阅读说明：本技术 一种嗜碱性粒细胞活化、脱颗粒的测试方法 (Test method for basophil activation and degranulation ) 是由 季江 焦晴晴 赵莹 魏钰倩 苏文星 于 2020-05-21 设计创作，主要内容包括：本发明一种嗜碱性粒细胞活化、脱颗粒的测试方法,包括：稀释待测过敏原制成标准待测过敏原,设立阳性对照和阴性对照；所有待测样本为肝素化的患者外周血,需封口孵育；随后加入缓冲液,离心；加入荧光染色混合液,轻混至沉淀物再悬浮,避光孵育；加入红细胞裂解液,漩涡混匀,避光孵育；稀释,离心,再悬沉淀物；过滤后添加缓冲液,采用流式细胞仪检测,得到活化、脱颗粒比例。本发明提供的嗜碱性粒细胞活化、脱颗粒的测试方法,无需分离纯化嗜碱性粒细胞,选用新的特异性嗜碱性粒细胞标记物组合,所测样本用量小,重复性好,误差小,有效分辨嗜碱性粒细胞的活化、脱颗粒程度,更好地为相关疾病临床鉴别诊断和科研项目提供检测和技术支持。(The invention relates to a test method for basophil activation and degranulation, which comprises the following steps: diluting the allergen to be tested to prepare standard allergen to be tested, and establishing a positive control and a negative control; all samples to be detected are heparinized peripheral blood of patients, and need to be sealed for incubation; then adding a buffer solution, and centrifuging; adding the fluorescent staining mixed solution, gently mixing until the precipitate is suspended again, and incubating in a dark place; adding erythrocyte lysate, mixing uniformly by vortex, and incubating in dark place; diluting, centrifuging and suspending the precipitate; and (3) filtering, adding a buffer solution, and detecting by using a flow cytometer to obtain the proportion of activated particles to the particles. According to the basophil activation and degranulation testing method provided by the invention, separation and purification of basophils are not needed, a new specific basophil marker combination is selected, the dosage of a tested sample is small, the repeatability is good, the error is small, the activation and degranulation degrees of the basophils are effectively distinguished, and detection and technical support are better provided for clinical differential diagnosis and scientific research projects of related diseases.)

1. A basophil activation and degranulation test method is characterized by comprising the following steps:

step (I), sample and related reagent preparation

S1, diluting the allergens to be detected in equal proportion, and numbering the allergens in sequence to be used as standard allergens to be detected;

s2, preparing a positive control and a negative control;

s3, diluting the erythrocyte lysate with sterile double-distilled water according to the proportion of 1:10-12 for later use;

step (II), sample incubation and detection

S4, transferring the positive control, the negative control and the standard allergen sample to be tested obtained in the step (I) into a flow cytometry sample loading tube respectively, adding 120 mu L of blood of a heparinized patient to be tested into each tube respectively, sealing and incubating;

s5, adding PBS/EDTA buffer solution into all the samples respectively, centrifuging and sucking out supernatant;

s6, adding 50-60 mu L of fluorescent staining mixed liquor into all samples respectively, lightly mixing until precipitates are suspended, and incubating at room temperature in a dark place;

s7, adding erythrocyte lysate into all samples respectively, whirling for 10-15S, and incubating in a dark place;

s8, adding a buffer solution into all samples, centrifuging, and removing supernatant;

s9, resuspending the precipitate, and then filtering to prepare a single cell suspension;

s10, adding 300-320 mu L MACS buffer solution into all samples, detecting by adopting a flow cytometer, and analyzing the ratio of basophil activation and degranulation according to the average fluorescence intensity of the staining markers in a gating mode.

2. The method according to claim 1, wherein in step S1, the RPMI 1640 medium is used as a dilution buffer, the extract of the sensitizer is selected as the allergen to be tested, the RPMI 1640 medium is used to dilute the allergen to be tested at a ratio of 1:7-9, and the diluted allergen is numbered in sequence as a standard allergen sample.

3. The method for testing basophil activation and degranulation according to claim 1, wherein in step S2, the step of preparing the positive control comprises:

step S21, diluting the human IgE antibody to 1: 10-12;

step S22, diluting 10-20 μ L of the human IgE antibody obtained in step S21 with 90-100 μ L of the culture medium to prepare a positive control.

4. The method for testing basophil activation and degranulation according to any of claims 1-3, wherein the culture medium is RPMI 1640, and the components of the culture medium comprise: 10% FCS, 100U penicillin, 100. mu.g/mL streptomycin.

