KR102869006B1 - A method for detecting a target protein in cytology sample using multiplex immunofluorescence staining - Google Patents

Abstract

The present invention relates to a method for detecting a target protein in a cell sample using multiplex immunofluorescence staining. According to a multiplex immunofluorescence staining method according to one aspect, there is an effect of being able to detect various types of target proteins inexpensively and quickly while reducing damage to a cell (cytology) sample.

Description

{A method for detecting a target protein in cytology sample using multiplex immunofluorescence staining}

This relates to a method for detecting a target protein in a cell sample using multiple immunofluorescence staining.

Immunohistochemistry is a widely used immunostaining technique for disease diagnosis and research, utilizing antibodies to maintain cell and tissue morphology while obtaining protein expression information. Recently, in medical fields such as pathology, the need for technologies capable of simultaneously detecting a variety of target proteins has increased for purposes such as disease diagnosis, research, and therapeutic target exploration. Research is underway on technologies that allow for the simultaneous staining of multiple proteins in a single tissue sample by attaching fluorescent substances that emit different colors to specific antibodies.

However, tissue samples are invasive to acquire, often requiring specialized medical facilities and techniques, and have limitations such as long processing times, including fixation. Therefore, there is a need for tests using cell (cytology) samples that are less invasive for patients, cheaper, and provide rapid results.

The multiplex immunofluorescence staining method designed for existing tissue samples is not suitable for application to cell (cytology) samples, and the currently known system using cell samples applies the PAP (Papanicolaou) staining method, but the PAP staining method has the limitation that it can only confirm morphological changes.

Therefore, there is a need to design a target protein multi-staining protocol optimized for cell samples to enable rapid detection of various types of target proteins while reducing damage to the sample after cytology collection.

A method for detecting a target substance of a cell is provided, comprising: a step of pretreating a separated biological sample (e.g., a cell sample); a step of treating the pretreated cell sample with a fluorescent dye conjugate that binds to a target substance of the cell; a step of performing antigen retrieval on the cell sample treated with the fluorescent dye conjugate; and a step of measuring a detectable signal from the cell treated with the fluorescent dye conjugate.

One aspect provides a method for detecting a target substance of a cell, comprising the steps of: pretreating a separated cell sample; treating the pretreated cell sample with a fluorescent dye conjugate that binds to a target substance of the cell; performing antigen retrieval on the cell sample treated with the fluorescent dye conjugate; and measuring a detectable signal from the cell treated with the fluorescent dye conjugate.

The term "sample" as used herein may refer to any biological sample in which target cells may be present. For example, the sample may be selected from the group consisting of a biopsy sample, a tissue sample, a cell suspension obtained by suspending isolated cells in a liquid medium, a cell culture, and combinations thereof. Furthermore, the sample may be a body fluid of an animal. The body fluid may be selected from the group consisting of blood, bone marrow fluid, lymph, saliva, tears, urine, mucosal fluid, amniotic fluid, and combinations thereof. For example, the sample may be blood containing circulating tumor cells.

In one specific example, the biological sample may be a cell. As used herein, the term "cell" means a basic structural and functional unit of a living organism. The "living organism" includes vertebrates such as mammals including humans (humans, monkeys, mice, rats, hamsters, cows, etc.), birds (chickens, ostriches, etc.), amphibians (frogs, etc.), and fish, or invertebrates such as insects (silkworms, moths, fruit flies, etc.), plants, microorganisms such as yeast, etc., and is preferably a mammal including humans, but is not limited thereto. In one specific example, the cell may be selected from the group consisting of circulating tumor cells, cancer stem cells, immune cells, fetal stem cells, fetal cells, cancer cells, and tumor cells. As used herein, the term "cancer" means a physiological condition in an animal that is typically characterized by abnormal or uncontrolled cell growth. The cancer may be associated with, for example, metastasis, interference with normally functioning surrounding cells, release of cytokines or other secreted products at abnormal levels, suppression or enhancement of inflammatory or immunological responses, neoplasia, premalignancy, malignancy, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc. In one specific example, the cancer may be a blood cancer or a solid cancer, and the solid cancer may be at least one selected from the group consisting of lung cancer, skin cancer, stomach cancer, intestinal cancer, colon cancer, pancreatic cancer, liver cancer, thyroid cancer, uterine cancer, cervical cancer, ovarian cancer, testicular cancer, prostate cancer, breast cancer, and oral cancer, but is not limited thereto.

