WO2023046111A1 - 一种嗜碱性粒细胞活化检测方法及其应用 - Google Patents

一种嗜碱性粒细胞活化检测方法及其应用 Download PDF

Info

Publication number
WO2023046111A1
WO2023046111A1 PCT/CN2022/121047 CN2022121047W WO2023046111A1 WO 2023046111 A1 WO2023046111 A1 WO 2023046111A1 CN 2022121047 W CN2022121047 W CN 2022121047W WO 2023046111 A1 WO2023046111 A1 WO 2023046111A1
Authority
WO
WIPO (PCT)
Prior art keywords
control group
positive control
group
basophils
activation
Prior art date
Application number
PCT/CN2022/121047
Other languages
English (en)
French (fr)
Inventor
彭霞
李莉
Original Assignee
上海市第一人民医院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海市第一人民医院 filed Critical 上海市第一人民医院
Priority to CN202280005696.XA priority Critical patent/CN116157672A/zh
Priority to CN202311231985.4A priority patent/CN117760942A/zh
Priority to CN202310651507.2A priority patent/CN117760939A/zh
Priority to CN202310292519.0A priority patent/CN117760938A/zh
Priority to CN202311231847.6A priority patent/CN117760941A/zh
Priority to CN202310649973.7A priority patent/CN117783519A/zh
Publication of WO2023046111A1 publication Critical patent/WO2023046111A1/zh

