Wherein the antibody is at least one selected from the group consisting of anti-CD4, anti-CD19, anti-CD3, anti-CD8, anti-CD14, anti-CD16, anti-CD25, anti-CD45, anti-CD56, and anti-CD127.
According to claim 1,
Characterized in that the concentration of the antibody in step (a) is 0.01 mg / mL to 3 mg / mL, the method.
According to claim 1,
In the step (a), the volume ratio of microbeads: antibody is 1 to 6: 1, characterized in that, the method.
According to claim 1,
The method, characterized in that the blood is at least one selected from the group consisting of whole blood and peripheral blood mononuclear cells (PBMC).
According to claim 1,
Characterized in that the microbeads have a diameter of 0.1 μm to 4 μm.
According to claim 1,
The PEG 8000 is characterized in that the concentration is 5% (v / v) to 25% (v / v), the method.
According to claim 1,
The step of centrifuging and concentrating the cells in step (d) is characterized in that using a cytospin, method.
According to claim 10,
Characterized in that the cell centrifugation using the cytospin is performed at a speed of 5000 rpm to 9000 rpm.
Chamber 1 (11, 21) in which the bead-antibody complex reacts with the blood separated from the subject;
Chamber 2 (12, 22) where the red blood cell lysis reaction takes place;
Chamber 3 (13, 23) containing a solution used for immune cell detection reaction including a red blood cell lysis solution;
Chamber 4 (14, 24) in which cell centrifugation and concentration are performed;
filter papers 16 and 25 made of paper that absorbs water;
Slides 17 and 26 made of a transparent material for microscopic image observation and in close contact with the filter paper; and
As an immune cell detection chip (10, 20) composed of barcodes (18, 27),
The bead-antibody complex is prepared by conjugating microbeads and antibodies,
The immune cell detection chip (10, 20) further includes a chamber 5 (15), and in the chamber 5, a reaction in which cell pellets are suspended by treatment with polyethylene glycol 8000 (PEG 8000) occurs, or
A chip for detecting immune cells, characterized in that a reaction in which the cell pellet is suspended by the polyethylene glycol 8000 treatment occurs in the chamber 4 (24).
delete According to claim 12,
A chip for detecting immune cells, characterized in that the material of the slides (17, 26) is transparent glass or plastic.
According to claim 12,
The chip for detecting immune cells (10, 20) is characterized in that it consists of a length of 7 cm to 13 cm, a height of 5 cm to 10 cm, and a width of 0.5 cm to 5 cm.

Images