CN111349683B

CN111349683B - Application of basophils of granulocyte group as allergic disease diagnosis marker

Info

Publication number: CN111349683B
Application number: CN202010084271.5A
Authority: CN
Inventors: 何韶衡; 何萍
Original assignee: Liaoning Huipuyuan Biomedical Technology Development Co ltd
Current assignee: Liaoning Huipuyuan Biomedical Technology Development Co ltd
Priority date: 2020-02-10
Filing date: 2020-02-10
Publication date: 2020-12-11
Anticipated expiration: 2040-02-10
Also published as: CN111349683A; WO2021160121A1

Abstract

The invention relates to the field of medical diagnostics, in particular to application of basophils of a granulocyte group as a diagnostic marker of allergic diseases. The inventors classified basophils into basophils of a mononuclear cell population and basophils of a granulocyte population, performed a systematic study on the basophils of the granulocyte population, and found that the basophils of the granulocyte population are specifically associated with allergic diseases and can be used for diagnosis of the allergic diseases. The diagnosis method has good specificity and easy detection, and is easy to popularize and apply clinically.

Description

Application of basophils of granulocyte group as allergic disease diagnosis marker

Technical Field

The invention relates to the field of medical diagnostics, in particular to application of basophils of a granulocyte group as a diagnostic marker of allergic diseases.

Background

The incidence of allergic (anaphylactic) reactive diseases accounts for more than 30% of the world's general population, and is classified as one of the four non-infectious diseases of the 21 st century by the world health organization. With the development of industrial economy and the change of ecological environment, the diseases are increasingly increased in recent years, become common diseases and frequently encountered diseases, and are important problems to be solved in the field of health and economic development of China.

However, to date, the problem of laboratory qualitative diagnosis of allergic diseases has not been solved, and the problem of "false negatives" often occurs clinically, i.e. a doctor tells a patient that he (she) is not allergic because he cannot detect the patient's allergens. The reasons for this are mainly the following: (1) the number of types of allergens is thousands, but only dozens of types are commonly used clinically, so that the patient can be missed if the allergens of the patient are not included in the dozens; (2) the quality of the current allergen preparation is low, Asia including China has no standardized allergen, and Europe and America are only the first generation of standardized allergen preparation defined more than 20 years ago. Since the first-generation standardized allergen preparation has only 24 species and even the first-generation standardized allergen preparation monitors only one major allergen (allergen protein capable of inducing allergic reactions in more than 50% of patients allergic to the species), even when the first-generation standardized allergen preparation is used, there is a problem of "missed diagnosis" due to the fact that the preparation does not contain specific allergen proteins inducing allergy in patients, in the case of species known to contain about 10 major allergens, such as mites, cockroaches, peanuts, and the like. If a certain dust mite allergic patient is only allergic to the main allergen Der p21 of the dust mites, but is not allergic to Der p1 monitored in the preparation, the negative result of mite detection appears clinically, so that doctors mistakenly think that the patient is not allergic to the dust mites and the diagnosis is missed; (3) there is no clinical method for "qualifying" allergic diseases, which is to determine whether a patient is allergic, then to screen the patient for an allergen (e.g., skin prick test, allergen-specific IgE detection, etc.), and to determine which allergen the patient is allergic to. This is in contrast to the clinical situation where a patient is first determined to be a bacterial infection and then which bacterial infection the patient is. Since the "qualitative" diagnosis of the disease is the best method to avoid "misdiagnosis" and "mistreatment", it is necessary to develop a "qualitative" diagnosis method for allergic diseases.

Disclosure of Invention

The traditional flow cytometry method for detecting basophils in human blood is to set a gate on a mononuclear cell population, namely to detect the mononuclear cell population. The present inventors have unexpectedly found that a gate is formed on a granulocyte population, and a large number of basophils are present in the granulocyte population. Further, the inventors classified basophils into basophils of a mononuclear cell population and basophils of a granulocyte population, and conducted a systematic study on the basophils of the granulocyte population, and found that the basophils of the granulocyte population are specifically associated with allergic diseases and can be used for diagnosis of the allergic diseases.

