CN111610326B

CN111610326B - Method for detecting in-vitro mast cell histamine release for allergen detection

Info

Publication number: CN111610326B
Application number: CN202010488782.3A
Authority: CN
Inventors: 焦晴晴
Original assignee: Suzhou Aileqi Biotechnology Co ltd
Current assignee: Suzhou Aileqi Biotechnology Co ltd
Priority date: 2020-06-02
Filing date: 2020-06-02
Publication date: 2023-10-13
Anticipated expiration: 2040-06-02
Also published as: CN111610326A

Abstract

The invention discloses a detection method for in vitro mast cell histamine release for allergen detection, which comprises the following steps: the three steps of in vitro isolation of cultured mast cells, induction of activated mast cells and in vitro histamine detection can quantitatively calculate the histamine release amount and evaluate the degree of allergy of patients, and help identify allergens. According to the method for detecting the histamine release of the in-vitro mast cells for allergen detection, the isolated and cultured mast cells can be cultured in vitro for 2 months at most, so that sample material taking and preparation time are greatly reduced; after the histamine is released by the induced mast cells, the histamine can be tightly adsorbed with the materials on the microtitration plate, can be stored and carried at normal temperature, can freely arrange the detection time and place at will, can rapidly and effectively identify the allergen, does not need to extract the histamine, has short preparation time, is simple and convenient to operate, saves the detection cost, is suitable for routine diagnosis and related clinical and scientific research projects, and has wide clinical and scientific research application values.

Description

Method for detecting in-vitro mast cell histamine release for allergen detection

Technical Field

The invention belongs to the technical field of allergen detection, and particularly relates to a detection method for in-vitro mast cell histamine release for allergen detection.

Background

With the ever-increasing global environmental pollution, chemical abuse, ionizing radiation, transgenic foods, and various food additives, etc., as a series of problems continue to occur, "allergies" become a further large "killer" for human health. The treatment of allergic diseases is a systematic engineering, the world allergic organization (WHO) recommends an optimal treatment regimen of "four-in-one", while the avoidance of allergy is the first principle of the treatment regimen, so allergen detection is the core for diagnosis and treatment of allergic diseases. Currently, the detection means of allergens are classified into in vivo methods (spot test, patch test, intradermal test, allergen challenge test, etc.) and in vitro methods (serum allergen screening, histamine release test, etc.). Spot prick, intradermal and allergen challenge tests are currently the most common detection means in clinical practice, but have limited allergen variety and may lead to serious complications such as anaphylactic shock. The ban test mainly detects chemical allergy in daily contact, and the detection range is limited. Serum allergen detection is the most recommended detection means for clinicians because of high reliability, good specificity, high sensitivity and optimal safety. In vitro serum allergen detection also suffers from a number of problems, one of which is "inaccurate detection". Although the existing means can accurately measure the content of total IgE or partially specific IgE in serum, the allergen cannot be accurately diagnosed: sometimes the clinical manifestation clearly suggests that an allergic reaction is likely, but no allergen is detected; while sometimes substances which are not allergic are clinically manifested, the allergen detection is positive; even sometimes the patient has never been exposed to the detected allergen. In view of the fact that the key step of allergy is the process of mast cell and basophil activation, degranulation and histamine release, in vitro histamine release detection is an effective in vitro allergen detection means. Basophils are relatively readily available in human peripheral blood relative to mast cells, and therefore, basophil activation experiments (Basophil activation test, BAT) are in vitro histamine release function assays currently being used by related laboratories internationally to date. Unfortunately, the sensitivity and specificity of BAT detection varies widely from individual to individual, and BAT detection is long-lasting and relies on fresh peripheral blood, and in view of the fact that mast cells are the key target cells for allergy, some allergic patients have lower peripheral blood basophils, and some allergic patients have higher IgE levels but the BAT detection results remain negative, possibly because basophils can also be activated through non-IgE-mediated pathways. Therefore, it has not been incorporated into conventional clinical detection means, but only applied to some clinical studies. Therefore, the allergen detection method based on the in-vitro mast cell histamine release is expected to effectively overcome the defects, and the aim of efficiently and accurately detecting the allergen is fulfilled.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention aims to provide a detection method for in-vitro mast cell histamine release for allergen detection.