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the invention relates to the field of medical detection, in particular to a method for detecting the activation of basophils and an application thereof.
  • Allergic diseases are common clinical diseases. According to WHO statistics, about 22% of the world's population is affected by allergic diseases, and the number of cases is increasing year by year, which brings a heavy economic burden to the society.
  • the total IgE level in the in vitro test is affected by many factors, and the specificity is low.
  • allergen sIgE detection is the most widely used detection method for the diagnosis of allergic diseases.
  • serum concentration of IgE is very low (about 30-70,000th of IgG)
  • the detection is difficult, and the allergens that can be detected are limited (about 30-40 kinds).
  • allergen-specific IgE results do not reflect the patient's internal status.
  • the object of the present invention is to provide an effective and reliable method for in vitro detection of allergic diseases.
  • the present invention provides a method for detecting basophil activation and corresponding detection reagents, which provide new solutions for the diagnosis of allergic diseases, screening of allergens and their components, and can be applied to Prediction of allergy severity and monitoring of efficacy.
  • a reagent combination for detecting basophil activation includes: a first antibody against human CD203c, a second antibody against human CD123, an anti- A third antibody against human CD45, a fourth antibody against human CD63, a positive control, and an allergen.
  • the reagent combination further includes a stimulation buffer and/or a stop buffer.
  • the reagent combination further includes one or more reagents selected from the group consisting of staining reagent diluent, erythrocyte lysate, and washing solution.
  • the stimulation buffer contains Hank's balanced salt buffer, NaHCO 3 and HEPES buffer.
  • the pH value of the stimulation buffer is 7.2-7.5 (preferably 7.35-7.45).
  • the storage condition used was 4°C.
  • the allergens include crude extracts of natural allergens, purified natural allergen components, recombinant allergen components, or combinations thereof.
  • the allergen is selected from the group consisting of dust mite, cockroach, German cockroach, mugwort, Aspergillus fumigatus, dog dander, cat dander, pollen, catkins, insect venom, fungus, shrimp , wheat, rye, barley, oats, corn, peanuts, fish, crab, eggs, milk, sesame, soybeans, common ragweed, quinoa, cocklebur, nuts, shiitake mushrooms, seafood, mango, kiwi, pineapple, antibiotics, biological products, or combinations thereof.
  • the allergen is selected from the group consisting of dust mites (such as dust mites, dust mites), cockroaches, German cockroaches, dog dander, cat dander, milk, eggs, antibiotics, or its combination.
  • dust mites such as dust mites, dust mites
  • cockroaches German cockroaches
  • dog dander dog dander
  • cat dander milk
  • eggs antibiotics
  • the allergens include dust mite allergens (such as dust mite allergen crude extract and recombinant dust mite allergen main component Der f2).
  • dust mite allergens such as dust mite allergen crude extract and recombinant dust mite allergen main component Der f2.
  • the concentration of the allergen is 0.01-20 ⁇ g/mL, preferably 0.05-15 ⁇ g/mL, more preferably 0.1-10 ⁇ g/mL.
  • the stop buffer includes PBS buffer containing EDTA or a salt thereof.
  • the concentration of EDTA or its salt in the stop buffer is 5-20mmol/L, and the pH is 7.2-7.5 (preferably 7.35-7.45).
  • the positive control substance is selected from the group consisting of anti-human IgE antibody, fMLP, or a combination thereof.
  • the human IgE is from the IgE (such as allergen-specific IgE (sIgE), or total serum IgE) of allergic patients.
  • IgE allergen-specific IgE
  • total serum IgE total serum IgE
  • the concentration of the anti-human IgE antibody is 0.1-20 ⁇ g/mL, preferably 0.5-15 ⁇ g/mL, more preferably 1-10 ⁇ g/mL.
  • the first antibody, the second antibody, the third antibody, and the fourth antibody have different fluorescent labels.
  • the detection antibodies with different fluorescent labels are respectively used as staining reagents.
  • the fluorescent marker molecule is selected from the group consisting of FITC, PE, ECD, PerCP-Cyanine5.5, PE-Cyanine, APC-Alexa 750, APC, Alexa Dyes, Pacific Blue, KO, or combinations thereof.
  • the staining reagent diluent is composed of PBS buffer containing BSA.
  • the erythrocyte lysate contains potassium bicarbonate, ammonium chloride and EDTA-Na2.
  • the washing solution is composed of PBS buffer containing BSA.
  • a method for in vitro detection of basophil activation comprises the following steps:
  • n is a positive integer ⁇ 3 (such as 3-10 parts, such as 3, 4, 5, 6 copies);
  • test conditions of the negative control group, the positive control group and the activation test group are the same, the difference is: in the positive control group, at least one part of the anticoagulated whole blood is incubated with the positive control substance; In the test group, at least one part of the anticoagulated whole blood is incubated with the allergen; while in the negative control group, at least one part of the anticoagulated whole blood is incubated in the absence of a positive control and in the absence of an allergen. Incubation;
  • the positive control group includes an anti-human IgE antibody positive control group and/or an fMLP positive control group;
  • n 4 described in step (a), and the positive control group described in step (b) includes positive control group a and positive control group b, wherein the positive control substance in positive control group a is anti-human IgE antibody, the positive control substance in the positive control group b is fMLP.
  • the stimulation incubation time t1 is 10-45 minutes, preferably 15-30 minutes, more preferably 15-25 minutes.
  • step (d) it includes calculating the first SI value of the positive control group and the second SI value of the activation test group, wherein,
  • SI percentage of activated basophils after stimulation in the activation test group/percentage of activated basophils in the negative control group;
  • the number of activated basophils is determined by the average fluorescence intensity of CD63 of basophils.
  • the first SI value average fluorescence intensity of CD63 basophils in the positive control group/average fluorescence intensity of CD63 basophils in the negative control group.
  • the second SI value average fluorescence intensity of CD63 basophils in the activation test group after stimulation/average fluorescence intensity of CD63 basophils in the negative control group.
  • step (d) it includes calculating the Z1 value of the positive control group and the Z2 value of the activation test group, wherein,
  • the qualitative and/or quantitative includes determining: whether the basophils in the sample to be detected are activated, and/or the degree of activation.
  • step (c) the target basophils in the negative control group, the positive control group and the activation test group are determined by flow cytometry.
  • step (c) it also includes: determining the number and/or ratio of target basophils in the negative control group, the positive control group and the activation test group.
  • the positive control substance is selected from the group consisting of anti-human IgE antibody, fMLP, or a combination thereof.
  • the human IgE is the IgE of allergic patients (such as allergen-specific IgE (sIgE), or total serum IgE).
  • allergen-specific IgE sIgE
  • total serum IgE total serum IgE
  • the anticoagulated whole blood is heparin anticoagulated whole blood.
  • step (b) in the negative control group, positive control group and activation test group, add 50-500 ⁇ L of anticoagulated whole blood to each test tube, and then add stimulation buffer respectively, or add positive control substance + Stimulation buffer, or add allergen + stimulation buffer;
  • the method includes the following steps:
  • Grouping and stimulation take flow tubes and divide them into 3 tubes, which are negative control tube, positive control tube and test tube respectively; heparin anticoagulant is added to each tube first, and then positive control substance and allergen are stimulated with buffer Dilute the solution to 0.1-10 ⁇ g/mL, then add the stimulation buffer, the diluted positive control substance and the allergen into the negative control tube, positive control tube and test tube respectively, and stimulate for a period of time t1 at 37 ⁇ 2°C (compared to The best place t1 is 15-30min);
  • Termination detection add termination buffer, centrifuge (200g, 10min), suck off the supernatant, and resuspend the cells;
  • Labeled antibody Dilute the fluorescently labeled antibody with the staining reagent diluent, add the fluorescently labeled antibody, and incubate in the dark for a period of time t2 (preferably t2 is 25-40min);
  • Lying red blood cells add red blood cell lysate, lyse red blood cells, add washing solution to wash, collect cell pellet after centrifugation, and the cell pellet is a cell group containing basophils;
  • the method includes the following steps:
  • Grouping and stimulation take flow tubes and divide them into 4 tubes, which are respectively negative control tube, positive control tube a, positive control tube b and test tube; first add heparin anticoagulant blood to each tube, and then add positive control substance a.
  • Positive control substance b and allergen are diluted to 0.1-10 ⁇ g/mL with stimulation buffer, and then respectively add stimulation buffer, diluted positive control substance a, diluted positive control substance b and allergen into negative control tube, In positive control tube a, positive control tube b and test tube, stimulate at 37 ⁇ 2°C for a period of time t1 (preferably t1 is 15-30min);
  • Termination detection add termination buffer, centrifuge (200g, 10min), suck off the supernatant, and resuspend the cells;
  • Labeled antibody Dilute the fluorescently labeled antibody with the staining reagent diluent, add the fluorescently labeled antibody, and incubate in the dark for a period of time t2 (preferably t2 is 25-40min);
  • Lying red blood cells add red blood cell lysate, lyse red blood cells, add washing solution to wash, collect cell pellet after centrifugation, and the cell pellet is a cell group containing basophils;
  • step (d) if the first SI value of the positive control group is ⁇ 2 and the absolute percentage of activated basophils is ⁇ 5%, the test is considered qualified for quality control; and if the activated A second SI value > 2 and an absolute percentage of activated basophils > 5% of the test group gives a positive test result, ie the basophils are activated or activatable.
  • the positive control group includes an anti-human IgE antibody positive control group and an fMLP positive control group; if the first SI value in any positive control group is ⁇ 2 and the absolute A percent activated basophils ⁇ 5% considers the test to be quality control acceptable; and a positive test is given if the second SI value of the activated test group is ⁇ 2 and the absolute percent activated basophils ⁇ 5% As a result, basophils are activated or activatable.
  • the absolute percentage of activated basophils the percentage of activated basophils after stimulation in the activation test group-the percentage of activated basophils in the negative control group .
  • the absolute percentage of activated basophils the percentage of activated basophils in the positive control group ⁇ the percentage of activated basophils in the negative control group.
  • the activated basophils include CD63 + basophils.
  • the method is non-diagnostic and non-therapeutic.
  • the reagent combination for detecting basophil activation as described in the first aspect of the present invention which is used to prepare a detection kit, which is used for
  • the allergic diseases include allergic asthma, rhinitis, dermatitis and other diseases related to allergies.
  • the kit further includes an instruction, which records the method described in the second aspect of the present invention.
  • Fig. 1 shows an example of steps of screening blood basophils using flow cytometry in the present invention. Circle the CD123c + CD203c + CD45 + SS low cell population, that is, basophils.
  • Figure 2 shows the number of basophils and the CD63 detection results of the negative control group, positive control group (anti-human IgE antibody) and recombinant dust mite allergen component Der f2 stimulation group, wherein Figure 2A is the negative control group, Figure 2B is the positive control group, and Figure 2C is the recombinant allergen component Der f2 stimulation group.
  • Figure 3 shows the negative control group, positive control group (anti-human IgE antibody) and recombinant dust mite allergen component Der f2 stimulation group were incubated with heparin-anticoagulated whole blood from dust mite allergen-specific IgE-positive patients, fluorescent antibodies Marker, flow cytometry analysis and comparison of the ratio of basophils to CD63 + cells.
  • Figure 4 shows the number of basophils in the negative control group, the positive control group a (anti-human IgE antibody), the positive control group b (fMLP) and the dust mite allergen crude extract stimulation group and their CD63 detection results, wherein Figure 4A is a negative control group, Figure 4B is a positive control group a (anti-human IgE antibody), Figure 4C is a positive control group b (fMLP), and Figure 4D is a dust mite allergen crude extract stimulation group.