Specifically, the present invention relates to the use of a detection agent for basophils of a granulocyte population for the preparation of a kit for diagnosing an allergic disease, wherein an increased number of basophils of the granulocyte population in blood is indicative for the development of an allergic disease.

The diagnosis method has good specificity and easy detection, and is easy to popularize and apply clinically.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows the detection of CCR3 in granulocyte populations by flow cytometry in one embodiment of the present invention⁺And CD123⁺HLA-DR^-A gating method of basophils;

FIG. 2 is a flow cytometry method for detecting CD123 in the mononuclear cell population of allergic rhinitis in one embodiment of the present invention⁺HLA-DR^-Cell population (1), CCR3⁺Cell population (2) and CCR3⁺CD123⁺HLA-DR^-The proportion and the count of the cell group (3) occupying mononuclear cells are not obviously increased compared with those of a healthy control group (note: AR ═ allergic rhinitis; sAR ═ seasonal allergic rhinitis; pAR ═ perennial allergic rhinitis; nAR ═ allergic rhinitis negative to allergen detection; HC ═ healthy control group);

FIG. 3 shows an example of the detection of CD63 in three basophil populations, CD123, by flow cytometry in an allergic rhinitis monocyte population in accordance with one embodiment of the present invention⁺HLA-DR^-Cell population (1), CCR3⁺Cell population (2) and CCR3⁺CD123⁺HLA-DR^-The expression level in the cell population (3) was not significantly increased as compared with the healthy control group (note: AR ═ allergic rhinitis; sAR ═ seasonal allergic rhinitis; pAR ═ perennial allergic rhinitis; nAR ═ allergic rhinitis negative to allergen detection; HC ═ healthy control group);

FIG. 4 is a graph showing CD123 in a blood granulocyte population for detecting allergic rhinitis by flow cytometry in accordance with one embodiment of the present invention⁺HLA-DR^-Cell population (1), CCR3⁺Cell population (2) and CCR3⁺CD123⁺HLA-DR^-The proportion and the count of the cell group (3) occupying the granulocyte are remarkably increased compared with those of the healthy control group (note: AR ═ allergic rhinitis; sAR ═ seasonal allergic rhinitis; pAR ═ perennial allergic rhinitis; nAR ═ allergic rhinitis negative to allergen detection; HC ═ healthy control group);

FIG. 5 is a diagram illustrating an exemplary embodiment of the present invention for flow cytometry to detect CD123 in the blood granulocyte population of patients with allergic rhinitis⁺HLA-DR^-(A),CCR3⁺(B),CD123⁺HLA-DR^-CCR3⁺(C) CD63 expression in basophil population was significant compared to healthy controlsIncreasing the number of patients (note: AR ═ allergic rhinitis; sAR ═ seasonal allergic rhinitis; pAR ═ perennial allergic rhinitis; nAR ═ allergic rhinitis negative to allergen assay; HC ═ healthy control group;

FIG. 6 is a diagram illustrating an exemplary embodiment of the present invention for flow cytometry to detect CD123 in the blood granulocyte population of patients with allergic rhinitis⁺HLA-DR^-(A),CCR3⁺(B),CD123⁺HLA-DR^-CCR3⁺(C) CD203c in cells⁺The percentage of cells; CD123 compared to healthy controls⁺HLA-DR^-CD203c in cell population⁺The percentage of cells was significantly increased (note: AR ═ allergic rhinitis; sAR ═ seasonal allergic rhinitis; pAR ═ perennial allergic rhinitis; nAR ═ allergic rhinitis negative to allergen assay; HC ═ healthy control);

FIG. 7 shows an example of the detection of CD123 in a blood mononuclear cell population in allergic asthma by flow cytometry⁺HLA-DR^-Cell population, CCR3⁺Cell population and CCR3⁺CD123⁺HLA-DR^-The proportion and the count of the cell population in the mononuclear cells are not obviously increased compared with those of a healthy control group (note: AS ═ allergic asthma; HC ═ healthy control group);