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

a method of detecting in vitro mast cell histamine release for allergen detection comprising the steps of:

step one, mast cell isolation

1. Removing fat tissue adhered to the isolated skin (all from excessive normal tissue excised by plastic surgery), cutting into pieces, and transferring into gel containing neutral protease and Ca ²⁺ 、Mg ²⁺ Incubation overnight at 4-6deg.C in PBS;

2. transferring to a culture dish, and removing epidermis;

3. continuously cutting skin, transferring the cut skin into a culture medium containing enzyme mixture, incubating for 1-1.5h in a shaking water bath, and setting the shaking frequency to 250-270Hz to avoid adhesion;

4. filtering the skin and enzyme mixture, and centrifuging the filtrate at 4-5deg.C and centrifugal force of 400g for 10-15min;

5. gently dispersing the precipitated cells by tapping and re-suspending with sterile cold PBS, washing the old test tube with sterile cold PBS, merging into a new test tube, filling the new test tube with PBS to 50mL, and centrifuging at 4-5deg.C under centrifugal force of 400g for 10-15min;

6. resuspension the pellet in 3-5mL MACS buffer, filtering twice, centrifuging at 4-5deg.C under centrifugal force 400g for 10-15min, and pouring out MACS buffer;

7. adding 200 μl of AB serum and 19-20 μl of intravenous immunoglobulin, adding MACS buffer to 1-2ml, and incubating at 4-5deg.C for 10-15min;

8. adding 50 mu L of CD117 immunomagnetic beads, and incubating for 10-15min at 4-5 ℃;

9. washing cells with MACS buffer, centrifuging at 4-5deg.C for 10-15min under the condition of centrifugal force of 400g, pouring out MACS buffer, and then re-suspending the precipitate in MACS buffer;

10. operating an immunomagnetic bead cell sorting system to sort out mast cells and other negative cells;

11. adding MC culture medium, counting MC and negative cells, obtaining purity and dead cell number, centrifuging at 4-5deg.C under centrifugal force 400g for 10-15min, collecting supernatant, and mixing with 5×10 ⁵ Resuspend cells in MC medium at individual cells/mL;

in the first step, no cytokine is added in the first 24 hours, and the cytokine is quantitatively added after 24 hoursThe cell density was maintained at 5X 10 at all times ⁵ individual/mL; the culture can be maintained for at least 2 months by changing the liquid 1-2 times per week depending on the cell culture condition.

Step two, mast cell activation

1. Centrifuging the sample obtained in the step one for 3-5min at room temperature under the condition of 500g centrifugal force, and collecting cells;

2. wash with Tyrode's desktop/0.1% bsa;

3. resuspended in Tyrode's desktop liquid/0.1% BSA, and activated mast cells were incubated with patient serum to be tested in an incubator for 1.5-2.5h while 1 μg/mL DNP-anti-IgE was used as positive control;

4. wash with Tyrode's desktop/0.1% bsa;

5. for allergen stimulation, activated mast cells were resuspended in 100 μl Tyrode's desk top liquid/0.1% bsa, and incubated at 37 ℃ for 30-45min;

step three, histamine determination

And (3) dripping the activated mast cell suspension to be detected obtained in the step (II) on a glass microfiber microtiter 96-well plate coated with an allergen extracting solution, incubating, washing, adding SDS (sodium dodecyl sulfate) for incubation, washing, coupling, performing fluorescence detection, quantitatively calculating the histamine release amount, and judging the anaphylactic degree of a patient.

Further, in the first step, all the adhered adipose tissues were removed from the isolated skin using scissors or a scalpel, and the skin was aseptically weighed, and then placed in a petri dish without adding PBS, cut into 1cm×1cm pieces, and cut in a woven mesh shape at intervals of 1-2mm using a scalpel.

Further, in the first step, the epidermis is removed by forceps, and the non-separated fragments of the epidermis are removed to remove CD117 ⁺ Melanocytes.

In the first step, the enzyme mixture is prepared by the following steps:

filtering and disinfecting collagenase and hyaluronidase respectively, dissolving in a separation culture medium, adding 14-15mg collagenase per gram of skin, adding 6.5-7.5mg hyaluronidase per gram of skin, preheating the whole separation culture medium in a 37 ℃ water bath, and adding 100 mu L of sterile DNase per 100mL of separation culture medium before adding skin homogenate to prepare a culture medium containing an enzyme mixture; 10-12mL of culture medium containing enzyme mixture is added to each gram of skin.