5. The method for testing basophil activation and degranulation according to claim 1, wherein in step S3, the erythrocyte lysate is: take 8.29g NH₄Cl、1.00g KHCO₃、37.2mg Na₂EDTA dissolved in 1000mL of distilled water.

6. The method as claimed in claim 1, wherein in step S4, the positive control, the negative control and the standard allergen to be tested obtained in step (A) are transferred to a flow cytometry sample loading tube with numbers in equal amount, each tube has a total volume of 120 μ L for 100-₂Incubating for 10-15min under the condition.

7. The method for testing basophil activation and degranulation according to claim 1, wherein 50-60 μ L of the mixture for fluorescent staining is added to all samples in step S6, and the samples are gently mixed, vortexed, gently resuspended, and incubated in the dark at room temperature for 30-45 min.

8. The method for testing basophil activation and degranulation according to claim 1, wherein in step S6, the fluorescence staining mixture is: the preparation method comprises the steps of taking 35-39 mu L of FACS buffer solution, and sequentially adding 1-2 mu L of gamma globulin, 2-3 mu L of VioBlue fluorescence labeled CD3, 5-6 mu L of FITC fluorescence labeled CD63, 2-3 mu L of PE fluorescence labeled CD203c and 3-4 mu L of APC fluorescence labeled CCR 3.

9. The method for testing basophil activation and degranulation according to claim 1, wherein in step S10, the MACS buffer solution is 1% BSA and 2mmol/L EDTA dissolved in PBS.

Technical Field

The invention belongs to the technical field of allergic disease diagnosis, and particularly relates to a method for testing basophil activation and degranulation.

Background

With the continuous emergence of serious environmental pollution, chemical abuse, ionizing radiation, transgenic food, various food additives and the like, the 'allergy' becomes another big 'killer' of human health. It is reported that 30-40% of people worldwide are afflicted with allergic problems involving multiple organs and systems, such as cutaneous allergic reactions, respiratory allergic reactions, gastrointestinal allergic reactions, anaphylactic shock, and the like. Epidemiological data in 2018 show that 3 million people worldwide suffer from asthma, 4 million people from eczema and atopic dermatitis, and more than 5 million people from allergic rhinitis. The natural course of allergic diseases is the increasing variety of allergens, such as newly synthesized food additives, industrial raw materials, chemical reagents, even part of novel drugs, etc.; the clinical symptoms are variable. Allergy is a complex disease of the immune system, has diversified clinical manifestations, such as allergic rhinitis, asthma, urticaria, allergic dermatitis, eczema and the like, and has life risks in severe anaphylactic shock, laryngeal edema, and sudden or persistent asthma. Allergy is not far away from us, mild people affect life quality, and severe people can have life threat, for example, one generation of postsinging danlijun is the outbreak of asthma, and 32-year-old female hollywood Brittany Murphy is the unexpected death due to ineffective rescue of drug allergy. The activation and degranulation of basophils, the major effector cells of allergic diseases, releases histamine, is a key step in the initiation of the allergic process by the biological function of basophils. In view of the relatively accessible nature of basophils in human peripheral blood, Basophil activation assay (BAT) is a relatively effective in vitro allergen detection means currently in use by relevant laboratories internationally to date. Unfortunately, practical results show that the currently commonly used biomarker combinations make the BAT detection have large differences in accuracy, sensitivity and specificity among different individuals, and some allergic patients have low peripheral blood basophil levels, so that the BAT is not included in the conventional clinical detection means at present and is only applied to partial clinical research. Therefore, a new and more specific basophil activation surface marker is searched for to improve the allergen detection method based on peripheral blood basophil histamine release, so that the defects can be effectively overcome, and the aim of efficiently and accurately detecting the allergen is fulfilled.

Disclosure of Invention

In order to solve the technical problems in the existing clinical and scientific research applications, the invention aims to provide a test method for basophil activation and degranulation.

In order to achieve the purpose and achieve the technical effects, the invention adopts the following technical scheme to realize:

a basophil activation and degranulation test method comprises the following steps:

step (I), sample and related reagent preparation

S1, diluting the allergens to be detected in equal proportion, and numbering the allergens in sequence to be used as standard allergens to be detected;

s2, preparing a positive control and a negative control;

s3, diluting the erythrocyte lysate with sterile double-distilled water according to the proportion of 1:10-12 for later use;

step (II), sample incubation and detection

S4, transferring the positive control, the negative control and the standard allergen sample to be tested prepared in the steps S1-S2 to a flow cytometry sample loading tube respectively, adding 100-;

s5, adding 2-4mL PBS/EDTA into all samples obtained in the above steps, centrifuging for 10-15min at 4 ℃ under the centrifugal force condition of 340g, and sucking out supernatant;