As used herein, the term "preprocessing step" includes a step of fixing a separated cell sample; or a step of blocking a cell sample. As used herein, the term "fixation" refers to a treatment method that preserves the original state, structure, etc. as much as possible without change by artificially manipulating a sample, etc. The above fixation includes physical fixation such as heating fixation, freezing fixation, microwave fixation, etc., chemical fixation such as immersion fixation, perfusion fixation, vapor fixation, etc., and preferably formaldehyde (HCHO), glutaraldehyde (Glutaraldehyde: (CH2) ₃ CHOCHO), Mercuric chloride (HgCl ₂ ), Potassium dichromate (K ₂ Cr ₂ O ₇ ), Chromic acid (H ₂ CrO ₄ ), Picric acid (C ₆ H ₂ (NO ₂ ) ₃ OH), Osmium tetroxide (OsO ₄ ), Acetic acid (CH ₃ COOH), Ethyl alcohol, A fixative such as acetone (CH ₃ COCH ₃ ) may be used, but is not limited thereto. The term "blocking" as used herein refers to blocking an antibody and a non-specific protein site to reduce non-specific binding of the antibody. The blocking method includes, but is not limited to, methods using normal serum, protein buffer, commercial mixes, endogenous biotin, etc. Specifically, protein buffer includes, but is not limited to, skim milk, bovine serum albumin (BSA), gelatin, etc.

As used herein, the term "fluorescent dye complex" refers to a complex that can identify a substance present in a cell using a fluorescent dye. In one specific example, the fluorescent dye complex may include a substance that binds to a target substance and a fluorescent dye. As used herein, the term "substance that binds to a target substance" refers to a substance that specifically binds to a target substance. In one specific example, the substance that binds to the target substance may be, but is not limited to, an antibody, an antigen, an aptamer, a receptor, a ligand, an enzyme substrate, an enzyme inhibitor, an enzyme cofactor, an enzyme, and the like.

As used herein, the term "antibody" refers to any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen. The antibody includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). The antibody also includes immunoglobulin molecules composed of four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, which are arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The above antibodies also include antigen-binding fragments of whole antibody molecules. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be derived from whole antibody molecules using any suitable standard technique, such as, for example, proteolytic digestion, or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optional constant domains. Such DNA is known and/or readily available, for example, from commercially available DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or using molecular biological techniques, for example, to arrange one or more variable domains and/or constant domains into a suitable configuration, to introduce codons, to create cysteine residues, to modify, add or delete amino acids, etc.

In one specific example, the antibody can be selected according to the type of tissue and cell origin. For example, antibodies that recognize various antigens, such as antibodies that are highly specific for cancer, can be used. Specifically, the antibody can be an antibody that specifically binds to an anticancer antigen - cancer cells or cancer tissue, and more specifically, an antitumor antibody, an antimucin antibody, an anti-stomach-related sugar chain antibody, an anti-tumor chain antibody that specifically reacts with CEA, AFP, CA19-9, NSE, DU-PAN-2, CA50, SPan-1, CA72-4, CA125, HCG, p53, and the like, and can be utilized for tumor antigens that specifically react with STN (sialyl Tn antigen), c-erbB-2 protein, esophageal cancer, colon cancer, rectal cancer, skin cancer, uterine cancer, and others. Anti-pathogenic protein antibodies, antitumor antigen antibodies, etc. can also be used that are coupled to an amplification system such as avidin and biotin. However, the antibodies described above are only given as examples, and the antibodies that can be used are not limited to those mentioned above.

In one embodiment, the antibody can be detectably labeled by coupling to a chemiluminescent compound. The presence of a chemiluminescent-tagged antigen-binding polypeptide is determined by detecting the presence of luminescence that occurs during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds include luminol, isoluminol, depleted acridinium esters, imidazoles, acridinium salts, and oxalate esters. In one embodiment, the antibody can be detectably labeled using a fluorescent metal, such as Eu, or others from the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

In one specific example, the substance that binds to the target substance may include primary antibodies that bind to the target substance of the cell; and a labeled secondary antibody that binds to the primary antibody. As used herein, the term "primary antibody" refers to an antibody that directly binds to an antigen or protein, and the term "secondary antibody" refers to an antibody that binds to another antibody that binds to an antigen or protein.

In one specific example, the substance binding to the target substance may be a primary antibody that specifically binds to the target substance of the cell; and a secondary antibody having an antibody-binding domain (ABD) that selectively and strongly binds to the Fc portion of the primary antibody. For example, a secondary antibody (immunogloblin) capable of specifically binding to the primary antibody in a specific animal species can be produced and used to recognize the primary antibody.