Further, in the first step, the step of filtering the skin and the enzyme mixture is as follows:

filtering the solution in two screens, wherein the mesh diameter of the upper screen is 300 μm, and the mesh diameter of the bottom screen is 100 μm, so as to remove skin residues; the solution was dispensed into a 50mL Falcon tube; centrifuging the filtrate at 4-5deg.C and centrifugal force of 400g for 10-15min; pouring out the enzyme mixture on the upper layer and collecting for reusing the unfiltered residual skin; re-trimming the remaining skin without filtering, and placing into enzyme mixture in water bath for 45-75min.

Further, in the first step, cytokines including hSCF and hIL-4 cells are added to the mast cell medium, no cytokines are added in the first 24 hours of culture, 100-120ng/mL of hSCF and 10-12ng/mL of hIL-4 cells are added after 24 hours, and the cell density is always 5×10 during cell culture ⁵ And each mL.

Further, in the first step, the components of the isolation medium are: 500mL of Ca-containing ²⁺ And Mg (magnesium) ²⁺ 5-6mL penicillin/streptomycin, 10-12mL fetal bovine serum, 4-5mL amphotericin B at a concentration of 2.5-3 μg/mL, and 2.5-3mL magnesium sulfate at a concentration of 4-5mmol/L.

Further, in the first step, the MC medium comprises the following components: 500mL of classical Iscove's medium, 50mL of fetal bovine serum at a concentration of 10-12%, 5-6mL of penicillin/streptomycin solution, 5-6mL of non-essential amino acid solution, and 10-12. Mu.L of alpha-monothioglycerol at a concentration of 11574mol/L.

Further, in step three, incubating the microtiter plate containing the allergen and mast cell suspension at 37 ℃ for 60-75min, washing the microtiter plate with deionized water to ensure that all mast cells are washed away, and carefully removing residues adhered to the inner walls of the plate by means of a cotton swab or facial tissue; 150-175. Mu.L of SDS at 0.4% concentration was added to each well of the microtiter plate, incubated at 37℃for 30-45min to remove allergen substances, followed by washing the microtiter plate with deionized water to ensure removal of all water; 70-75. Mu.L of coupling agent was added to each well of the microtiter plate, the reaction was stopped by room temperature reaction for 10-15min followed by 0.6% HClO4 for subsequent fluorescence detection.

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

the invention discloses a detection method for in vitro mast cell histamine release for allergen detection, which comprises the following steps: the three steps of in vitro isolation of cultured mast cells, induction of activated mast cells and in vitro histamine detection can quantitatively calculate the histamine release amount and evaluate the degree of allergy of patients, and help identify allergens. The method for detecting the histamine release of the in-vitro mast cells for allergen detection provided by the invention has the advantages that the isolated and cultured mast cells can be cultured in vitro for 2 months at most, so that the sample material taking and preparing time are greatly reduced; after the mast cells are induced to release the histamine, the histamine can be tightly adsorbed with the materials on the microtiter plate, can be stored and carried at normal temperature, if the problems of instrument failure, shortage of laboratory conditions and the like exist in experiments, the microtiter plate can be stored in a dark place, and the like, is recovered to be normal or is transferred to a proper laboratory for detection, and is detected after delay, so that the operator is greatly facilitated, and the detection duration and the detection place can be freely arranged at will; different cell holes are formed in the titration plate, so that different allergens can be detected, the relation between the allergens with different concentrations and histamine release can be studied, and the allergens can be rapidly and effectively identified; the histamine is not required to be extracted, the preparation time is short, and the operation is simple and convenient; the fluorescent reagent with higher sensitivity is selected, the detection can be completed by the common enzyme-labeled instrument, no special instrument is needed, and the detection cost is greatly saved, so that the kit is more suitable for routine diagnosis and related clinical and scientific research projects, and has wide clinical and scientific research application values.

Drawings

FIG. 1 is a schematic view of woven screen surface skin after cutting according to the present invention;

FIG. 2 is a schematic diagram of a 96-well plate type detection plate structure of the present invention; FIG. 2A is a negative control, FIG. 2B is a positive control, and FIG. 2C is a sample to be tested;

FIG. 3 is a schematic diagram of the structure of a glass microfiber microtiter 96-well plate test strip coated with an allergen extract of the present invention; FIG. 3a is a food class, FIG. 3b is an inhalation class, and FIG. 3c is an autoantibody class; FIG. 3d shows other allergens;

fig. 4 is a diagram of a material dilution scheme according to the invention.

Detailed Description

The embodiments of the present invention will be described in detail below with reference to the drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making clear and unambiguous the scope of the present invention.