s6, adding 50-60 mu L of fluorescent staining mixed liquor into all samples respectively, lightly mixing until precipitates are suspended, and incubating at room temperature in a dark place;

s7, adding erythrocyte lysate into all samples respectively, whirling for 10-15S, and incubating in a dark place;

s8, adding PBS buffer solution into all samples, centrifuging, and removing supernatant;

s9, resuspending the precipitate, and then filtering to prepare a single cell suspension;

s10, adding 300-320 mu L of MACS buffer solution into all samples, storing and waiting for detection;

and detecting the obtained sample by adopting a flow cytometer, and analyzing the proportion of basophil activation and degranulation according to the average fluorescence intensity of the staining marker in a gating mode.

Further, in step S1, an RPMI 1640 medium is used as a dilution buffer, an extract of easily allergenic substances such as milk, pollen, dust mites, peanuts and the like is selected as the allergen to be tested, the allergen to be tested is diluted in an equal ratio of 1:7-9 by using the RPMI 1640 medium, and the diluted allergen is numbered in sequence to serve as a standard allergen sample to be tested.

Further, in step S2, the step of preparing a positive control is:

step S21, diluting the human IgE antibody to 1: 10-12;

step S22, diluting 10-20 μ L of the human IgE antibody obtained in step S21 with 90-100 μ L of the culture medium to prepare a positive control.

Further, the culture medium is RPMI 1640 culture medium, and the components of the culture medium comprise: 10% FCS, 100U penicillin, 100. mu.g/mL streptomycin.

Further, in step S3, the erythrocyte lysate is: take 8.29g NH₄Cl、1.00g KHCO₃、37.2mgNa₂EDTA dissolved in 1000mL of distilled water.

Further, in step S4, the positive control, the negative control and the standard allergen to be tested obtained in steps S1-S2 in step (I) are respectively transferred to the numbered flow cytometry sample application tubes with equal volume, wherein the total volume of each tube is 120 μ L of 100-₂Incubating for 10-15min under the condition.

Further, in step S6, 50-60 μ L of the fluorescent staining mixture is added to all samples, mixed gently without vortexing, and resuspended in cells gently, and incubated in the dark at room temperature for 30-45 min.

Further, 50-60 μ L of the fluorescent staining mixture (CD3, CD63, CD203c, and CCR3) was added to each sample, gently mixed, not vortexed, gently resuspended, incubated the sample in the dark at room temperature for 30-45 minutes, added to the red blood cell lysate, vortexed on a vortexer at low speed for 10-15 seconds, and incubated in the dark at room temperature for 5-10 min.

Further, in step S6, the fluorescent staining mixture solution is: the preparation method comprises the steps of taking 35-39 mu L of FACS buffer solution, and sequentially adding 1-2 mu L of gamma globulin, 2-3 mu L of VioBlue fluorescence labeled CD3, 5-6 mu L of FITC fluorescence labeled CD63, 2-3 mu L of PE fluorescence labeled CD203c and 3-4 mu L of APC fluorescence labeled CCR 3.

Further, in step S10, 2 to 4mL of PBS is added to all the samples obtained above, the samples are centrifuged at 4 ℃ and 340g for 10 to 15min under centrifugal force, the supernatant is discarded, re-suspended, and repeated centrifugation is performed if the red color is still visible to the naked eye, and multiple centrifugation and suspension are performed until no red precipitate appears; all samples were filtered through a sterile cell filter with a pore size of 30 μm to prepare single cell suspensions, and then 300-320 μ L of MACS buffer was added to all samples, stored at 4 ℃ and awaited detection.

Further, in step S10, the MACS buffer is 1% BSA and 2mmol/L EDTA in PBS.

Compared with the prior art, the invention has the beneficial effects that:

the invention discloses a method for testing basophil activation and degranulation, which comprises the following steps: diluting the allergen to be detected as standard allergen to be detected, and simultaneously setting human IgE antibody as positive control and culture medium as negative control; all samples to be detected are heparinized peripheral blood of patients, and need to be sealed for incubation; then adding PBS/EDTA, centrifuging, and sucking out supernatant; then adding a fluorescent staining mixed solution (CD3, CD63, CD203c and CCR3), gently mixing until the precipitate is resuspended, and incubating in the dark; adding the erythrocyte lysate again, mixing uniformly by vortex, and incubating in a dark place; diluting, centrifuging, sucking out supernatant, and suspending precipitate; and (3) filtering, adding MACS buffer solution, detecting by using a flow cytometer, and analyzing the ratio of basophil activation and degranulation according to the average fluorescence intensity of the staining marker in a gating mode. According to the basophil activation and degranulation testing method provided by the invention, separation and purification of basophils are not needed, a new specific basophil marker combination is selected, the used amount of a tested sample is small, the repeatability is good, the error is small, the activation and degranulation degrees of the basophils can be effectively distinguished, and detection and technical support are better provided for clinical differential diagnosis and scientific research projects of related diseases.