In one specific example, the secondary antibody may be an antibody labeled with a fluorescent dye by covalent bonding. Specifically, the fluorescent dye is not particularly limited, but may include Fluorescein, CR110: Carboxyrhodamine 110: Rhodamine Green (trade name), TAMRA: Carboxytetramethylrhodamine: TMR, Carboxyrhodamine 6G: CR6G, BODIPY FL (trade name): 4,4-Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, BODIPY 493/503 (trade name): 4,4-Difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-propionic acid, BODIPY R6G (trade name): 4,4-Difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, BODIPY 558/568 (trade name): 4,4-Difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, BODIPY 564/570 (trade name): 4,4-Difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, BODIPY 576/589 (trade name): 4,4-Difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, BODIPY 581/591 (trade name): 4,4-Difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, EvoBlue10 (trade name), EvoBlue30 (trade name), MR121, ATTO 655 (trade name), ATTO 680 (trade name), ATTO 700 (trade name), ATTO MB2 (trade name), Alexa Fluor 350 (trade name), Alexa Fluor 405 (trade name), Alexa Fluor 430 (trade name), Alexa Fluor 488 (trade name), Alexa Fluor 532 (trade name), Alexa Fluor 546 (trade name), Alexa Fluor 555 (trade name), Alexa Fluor 568 (trade name), Alexa Fluor 594 (trade name), Alexa Fluor 633 (trade name), Alexa Fluor 680 (trade name), Alexa Fluor 700 (trade name), Alexa Fluor 750 (trade name), Alexa Fluor 790 (trade name), Flamma 496 (trade name), Flamma 507 (trade name), Flamma 530 (trade name), Flamma 552 (trade name), Flamma 560 (trade name), Flamma 575 (trade name), Flamma 581 (trade name), Flamma 648 (trade name), Flamma 675 (trade name), Flamma 749 (trade name), Flamma 774 (trade name), Flamma 775 (trade name), Rhodamine Red-X (trade name), Texas Red-X (trade name), 5(6)-TAMRA-X (trade name), It may be selected from the group consisting of 5TAMRA (trade name), indocyanine green (ICG) and 2-(((E)-2-(4-(2-carboxyethyl)phenoxy)-3-((E)-2-(3,3-dimethyl-5-sulfonato-1-(3-(trimethylammonio)-propyl)indolin-2-ylidene)ethylidene)cyclohex-1-enyl)vinyl)-3,3-dimethyl-1-(3-(trimethylammonio)-propyl)-3H-indolium-5-sulfonate disodium bromide (ZW800-1).

In one specific example, the processing step may be processing a fluorescent dye complex comprising a primary antibody that binds to a target substance of the cell and a labeled secondary antibody that binds to the primary antibody.

In one specific example, the target material may be selected from the group consisting of, but not limited to, proteins, sugars, lipids, nucleic acids, and combinations thereof.

As used herein, the term "antigen retrieval" refers to a process of exposing an epitope masked during a sample fixation process. In one specific example, the antigen retrieval step may include a heat treatment step. For example, the antigen retrieval process may be heat-induced epitope retrieval (HIER). The HIER uses heat to restore the binding of an antibody to an epitope. For example, heat sources used in the HIER may include, but are not limited to, a microwave oven, a pressure cooker, a vegetable steamer, a water bath, or an autoclave.

In one specific embodiment, the antigen retrieval step includes a first heat treatment step and a second heat treatment step, wherein the power of the first heat treatment step may be higher than that of the second heat treatment step, and the treatment time of the first heat treatment step may be shorter than that of the second heat treatment step. In one specific embodiment, the first heat treatment step and the second heat treatment step may be performed using a microwave oven.

In one specific example, the microwave oven can be used at a power of at least 200 W and at most 1000 W. In addition, in one specific example, when using the microwave oven, a process of processing the dyed sample at maximum power and then processing it at reduced power may be included.

When processing at the above maximum power, the processing can be performed for 20 to 120 seconds (until the buffer boils), preferably 40 to 100 seconds, and more preferably 60 to 90 seconds.

When processing at the above reduced power, processing can be done for 3 to 12 minutes, preferably 4 to 9 minutes, and more preferably 5 to 10 minutes.

In one specific example, the antigen retrieval process may be Proteolytic-induced epitope retrieval (PIER). PIER uses an enzyme to restore the binding of an antibody to an epitope. Examples of the enzyme used include, but are not limited to, Proteinase K, Trypsin, or Pepsin.

In one specific embodiment, the measuring step may include performing an in situ hybridization or immunohistochemical analysis on the target cell. As used herein, the term "measurement" refers to identifying the location of a target substance within the biological sample through a color detected from the fluorescent dye complex specifically bound to the target substance. The term "in situ hybridization (ISH)" as used herein refers to a technique for directly detecting a specific target nucleic acid in a cell or tissue fragment. Specifically, it may refer to identifying the location of a target substance in a target cell using a fluorescently labeled probe. For example, it may be fluorescence in situ hybridization (FISH), but is not limited thereto. The term "fluorescence in situ hybridization (FISH)" as used herein refers to a method of observing mutations in chromosomes or genes using a fluorescence microscope by fixing cells on a slide while maintaining the shape of the chromosome or nucleus intact, and reacting them with various types of probes that have fluorescent materials attached to DNA complementary to a specific base sequence of the target nucleic acid to confirm the presence or absence and location of the target nucleic acid. The term "immunohistochemical analysis" as used herein may identify a protein expressed within a target cell through an antigen-antibody reaction. For example, it may include an immunofluorescence staining method. The term "immunofluorescence staining method" as used herein refers to a method of detecting an antigen within a specimen using a fluorescently labeled antibody with a fluorescence microscope, etc. Specifically, it may refer to an immunostaining method that identifies a substance present within a cell or tissue by utilizing the reaction between an antigen and an antibody. There are, but are not limited to, a direct fluorescent antibody method that uses a fluorescently labeled specific antibody for an antigen, and an indirect fluorescent antibody method that binds an unlabeled specific antibody (primary antibody) to an antigen and then binds a fluorescently labeled secondary antibody (anti-immunoglobulin antibody).