Noun interpretation:

falcon tube: a flow cytometer tube;

MC: mast cells;

sandoglobin: intravenous immunoglobulin.

step one, mast cell isolation

2. transferring to a culture dish, and removing epidermis;

4. filtering the skin and enzyme mixture, and centrifuging the filtrate at 4-5deg.C for 10-15min;

5. gently dispersing the precipitated cells by tapping and re-suspending with sterile cold PBS, transferring into a new test tube, and centrifuging at 4-5deg.C for 10-15min;

6. resuspension the pellet in 3-5mL MACS buffer, filtering, centrifuging at 4-5deg.C for 10-15min, and pouring out MACS buffer;

7. adding AB serum and intravenous immunoglobulin, adding MACS buffer to 1-2mL, and incubating at 4-5deg.C for 10-15min;

8. adding CD117 immunomagnetic beads, and incubating at 4-5deg.C for 10-15min;

9. washing cells with MACS buffer, centrifuging at 4-5deg.C for 10-15min, pouring out MACS buffer, and re-suspending the precipitate in MACS buffer;

11. adding MC culture medium, counting MC and negative cells, obtaining purity and dead cell number, centrifuging at 4-5deg.C for 10-15min, pouring out supernatant, and re-suspending cells in MC culture medium;

in the first step, no cytokine is added in the first 24 hours, and the cytokine is quantitatively added after 24 hours, so that the cell density is kept to be a fixed value; the culture can be maintained for at least 2 months by changing the liquid 1-2 times per week depending on the cell culture condition.

Step two, mast cell activation

1. Centrifuging the sample obtained in the step one at room temperature for 3-5min, and collecting cells;

2. wash with Tyrode's desktop/0.1% bsa;

4. wash with Tyrode's desktop/0.1% bsa;

5. for allergen stimulation, activated mast cells were resuspended in Tyrode's desk-top liquid/0.1% bsa and incubated at 37 ℃ for 30-45min;

step three, histamine determination

Dropping the DNP-anti-IgE activated mast cell suspension obtained in the second step on a glass microfiber micro-titration plate coated with allergen extracting solution, performing fluorescence detection after incubation, washing, SDS incubation, washing and coupling, quantitatively calculating histamine release amount, and judging the anaphylaxis degree of a patient.

In step one, all adherent adipose tissue was removed from the isolated skin using scissors or a scalpel, the skin was then aseptically weighed, and then placed in a petri dish without adding PBS, cut into 1cm×1cm pieces, and cut in a woven mesh-like shape at 1-2mm intervals using a scalpel.

In step one, the epidermis is removed with forceps, and the non-separated pieces of the epidermis are removed to remove CD117 ⁺ Melanocytes.

In the first step, the enzyme mixture is prepared by the following steps:

In the first step, the step of filtering the skin and enzyme mixture is as follows:

In the first step, the cytokines added into the mast cell culture medium comprise human SCF and human IL-4 cells, no cytokines are added in the first 24 hours of culture, 100-120ng/mL of human SCF and 10-12ng/mL of human IL-4 cells are respectively added after 24 hours, and the cell density is always 5 multiplied by 10 in the process of cell culture ⁵ And each mL.

In the first step, the components of the separation medium are as follows: 500mL of Ca-containing ²⁺ And Mg (magnesium) ²⁺ 5-6mL penicillin/streptomycin, 10-12mL fetal bovine serum, 4-5mL amphotericin B at a concentration of 2.5-3 μg/mL, and 2.5-3mL magnesium sulfate at a concentration of 4-5mmol/L.

In the first step, the MC medium comprises the following components: 500mL of classical Iscove's medium, 50mL of fetal bovine serum at a concentration of 10-12%, 5-6mL of penicillin/streptomycin solution, 5-6mL of non-essential amino acid solution, and 10-12. Mu.L of alpha-monothioglycerol at a concentration of 11574mol/L.

Incubating the microtiter plate containing the allergen and mast cell suspension at 37 ℃ for 60-75min, washing the microtiter plate with deionized water to ensure that all mast cells are washed away, and carefully removing residues adhered to the inner wall of the plate by using a cotton swab or facial tissue; 150-175. Mu.L of SDS at 0.4% concentration was added to each well of the microtiter plate, incubated at 37℃for 30-45min to remove allergen substances, followed by washing the microtiter plate with deionized water to ensure removal of all water; 70-75. Mu.L of coupling agent was added to each well of the microtiter plate, reacted at room temperature for 10-15min followed by 0.6% HClO ₄ Terminating the reaction, and storing in a shading way for subsequent fluorescence detection.