FIG. 8 is a graph showing the detection of CD123 in a blood granulocyte population in allergic asthma by flow cytometry in accordance with one embodiment of the present invention⁺HLA-DR^-Cell population, CCR3⁺Cell population and CCR3⁺CD123⁺HLA-DR^-The proportion and the count of the cell population occupying the granulocyte are obviously increased compared with those of a healthy control group (note: AS ═ allergic asthma; HC ═ healthy control group);

FIG. 9 is a schematic representation of flow cytometry differentiation of a cell population into mononuclear cell and granulocyte populations in accordance with one embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.

It is therefore intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention are disclosed in or are apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention.

The present invention relates to the use of a detection agent for basophils of a granulocyte population for the preparation of a kit for diagnosing an allergic disease, wherein an increased number of basophils of the granulocyte population in blood is indicative for the development of an allergic disease.

In some embodiments, the population of granulocytes and the population of monocytes basophils differentiate the cells by two indicators, forward angle scattering and side angle scattering; the cells can be divided into two substantially different cell populations by the two criteria.

As shown in FIG. 9, SSC intensity was about 50K to 100K, FSC intensity was about 50K to 250K and was attributed to basophils of the granulocyte group, SSC intensity was about 0K to 100K and FSC intensity was about 50K to 250K and was attributed to basophils of the monocyte group. Such differentiation is readily apparent to those skilled in the art.

In the present invention, "allergic disease" may refer to an auto-allergic disease or a disease induced by some external allergens.

In a preferred embodiment, the allergen is grass pollen, tree pollen, animal dander, animal hair (feathers), cockroaches or their secretions or sheds, moulds or their spores.

In a preferred embodiment, the allergen is a pharmaceutical substance selected from the group consisting of: an antibiotic, preferably a beta-lactam, more preferably selected from the group comprising: penicillin G, penicillin V, PPL, MDM, amoxicillin, and ampicillin; cephalosporin, more preferably selected from the group comprising: ceftizoline, cefamandole, cefaclor, Cefonaxim, ceftazidime, cefotaxime, ceftazidime, cefepime, carbapenem, monobactam, beta-lactamase-inhibitory agent, more preferably clavulanic acid; macrolides, aminoglycosides, rifamycins, glycopeptides, polypeptides, tetracyclines, imidazoles, fluoroquinolones, pyrazolones, more preferably sulfomethoxazols; streptogramins, nitrofurans, isoniazid, pentamidine; preservatives, preferably chlorhexidine, fungicides, antiviral agents, antimalarial-agents, analgesics, COX-2-inhibiting agents and non-steroidal anti-inflammatory drugs, preferably selected from the group comprising: aspirin, Lys-aspirin, ibuprofen, ketoprofen, diclofenac, naproxen, acetaminophen, analgin, indomethacin, mefenamic acid, phenylbutazone, and isopropanamiline; neuromuscular blocking agents, preferably selected from the group comprising: succinylcholine, atracurium, cis-atracurium, mevinolinic, bavulong, rocuronium, vecuronium, and succinylcholine; hypnotics and local anesthetics, preferably selected from the group comprising: midazolam, propofol, thiopental, fentanyl, and lidocaine; a sedative; (ii) an opioid; a radio-contrast agent, preferably selected from the group comprising: ionic iodine contrast agents, non-ionic iodinated contrast agents, isothio blue, patent blue and methylene blue; a proton pump inhibiting agent; an anti-tic agent (Antikonvultiva) and an antipsychotic agent, preferably selected from the group comprising: carbamazepine, phenytoin, and valproic acid; an antipsychotic agent; an antidepressant; (ii) dopamine; an antihistamine; corticosteroids and glucocorticoids; chemotherapeutic and immunosuppressive agents; diuretic agents; an anticoagulant; vasoconstrictors and vasodilators; a cardiac drug further selected from the group consisting of: statin, ACE-inhibiting agent, alpha-receptor blocker, and BETA-receptor blocker; calcium antagonists and antihypertensive agents; (anti) ulcer agents; (anti) thyroid agents; an estrogen; heparin and derivatives thereof; insulin; streptokinase and urokinase.