Drawings

FIG. 1 is a schematic view of the present invention with the door set to be CD3 negative;

FIG. 2 is a schematic representation of the present invention setting the gate positive for CCR 3;

FIG. 3 is a positive control activated basophil of the present invention;

FIG. 4 shows a negative control activated basophil according to the present invention;

FIG. 5 is a graph showing the mean fluorescence intensity of all CD203c positive cells expressing CD203c in the positive control of the present invention;

FIG. 6 is a dot-matrix plot of degranulated cells and non-degranulated cells of a positive control of the invention;

FIG. 7 is a dot-matrix diagram of degranulated cells and non-degranulated cells of a negative control of the present invention.

Detailed Description

The embodiments of the present invention will be described in detail with reference to the accompanying drawings so that the advantages and features of the invention can be more easily understood by those skilled in the art, and the scope of the invention will be clearly and clearly defined.

As shown in fig. 1-7, a method for testing basophil activation and degranulation comprises the following steps:

step (I), sample and related reagent preparation

S1, diluting the allergens to be detected in equal proportion, and numbering the allergens in sequence to be used as standard allergens to be detected;

s2, preparing a positive control and a negative control;

s3, diluting the erythrocyte lysate with sterile double-distilled water according to the proportion of 1:10-12 for later use;

step (II), sample incubation and detection

S4, transferring the positive control, the negative control and the standard allergen sample to be tested obtained in the step (I) into a flow cytometry sample loading tube respectively, adding 120 mu L of blood of a heparinized patient to be tested into each tube respectively, sealing and incubating;

s5, adding PBS/EDTA buffer solution into all the samples respectively, centrifuging and sucking out supernatant;

s6, adding 50-60 mu L of fluorescent staining mixed liquor into all samples respectively, lightly mixing until precipitates are suspended, and incubating at room temperature in a dark place;

s7, adding erythrocyte lysate into all samples respectively, whirling for 10-15S, and incubating in a dark place;

s8, adding PBS buffer solution into all samples, centrifuging, and removing supernatant;

s9, resuspending the precipitate, and then filtering to prepare a single cell suspension;

s10, adding 300-320 mu L MACS buffer solution into all samples, detecting by adopting a flow cytometer, and analyzing the ratio of basophil activation and degranulation according to the average fluorescence intensity of the staining markers in a gating mode.

In step S1, the RPMI 1640 culture medium is used as a dilution buffer solution, the extract of the allergenic substance is selected as the allergen to be detected, the allergen to be detected is diluted by the RPMI 1640 culture medium in an equal proportion of 1:7-9, and the diluted allergen to be detected is numbered in sequence and used as a standard allergen sample to be detected.

In step S2, the step of preparing a positive control is:

step S21, diluting the human IgE antibody to 1: 10-12;

step S22, diluting 10-20 μ L of the human IgE antibody obtained in step S21 with 90-100 μ L of the culture medium to prepare a positive control.

The culture medium is RPMI 1640 culture medium, and comprises the following components: 10% FCS, 100U penicillin, 100. mu.g/mL streptomycin.

In step S3, the erythrocyte lysate is: take 8.29g NH₄Cl、1.00g KHCO₃、37.2mg Na₂EDTA dissolved in 1000mL of distilled water.

In step S4, the positive control, the negative control and the standard allergen to be tested obtained in step (I) are respectively transferred to flow cytometry sample loading tubes with numbers in equal amount, the total volume of each tube is 100-₂Incubating for 10-15min under the condition.

In step S6, 50-60 μ L of the fluorescent staining mixture was added to each sample, gently mixed without vortexing, gently resuspended cells, and incubated at room temperature in the dark for 30-45 min.

In step S6, the fluorescent staining mixed solution is: the preparation method comprises the steps of taking 35-39 mu L of FACS buffer solution, and sequentially adding 1-2 mu L of gamma globulin, 2-3 mu L of VioBlue fluorescence labeled CD3, 5-6 mu L of FITC fluorescence labeled CD63, 2-3 mu L of PE fluorescence labeled CD203c and 3-4 mu L of APC fluorescence labeled CCR 3.

In step S10, the MACS buffer was 1% BSA and 2mmol/L EDTA in PBS.