  • Figure 4A is a negative control group
  • Figure 4B is a positive control group a (anti-human IgE antibody)
  • Figure 4C is a positive control group b (fMLP)
  • Figure 4D is a dust mite allergen crude extract stimulation group.
  • Figure 5 shows the negative control group, positive control group a (anti-human IgE antibody), positive control group b (fMLP) and dust mite allergen crude extract stimulation group, respectively, compared with dust mite allergen-specific IgE positive patients with heparin antibody
  • positive control group a anti-human IgE antibody
  • fMLP positive control group b
  • dust mite allergen crude extract stimulation group respectively
  • the inventors After extensive and in-depth research and a large number of screenings, the inventors first developed a detection method for basophil activation and corresponding detection reagents. Specifically, the inventors found that CD63 + positive cells in a specific target basophil population can be used as detection indicators for allergic diseases or their susceptibility in vitro.
  • target basophils or “target basophils of the present invention” are used interchangeably and refer to basophils with characteristics of CD203c + CD123 + CD45 + SS low .
  • basophil generally refers to a type of white blood cells that originate from hematopoietic pluripotent stem cells in the bone marrow and enter the bloodstream after differentiation and maturation in the bone marrow. Basophils and mast cells are effector cells that contribute to the development of allergic diseases. Allergen-bound IgE is activated by bridging basophils and mast cell membrane receptors. Once the effector cells are activated, they immediately release pre-synthesized granules in the cytoplasm.
  • Chemical factors, etc. are active factors that lead to telangiectasia, increased vascular permeability, smooth muscle contraction, and increased glandular secretion, causing local or systemic allergic symptoms, including allergic rhinitis, asthma, conjunctivitis, eczema, food allergy, drug allergies etc.
  • Basophils are effector cells in blood.
  • the gating method for detecting basophils in blood by flow cytometry is defined as CD203c + CD123 + CD45 + SS low cell population.
  • CD203c is mainly expressed on the surface of basophils, and CD123 is expressed on the surface of monocytes, neutrophils, basophils, eosinophils and other cells.
  • the fluorescence intensity of CD203c is taken as the X axis
  • CD123 fluorescence intensity as the Y axis draw a scatter diagram, circle the CD203c + CD123 + cell population, and obtain basophils and a small number of neutrophils and other cells.
  • CD45 is the common antigen of leukocytes, and side scattered light (SS) reflects internal information such as nuclei and granules.
  • SS side scattered light
  • the CD45 + SS low cell population circled in the CD203c + CD123 + cell population was defined as basophils.
  • the invention provides a reagent combination for detecting basophil activation.
  • the reagent combination of the present invention includes: a primary antibody against human CD203c, a secondary antibody against human CD123, a third antibody against human CD45, a fourth antibody against human CD63, a positive control substance, and an allergen.
  • the reagent combination of the present invention further includes: a stimulation buffer and/or a stop buffer.
  • a typical positive control substance is selected from the following group: anti-human IgE antibody, fMLP, or a combination thereof; wherein, human IgE is from IgE (such as allergen-specific IgE (sIgE) or serum total IgE) of allergic patients. ), fMLP is a chemotactic peptide/chemokine N-formylmethionyl-leucyl-phenylalanine (also known as CHO-Met-Ile-Phe), N-formylmethionyl-leucyl-phenylalanine.
  • the positive control substances include anti-human IgE antibody and fMLP, which are respectively positive control substance a and positive control substance b.
  • the first antibody, the second antibody, the third antibody, and the fourth antibody have different fluorescent labels.
  • the invention also provides an in vitro detection method for basophil activation.
  • the inventive method comprises the following steps:
  • n is a positive integer ⁇ 3 (such as 3-6 parts);
  • test conditions of the negative control group, the positive control group and the activation test group are the same, the difference is: in the positive control group, at least one part of the anticoagulated whole blood is incubated with the positive control substance; In the test group, at least one part of the anticoagulated whole blood is incubated with the allergen; while in the negative control group, at least one part of the anticoagulated whole blood is incubated in the absence of a positive control and in the absence of an allergen. Incubation;
  • the present inventors unexpectedly found that the target basophils of the present invention (i.e. CD203c + CD123 + CD45 + SS low cells) were first selected by flow cytometry, and then the negative control group, positive The data analysis between the control group and the activation test group can more accurately, quickly and reliably obtain the detection results of basophil activation in vitro.
  • the target basophils of the present invention i.e. CD203c + CD123 + CD45 + SS low cells
  • the positive control group can be set to 1 group (anti-human IgE antibody positive control group or fMLP positive control group) or 2 groups (anti-human IgE antibody positive control group and fMLP positive control group), all of which can achieve high specificity , High sensitivity detection effect. Furthermore, when two sets of positive controls are set at the same time, not only IgE-mediated basophil activation can be covered, but also non-IgE-mediated basophil activation can be covered, thereby avoiding non-response to anti-human IgE stimulation There may be invalid detection in a small part of the population. Therefore, when performing large-scale population testing, it is preferable to set the positive control group into two groups.
  • the test group can be equipped with multi-tube parallel test groups at the same time to detect different allergen types or components, thereby realizing the test of the activation level of basophils to various allergens under the same system .
  • the detection method of the present invention can simultaneously detect multiple allergens, thereby improving the efficiency of diagnosing allergic diseases.
  • the detection method of the present invention has a high correlation with allergy diagnosis, and the allergens applicable to the present invention are not particularly limited, no matter what type of allergen it is, it can stimulate the activation of basophils, Therefore, whether the subject is allergic, the specific allergen of the subject, the severity of the subject's allergy, etc. can be identified through the detection method of the present invention.
  • representative allergens include, but are not limited to: dust mite, cockroach, German cockroach, mugwort, Aspergillus fumigatus, dog dander, cat dander, pollen, catkins, insect venom, fungus, shrimp, wheat , rye, barley, oats, corn, peanuts, fish, crab, eggs, milk, sesame, soybeans, common ragweed, quinoa, cocklebur, nuts, shiitake mushrooms, seafood, mango, kiwi, pineapple, antibiotics, biological products , or a combination thereof.
  • Allergens and their component raw materials can be quickly put into the detection system of the present invention for application, and the types and components of allergens to be tested can be freely matched, with high flexibility.
  • the detection method of the present invention does not require the cumbersome development and verification process of specific IgE detection, and can also be applied to patients who are allergic to uncommon allergens.
  • the reagent combination or kit and detection method of the present invention can be typically used for one or more applications selected from the following groups:
  • the basophil activation detection method of the present invention does not have the risk of inducing severe allergic reactions, has higher safety, and can reduce the pain of patients, especially children, during detection.
  • the basophil activation detection method of the present invention reduces the detection technical requirements and expands the range of allergen types that can be used for screening.
  • the basophil activation detection method of the present invention does not need to be imported from abroad, which can reduce the price and cost. At the same time, it has been simplified in terms of operational technical difficulty and result interpretation difficulty, which is more conducive to clinical application.
  • the method for detecting basophil activation of the present invention is more concise in gating the basophil population, reduces the difficulty and tediousness of detection, and improves the detection efficiency.
  • the basophil activation detection method of the present invention has better consistency with the gold standard (in vivo challenge test) for the diagnosis of allergic diseases than the existing method, and reduces the probability of misdiagnosis and missed diagnosis compared with the existing method.
  • the basophil activation detection method of the present invention has high specificity and can distinguish the reaction results when multiple allergens exist, so that multiple allergen types can be screened in one detection, Improve the efficiency of the diagnosis of allergic diseases.
  • the detection method for basophil activation described in the present invention has relatively high sensitivity, and can predict the severity of allergic symptoms through the degree of activation of basophils, and can also be used for monitoring the curative effect of desensitization treatment .
  • the detection method and detection system of the present invention can reflect the in vivo state of the subject. Almost all common or uncommon allergens and their components can be quickly put into use in the detection method and detection system of the present invention, and the allergens to be tested and their components can also be freely matched according to the patient's condition, with high flexibility.
  • PBS buffer pH 7.2-7.5
  • 5-20mM EDTA 5-20mM EDTA
  • the materials and volumes used for incubation in each group can be as shown in Table 1.
  • the SI value in the table is calculated based on the average fluorescence intensity of cells; it can also be directly converted by the number or percentage of cells.
  • the SI value in the table is calculated based on the average fluorescence intensity of cells; it can also be directly converted by the number or percentage of cells.
  • the patient female, 25 years old, had obvious allergic symptoms on the face. She reported that she was allergic to penicillin. Blood was collected from the patient (heparin anticoagulant), and flow cytometry was used to detect the activation level of basophils. A negative control group and a positive control group were set up.
  • Group a anti-human IgE antibody
  • positive control group b fMLP
  • test group 3a dust mite allergen crude extract
  • test group 3b milk
  • test group 3c egg
  • test group 3d penicillin
  • the patient was rechecked with conventional allergen-specific IgE and skin tests.
  • the results showed that the patient was positive for dust mite allergen-specific IgE (Dermatophex farinae: 1.69 KU/L) and negative for egg and milk allergens.
  • the results of the skin test showed that he was allergic to dust mites, but not allergic to penicillin and other allergens.
  • the detection method of the present invention can simultaneously perform parallel detection and identification of multiple allergens in the same detection system.
  • the detection method of the invention can rapidly and massively identify allergens; on the other hand, it has high detection specificity and high sensitivity, and the detection result for this patient is completely consistent with the specific IgE and skin test.
  • Heparin anticoagulated blood samples were collected from 61 patients with respiratory diseases, and the method in Example 3 or Example 4 was used for detection.
  • 42 examples adopt 1 positive control (anti-human IgE antibody) (method of embodiment 3)
  • 38 examples adopt 2 positive controls (anti-human IgE antibody and fMLP) (method of embodiment 4)
  • two positive quality control If at least one of them is positive, it means that the quality control is qualified.
  • Example 3 Using the method of Example 3: Among the 42 cases, 39 cases passed the quality control test, and the pass rate was 92.8%. The other 3 cases were unqualified for quality control (possibly for patients who did not respond to anti-IgE stimulation), and it was impossible to judge whether the basophils were activated.
  • Adopt the method of embodiment 4 in 38 routine detection specimens, the qualified specimen of test quality control is 37 examples (contains two positive control quality control qualified of 27 routine samples; 7 routine specimen anti-human IgE antibody positive quality control is unqualified, but fMLP positive quality control qualified), the total pass rate was 97.37%.
  • Heparin anticoagulated blood was collected from dust mite allergen-specific IgE-positive patients, and flow cytometry was used to detect the activation level of basophils.