In some embodiments, the allergen is a colloid, a plasma expander or an adjuvant, further selected from the group comprising: albumin, dextran, gelatin, hydroxyethyl starch, penta-starch, levulose, polidocanol 600, lactose, carboxymethyl cellulose, hydroxypropyl cellulose, protamine and aprotinin.

In some embodiments, the allergen is a food supplement, further selected from the group comprising: food preservatives, food colors, food refiners, antioxidants and emulsifiers.

In some embodiments, the allergen is an environmental agent or a hazardous agent, further selected from the group consisting of: isocyanates, isothiazolinones, formaldehyde, ethylene oxide, phthalic anhydride, chloramine T, DMSO, emulsions and enzymes used in baking processes, food processing and laundry industries.

In some embodiments, the allergen is selected from the group consisting of: a culture of mites and/or feces.

In some embodiments, the allergen is selected from the group consisting of: shrimp or shrimp-containing food of the taxonomic arthropoda phylum; crayfish or food containing crayfish from the taxonomic arthrodle phylum; fruits, pods, grains or legumes derived from the taxonomic plant Liliaceae (Plantae Liliopsidae), or food products containing such fruits, pods, grains and/or legumes; fruits, pods, grains or legumes derived from the taxonomic class magnoliidae (Plantae Magnoliopsidae) plant, or food products containing such fruits, pods, grains and/or legumes; taxonomic plant of Magnoliidae or a food containing nuts.

In some embodiments, the allergen is selected from the group consisting of: milk, milk-containing food, chicken protein-containing food, fish or fish-containing food.

In some embodiments, the allergen is selected from the group consisting of: hordeum (Hordeum), Oryza (Oryza), Secale (Secale), Triticum (Triticum), Zea (Zea), Arachis (Arachis), Coriolis, Juglans (Juglans), Prunus (Prunus), Anacardia (Anacardium), Pistacia (Pistacia) and Glycine (Glycine).

In some embodiments, the allergic disease comprises allergic rhinitis, allergic asthma, allergic conjunctivitis, atopic dermatitis, severe allergy, pollinosis, food allergy, urticaria, allergic purpura, eosinophilic gastroenteritis, and drug allergy.

In some specific embodiments, the allergic disease comprises allergic rhinitis and allergic asthma.

The allergic rhinitis may further include simple allergic rhinitis, seasonal allergic rhinitis, perennial allergic rhinitis, and allergic rhinitis negative to allergen detection.

Allergic asthma may further include allergic asthma that is positive for total IgE and/or negative for total IgE.

In some embodiments, the population of granulocytes is characterized by counting the number of viable leukocytes relative to the number of any one of the cells selected from the following subpopulations:

CCR3⁺cell, CCR3⁺CD63⁺Cell, CD123⁺HLA-DR^-Cell, CD123⁺HLA-DR^-CD203c⁺Cell, CD123⁺HLA-DR^-CD63⁺Cell, CD49f⁺A cell.

The purpose of detection of these cell subsets is generally:

serial number	Combination regimens for anti-human antibodies	Purpose(s) to
			1	CCR3⁺	Detection of basophil number
2	CCR3⁺CD63⁺	Detection of the number of activated basophils (challenge test)
			3	CD123⁺HLA-DR^-	Detection of basophil number
4	CD123⁺HLA-DR^-CD203c⁺	Detection of the number of activated basophils (challenge test)
			5	CD123⁺HLA-DR^-CD63⁺	Detection of the number of activated basophils (challenge test)
6	CD49f⁺	Detection of basophil number

In some embodiments, the blood is an ex vivo sample.

In some embodiments, the blood is peripheral blood.

In some embodiments, the detection agent specifically measures basophils of the population of granulocytes by an immunological method; in particular to measure cell surface markers of basophils of the granulocyte population.