In one specific example, a method of binding an antigen to a tissue and then binding a fluorescently labeled antibody (secondary antibody) can be used for the purpose of detecting a partial distribution of a specific antibody (primary antibody) for the antigen. The fluorescent labels of the above antibodies include Fluorescein, CR110: Carboxyrhodamine 110: Rhodamine Green (trade name), TAMRA: Carboxytetramethylrhodamine: TMR, Carboxyrhodamine 6G: CR6G, BODIPY FL (trade name): 4,4-Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, BODIPY 493/503 (trade name): 4,4-Difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-propionic acid, BODIPY R6G (trade name): 4,4-Difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, BODIPY 558/568 (trade name): 4,4-Difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, BODIPY 564/570 (trade name): 4,4-Difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, BODIPY 576/589 (trade name): 4,4-Difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, BODIPY 581/591 (trade name): 4,4-Difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, EvoBlue10 (trade name), EvoBlue30 (trade name), MR121, ATTO 655 (trade name), ATTO 680 (trade name), ATTO 700 (trade name), ATTO MB2 (trade name), Alexa Fluor 350 (trade name), Alexa Fluor 405 (trade name), Alexa Fluor 430 (trade name), Alexa Fluor 488 (trade name), Alexa Fluor 532 (trade name), Alexa Fluor 546 (trade name), Alexa Fluor 555 (trade name), Alexa Fluor 568 (trade name), Alexa Fluor 594 (trade name), Alexa Fluor 633 (trade name), Alexa Fluor 680 (trade name), Alexa Fluor 700 (trade name), Alexa Fluor 750 (trade name), Alexa Fluor 790 (trade name), Flamma 496 (trade name), Flamma 507 (trade name), Flamma 530 (trade name), Flamma 552 (trade name), Flamma 560 (trade name), Flamma 575 (trade name), Flamma 581 (trade name), Flamma 648 (trade name), Flamma 675 (trade name), Flamma 749 (trade name), Flamma 774 (trade name), Flamma 775 (trade name), Rhodamine Red-X (trade name), Texas Red-X (trade name), 5(6)-TAMRA-X (trade name), 5TAMRA (trade name), indocyanine green (ICG) and 2-(((E)-2-(4-(2-carboxyethyl)phenoxy)-3-((E)-2-(3,3-dimethyl-5-sulfonato-1-(3-(trimethylammonio)-propyl)indolin-2-ylidene)ethylidene)cyclohex-1-enyl)vinyl)-3,3-dimethyl-1-(3-(trimethylammonio)-propyl)-3H-indolium-5-sulfonate disodium bromide (ZW800-1) may be selected from the group consisting of, but is not limited thereto.

In one specific example, a multiplex immunofluorescence staining method can be performed to detect multiple antigens in the same sample. As used herein, the term "multiplex immunofluorescence staining method" refers to a method capable of simultaneously detecting multiple targets using individual target-specific antibodies on a single slide. Specifically, eight or more multiple targets can be detected simultaneously using the antibodies mentioned above. Preferably, seven or more multiple targets can be detected simultaneously, more preferably six or more multiple targets can be detected simultaneously, and at least five or more multiple targets can be detected simultaneously.

In one specific embodiment, a method for detecting a target substance of a cell, comprising the steps of: pretreating a separated cell sample; treating the pretreated cell sample with a fluorescent dye conjugate that binds to a target substance of the cell; performing antigen retrieval on the cell sample treated with the fluorescent dye conjugate; and measuring a detectable signal from the cell treated with the fluorescent dye conjugate; may not include a separate step of performing antigen retrieval between the pretreating step and the treating step.

In one specific embodiment, a method for detecting a target substance of a cell, comprising the steps of: pretreating a separated cell sample; treating the pretreated cell sample with a fluorescent dye conjugate that binds to a target substance of the cell; performing antigen retrieval on the cell sample treated with the fluorescent dye conjugate; and measuring a detectable signal from the cell treated with the fluorescent dye conjugate; After the antigen retrieval step, the treating step may be repeated at least once.

In one specific example, a method for detecting a target substance of a cell, comprising: a step of pretreating a separated cell sample; a step of treating the pretreated cell sample with a fluorescent dye conjugate that binds to a target substance of the cell; a step of performing antigen retrieval on the cell sample treated with the fluorescent dye conjugate; and a step of measuring a detectable signal from the cell treated with the fluorescent dye conjugate; After the antigen retrieval step, the processing step and the antigen retrieval step may be sequentially repeated at least once, and then a staining and mounting step may be performed instead of the antigen retrieval step in the last step.

The term "staining" in this specification means making it possible to easily distinguish the components of target cells by using dyes, etc. For example, there are methods such as vital staining, staining by selective dissolution, staining by chemical color reaction, metallic impregnation, and staining by dyes, but are not limited thereto. More specifically, DAPI(4 Dyeing can be performed.