Example 1

As shown in fig. 1-4, a method for detecting in vitro mast cell histamine release for allergen detection comprising the steps of:

step one, isolation of mast cells

The first day:

1. removing all adhered adipose tissue from the skin using scissors or a scalpel;

2. aseptically weighing the skin obtained in the step 1;

3. placing skin in a culture dish with diameter of 14cm without PBS, cutting skin into pieces of 1cm×1cm, and cutting with a scalpel at 1-2mm intervals to obtain skin graph shown in figure 1;

4. transferring the fragments to a medium protease containing 2-3mL and 50U/mL and Ca ²⁺ 、Mg ²⁺ In 24-25mL PBS;

5. incubate overnight at 4-5 ℃.

The following day:

6. transferring the sample of step 5 of the first day into a petri dish, removing epidermis with forceps, removing non-separated fragments of epidermis to remove CD117 ⁺ Melanocytes;

7. respectively filtering and sterilizing collagenase and hyaluronidase, dissolving in a separation medium, adding 14-15mg collagenase per gram of skin, adding 6.5-7.5mg hyaluronidase per gram of skin, specifically, filtering in a filter with pore diameter of 0.22 μm before adding into the separation medium, sterilizing, and preheating the whole separation medium in a water bath at 37deg.C; before skin homogenization, 100. Mu.L of sterile DNase with concentration of 10. Mu.g/mL is added to 100mL of culture medium, and DNA agglomeration in damaged cells can be prevented when the culture medium is used together with breast or abdomen skin, so as to prepare culture medium containing enzyme mixture;

8. cutting the skin sample very finely with scissors, the smaller the fragments, the higher the cell yield;

9. transferring the sheared skin to the culture medium containing the enzyme mixture obtained in the step 7, adding 10-12mL of the culture medium containing the enzyme mixture to each gram of skin, incubating for 1-1.5h in a shaking water bath, and setting the shaking frequency to 250-270Hz so that all the skin moves normally and cannot stick together, and avoiding adhesion.

10. After the first separation period, the skin and enzyme mixture (enzyme cocktail) is filtered to avoid excessive time of the skin in the enzyme so as not to reduce the function of the cells, as follows:

filtering the solution in two screens, wherein the mesh diameter of the upper screen is 300 μm, and the mesh diameter of the bottom screen is 100 μm, so as to remove skin residues;

the filtered solution was dispensed into a 50mL Falcon tube;

centrifuging at 4-5deg.C and centrifugal force of 400g for 10-15min; collecting the enzyme mixture at the upper layer for skin in the second separation period, and during centrifugation, pruning the rest skin without filtration, and putting into use;

pouring out the enzyme mixture from the upper layer and collecting the enzyme mixture in the second separation period for re-cutting skin to be reused, and separating the skin as much as 100%; then water bath is carried out for 45-75min;

11. the pelleted cells were gently dispersed by tapping, with sterile cold PBS (Ca free ²⁺ 、Mg ²⁺ ) Resuspended and pooled into a new 50mL capacity Falcon tube, each old Falcon tube was rinsed several times with PBS and pooled into the new tube; fill fresh Falcon tubes with PBS to 50mL at 4-5Centrifuging at 400deg.C for 10-15min.

12. The pellet was resuspended in 3-5mL MACS buffer:

the solution was filtered through a 40 μm filter into a 15mL Falcon tube;

the old Falcon tube was rinsed with 3-5mL MACS buffer and added to a 40 μm filter for re-filtration;

the filtrate was poured into a 30 μm filter and filtered into a new 15mL Falcon tube;

wash old 15mL tube with 3-5mL MACS buffer and add it to 30 μm filter for filtration, then go to step 13;

13. centrifuging at 4-5deg.C under centrifugal force of 400g for 10-15min, and pouring out MACS buffer.