In some embodiments, the reagent (specific binding agent) required to specifically measure a cell surface marker is, for example, a cell surface markerA ligand or receptor (if present), a lectin that binds to a cell surface marker, an aptamer that binds to a cell surface marker, or an antibody that binds to a cell surface marker. The specific binding agent has at least 10 for its corresponding target molecule⁷Affinity of l/mol. The specific binding agent preferably has 10 to its target molecule⁸l/mol, or more preferably 10⁹Affinity of l/mol. The skilled person will understand that the use of the term "specific" means that other biomolecules present in the sample do not significantly bind to the specific binding agent of the cell surface marker that it is desired to detect.

Preferably, the level of binding to biomolecules other than the target molecule results in a binding affinity that is at most only 10% or less, only 5% or less, only 2% or less, or only 1% or less, respectively, of the affinity to the target molecule. Preferred specific binding agents will meet the above minimum criteria for both affinity and specificity.

In some embodiments, the detection agent is an antibody.

In the present invention, "antibody" generally refers to all proteins/protein fragments comprising CDR regions, in particular full-length antibodies or functional fragments of antibodies. The term "full-length antibody" includes both polyclonal and monoclonal antibodies, and the term "antibody functional fragment" is a substance that comprises part or all of the CDRs of an antibody, which lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to an antigen. Such fragments are biologically active in that they bind to a target antigen and bind to a given epitope. The functional antibody fragment may be selected from, for example, F (ab') 2, Fab, Fv, scFv.

In some embodiments, the antibody has a detectable label.

In some embodiments, the detectable label is a fluorescent label.

In some embodiments, the fluorescent label is selected from one or more of Alexa Fluor 488, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 700, APC/Cy7, APC/H7, APC/R700, BB 515, BB 700, BD IMag/DM, BUV 395, BUV 496, BUV 563, BUV 661, BUV 737, BUV 805, Brilliant Violet 421, Brilliant Violet 480, Brilliant Violet 510, Brilliant Blue 515, Brilliant Violet 570, Brilliant Violet 605, Brilliant Violet 650, Brilliant Violet 711, Brilliant Violet 750, Brilliant Violet785, Brilliant Violet 786, FITC, NA/LE, LEC, Pafil, PE, CF/PE, CyzPE 6335, CyzPE 35V/CP 35, CyzPE 35, CyzV 6335, CyzPE 5, Cyzpe, and Cyzpe.

The kit is preferably a kit for performing flow cytometry.

In some embodiments, the kit further comprises one or more of an FcR blocker, a dye for labeling dead cells, an erythrocyte lysate, and a cell wash buffer.

FcR is widely expressed on B cells, monocytes, macrophages, DC cells, granulocytes, etc., and readily binds to the Fc fragment of antibodies, resulting in false positive results. In the process of cell staining of high FcR expression, it is recommended to use a commercial FcR blocker or serum of the corresponding species to block the FcR from being combined with an antibody, reduce the detection background and eliminate false positive.

Flow cytometry is a technique that is easily interfered with by dead cells because: 1. dead cells readily take up antibody and probe, resulting in significant non-specific staining. 2. Dead cells are particularly strongly autofluorescent.

This causes an increase in background fluorescence, which makes it impossible for the experimenter to observe weak positive expression of some markers. Dead cells may also release DNA, which is very viscous and therefore eventually causes cell clumping, which both affects the results and tends to clog the tubing. In flow sorting, there is generally a reluctance to sort poorly viable cells. This interference can thus be reduced by using dyes for labelling dead cells which, in a preferred embodiment,

the dye for labeling dead cells is selected from one or more of 7-AAD (7-Aminoactinomycin D ), PI (propidium iodide), DAPI, TO-PRO-3, Zombie Dyes and SYTOX. The dyes typically enter cells with damaged cell membranes and bind to the DNA of the cells, thereby allowing dead cells to be readily distinguished upon detection.