As used herein, the term "DAPI(4 "Staining" refers to a method of detecting DNA using DAPI (4,6-diamidino-2-phenylindole) in a fluorescence microscope. The DAPI is a fluorescent dye that specifically binds to the A (adenine): T (thymine) base pair of DNA. When a chromosome is stained with DAPI and observed under an ultraviolet ray (300-400 nm) in a fluorescence microscope, a portion with a large number of A: T pairs generates blue fluorescence (400-500 nm). In one specific example, DAPI can be used as a standard for determining whether a cell is damaged.

As used herein, the term "mounting" refers to a step of sealing by covering with a cover glass using a mounting agent to protect against mechanical damage, drying in the air, fading of dye, etc. Specifically, the mounting agent may be a water-soluble mounting agent such as glycerin, glycerin-jelly, apathy gum syrup, crystal mount, etc., or an insoluble mounting agent such as canada balsam, permount, HSR, Enkitt, cedar wood oil, etc., but is not limited thereto.

In one specific example, a method for detecting a target substance of a cell, comprising: a step of pretreating a separated cell sample; a step of treating the pretreated cell sample with a fluorescent dye conjugate that binds to a target substance of the cell; a step of performing antigen retrieval on the cell sample treated with the fluorescent dye conjugate; and a step of measuring a detectable signal from the cell treated with the fluorescent dye conjugate; the method may include, after the antigen retrieval step, sequentially repeating the processing step and the antigen retrieval step at least once, and then performing an in situ hybridization or immunohistochemical analysis step in the final step.

In one specific example, the in situ hybridization or immunohistochemical analysis may be performed using a fluorescence scanner or fluorescence microscopy, but is not limited thereto, and any device capable of acquiring fluorescence images may be used.

According to a method for detecting target proteins in cell samples using multiple immunofluorescence staining according to a single aspect, there is an effect of detecting various types of target proteins while reducing damage to cell (cytology) samples.

Figure 1 is a schematic diagram of a method for detecting a target protein using a cell sample.
Figure 2 is a photograph confirming whether cluster loss occurred when the antigen retrieval step was performed: Figure 2a is a photograph confirming cell clusters after the first fixation, and Figure 2b is a photograph confirming cell clusters after performing the first antigen retrieval.
Figure 3 is a photograph confirming whether antibody expression occurs when antigen retrieval is omitted: Figure 3a is a photograph confirming whether antibody expression occurs when antigen retrieval is performed, and Figure 3b is a photograph confirming whether antibody expression occurs when antigen retrieval is omitted.
Figure 4 is a photograph confirming the effect of improving the ease of detection of antibody expression signals when the antigen retrieval process at a specific time is omitted: Figure 4a is a photograph when the antigen retrieval process is omitted only in the first antibody processing step, and Figure 4b is a photograph of antibody expression when all antigen retrieval processes are omitted.
Figure 5 is a photograph confirming the reduction in sample damage rate by controlling the antigen retrieval time: Figure 5a is a photograph confirming sample damage when antigen retrieval was performed under the condition that the first heat treatment step was performed at the maximum power (100% power) of a 1000 W microwave oven for 1 to 1 minute and 30 seconds (until the buffer boils) and then the second heat treatment step was performed at the reduced power (20% power) for 15 minutes, and Figure 5b is a photograph confirming whether the sample was damaged when antigen retrieval was performed under the condition that the first heat treatment step was performed at the maximum power (100% power) of a 1000 W microwave oven for 1 to 1 minute and 30 seconds (until the buffer boils) and then the second heat treatment step was performed at the reduced power (20% power) for 5 minutes, by shortening the heat treatment time. 5c is a photograph confirming whether fluorescence expression occurred when antigen retrieval was performed by shortening the heat treatment time, and Fig. 5d is a graph comparing the DAPI exposure time values of Figs. 5a and 5b.
Figure 6 is a photograph of multiple immunofluorescence staining performed with three antibodies on a lymph node.
Figure 7 is a photograph of multiple immunofluorescence staining performed with three antibodies on a lymph node.
Figure 8 is a photograph of multiple immunofluorescence staining performed with four antibodies on ovarian cancer.
Figure 9 is a photograph of multiple immunofluorescence staining performed with four antibodies on ovarian cancer.
Figure 10 is a photograph of multiple immunofluorescence staining performed with five antibodies on ovarian cancer.

The present invention will be described in more detail through the following examples. However, these examples are provided for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1. Multiplex immunofluorescence staining process in liquid-based cytology (LBC)

1-1. Preparation and fixation of cell samples

To utilize the collected cells as a sample, large cell clusters require removal, as they exhibit low fluorescence and are more likely to be lost during the staining process. Therefore, the sample was filtered through a 200 μm mesh to remove foreign substances and cell clusters.

Additionally, a fixation process was performed to minimize cell degeneration and prevent cells attached to the slide glass from falling off.