Abstract

一种嗜碱性粒细胞活化试剂组合、检测方法及其应用。该试剂组合包括：抗人CD203c的第一抗体、抗人CD123的第二抗体、抗人CD45的第三抗体、抗人CD63的第四抗体、阳性对照品和过敏原。该检测方法包括血液采集、活化试剂配置、激活、终止活化、抗体标记、红细胞裂解和流式细胞术检测、结果分析与判断，可快速、准确地进行嗜碱性粒细胞活化检测。该试剂组合及检测方法可用于诊断过敏性疾病、明确过敏原和其组分，预测过敏严重程度并监测疗效。

Description

一种嗜碱性粒细胞活化检测方法及其应用 技术领域
本发明涉及医学检测领域，具体地涉及一种嗜碱性粒细胞活化检测方法及其应用。
背景技术
过敏性疾病是临床常见病。据WHO统计，全球约22％人群受到过敏性疾病的干扰，且发病人数呈逐年上升趋势，给社会带来了沉重的经济负担。
过敏性疾病的临床症状常常缺乏特异性，这给临床诊断带来了困难。目前临床上对于过敏性疾病的诊断，除了病史、体格检查外，实验室检查是重要辅助诊断手段。
目前常用的实验室检查主要是检测过敏原，包括体内激发试验和体外过敏原特异性IgE检测(sIgE)以及血清总IgE检测。体内试验虽然准确性较高，但存在诱发严重过敏反应的风险，多种抗原过敏时的交叉反应结果也难以判断，而且检测时患者尤其是儿童比较痛苦。
体外试验中总IgE水平受多种因素影响，特异性较低。目前，过敏原sIgE检测是过敏性疾病诊断应用最广泛的检测手段。但是，由于IgE的血清浓度很低(大约为IgG的3-7万分之一)，因此，检测难度大，且能够检测的过敏原有限(约30-40种)。此外，过敏原特异性IgE结果并不能反应患者的体内状态。
因此，临床迫切需要开发有效和可靠地对过敏性疾病进行体外检测的方法(包括实验室检查方法)。
发明内容
本发明的目的就是提供了一种有效和可靠地对过敏性疾病进行体外检测的方法。
具体地，本发明提供了一种嗜碱性粒细胞活化检测方法和相应的检测试剂，为过敏性疾病诊断、过敏原和其组分的筛查等提供了新的解决方案，并可应用于过敏严重程度的预测和疗效的监测。
在本发明的第一方面，提供了一种用于嗜碱性粒细胞活化检测的试剂组合，其中，所述试剂组合包括：抗人CD203c的第一抗体、抗人CD123的第二抗体、抗人CD45的第三抗体、抗人CD63的第四抗体、阳性对照品，和过敏原。
在另一优选例中，所述试剂组合还包括刺激缓冲液和/或终止缓冲液。
在另一优选例中，所述的试剂组合还包括选自下组的一种或多种试剂：染色试剂稀释液、红细胞裂解液、洗涤液。
在另一优选例中，所述刺激缓冲液含有Hank's平衡盐缓冲液、NaHCO 3和HEPES缓冲液。
在另一优选例中，所述的刺激缓冲液pH值为7.2-7.5(较佳地7.35-7.45)。采用的保存条件为4℃。
在另一优选例中，所述的过敏原包括已发现的天然过敏原粗提物、提纯的天然过敏原组分、重组的过敏原组分，或其组合。
在另一优选例中，所述的过敏原选自下组：尘螨、蟑螂、德国小蠊、艾蒿、烟曲霉、狗毛屑、猫皮屑、花粉、柳絮、昆虫毒液、真菌、虾、小麦、黑麦、大麦、燕麦、玉米、花生、鱼、蟹、鸡蛋、牛奶、芝麻、大豆、普通豚草、藜、苍耳、坚果类、香菇、海鲜、芒果、猕猴桃、菠萝、抗生素、生物制品，或其组合。
在另一优选例中，所述的过敏原选自下组：尘螨(如户尘螨、粉尘螨)、蟑螂、德国小蠊、狗毛屑、猫皮屑、牛奶、鸡蛋、抗生素，或其组合。
在另一优选例中，所述的过敏原包括尘螨过敏原(如尘螨过敏原粗提物和重组尘螨过敏原主要组分Der f2)。
在另一优选例中，所述的过敏原的浓度为0.01-20μg/mL，较佳地0.05-15μg/mL，更佳地0.1-10μg/mL。
在另一优选例中，所述终止缓冲液包括含有EDTA或其盐的PBS缓冲液。
在另一优选例中，所述的终止缓冲液中EDTA或其盐的浓度为5-20mmol/L，pH为7.2-7.5(较佳地7.35-7.45)。
在另一优选例中，所述的阳性对照品选自下组：抗人IgE抗体、fMLP，或其组合。
在另一优选例中，所述的人IgE来自过敏患者的IgE(如过敏原特异性IgE(sIgE)、或血清总IgE)。
在另一优选例中，所述的抗人IgE抗体浓度为0.1-20μg/mL，较佳地0.5-15μg/mL，更佳地1-10μg/mL。
在另一优选例中，所述的第一抗体、第二抗体、第三抗体、和第四抗体带有不同的荧光标记。
在另一优选例中，所述的带有不同荧光标记的检测抗体(即第一抗体、第二抗体、第三抗体、和第四抗体)分别作为染色试剂。
在另一优选例中，所述的荧光标记分子选自下组：FITC、PE、ECD、 PerCP-Cyanine5.5、PE-Cyanine、APC-Alexa
Figure PCTCN2022121047-appb-000001
750、APC、Alexa
Figure PCTCN2022121047-appb-000002
染料、Pacific Blue、KO、或其组合。
在另一优选例中，所述的染色试剂稀释液由含有BSA的PBS缓冲液组成。
在另一优选例中，所述的红细胞裂解液含有碳酸氢钾、氯化氨和EDTA-Na2。
在另一优选例中，所述的洗涤液由含有BSA的PBS缓冲液组成。
在本发明的第二方面，提供了一种嗜碱性粒细胞活化的体外检测方法，其中，所述方法包括如下步骤：
(a)提供一待检测的样品，所述的样品为抗凝全血，并将所述抗凝全血分为n份，n为≥3的正整数(如3-10份，例如3、4、5、6份)；
(b)设置阴性对照组、阳性对照组和活化测试组，并添加所述的抗凝全血，形成测试混合物，并对所述测试混合物进行刺激孵育；
其中，所述阴性对照组、阳性对照组和活化测试组的测试条件相同，不同点在于：在阳性对照组中，至少一份所述的抗凝全血与阳性对照品一起进行孵育；在活化测试组中，至少一份所述的抗凝全血与过敏原一起进行孵育；而在阴性对照组中，至少一份所述的抗凝全血在无阳性对照品和无过敏原存在下进行孵育；
(c)对所述阴性对照组、阳性对照组和活化测试组中各自的测试混合物进行测定，以确定阴性对照组、阳性对照组和活化测试组中的目标嗜碱性粒细胞，其中，所述的目标嗜碱性粒细胞为CD203c +CD123 +CD45 +SS low型细胞；并确定所述目标嗜碱性粒细胞中CD63 +的细胞数量和/或比例；和
(d)对阴性对照组、阳性对照组和活化测试组的CD63 +的细胞数量和/或比例进行比较，从而定性和/或定量地确定待检测的样品中的嗜碱性粒细胞的活化程度。
在另一优选例中，所述阳性对照组包括抗人IgE抗体阳性对照组和/或fMLP阳性对照组；
较佳地，步骤(a)中所述n＝4，并且步骤(b)中所述阳性对照组包括阳性对照组a和阳性对照组b，其中阳性对照组a中的阳性对照品为抗人IgE抗体，阳性对照组b中的阳性对照品为fMLP。
在另一优选例中，在步骤(b)中，所述刺激孵育的时间t1为10-45分钟，较佳地15-30分钟，更佳地15-25分钟。
在另一优选例中，在步骤(d)中，包括计算阳性对照组的第一SI值和活化测试组的第二SI值，其中，
第一SI值如下计算：SI＝阳性对照组的活化嗜碱性粒细胞百分比/阴性对照组活 化嗜碱性粒细胞百分比；而且
第二SI值如下计算：SI＝活化测试组中刺激后的活化嗜碱性粒细胞百分比/阴性对照组活化嗜碱性粒细胞百分比；
或者
第一SI值如下计算：SI＝阳性对照组的活化嗜碱性粒细胞数量/阴性对照组活化嗜碱性粒细胞数量；而且
第二SI值如下计算：SI＝活化测试组中刺激后的活化嗜碱性粒细胞数量/阴性对照组活化嗜碱性粒细胞数量。
在另一优选例中，所述活化嗜碱性粒细胞数量由嗜碱性粒细胞CD63平均荧光强度确定。
在另一优选例中，所述第一SI值＝阳性对照组的嗜碱性粒细胞CD63平均荧光强度/阴性对照组的嗜碱性粒细胞CD63平均荧光强度。
在另一优选例中，所述第二SI值＝活化测试组中刺激后的嗜碱性粒细胞CD63平均荧光强度/阴性对照组的嗜碱性粒细胞CD63平均荧光强度。
在另一优选例中，在步骤(d)中，包括计算阳性对照组的Z1值和活化测试组的Z2值，其中，
Z1值如下计算：Z1＝阳性对照组的活化嗜碱性粒细胞百分比-阴性对照组活化嗜碱性粒细胞百分比；而且
Z2值如下计算：Z2＝活化测试组中刺激后的活化嗜碱性粒细胞百分比-阴性对照组活化嗜碱性粒细胞百分比。
在另一优选例中，当Z1≥5％且第一SI值SI-1≥2时，认定实验检测有效，否则实验无效。
在另一优选例中，当Z2≥5％且第二SI值SI-2≥2时，判断作为所述样品来源的受试者为过敏患者或过敏高风险群体，否则判断为非过敏患者或过敏低风险群体。
在另一优选例中，所述的定性和/或定量包括确定：待检测的样品中的嗜碱性粒细胞是否活化，和/或活化程度。
在另一优选例中，在步骤(c)中，通过流式细胞术确定阴性对照组、阳性对照组和活化测试组中的目标嗜碱性粒细胞。
在另一优选例中，在步骤(c)中，还包括：确定阴性对照组、阳性对照组和活化测试组中的目标嗜碱性粒细胞数量和/或比例。
在另一优选例中，所述的阳性对照品选自下组：抗人IgE抗体、fMLP，或其组合。
在另一优选例中，所述的人IgE为过敏患者的IgE(如过敏原特异性IgE(sIgE)、或血清总IgE)。
在另一优选例中，所述的抗凝全血为肝素抗凝全血。
在步骤(b)中，在阴性对照组、阳性对照组和活化测试组中，在各测试管中分别添加50-500μL的抗凝全血,然后分别添加刺激缓冲液，或添加阳性对照品+刺激缓冲液，或添加过敏原+刺激缓冲液；
在另一优选例中，所述方法包括以下步骤：
(1)提供样品：新鲜的肝素抗凝全血(最好不超过24h)，较佳地体积为1-5mL；
(2)分组并进行刺激：取流式管，分3管，分别为阴性对照管、阳性对照管和测试管；各管先加入肝素抗凝血，再将阳性对照品和过敏原用刺激缓冲液稀释到0.1-10μg/mL，再分别将刺激缓冲液、稀释的阳性对照品和过敏原分别加入阴性对照管、阳性对照管和测试管中，在37±2℃下刺激一段时间t1(较佳地t1为15-30min)；