According to yet another aspect of the present invention, the present invention also relates to a method for diagnosing an allergic disease, which comprises quantifying basophils of a granulocyte population in blood.

Further, the method includes calculating a ratio of basophils of the granulocyte population to a number of leukocytes.

The typical method for diagnosing allergic diseases in the present invention is selected from the group consisting of flow cytometry, which may comprise the steps of:

(1) FcR blocker and dead cell removal dye incubation of blood samples;

(2) incubating the blood sample with the fluorescence-coupled flow antibody;

(3) lysing erythrocytes in the blood sample;

(4) washing the cells;

(5) detecting by a flow cytometer;

(6) the ratio of basophils of the granulocyte population to the number of leukocytes was calculated.

In some embodiments, the compound is selected from the group consisting of CCR3, as determined by the presence of CCR in the granulocyte population⁺Proportion or absolute number of leukocytes, or CCR3⁺CD63⁺Proportion or absolute number of leukocytes, or CD123⁺HLA-DR^-Proportion or absolute number of leukocytes, or CD123⁺HLA-DR^-CD203c⁺Proportion or absolute number of leukocytes, or CD123⁺HLA-DR^-CD63⁺Proportion or absolute number of leukocytes, or CD49f⁺The proportion or absolute number of white blood cells.

In some embodiments, the compound is selected from the group consisting of CCR3, as determined by the presence of CCR in the granulocyte population⁺CD63⁺Proportion or absolute number of leukocytes, or CD123⁺HLA-DR^-Proportion or absolute number of leukocytes, or CD123⁺HLA-DR^-CD63⁺The proportion or absolute number of white blood cells.

In some embodiments, the lysing red blood in the blood sampleThe cell method is preferably to add erythrocyte lysate, incubate blood sample after mixing evenly; preferably, the erythrocyte lysate is a hypotonic solution with osmotic pressure lower than that of human plasma crystals, such as distilled water, or contains NH₄A hypotonic solution of Cl; the temperature of the incubated blood sample is preferably 2-40 ℃, and more preferably 4-37 ℃; the incubation time is preferably 30 s-60 min, and more preferably 1 min-30 min; the environment of the incubation is preferably protected from light.

In some embodiments, the method for washing the cells is preferably to centrifuge the blood sample after red blood cell lysis, discard the supernatant, and add a cell washing buffer; the centrifugation step was repeated and the supernatant was discarded. The centrifugal force of the blood specimen centrifugation is preferably 90g to 600g, more preferably 100g to 400g, and most preferably 150g to 300 g; the centrifugal temperature is preferably 4-37 ℃; the cell washing buffer is preferably an isotonic solution equal to the osmotic pressure of human plasma crystals, such as PBS buffer, or physiological saline containing 0.9% NaCl, or the like.

In some embodiments, the flow cytometry detection method is preferably to add a cell washing buffer, resuspend the cells and then detect the number of the cells of interest in the granulocyte population by flow cytometry. The method for detecting the number of the target cells in the granulocyte colony is preferably to collect at least 100000-1000000 viable leukocytes by a flow cytometer.

Embodiments of the present invention will be described in detail with reference to examples.

Examples

1. Flow cytometry detection method

Addition of FcR blocker and dead cell removal dye: putting 100 mu L of blood specimen at the bottom of a flow-type sample loading tube, adding 5 mu L of human FcR blocker and 5 mu L of dead cell removal dye (7-AAD) into the blood specimen, lightly blowing the blood specimen for 5 times by using a pipette, and incubating for 10min in a dark place at room temperature (18-22 ℃);

addition of fluorescently-coupled flow antibody: after the incubation, 5. mu.L of each of the following fluorescence-coupled antibodies was added to each tube according to the number in Table 1, and the blood specimen was gently pipetted 5 times and incubated for 15min at room temperature (18 ℃ to 22 ℃) in the dark.