14. 200. Mu.L of type AB serum, 19-20. Mu.L of intravenous immunoglobulin Sandoglobin was added and 1-2mL of MACS buffer was added;

15. incubating at 4-5deg.C for 10-15min;

16. add 50. Mu.L of CD117 immunomagnetic beads;

17. incubating at 4-5deg.C for 10-15min;

18. washing cells with 8-9mL MACS buffer, centrifuging at 4-5deg.C for 10-15min at a centrifugal force of 400g, pouring out the buffer, and then re-suspending the precipitate in 1700-1800 μl MACS buffer;

19. operating a full-automatic immunomagnetic bead technology cell sorting system to sort out mast cells and other negative cells;

20. adding 8-9mL of MC medium, counting MC and negative cells while staining with tololidinblue for purity while staining MC with Trypanblue for dead cell numbers;

21. centrifuging at 4-5deg.C for 10-15min under the condition of centrifugal force of 400g, and collecting supernatant at 5×10 ⁵ Resuspend cells in MC medium at individual cells/mL;

22. then long-term culture was performed, without hSCF and hIL-4 culture cells were added for the first 24 hours, 100-120ng/mL hSCF and 10-12ng/mL hIL-4 were added after 24 hours, fed and counted 2 times a week, and the density was always set to 5X 10 ⁵ Individual cells/mL.

In the first step, the components of the separation medium are:

500mL of Ca-containing ²⁺ 、Mg ²⁺ PBS of (2)

5-6mL penicillin/streptomycin (10.000U/mL, 10.000 μg/mL)

10-12mL fetal bovine serum FBS

4-5mL amphotericin B at a concentration of 2.5-3 μg/mL

2.5-3mL of magnesium sulfate with a concentration of 4-5mmol/L.

The MC medium comprises the following components:

500mL classical Iscove's medium

50mL fetal bovine serum FBS (heat-inactivated, concentration 10-12%)

5-6mL penicillin/streptomycin solution (10.000U/mL, 10.000 μg/mL)

5-6mL of the NEAA 100x non-essential amino acid solution

10-12 mu L of alpha-monothioglycerol with the concentration of 11574mol/L.

Whether 1.25-1.5mg amphotericin B is added or not can be selected according to actual requirements in the MC culture medium, and the flexibility and the practicability are high.

The MACS running buffer had the following composition:

500mL of calcium-magnesium free PBS

25mL sterile fetal bovine serum FBS

5-6mL amphotericin B (2.5 μg/mL)

5-6mL penicillin/streptomycin (10.000U/mL, 10.000 μg/mL).

The MACS storage solution had the following composition: 500mL of PBS without Ca, mg+0.1% azide.

The sources and types of raw materials used in the present invention are shown in table 1.

TABLE 1

Step two, inducing mast cell activation

1. Centrifuging the sample obtained in the step one for 3-5min at room temperature under the centrifugal force of 500g, and collecting mast cells;

2. wash with Tyrode's desktop/0.1% bsa;

3. resuspended in Tyrode's desktop/0.1% BSA and mast cells activated with patient C serum and 1 μg/mL DNP-anti-IgE in an incubator for 1.5-2.5h;

4. again using Tyrode's desktop/0.1% bsa wash;

DNP-anti-IgE is stored at-20 ℃;

tyrode's desk top liquid (Ca ²⁺ The composition of +) is shown in table 2.

TABLE 2

Reagent(s)	Concentration of stock solution		Final concentration
				Sodium chloride	5mol/L	27mL	135mmol/L
Potassium chloride	1mol/L	5mL	5mmol/L
				Magnesium chloride	1mol/L	1mL	1mmol/L
Calcium chloride	100mmol/L	18mL	1.8mmol/L
				Glucose		1g	5.6mmol/L
HEPES buffer	1mol/L	20mL	20mmol/L
				Bovine serum albumin BSA		0.5g	0.05％
Ultra light water DDW	pH＝7.4	1000mL

Step three, histamine determination

The suspension of the mast cells which are activated by the serum of the patient C and DNP-anti-IgE obtained in the step two is dripped on a micro-titration plate coated with glass microfiber of allergen extracting solution, histamine released by the mast cells is adsorbed on a glass microfiber substrate with high affinity and selectivity, fluorescence detection is carried out after incubation, washing, SDS incubation, washing and coupling, the histamine release amount is quantitatively calculated, the anaphylaxis degree of the patient is judged, different standard plates can be used for classifying the results into different grades from 0 to 10, and the test of the sensitized substances can be classified as positive or negative.

The scoring system is based on the lowest allergen concentration that can induce histamine release.

Prior to fluorescence testing, allergen coated standard plates, allergen coated standard strips, calibration plates, coupling agent, pipes buffer, HAS at 20% concentration, IL-3 at 10ng/mL, naOH at 0.05M, HClO at 0.6% ₄ SDS, deionized water and distilled water with concentration of 0.4%.