(3)终止检测：加入终止缓冲液，离心(200g，10min)，吸去上清，重悬细胞；
(4)标记抗体：用染色试剂稀释液稀释荧光标记抗体，加入荧光标记抗体，避光孵育一段时间t2(较佳地t2为25-40min)；
(5)裂解红细胞：加入红细胞裂解液，裂解红细胞，加入洗涤液洗涤，离心后收集细胞沉淀，所述细胞沉淀为含有嗜碱性粒细胞的细胞群；
(6)加入洗涤液重悬细胞沉淀，并对重悬的细胞进行流式细胞术检测，从而确定目标嗜碱性粒细胞，所述的目标嗜碱性粒细胞为CD203c +CD123 +CD45 +SS low型细胞；
(7)确定所述目标嗜碱性粒细胞中CD63 +的细胞数量和/或比例；和
(8)对阴性对照组、阳性对照组和活化测试组的CD63 +的细胞数量和/或比例进行比较，从而定性和/或定量地确定待检测的样品中的嗜碱性粒细胞的活化程度。
在另一优选例中，所述方法包括以下步骤：
(1)提供样品：新鲜的肝素抗凝全血(最好不超过24h)，较佳地体积为1-5mL；
(2)分组并进行刺激：取流式管，分4管，分别为阴性对照管、阳性对照管a、阳性对照管b和测试管；各管先加入肝素抗凝血，再将阳性对照品a、阳性对照品b和过敏原用刺激缓冲液稀释到0.1-10μg/mL，再分别将刺激缓冲液、稀释的阳性对照品a、稀释的阳性对照品b和过敏原分别加入阴性对照管、阳性对照管a、阳性对照管b和测试管中，在37±2℃下刺激一段时间t1(较佳地t1为15-30min)；
(3)终止检测：加入终止缓冲液，离心(200g，10min)，吸去上清，重悬细胞；
(4)标记抗体：用染色试剂稀释液稀释荧光标记抗体，加入荧光标记抗体，避光孵育一段时间t2(较佳地t2为25-40min)；
(5)裂解红细胞：加入红细胞裂解液，裂解红细胞，加入洗涤液洗涤，离心后收集细胞沉淀，所述细胞沉淀为含有嗜碱性粒细胞的细胞群；
(6)加入洗涤液重悬细胞沉淀，并对重悬的细胞进行流式细胞术检测，从而确定目标嗜碱性粒细胞，所述的目标嗜碱性粒细胞为CD203c +CD123 +CD45 +SS low型细胞；
(7)确定所述目标嗜碱性粒细胞中CD63 +的细胞数量和/或比例；和
(8)对阴性对照组、阳性对照组a、阳性对照组b和活化测试组的CD63 +的细胞数量和/或比例进行比较，从而定性和/或定量地确定待检测的样品中的嗜碱性粒细胞的活化程度。
在另一优选例中，在步骤(d)中，如果阳性对照组的第一SI值≥2且绝对活化嗜碱性粒细胞百分比≥5％，则认为测试是质控合格的；并且如果活化测试组的第二SI值≥2且绝对活化嗜碱性粒细胞百分比≥5％，则给出阳性测试结果，即嗜碱性粒细胞是活化的或可活化的。
在另一优选例中，在步骤(d)中，所述阳性对照组包括抗人IgE抗体阳性对照组和fMLP阳性对照组；如果其中任一阳性对照组中的第一SI值≥2且绝对活化嗜碱性粒细胞百分比≥5％，则认为测试是质控合格的；并且如果活化测试组的第二SI值≥2且绝对活化嗜碱性粒细胞百分比≥5％，则给出阳性测试结果，即嗜碱性粒细胞是活化的或可活化的。
在另一优选例中，在活化测试组中，所述的绝对活化嗜碱性粒细胞百分比＝活化测试组中刺激后的活化嗜碱性粒细胞百分比-阴性对照组活化嗜碱性粒细胞百分比。
在另一优选例中，在阳性对照组中，所述的绝对活化嗜碱性粒细胞百分比＝阳性对照组的活化嗜碱性粒细胞百分比-阴性对照组活化嗜碱性粒细胞百分比。
在另一优选例中，活化嗜碱性粒细胞包括CD63 +嗜碱性粒细胞。
在另一优选例中，所述的方法是非诊断和非治疗性的。
在本发明的第三方面，提供了如本发明第一方面所述的用于嗜碱性粒细胞活化检测的试剂组合的用途，它被用于制备一检测试剂盒，所述试剂盒用于
(a)过敏性疾病诊断或过敏原的鉴别；
(b)用于过敏严重程度的分析；
(c)对于过敏疾病或其易感性的筛查；和/或
(d)对过敏疾病的疗效的监测。
在另一优选例中，所述的过敏性疾病包括过敏性哮喘、鼻炎、皮炎等各种与过敏相关的疾病。
在另一优选例中，所述试剂盒中还包括一说明书，所述说明书中记载了本发明第二方面所述的方法。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了本发明中使用流式细胞术筛选血液中嗜碱性粒细胞的步骤示例。圈出CD123c +CD203c +CD45 +SS low细胞群，即为嗜碱性粒细胞。
图2显示了阴性对照组、阳性对照组(抗人IgE抗体)和重组尘螨过敏原组分Der f2刺激组的嗜碱性粒细胞数量及其CD63检测结果，其中图2A为阴性对照组，图2B为阳性对照组，图2C为重组过敏原组分Der f2刺激组。
图3显示了阴性对照组、阳性对照组(抗人IgE抗体)和重组尘螨过敏原组分Der f2刺激组，分别与尘螨过敏原特异性IgE阳性患者肝素抗凝全血孵育，荧光抗体标记，流式细胞术分析嗜碱性粒细胞CD63 +细胞比例的比较结果。
图4显示了阴性对照组、阳性对照组a(抗人IgE抗体)、阳性对照组b(fMLP)和尘螨过敏原粗提物刺激组的嗜碱性粒细胞数量及其CD63检测结果，其中图4A为阴性对照组，图4B为阳性对照组a(抗人IgE抗体)，图4C为阳性对照组b(fMLP)，图4D为尘螨过敏原粗提物刺激组。
图5显示了阴性对照组、阳性对照组a(抗人IgE抗体)、阳性对照组b(fMLP)和尘螨过敏原粗提物刺激组，分别与尘螨过敏原特异性IgE阳性患者肝素抗凝全血孵育，荧光抗体标记，流式细胞术分析嗜碱性粒细胞CD63 +细胞比例的比较结果。
具体实施方式
本发明人经过广泛而深入的研究，经过大量的筛选，首次开发了一种嗜碱性粒细胞活化检测方法及相应的检测试剂。具体地，本发明人发现在一类特定的目标嗜碱性粒细胞群中的CD63 +阳性细胞，可作为过敏疾病或其易感性体外检测的检测指标。具体地，当使用流式细胞术对所述的CD203c +CD123 +CD45 +SS low嗜碱性粒细胞群的细胞表面活化标志物分子的表达水平进行检测时，根据发明人开发的嗜碱性细胞活化阳性判断标准——刺激指数(SI)≥2和绝对活化嗜碱性粒细胞百分比≥5％，可评估过敏原刺激下嗜碱性粒细胞的活化状态，对于过敏的诊断具有较高的特异度和敏感度，且与过 敏疾病诊断的金标准(体内激发试验)有较好的一致性。该检测结果可以反应体内状态，对于过敏症状严重程度预测、脱敏治疗疗效监测及判断预后都具有重要价值，在此基础上完成了本发明。
术语
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。
如本文所用，术语“目标嗜碱性粒细胞”或“本发明的目标嗜碱性粒细胞”可互换使用，指具有CD203c +CD123 +CD45 +SS low特征的嗜碱性粒细胞。
嗜碱性粒细胞
如本文所用，术语“嗜碱性粒细胞”一般指的是白细胞中的一种，起源于骨髓造血多能干细胞，在骨髓内分化成熟后进入血流。嗜碱性粒细胞和肥大细胞是导致过敏性疾病发生的效应细胞。结合了过敏原的IgE通过桥联嗜碱性粒细胞和肥大细胞膜受体而使之激活，效应细胞一旦活化立刻释放胞浆内预先合成的颗粒，颗粒富含的释放炎症介质、细胞因子和趋化因子等，是导致毛细血管扩张、血管通透性增加、平滑肌收缩、腺体分泌增加的活性因子，引起局部或全身过敏反应症状，包括过敏性鼻炎、哮喘、结膜炎、湿疹、食物过敏、药物过敏等。
嗜碱性粒细胞是血液中的效应细胞，在本发明中，利用流式细胞术检测血液中嗜碱性粒细胞的设门方法定义为CD203c +CD123 +CD45 +SS low细胞群。
CD203c主要表达在嗜碱性粒细胞表面，CD123表达在单核细胞、中性粒细胞、嗜碱性粒细胞、嗜酸性粒细胞等细胞表面，如图1A所示，将CD203c荧光强度作为X轴，CD123荧光强度作为Y轴，绘制散点图，圈出CD203c +CD123 +细胞群，可获得嗜碱性粒细胞和少部分中性粒细胞等细胞。
CD45是白细胞共同抗原，侧向散射光(SS)反映细胞核和颗粒等内部信息。圈出CD203c +CD123 +细胞群后，再以CD45荧光强度作为X轴，SS大小作为Y轴绘制散点图，如图1B所示，可见细胞根据CD45荧光强度和SS明显成群分布，嗜碱性粒细胞相对其他白细胞CD45偏弱，且SS偏低即SS low(侧向散射光在白细胞里偏低的细胞群)。将CD203c +CD123 +细胞群中圈出的CD45 +SS low细胞群，定义为嗜碱性粒细胞。
用于嗜碱性粒细胞活化检测的试剂组合
本发明提供了一种用于嗜碱性粒细胞活化检测的试剂组合。典型地，本发明的试剂组合包括：抗人CD203c的第一抗体、抗人CD123的第二抗体、抗人CD45的第三抗体、抗人CD63的第四抗体、阳性对照品，和过敏原。优选地，本发明的试剂组合还包括：刺激缓冲液和/或终止缓冲液。
在本发明中，典型的阳性对照品选自下组：抗人IgE抗体、fMLP，或其组合；其中，人IgE来自过敏患者的IgE(如过敏原特异性IgE(sIgE)、或血清总IgE)，fMLP是一种趋化肽/趋化因子N-formylmethionyl-leucyl-phenylalanine(也即CHO-Met-Ile-Phe)，N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸。优选地，阳性对照品包括抗人IgE抗体和fMLP，分别为阳性对照品a、阳性对照品b。
优选地，在本发明的试剂组合中，所述的第一抗体、第二抗体、第三抗体、和第四抗体带有不同的荧光标记。
嗜碱性粒细胞活化的检测方法
本发明还提供了一种嗜碱性粒细胞活化的体外检测方法。
典型地，本发明方法包括如下步骤：
(a)提供一待检测的样品，所述的样品为抗凝全血，并将所述抗凝全血分为n份，n为≥3的正整数(如3-6份)；
(b)设置阴性对照组、阳性对照组和活化测试组，并添加所述的抗凝全血，形成测试混合物，并对所述测试混合物进行刺激孵育；