Specifically, a slide glass was placed on a slide plate and a chamber was fixed on top. An LBC (Liquid-based cytology) sample was placed in the chamber and incubated at room temperature. The supernatant was discarded from the chamber, washed five times with 95% EtOH, and incubated for 15 minutes in the final EtOH washing step before removal. After removing the chamber, it was lightly rinsed with DW and washed twice with 1X TBST.

1-2. Blocking process

A blocking process was performed to remove the background by blocking nonspecific binding of the detection antibody.

Specifically, after placing the slide on a humidity chamber, the area was drawn with a PAP pen. Antibody diluent (blocking solution) was sprayed on the tissue and incubated for 10 minutes.

1-3. Antibody (Target antibody) processing process

The sample was treated with an antibody (target antibody) to detect the protein to be observed.

Specifically, the primary antibody was diluted in 1x TBST and then treated. The antibody was sprayed on the cell sample (at least 100-400 μl depending on the sample size) and incubated at RT for 1 hour. The sample was washed twice by pipetting 1x TBST, and then washed twice for 3 minutes. The sample treated with the primary antibody was treated with the secondary antibody. Specifically, the secondary antibody solution was sprayed on the sample and incubated at RT for 10 minutes. The sample was washed twice by pipetting 1x TBST, and then washed twice for 3 minutes. The Opal 6-Plex Manual Detection Kit (Akoya Biosciences) was used.

1-4. Signal amplification process

To amplify the signal of the fluorescent sample, it was diluted 1:100 in Amplification Diluent. After spreading it on the sample (same volume as the primary antibody), it was incubated for 10 minutes. The sample was rinsed with 1x TBST for 3 minutes and then rinsed again for 2 minutes.

1-5. Antigen retrieval process

After one antibody staining, antigen retrieval was performed after fluorescent sample treatment for the next antibody staining. Specifically, the antigen retrieval (AR) solution was filled into a jar and microwaved in a microwave oven with a minimum of 200 W and a maximum of 1000 W. The microwave was turned on at full power (100% power) until boiling, then reduced to 20% power for 5 min. The sample was rinsed briefly with autoclaved distilled water (DW) and washed twice with 1X TBST.

1-6. DAPI staining & mounting process

To stain the nucleus (DNA) within the cell, after the final antibody fluorescence sample treatment (after the signal amplification process) was completed, DAPI (4 Staining was performed. This allowed for the precise determination of the expression of various antibodies to be observed, and the degree of sample damage could be identified through DAPI expression patterns (e.g., nuclear rupture or cell bursting) and exposure time values. Furthermore, a mounting process was performed to protect against mechanical damage, air drying, and stain fading.

Specifically, DAPI was diluted by dropping 2 drops in 1 ml of 1x TBST, sprayed on the sample, and incubated for 5 min. After washing with 1x TBST for 3 min, the sample was washed with distilled water (DW) for 2 min, and after removing the DW (distilled water), mounting solution was added, covered with a coverslip, and dried.

1-7. Fluorescence scanner shooting in progress

Through the above process, fluorescence scanner photography was performed to measure whether each antibody expressed fluorescence.

Specifically, we used the Vectra Polaris (now PhenoImager HT) image scanner from Akoya Biosciences, which can capture both brightfield and fluorescence images and scan at a minimum of 10x (1.0 μm/pixel) and a maximum of 40x (0.25 μm/pixel). To confirm the results, images were acquired after scanning at a resolution of 20x (0.5 μm/pixel).

The fluorescent staining method through Example 1 is shown in Fig. 1.

Figure 1 is a schematic diagram of a method for detecting a target protein using a cell sample.

As shown in Fig. 1, a method according to one embodiment includes a method for detecting a target substance of a cell, including the steps of preparing and fixing a cell sample; blocking the fixed sample; treating the blocked sample with an antibody; amplifying a signal of the sample treated with the antibody; and performing antigen retrieval on the sample whose signal has been amplified. By repeating the antibody treatment step and the antigen retrieval step multiple times, various types of target proteins can be detected simultaneously.

Example 2. Antigen retrieval process

2-1. Antigen recovery process

In order to expose the masked epitope during the sample fixation process, an antigen retrieval process was performed after the antibody treatment and signal amplification process of Example 1.

Specifically, the jar was filled with Antigen retrieval (AR) solution and microwaved at 1000 W (100% power) for 1 minute and then at 200 W (20% power) for 5 minutes. After lightly rinsing with autoclaved DW (distilled water), it was washed twice with 1X TBST.

The fluorescence staining method through Example 2-1 is illustrated in Fig. 1. As illustrated in Fig. 1, an antigen retrieval process was performed after the signal amplification process of Example 1. In the following examples, the effectiveness of the immunofluorescence staining method was confirmed depending on the presence or absence of a primary antigen retrieval process after the step of fixing a cell sample of Example 1 or a secondary antigen retrieval process after the step of amplifying the signal of a sample treated with an antibody of Example 1.