Coupling reagents corresponding to the number of plates to be analyzed (50 mL of reagent corresponds to 5 plates) were prepared:

before use, the coupling agent must be soaked in the diluent for at least 45 minutes and then mixed with stirring. Note that the coupling agent is insoluble in the diluent. By detecting the calibration plate in the allergen detector, the coupling agent can be calibrated in advance, and the coupling agent can only be used on the same day.

IL-3 factor freeze-dried is stored in test tubes at-20deg.C, each test tube contains 0.1-0.2 μg IL-3 factor, and IL-3 factor is used for enhancing histamine release of mast cells. The IL-3 factor is prepared by the following steps:

each tube contains 0.1-0.2 mug IL-3 freeze-dried powder, and 1000 mug Pipes buffer solution is added to prepare 1 bottle;

15-25uL IL-3 factor is added per 1mL mast cell suspension, carefully mixed 5-10 times or mixed on a shaker.

Standard plate and extract preparation, comprising the steps of:

(1) Standard panel

Allergens are pre-coated and stored in standard panels in glycerol, classified as three standard panels of inhalation, food, autoantibody class, stored at 5 ℃, as shown in figures 3a-3 d; standard panels were reconstituted by adding 25 μl of pins buffer per well prior to incubation with mast cell suspension.

(2) Standard belt

Pre-coated with an allergen and stored in glycerol in the form of strips (microwells 1X 8) and stored at 5 ℃; the strips are snapped into a frame and marked with the correct identification. Wells were reconstituted by adding 25 μl of buffer to each well prior to incubation with the patient mast cell suspension, as shown in figure 2.

(3) A single allergen cannot be used for standard plates and standard bands.

Some allergens are unstable after being coated and dried onto a plate or strip and are therefore lyophilized and stored in vials at-20 ℃. Upon analysis, the allergen is reconstituted and transferred to the material strip:

the strip of material enters the frame; add 25. Mu.L buffer per well to the material strip; priming according to the volume indicated on the label; pipetting 10 μl allergen into the E well; mix and transfer 10 μl from E well to F well; mix and transfer 10 μl from F well to G well; mix and transfer 10 μl from G well to H well; mix and transfer 10 μl from H-well into waste as shown in fig. 2.

Non-standardized allergen materials (e.g. environmental samples, pharmaceuticals, fresh foods) may contain different amounts of allergens, as shown in fig. 3a-3d, and thus the amount of allergen extracted may also be different. To reduce errors caused by these factors, a range of dilutions, preferably 12 dilutions, must be used for testing.

Extracting solid material or dust or hair sample for 30-45min, and adjusting extract to 100mg in 1mL buffer solution at room temperature; after extraction, the liquid phase is removed and used at maximum concentration C1.

The tablet medicine contains 50-500mg of active compound. The tablets were crushed and suspended in 5mL buffer, followed by 30-45min of slurry extraction, and then the samples were centrifuged, the liquid phase removed and used at maximum concentration C1.

Based on the assumption that the active ingredient in the tablet drug is temporarily distributed in the blood compartment (5L), the maximum concentration C1 is 1000 times stronger than the concentration actually exposed by the patient, and a very wide test range can be ensured by using 12 concentrations (3.5 times dilution).

Crushing 100-120mg of fresh food in 1-2mL buffer, extracting crushed food for 30-45min, and removing liquid phase for use at maximum concentration of C1.

The liquid drug (i.e., injectable drug or other liquid form) is diluted with buffer at a ratio of 1:10, with a maximum concentration of C1.

Based on the maximum concentration C1, the material was diluted (4-fold dilution) 12 times in total and the dilution scheme is shown in fig. 4.

Subsequently 25 μl of the above allergen dilutions were transferred to the assay 96 wells, as shown in table 3 below, table 3 is a configuration table of 96 well plates for testing one allergen material/sample, table 4 is a configuration table of 96 well plates for testing two allergen materials/samples, where Al-C1, al-C2, al-C3 represent different concentrations of allergens.

TABLE 3 Table 3

TABLE 4 Table 4

To the remaining wells (columns 1 and 2 and rows a and B) 25 μl buffer was added per well. Note that:

column 1: column histamine criteria (used to calculate histamine in unknown samples) and column 2: anti-IgE (positive control). Row a and row B: corresponding to the background of spontaneous release.

For multiple allergens, up to five allergens can be transferred to one plate. Two identical plates were prepared: one for patient testing and the other for negative control.