其中，所述阴性对照组、阳性对照组和活化测试组的测试条件相同，不同点在于：在阳性对照组中，至少一份所述的抗凝全血与阳性对照品一起进行孵育；在活化测试组中，至少一份所述的抗凝全血与过敏原一起进行孵育；而在阴性对照组中，至少一份所述的抗凝全血在无阳性对照品和无过敏原存在下进行孵育；
(c)对所述阴性对照组、阳性对照组和活化测试组中各自的测试混合物进行测定，以确定阴性对照组、阳性对照组和活化测试组中的目标嗜碱性粒细胞，其中，所述的目标嗜碱性粒细胞为CD203c +CD123 +CD45 +SS low型细胞；并确定所述目标嗜碱性粒细胞中CD63 +的细胞数量和/或比例；和
(d)对阴性对照组、阳性对照组和活化测试组的CD63 +的细胞数量和/或比例进行比较，从而定性和/或定量地确定待检测的样品中的嗜碱性粒细胞的活化程度。
本发明人通过筛选和优化，意外地发现，通过流式细胞术先选出本发明的目的嗜碱性粒细胞(即CD203c +CD123 +CD45 +SS low型细胞)，再通过阴性对照组、阳性对照组和活化测试组三者之间的数据分析，可以更准确、快速和可靠地在体外获得 嗜碱性粒细胞活化的检测结果。
在本发明中，阳性对照组可设置1组(抗人IgE抗体阳性对照组或fMLP阳性对照组)或2组(抗人IgE抗体阳性对照组和fMLP阳性对照组)，均可实现高特异性、高敏感度的检测效果。进一步地，当同时设置2组阳性对照时，不仅可以覆盖IgE介导的嗜碱性粒细胞活化，还可以额外覆盖非IgE介导的嗜碱性粒细胞活化，从而避免抗人IgE刺激无应答的小部分人群中可能存在的检测无效现象。因此，当进行大规模人群检测时，优选地可将阳性对照组设置为2组。
在本发明的检测方法中，测试组可同时设置多管平行测试组，分别检测不同的过敏原类型或者组分，从而实现同一体系下对多种过敏原的嗜碱性粒细胞活化水平的测试。相比于特异性IgE检测种类的限制，本发明的检测方法能同时进行多种过敏原的检测，提高了对过敏性疾病诊断的效率。
应理解，本发明的检测方法与过敏诊断之间的相关性高，且适用于本发明的过敏原没有特别的限制，不论是何种类型的过敏原均能刺激嗜碱性粒细胞的活化，从而能够通过本发明的检测方法鉴别出受试者是否过敏、受试者的具体过敏原以及受试者过敏的严重程度等。
在本发明中，代表性的过敏原包括但不限于：尘螨、蟑螂、德国小蠊、艾蒿、烟曲霉、狗毛屑、猫皮屑、花粉、柳絮、昆虫毒液、真菌、虾、小麦、黑麦、大麦、燕麦、玉米、花生、鱼、蟹、鸡蛋、牛奶、芝麻、大豆、普通豚草、藜、苍耳、坚果类、香菇、海鲜、芒果、猕猴桃、菠萝、抗生素、生物制品，或其组合。
过敏原及其组分原料可快速投入本发明的检测体系中应用，且待测过敏原的种类和组分可自由搭配，灵活性高。本发明的检测方法无需特异性IgE检测的繁琐研发和验证流程，还能适用于对非常见过敏原产生过敏的患者。
本发明的试剂组合或试剂盒以及检测方法，代表性地可用于选自下组的一个或多个应用：
(a)过敏性疾病诊断或过敏原的鉴别；
(b)用于过敏严重程度的分析；
(c)对于过敏疾病或其易感性的筛查；和/或
(d)对过敏疾病的疗效的监测。
本发明的主要优点
1.本发明所述的嗜碱性粒细胞活化检测方法相对体内激发试验而言，不存在诱发严重过敏反应的风险，具有更高的安全性，且能够减轻检测时患者尤其是儿童的痛 苦。
2.本发明所述的嗜碱性粒细胞活化检测方法相对过敏原sIgE检测方法而言，降低了检测技术要求，且扩大了可用于筛检的过敏原种类范围。
3.本发明所述的嗜碱性粒细胞活化检测方法相对市售的嗜碱性粒细胞活化试剂盒而言，无需从国外进口，可以降低价格成本。同时在操作技术难度和结果解读难度等方面进行了易化，更利于开展临床应用。
4.本发明所述的嗜碱性粒细胞活化检测方法对嗜碱性粒细胞群的设门方法更简明，降低了检测的难度及繁琐程度，提高了检测效率。
5.本发明所述的嗜碱性粒细胞活化检测方法，与过敏疾病诊断的金标准(体内激发试验)的结果一致性优于现有方法，相对现有方法减少了误诊、漏诊的概率。
6.本发明所述的嗜碱性粒细胞活化检测方法具有较高的特异性，能够分辨多种过敏原存在时的反应结果，因而能够在一次检测中进行多种过敏原种类的筛检，提高了对过敏性疾病诊断的效率。
7.本发明所述的嗜碱性粒细胞活化检测方法具有较高的敏感度，可通过对嗜碱性粒细胞的活化程度高低对过敏症状严重程度进行预测，也可用于脱敏治疗疗效监测。
8.本发明的检测方法及检测体系能反映受试者的体内状态。几乎所有常见或非常见过敏原及其组分均能快速地在本发明的检测方法及检测体系中投入使用，待测过敏原及其组分还可根据患者情况自由搭配，灵活性高。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如《分子克隆：实验室手册》(Sambrook等人，New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例1刺激缓冲液的配制
将Hank'balanced salt solution 1×(HBSS)与HEPES缓冲液(体积比为15：4～25：4)混合并添加NaHCO 3(终浓度为0.1-0.25wt％)，调pH至7.2-7.5，配制成刺激缓冲液，0.22μm过滤，4度冰箱保存。
实施例2终止缓冲液的配制
将EDTA溶解于PBS，调pH，配制成含5-20mM EDTA的PBS缓冲液(pH 7.2-7.5)，即为终止缓冲液。
实施例3嗜碱性粒细胞活化方法的操作流程
设置阴性对照组(刺激缓冲液刺激)、阳性对照组(抗人IgE抗体刺激+刺激缓冲液)和尘螨过敏原Der f2刺激组(重组尘螨过敏原+刺激缓冲液)，分别与3个尘螨过敏原特异性IgE阳性患者及3个正常对照的肝素抗凝全血100μL孵育20min。
例如，各组孵育所用材料及体积可以如表1所示。
表1
Figure PCTCN2022121047-appb-000003
各组均加入终止缓冲液，200g离心10min，去除上清。细胞沉淀重悬，与FITC-抗人CD63、PE-抗人CD203c、PerCP-Cyanine5.5-抗人CD123和KO-抗人CD45避光孵育30min，加入红细胞裂解液裂解5min，加入稀释缓冲液终止裂解，离心后重悬细胞，流式细胞术检测嗜碱性粒细胞(CD203c +CD123 +CD45 +SS low设门)CD63 +细胞比例，与阴性对照组相比，刺激指数(SI)≥2且活化CD63 +细胞比例的绝对差值超过5％即认为是阳性。
如表2结果显示，与阴性对照组相比，阳性对照组和重组尘螨过敏原组分Der f2刺激组嗜碱性粒细胞CD63 +细胞比例显著增高，与阴性对照组的差异值分别为(90.6％、55.3％、19.2％)和(88.3％、48.37％、71.9％)，SI分别为(146.5、52.9、12.3)和(133.8、39.2、110.1)，符合阳性判断标准，建立了流式细胞术检测嗜碱性粒细胞活化方法(图2及图3)。
表2
Figure PCTCN2022121047-appb-000004
Figure PCTCN2022121047-appb-000005
*表中的SI值是基于细胞的平均荧光强度进行计算；也可直接采用细胞的数量或百分比进行换算。
嗜碱性粒细胞活化检测方法的判断标准：
1.鉴别嗜碱性粒细胞：CD203c +CD123 +CD45 +SS low；
2.检测所述各组嗜碱性粒细胞中CD63 +的比例及其平均荧光强度，计算相应的差值与SI；
3.判定实验是否有效：当Z1≥5％且SI-1≥2被认定实验检测有效，否则实验无效；
4.判定过敏：当实验被判定有效的情况下，Z2≥5％且SI-2≥2被认为过敏风险高，结合临床症状，可判断为过敏；否则，不能认定为过敏高风险或者过敏。
根据以上判断标准，表明：
对病例1、病例2、病例3的实验检测有效，并且结果显示3人均为过敏患者或过敏高风险人群，与预期值相符；
对正常对照1、正常对照2、正常对照3的实验检测有效，并且结果显示3人都不是过敏患者，或都不是过敏高风险人群，与预期值相符。
实施例4双阳性对照的嗜碱性粒细胞活化方法的操作流程
设置阴性对照组(刺激缓冲液刺激)、阳性对照组a(抗人IgE抗体刺激+刺激缓冲液)、阳性对照组b(fMLP+刺激缓冲液)和尘螨过敏原粗提物刺激组(尘螨过敏原粗提物+刺激缓冲液)，分别与3个尘螨过敏原特异性IgE阳性患者及3个正常对照的肝素抗凝全血100μL孵育20min。
例如，各组孵育所用材料及体积可以如表3所示。
表3
  刺激缓冲液 抗人IgE抗体 fMLP 尘螨过敏原 肝素抗凝全血
  (μL) (μL) (μL) 粗提物(μL) (μL)
阴性对照组 100 0 0 0 100
阳性对照组a 99.68 0.32 0 0 100
阳性对照组b 99.6 0 0.4 0 100
尘螨过敏原刺激组 99 0 0 1 100
各组均加入终止缓冲液，200g离心10min，去除上清。细胞沉淀重悬，与FITC-抗人CD63、PE-抗人CD203c、PerCP-Cyanine5.5-抗人CD123和KO-抗人CD45避光孵育30min，加入红细胞裂解液裂解5min，加入稀释缓冲液终止裂解，离心后重悬细胞，流式细胞术检测嗜碱性粒细胞(CD203c +CD123 +CD45 +SS low设门)CD63 +细胞比例，与阴性对照组相比，刺激指数(SI)≥2且活化CD63 +细胞比例的绝对差值超过5％即认为是阳性。
如表4结果显示，在尘螨患者中，与阴性对照组相比，阳性对照组a、阳性对照组b和尘螨过敏原粗提物刺激组嗜碱性粒细胞CD63 +细胞比例显著增高，与阴性对照组的差异值分别为(22.11％、54.96％、51.48％)、(45.75％、39.57％、8.55％)(37.64％、26.74％、70.19％)，SI分别为(16.02、39.82、47.08)、(67、21.5、2.36)和(50.82、16.64、91.84)，符合阳性判断标准，建立了双阳性对照的流式细胞术检测嗜碱性粒细胞活化方法(图4及图5)。