2-2. Confirmation of the effect of omitting the antigen retrieval process

To determine the effect of the antigen retrieval process on cell clusters, immunofluorescence staining was performed on cell samples using the same process as in Example 1 and Example 2-1, except that the first antigen retrieval was additionally performed in Example 1. Cell clusters were measured after the fixation step of Example 1 and after the first antigen retrieval.

Specifically, the cell sample attached to the slide glass was photographed with a cell phone camera at the same location and distance and then confirmed with the naked eye.

Figure 2a is a photograph confirming cell clusters after the first fixation, and Figure 2b is a photograph confirming cell clusters after the first antigen retrieval. As shown in Figures 2a and 2b, it was confirmed that cell clusters were lost due to the first antigen retrieval process.

2-3. Confirmation of differences in antibody expression depending on the presence or absence of the antigen retrieval process

In order to confirm the difference in antibody expression depending on the presence or absence of the antigen retrieval process, the antigen retrieval process of Example 2-1 was omitted, and the presence or absence of antibody expression depending on the presence or absence of the antigen retrieval process was confirmed.

Specifically, two cell samples were attached to each slide glass, fixed, and then primary antigen retrieval was performed on one sample, while the other was omitted. All subsequent steps were performed identically, and the target antibody pan-cytokeratin (CK) was attached, and after treatment with the opal570 fluorescent reagent, images were taken with a fluorescent scanner for comparison.

Figure 3a is a photograph showing antibody expression when antigen retrieval was performed, and Figure 3b is a photograph showing antibody expression when antigen retrieval was omitted.

As shown in Figures 3a and 3b, it was confirmed that antibody expression was evident regardless of whether an antigen retrieval process was performed.

2-4. Confirmation of increased antibody binding efficiency when only the primary antigen retrieval process is omitted.

The presence or absence of antibody expression was confirmed in cases where only the first antigen retrieval process after the step of fixing the cell sample was omitted in the same process as Example 1, and in cases where all antigen retrieval processes in Example 1 were omitted.

Specifically, two cell samples were attached to each slide glass, fixed, and then one sample was omitted only for the primary antigen retrieval process, while the other sample was omitted for all antigen retrieval processes. All processes except the step of omitting the antigen retrieval process were performed identically, and target antibodies CD4, CD8, and CK were attached, and after treatment with opal480, 570, and 690 fluorescent reagents, respectively, images were taken with a fluorescent scanner for comparison.

Figure 4a is a photograph of a case where the antigen retrieval process is omitted only in the first antibody processing step, and Figure 4b is a photograph of antibody expression when all antigen retrieval processes are omitted.

As shown in Figures 4a and 4b, it was confirmed that when only the primary antigen retrieval process was omitted, the antibody binding efficiency increased compared to when all antigen retrieval processes were omitted, making antibody expression analysis easier.

Example 3. Control of antigen retrieval time

The effect of heat treatment time on antibody fluorescence expression during the antigen retrieval process was confirmed.

Specifically, immunofluorescence staining was performed in the same manner as in Examples 1 and 2, except that the antigen retrieval process was performed using a microwave oven in Examples 1 and 2. The experiment was performed using a microwave oven with a maximum power of 1000 W. In addition, the exposure time of DAPI was compared to confirm the degree of damage to the cell sample due to heat treatment. DAPI is an indicator that can objectively confirm the degree of damage to the cell sample by staining the cell nucleus, and the more damaged the sample, the weaker the fluorescence expression, and the longer the exposure time of the scanner to recognize it.

Figure 5a is a photograph confirming whether a sample was damaged when antigen retrieval was performed under the condition that the first heat treatment step was performed at the maximum power (100% power) of a 1000 W microwave oven for 1 minute to 1 minute 30 seconds (until the buffer boils) and then at the reduced power (20% power) for 15 minutes, and Figure 5b is a photograph confirming whether a sample was damaged when antigen retrieval was performed under the condition that the first heat treatment step was performed at the maximum power (100% power) of a 1000 W microwave oven for 1 minute to 1 minute 30 seconds (until the buffer boils) and then at the reduced power (20% power) for 5 minutes, and Figure 5c is a photograph confirming whether fluorescence was expressed when antigen retrieval was performed by shortening the heat treatment time. Figure 5d is a graph comparing the DAPI exposure time values of Figures 5a and 5b.

As shown in Figures 5a to 5d, it was confirmed that the damage to the sample was significantly lower in the case of treatment at the maximum power of 1000 W (100% power) for 1 minute followed by treatment at the reduced power of 200 W (20% power) for 5 minutes than in the case of treatment at the maximum power of 1000 W (100% power) for 1 minute followed by treatment at the reduced power of 200 W (20% power) for 15 minutes, and that there was no effect on the fluorescence expression of the antibody after staining.

Example 4. Measurement of fluorescence signal according to the number of antibody treatments

4-1. Confirmation of fluorescence signal measurement results for three antibodies

To simultaneously detect three or more targets in a single sample, multiplex immunofluorescence staining was performed using the same method as in Example 1. Specifically, multiplex immunofluorescence staining of CD4 (opal480), CD8 (opal570), and CK (opal690) was performed in lymph nodes, and the results are shown in Figs. 6 and 7.