And (3) cracking red blood cells: after the incubation is finished, adding 1.5mL of erythrocyte lysate, gently blowing the blood sample for 5 times by using a pipette, and incubating for 12min in a dark place at room temperature (18-22 ℃);

and (3) washing the cells: after incubation, placing a flow-type sample loading tube containing a blood sample in a centrifuge, and setting centrifugation conditions: centrifugal force: 200g, centrifuge temperature: 20 ℃, centrifugation time: centrifuging for 6 min; after the centrifugation is finished, carefully sucking the supernatant by a pipettor under the condition of not touching the bottom cells of the tube, and then discarding the supernatant, and keeping about 150 mu L of the supernatant; then adding 1mL of PBS buffer solution, uniformly mixing by using a pipettor, and repeating the steps of centrifuging and discarding the supernatant;

flow cytometry detection: adding 300 mu L of PBS buffer solution, uniformly mixing by using a pipettor, collecting 100000 live white blood cells by using a flow cytometer, dividing the cells into a granulocyte group and a monocyte group, and respectively detecting the number of target cells.

2. And (3) distinguishing and detecting basophils of the granulocyte group and the monocyte group.

Flow cytometry detection of cells are distinguished by two indicators, forward angle scattering and side angle scattering: the intensity of forward angle scattering (FSC) is related to the size of the cells, and for the same population of cells, strong FSC means large cells, and weak FSC means small cells. The intensity of Side Scatter (SSC) is related to the complexity of the internal structure of the cell, which is sensitive to the refractive indices of the cell membrane, cytoplasm, nuclear membrane, and also to the response of larger particles within the cell. Therefore, the intensity of the scattered light at the lateral angle reflects the fine structure and particle properties in the cell.

The flow cytometry detection of peripheral blood leukocytes as described above allowed the differentiation of cell populations into mononuclear cells and granulocyte populations (FIG. 9), and detection of CCR3 and CD123 positive cells in both, respectively, with results as shown in FIG. 1.

3. The number of basophils in the granulocyte population is specifically increased in allergic diseases.

Detection of CD123 in allergic rhinitis mononuclear cell population by flow cytometry⁺HLA-DR^-A cell population,CCR3⁺Cell population and CCR3⁺CD123⁺HLA-DR^-The expression level of CD63 in the cell population, or in all three cells, was not significantly increased compared to the healthy control group (fig. 2, fig. 3). However, flow cytometry was used to detect CD123 in the allergic rhinitis granulocyte population⁺HLA-DR^-Cell population, CCR3⁺Cell population and CCR3⁺CD123⁺HLA-DR^-The expression level of CD63 in the cell population, or in all three cells, was significantly increased compared to the healthy control group (fig. 4, fig. 5). Further, the inventors detected CD123 in the blood granulocyte population of patients with allergic rhinitis⁺HLA-DR-(A),CCR3⁺(B),CD123⁺HLA-DR^-CCR3⁺(C) CD203c in cells⁺The percentage of cells; CD123 compared to healthy controls⁺HLA-DR^-CD203c in cell population⁺The percentage of cells was significantly increased, and other groups were not significantly increased (fig. 6).

A similar phenomenon also occurs in the detection of allergic asthma, CD123 in the population of mononuclear cells of allergic asthma⁺HLA-DR^-Cell population, CCR3⁺Cell population and CCR3⁺CD123⁺HLA-DR^-The proportion and count of the cell population in mononuclear cells were not significantly increased compared to the healthy control group, while a significant increase occurred in the granulocyte population (fig. 7, fig. 8).

The numbers of basophils and activated basophils (unit: one/100000 leukocytes) in the blood granulocyte population of healthy human (n-24) allergic rhinitis patients (n-182) and allergic asthma patients (n-125) in the above test; allergic rhinitis: sAR (n-30); pAR (n-79); nAR (n-73); allergic asthma: AS (IgE)^-)(n＝82)；AS(IgE⁺)(n＝43)

AR ═ allergic rhinitis; sAR seasonal allergic rhinitis; pAR ═ perennial allergic rhinitis; allergic rhinitis negative to allergen detection; AS ═ allergic asthma; HC was healthy control group.