表4
Figure PCTCN2022121047-appb-000006
Figure PCTCN2022121047-appb-000007
*表中的SI值是基于细胞的平均荧光强度进行计算；也可直接采用细胞的数量或百分比进行换算。
嗜碱性粒细胞活化检测方法的判断标准：
1.鉴别嗜碱性粒细胞：CD203c +CD123 +CD45 +SS low；
2.检测所述各组嗜碱性粒细胞中CD63 +的比例及其平均荧光强度，计算相应的差值与SI；
3.判定实验是否有效：当满足(Z1(a)≥5％且SI-1(a)≥2)与(Z1(b)≥5％且SI-1(b)≥2)中的任一条件即被认定实验检测有效，否则实验无效；
4.判定过敏：当实验被判定有效的情况下，Z2≥5％且SI-2≥2被认为过敏风险高，结合临床症状，可判断为过敏；否则，不能认定为过敏高风险或者过敏。
根据以上判断标准，表明：
对病例1、病例2、病例3的实验检测有效，并且结果显示3人均为过敏患者或过敏高风险人群，与预期值相符；
对正常对照1、正常对照2、正常对照3的实验检测有效，并且结果显示3人都不是过敏患者，或都不是过敏高风险人群，与预期值相符。
实施例5通过嗜碱性粒细胞活化实验发现的尘螨过敏患者
患者，女，25岁，脸部过敏症状明显，自述对可能青霉素过敏，采集患者血液(肝素抗凝)，采用流式细胞术检测其嗜碱性粒细胞活化水平，设置阴性对照组，阳性对照组a(抗人IgE抗体)、阳性对照组b(fMLP)，测试组3a(尘螨过敏原粗提物)、测试组3b(牛奶)、测试组3c(鸡蛋)、测试组3d(青霉素)。
检测结果显示，该患者尘螨过敏(Z2＝73.78％，SI-2＝211.25)，针对其他过敏原不过敏。
为了进一步验证，对该患者还采用常规的过敏原特异性IgE和皮试进行复核。 结果显示，该患者的尘螨过敏原特异性IgE呈阳性(粉尘螨：1.69KU/L)，鸡蛋和牛奶过敏原呈阴性。皮试结果显示，尘螨过敏，青霉素等过敏原不过敏。
上述结果表明，本发明的检测方法可在同一检测体系中同时进行多种过敏原的平行检测与鉴别。本发明的检测方法一方面能快速、大量地鉴别过敏原，另一方面其检测特异性高、灵敏度高，针对该患者的检测结果与特异性IgE和皮试完全一致。
实施例6嗜碱性粒细胞活化实验性能分析
收集61例呼吸病患者的肝素抗凝血样本，采用实施例3或实施例4的方法进行检测。其中42例采用1个阳性对照(抗人IgE抗体)(实施例3的方法)，38例采用2个阳性对照(抗人IgE抗体和fMLP)(实施例4的方法)，两个阳性质控中至少一个为阳性，则表示质控合格。
上述检测结果如表5所示，结果显示：
采用实施例3的方法：42例中，测试质控合格的标本为39例，合格率为92.8％。另外3例质控不合格(可能为抗IgE刺激不反应患者)，无法判断嗜碱性粒细胞是否活化。
采用实施例4的方法：38例检测标本中，测试质控合格的标本为37例(含27例样本的两个阳性对照质控合格；7例标本抗人IgE抗体阳性质控不合格，但fMLP阳性质控合格)，总合格率97.37％。
表5
  质控合格 质控不合格
42 39(39+) 3(-)
38 37(27++；3+-；7-+) 1(--)
其中，“+”表示采用单个阳性对照时，阳性质控抗人IgE抗体为阳性，质控合格；
“++”表示采用两个阳性对照时，两个阳性质控抗人IgE抗体和fMLP均为阳性，质控合格；
“-+”表示采用两个阳性对照时，抗人IgE抗体质控为阴性，fMLP质控为阳性，质控合格；
“+-”表示采用两个阳性对照时，抗人IgE抗体质控为阳性，fMLP质控为阴性，质控合格；
“-”表示采用单个阳性对照时，阳性质控抗人IgE抗体为阴性，质控不合格；
“--”表示采用两个阳性对照时，两个阳性质控抗人IgE抗体和fMLP均为阴性，质控不合格。
实施例7嗜碱性粒细胞活化结果与特异性IgE的比较
收集尘螨过敏原特异性IgE阳性患者的肝素抗凝血，采用流式细胞术检测嗜碱性粒细胞活化水平。比较分析了9例志愿者(包括尘螨过敏患者)尘螨过敏原特异性IgE结果、嗜碱性粒细胞活化检测结果(由检测的CD63 +细胞百分比例计算出相应差值与SI)以及金标准(皮内激发试验)结果的相关性(表6)。
结果表明，基于过敏原特异性IgE的方法与皮内激发试验两者弱相关，与此相反，本发明的方法的检测结果与金标准更为一致，相关性远高于基于过敏原特异性IgE的分析方法。
表6
Figure PCTCN2022121047-appb-000008
上述结果表明，本发明的基于特定的目标嗜碱性粒细胞的体外分析方法，可快速、准确地进行嗜碱性粒细胞活化检测。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种用于嗜碱性粒细胞活化检测的试剂组合，其特征在于，所述试剂组合包括：抗人CD203c的第一抗体、抗人CD123的第二抗体、抗人CD45的第三抗体、抗人CD63的第四抗体、阳性对照品，和过敏原。
  2. 如权利要求1所述的试剂组合，其特征在于，所述试剂组合还包括：刺激缓冲液和/或终止缓冲液。
  3. 如权利要求2所述的试剂组合，其特征在于，所述刺激缓冲液含有Hank's平衡盐缓冲液、NaHCO 3和HEPES缓冲液。
  4. 如权利要求1所述的试剂组合，其特征在于，所述的过敏原选自下组：已发现的天然过敏原粗提物、提纯的天然过敏原组分、重组的过敏原组分，或其组合。
  5. 如权利要求1所述的试剂组合，其特征在于，所述的过敏原选自下组：尘螨、蟑螂、德国小蠊、艾蒿、烟曲霉、狗毛屑、猫皮屑、花粉、柳絮、昆虫毒液、真菌、虾、小麦、黑麦、大麦、燕麦、玉米、花生、鱼、蟹、鸡蛋、牛奶、芝麻、大豆、普通豚草、藜、苍耳、坚果类、香菇、海鲜、芒果、猕猴桃、菠萝、抗生素、生物制品，或其组合。
  6. 如权利要求2所述的试剂组合，其特征在于，所述终止缓冲液包括含有EDTA或其盐的PBS缓冲液。
  7. 如权利要求1所述的试剂组合，其特征在于，所述的阳性对照品选自下组：抗人IgE抗体、fMLP，或其组合。
  8. 如权利要求1所述的试剂组合，其特征在于，所述的第一抗体、第二抗体、第三抗体、和第四抗体带有不同的荧光标记。
  9. 一种嗜碱性粒细胞活化的体外检测方法，其特征在于，所述方法包括如下步骤：
    (a)提供一待检测的样品，所述的样品为抗凝全血，并将所述抗凝全血分为n份，n为≥3的正整数；
    (b)设置阴性对照组、阳性对照组和活化测试组，并添加所述的抗凝全血，形成测试混合物，并对所述测试混合物进行刺激孵育；
    其中，所述阴性对照组、阳性对照组和活化测试组的测试条件相同，不同点在于：在阳性对照组中，至少一份所述的抗凝全血与阳性对照品一起进行孵育；在活化测试组中，至少一份所述的抗凝全血与过敏原一起进行孵育；而在阴性对照组中，至少一份所述的抗凝全血在无阳性对照品和无过敏原存在下进行孵育；
    (c)对所述阴性对照组、阳性对照组和活化测试组中各自的测试混合物进行测定，以确定阴性对照组、阳性对照组和活化测试组中的目标嗜碱性粒细胞，其中，所述的目标嗜碱性粒细胞为CD203c +CD123 +CD45 +SS low型细胞；并确定所述目标嗜碱性粒细胞中CD63 +的细胞数量和/或比例；和
    (d)对阴性对照组、阳性对照组和活化测试组的CD63 +的细胞数量和/或比例进行比较，从而定性和/或定量地确定待检测的样品中的嗜碱性粒细胞的活化程度。
  10. 如权利要求9所述的方法，其特征在于，所述阳性对照组包括抗人IgE抗体阳性对照组和/或fMLP阳性对照组；
    较佳地，步骤(a)中所述n＝4，并且步骤(b)中所述阳性对照组包括阳性对照组a和阳性对照组b，其中阳性对照组a中的阳性对照品为抗人IgE抗体，阳性对照组b中的阳性对照品为fMLP。
  11. 如权利要求9所述的方法，其特征在于，在步骤(d)中，包括计算阳性对照组的第一SI值和活化测试组的第二SI值，其中，
    第一SI值如下计算：SI＝阳性对照组的活化嗜碱性粒细胞百分比/阴性对照组活化嗜碱性粒细胞百分比；而且
    第二SI值如下计算：SI＝活化测试组中刺激后的活化嗜碱性粒细胞百分比/阴性对照组活化嗜碱性粒细胞百分比；
    或者
    第一SI值如下计算：SI＝阳性对照组的活化嗜碱性粒细胞数量/阴性对照组活化嗜碱性粒细胞数量；而且
    第二SI值如下计算：SI＝活化测试组中刺激后的活化嗜碱性粒细胞数量/阴性对照组活化嗜碱性粒细胞数量。
  12. 如权利要求11所述的方法，其特征在于，所述第一SI值＝阳性对照组的嗜碱性粒细胞CD63平均荧光强度/阴性对照组的嗜碱性粒细胞CD63平均荧光强度，所述第二SI值＝活化测试组中刺激后的嗜碱性粒细胞CD63平均荧光强度/阴性对照组的嗜碱性粒细胞CD63平均荧光强度。
  13. 如权利要求9所述的方法，其特征在于，在步骤(d)中，包括计算阳性对照组的Z1值和活化测试组的Z2值，其中，
    Z1值如下计算：Z1＝阳性对照组的活化嗜碱性粒细胞百分比-阴性对照组活化嗜碱性粒细胞百分比；而且
    Z2值如下计算：Z2＝活化测试组中刺激后的活化嗜碱性粒细胞百分比-阴性对照组活化嗜碱性粒细胞百分比。
  14. 如权利要求9所述的方法，其特征在于，在步骤(c)中，通过流式细胞术确定阴性对照组、阳性对照组和活化测试组中的目标嗜碱性粒细胞。
  15. 如权利要求1所述的用于嗜碱性粒细胞活化检测的试剂组合的用途，其特征在于，用于制备一检测试剂盒，所述试剂盒用于
    (a)过敏性疾病诊断或过敏原的鉴别；
    (b)用于过敏严重程度的分析；
    (c)对于过敏疾病或其易感性的筛查；和/或
    (d)对过敏疾病的疗效的监测。
PCT/CN2022/121047 2021-09-23 2022-09-23 一种嗜碱性粒细胞活化检测方法及其应用 WO2023046111A1 (zh)