Figure 6 is a photograph showing multiple immunofluorescence staining with three antibodies in a lymph node, and Figure 7 is a photograph showing multiple immunofluorescence staining with three antibodies in a lymph node.

As shown in Figures 6 and 7, it was confirmed that the fluorescence signals for the three antibodies were specifically and well detected.

4-2. Confirmation of fluorescence signal detection effect for four antibodies

To simultaneously detect four or more targets in a single sample, multiplex immunofluorescence staining was performed using the same method as in Example 1. Specifically, multiplex immunofluorescence staining of CD8 (opal520), CD3 (opal570), CD20 (opal620), and CK (opal690) was performed in ovarian cancer, and the results are shown in Figs. 8 and 9.

Figure 8 is a photograph of multiple immunofluorescence staining with four antibodies in ovarian cancer, and Figure 9 is a photograph of multiple immunofluorescence staining with four antibodies in ovarian cancer.

As shown in Figures 8 and 9, it was confirmed that the fluorescence signals for the four antibodies were specifically and well detected.

4-3. Confirmation of the fluorescence signal detection effect for five antibodies

To simultaneously detect five or more targets in a single sample, multiplex immunofluorescence staining was performed using the same method as in Example 1. Specifically, multiplex immunofluorescence staining of CD8 (opal520), CD3 (opal570), ER (opal620), Ki67 (opal690), and CK (opal780) was performed in ovarian cancer, and the results are shown in Fig. 10.

Figure 10 is a photograph of multiplex immunofluorescence staining performed with five antibodies in ovarian cancer. As shown in Figure 10, the fluorescent signals for the five antibodies were specifically detected, and staining was confirmed to be possible in all areas of the cell, including the cell membrane (CD8, CD3, CK) and cytoplasm (CD3), as well as the cell nucleus (ER, Ki67).

As a result of the above, according to the method for detecting a target protein in a cell sample using multiple immunofluorescence staining according to one specific example, there is an effect of simultaneously detecting various types of target proteins in a single cell sample while reducing damage to the cell (cytology) sample.

Claims (17)

A step of preprocessing the separated cell sample;
A step of treating the pretreated cell sample with a fluorescent dye complex that binds to a target substance of the cell;
A step of performing antigen retrieval on a cell sample treated with the above fluorescent dye complex; and
A method for detecting a target substance of a cell, comprising: measuring a detectable signal from a cell treated with the fluorescent dye complex;
A method that does not include a step of performing a separate antigen recovery between the above preprocessing step and the above processing step. A method according to claim 1, wherein the preprocessing step includes a step of fixing the separated cell sample; or a step of blocking the cell sample. A method according to claim 1, wherein the fluorescent dye complex comprises a substance that binds to a target substance and a fluorescent dye. A method according to claim 3, wherein the substance binding to the target substance is an antibody, an antigen, an aptamer, a receptor, a ligand, an enzyme substrate, an enzyme inhibitor, an enzyme cofactor, and an enzyme. A method according to claim 4, wherein the target material is selected from the group consisting of proteins, sugars, lipids, nucleic acids, and combinations thereof. The method according to claim 1, wherein the cell is selected from the group consisting of circulating tumor cells, cancer stem cells, immune cells, fetal stem cells, fetal cells, cancer cells, and tumor cells. A method according to claim 1, wherein the processing step comprises processing a fluorescent dye complex comprising a primary antibody that binds to a target substance of the cell and a labeled secondary antibody that binds to the primary antibody. In claim 1, the method wherein the antigen retrieval step includes a heat treatment step. A method according to claim 1, wherein the antigen retrieval step comprises a first heat treatment step and a second heat treatment step, wherein the power of the first heat treatment step is higher than that of the second heat treatment step, and the treatment time of the first heat treatment step is shorter than that of the second heat treatment step. A method according to claim 9, wherein the first heat treatment step is performed in a microwave oven for 60 to 90 seconds. A method according to claim 9, wherein the second heat treatment step is performed in a microwave oven for 5 to 10 minutes. A method according to claim 1, wherein the measuring step comprises performing an in situ hybridization or immunohistochemical analysis on cells treated with the fluorescent dye complex. delete A method according to claim 1, comprising a step of repeating the processing step at least once after the antigen recovery step. A method according to claim 1, wherein after the antigen retrieval step, the processing step and the antigen retrieval step are sequentially repeated at least once, and then, in the last step, a staining and mounting step is performed instead of the antigen retrieval step. A method according to claim 1, wherein after the antigen recovery step, the processing step and the antigen recovery step are sequentially repeated at least once, and then in the last step, a step of performing in situ hybridization or immunohistochemical analysis is performed. A method according to claim 1, wherein the step of measuring the signal is to measure a signal in at least one selected from the group consisting of the cytoplasm, cell membrane, cell organelles, and cell nucleus of a cell sample.