Thus, the inventors found that the following sub-populations of cells in the granulocytic population can be used for the detection of allergic diseases:

CCR3⁺cell, CCR3⁺CD63⁺Cell, CD123⁺HLA-DR^-Cell, CD123⁺HLA-DR^-CD203c⁺Cell, CD123⁺HLA-DR^-CD63⁺Cell, CD49f⁺Cells (data in table 2, not shown).

The combination scheme of the anti-human fluorescence coupling antibody during detection is shown in table 1, and the positive detection result is shown in table 2.

TABLE 1

Serial number	Combination protocol for anti-human fluorescence coupled antibodies
		1	APC-CCR3 ⁺
2	APC-CCR3⁺FITC-CD63 ⁺
		3	FITC-CD123⁺APC/CY7-HLA-DR^-
4	FITC-CD123⁺APC/CY7-HLA-DR^-PE-CD203c⁺
		5	FITC-CD123⁺APC/CY7-HLA-DR^-PE-CD63⁺
6	PE-CD49f⁺

TABLE 2

From the above embodiments, the kit for diagnosing allergic rhinitis and allergic asthma by detecting the number of basophils in human blood granulocyte colony by flow cytometry and the kit for the detection method provided by the invention have the characteristics of simple composition, simple and easy detection method, and capability of quickly and accurately diagnosing whether rhinitis and asthma patients are allergic rhinitis and allergic asthma on the basis of saving medical cost and blood samples.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. Use of a detection agent for basophils of a granulocyte population for the preparation of a kit for diagnosing an allergic disease, wherein an increased number of basophils of the granulocyte population in blood is indicative of the suffering of the allergic disease; the detection agent is an antibody, and the allergic diseases are allergic rhinitis and allergic asthma;

through flow sorting, the SSC intensity is more than 50K-100K, and the FSC intensity is in the range of 50K-250K and is the group of basophils of the granulocyte group;

when the allergic disease is allergic rhinitis, the population of granulocytes is characterized by counting the proportion of viable leukocytes by the number of any one of the cells selected from the following subpopulations:

CCR3⁺cell, CCR3⁺CD63⁺Cell, CD123⁺HLA-DR^-Cell, CD123⁺HLA-DR^-CD203c⁺Cell, CD123⁺HLA-DR^- CD63⁺Cell, CD49f⁺A cell;

when the allergic disease is allergic asthma, the population of granulocytes is characterized by counting the proportion of viable leukocytes by the number of any one of the cells selected from the following subpopulations:

CCR3⁺cell population, CD123⁺HLA-DR^-A cell.

2. The use of claim 1, wherein the blood is an ex vivo sample.

3. The use of claim 2, wherein the blood is peripheral blood.

4. The use of claim 1, wherein the antibody has a detectable label.

5. The use of claim 4, wherein the detectable label is a fluorescent label.

6. The use according to claim 5, wherein the fluorescent label is selected from the group consisting of Alexa Fluor 488, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 700, APC/Cy7, APC/H7, APC/R700, BB 515, BB 700, BD IMag/DM, BUV 395, BUV 496, BUV 563, BUV 661, BUV 737, BUV 805, Brilliant Violet 421, Brilliant Violet 480, Brilliant Violet 510, Brilliant Blue 515, Brilliant Violet 570, Brilliant Violet 605, Brilliant Violet 650, Brilliant Violet 711, Brilliant Violet 750, Brilliant Violet785, Brilliant Violet 786, FITC, Brilliant/LE, AF, Pafil LEc, Cy/PE, CyzPE 6335, CyzPE 35/CP 35, CyzPE 635, CyzPE 35, CyzPE or CyzV 6335.

7. Use according to claim 1, further comprising one or more of an FcR blocker, a dye for labelling dead cells, an erythrocyte lysate and a cell washing buffer.

8. Use according TO claim 7, wherein the dye for marking dead cells is selected from one or more of 7-AAD, PI, DAPI, TO-PRO-3, Zombie Dyes and SYTOX.