Priority Applications (6)

Application Number Priority Date Filing Date Title
CN202280005696.XA CN116157672A (zh) 2021-09-23 2022-09-23 一种嗜碱性粒细胞活化检测方法及其应用
CN202311231985.4A CN117760942A (zh) 2022-09-23 2023-03-23 注入性过敏原嗜碱性粒细胞活化检测方法及其应用
CN202310651507.2A CN117760939A (zh) 2022-09-23 2023-03-23 食物过敏原嗜碱性粒细胞活化检测方法及其应用
CN202310292519.0A CN117760938A (zh) 2021-09-23 2023-03-23 一种嗜碱性粒细胞活化检测方法及其应用
CN202311231847.6A CN117760941A (zh) 2022-09-23 2023-03-23 药物过敏原嗜碱性粒细胞活化检测方法及其应用
CN202310649973.7A CN117783519A (zh) 2021-09-23 2023-03-23 吸入性过敏原嗜碱性粒细胞活化检测方法及其应用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111115531.1 2021-09-23
CN202111115531 2021-09-23

Publications (1)

Publication Number Publication Date
WO2023046111A1 true WO2023046111A1 (zh) 2023-03-30

Family

ID=85720125

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/121047 WO2023046111A1 (zh) 2021-09-23 2022-09-23 一种嗜碱性粒细胞活化检测方法及其应用

Country Status (2)

Country Link
CN (2) CN116157672A (zh)
WO (1) WO2023046111A1 (zh)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009033691A1 (en) * 2007-09-11 2009-03-19 Bühlmann Laboratories Ag Allergy test based on flow cytometric analysis
CN104677810A (zh) * 2015-01-30 2015-06-03 广东医学院附属医院 嗜碱性粒细胞活化的检测试剂盒及其使用方法
CN104777303A (zh) * 2015-04-29 2015-07-15 辽宁医学院附属第一医院 一种嗜碱性粒细胞激活、脱颗粒的鉴定方法
WO2018223149A1 (en) * 2017-06-03 2018-12-06 Flowmetric Life Sciences, Inc. Systems and methods for determining the risk of severe allergic reaction
CN113049815A (zh) * 2019-12-26 2021-06-29 上海益诺思生物技术股份有限公司 一种食蟹猴淋巴细胞的流式圈门方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009033691A1 (en) * 2007-09-11 2009-03-19 Bühlmann Laboratories Ag Allergy test based on flow cytometric analysis
CN104677810A (zh) * 2015-01-30 2015-06-03 广东医学院附属医院 嗜碱性粒细胞活化的检测试剂盒及其使用方法
CN104777303A (zh) * 2015-04-29 2015-07-15 辽宁医学院附属第一医院 一种嗜碱性粒细胞激活、脱颗粒的鉴定方法
WO2018223149A1 (en) * 2017-06-03 2018-12-06 Flowmetric Life Sciences, Inc. Systems and methods for determining the risk of severe allergic reaction
CN113049815A (zh) * 2019-12-26 2021-06-29 上海益诺思生物技术股份有限公司 一种食蟹猴淋巴细胞的流式圈门方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHANG MIN, CUI ZHEN-ZE; LI YUE; XU CHAO: "Research progress of basophil activation test and its application in allergic diseases", ZHONGGUO MIANYIXUE ZAZHI = CHINESE JOURNAL OF IMMUNOLOGY, JILIN SHENG YIXUE QIKANSHE, CN, no. 22, 1 July 2021 (2021-07-01), CN , pages 2803 - 2809, XP093055079, ISSN: 1000-484X, DOI: 10.3969/j.issn.1000-484X.2021.22.021 *

Also Published As

Publication number Publication date
CN117760938A (zh) 2024-03-26
CN116157672A (zh) 2023-05-23

Similar Documents

Publication Publication Date Title
Oshita et al. Semi-quantitative procalcitonin test for the diagnosis of bacterial infection: clinical use and experience in Japan
US20120083007A1 (en) Basophil Activation Based Allergy Diagnostic Test
US9891213B2 (en) Granulocyte-based methods for detecting and monitoring immune system disorders
WO2021160121A1 (zh) 粒细胞群嗜碱性粒细胞作为过敏性疾病诊断标记物的应用
CN106501530A (zh) 一种诊断结核杆菌感染的生物标志物及其相关试剂盒
WO2008143890A2 (en) Biomarkers for septic shock patients
CN116699131B (zh) Hla-dr+cd14+cd56+单核细胞在hlh中诊断的应用
CN111549091B (zh) 一种嗜碱性粒细胞活化、脱颗粒的测试方法
Kuethe et al. Assessing the immune status of critically ill trauma patients by flow cytometry
Troia et al. Cytokine and chemokine profiling in cats with sepsis and septic shock
Pandey et al. Comparison of neutrophil CD64 and monocytic HLA-DR with existing biomarkers for the diagnosis and prognosis of sepsis
Stelmaszczyk-Emmel et al. Th1, Th2, Th17, and regulatory cytokines in children with different clinical forms of allergy
WO2023046111A1 (zh) 一种嗜碱性粒细胞活化检测方法及其应用
CN109991417B (zh) 一种结核病的免疫标志物及应用
EP1430305A2 (en) A method and kit for the measurement of the activation of basophils induced by allergen to determine hypersensitivity
RU2289138C2 (ru) Способ аллергодиагностики по показателям хемилюминесцентного свечения нейтрофилов
Shahid et al. Evaluation of mean neutrophil volume and immature to total neutrophil ratio as a biomarker for bacterial sepsis in adult patients
CN117760939A (zh) 食物过敏原嗜碱性粒细胞活化检测方法及其应用
CN117783519A (zh) 吸入性过敏原嗜碱性粒细胞活化检测方法及其应用
Shokouhi et al. Diagnostic value of the leukocyte esterase test for early detection of pleocytosis in cerebrospinal fluid of patients with suspected acute bacterial meningitis
CN109900904B (zh) 包含cd14检测试剂的神经梅毒检测试剂盒及该试剂在神经梅毒检测中的应用
Zhai et al. Development of prediction models for new integrated models and a bioscore system to identify bacterial infections in systemic lupus erythematosus
CN107860907A (zh) Pru p 3蛋白在制备蒿花粉相关桃过敏检测试剂盒中的应用
Woodrow et al. Initial investigation of molecular phenotypes of airway mast cells and cytokine profiles in equine asthma
WO2024041257A1 (zh) 一种通过检测尿液迁移体来判断慢性肾病的方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22872155

Country of ref document: EP

Kind code of ref document: A1