In the third step, the incubation step is as follows: incubating a 96-well plate containing allergen and mast cell suspension at 37 ℃ for 1-1.5h;

the washing steps are as follows: washing the microtiter plate with deionized water and washing with a hand-held plate washer or ELISA plate washer to ensure that all mast cells are washed away and that residues adhering to the inner walls of the plate can be carefully removed with a cotton swab or facial tissue;

analysis pauses: at this stage of the analysis, the fluorescence detection may be stopped, or the histamine detection may be delayed. 96-well plates can be stored in the dark at room temperature for up to 1 month without affecting the histamine binding to glass microfibers. Note that 96-well plates should be placed in plastic bags to remain dry;

the test is completed: adding 150-175 μl SDS at 0.4% concentration into each well of 96-well plate, incubating at 37deg.C for 30-45min, and removing binding substances; washing the 96-well plate with deionized water, and inverting the 96-well plate on the adsorption paper to ensure that all water is removed;

the coupling steps are as follows: 70-75. Mu.L of coupling agent was added to each well of the microtiter plate, reacted at room temperature for 10-15min, and then added with 0.6% HClO ₄ After the reaction is terminated and the coupling step is completed, the operation should be protected from light, and then fluorescence detection can be performed by a common enzyme-labeled instrument, the amount of histamine release can be quantitatively calculated, and the degree of allergy of the patient can be evaluated and the allergen can be identified.

The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes or direct or indirect application in other related technical fields are included in the scope of the present invention.

Claims

1. A method for detecting histamine release from a mast cell in vitro for non-diagnostic purposes, comprising the steps of:

step one, mast cell isolation

Removing fat tissue adhered on the isolated skin, cutting into pieces, and transferring into gel containing neutral protease and Ca ²⁺ And Mg (magnesium) ²⁺ Incubation overnight at 4-6deg.C in PBS;

removing epidermis, continuously cutting skin, transferring the cut skin into a culture medium containing enzyme mixture, and incubating for 1-1.5h under shaking;

filtering the skin and enzyme mixture, and centrifuging at 4-5deg.C;

adding AB serum and intravenous immunoglobulin, adding MACS buffer to 1-2mL, and incubating at 4-5deg.C for 10-15min;

adding CD117 immunomagnetic beads, and incubating at 4-5deg.C for 10-15min;

washing the cells, centrifuging, and resuspending the pellet in buffer;

operating an immunomagnetic bead cell sorting system to sort out mast cells and other negative cells;

step two, mast cell activation

Centrifuging the sample obtained in the step one at room temperature, collecting mast cells, washing and suspending in Tyrode's desk top liquid/0.1% BSA;

mast cells were incubated with test serum in an incubator for 1.5-2.5h, followed by washing with Tyrode's desk top/0.1% BSA;

for allergen stimulation, activated mast cells were resuspended in Tyrode's desk-top liquid/0.1% bsa and incubated for 30-45min;

step three, histamine determination

Dropping the activated mast cell suspension obtained in the step two on a glass microfiber micro-titration plate coated with an allergen extracting solution, incubating, washing, adding SDS for incubation, washing, coupling, performing fluorescence detection, and quantitatively calculating the histamine release amount;

2. The method according to claim 1, wherein in the first step, all adhered adipose tissues are removed from the isolated skin using scissors or a scalpel, the skin is aseptically weighed again, then placed in a petri dish without PBS, sheared into 1cm x 1cm pieces, cut into a woven screen surface shape at 1-2mm intervals using a scalpel, and the non-separated pieces of epidermis are removed using forceps after incubation to remove CD117 ⁺ Melanocytes.

3. The method according to claim 1, wherein in the first step, the step of filtering the skin and the enzyme mixture comprises:

filtering the solution in two screens, wherein the mesh diameter of the upper screen is 300 mu m, and the mesh diameter of the lower screen is 100 mu m, so as to remove skin residues; the solution was dispensed into a 50mL Falcon tube; centrifuging the filtrate at 4-5deg.C and centrifugal force of 400g for 10-15min; pouring out the enzyme mixture on the upper layer and collecting for reusing the unfiltered residual skin; re-trimming the remaining skin without filtering, and placing into enzyme mixture in water bath for 45-75min.

4. The method according to claim 1, wherein after filtering the skin and enzyme mixture, centrifuging the filtrate at 4-5 ℃ for 10-15min, then gently scattering the precipitated cells and resuspension with sterile cold PBS, transferring into a new tube, and centrifuging at 4-5 ℃ for 10-15min; the pellet was resuspended in 3-5mL MACS buffer, filtered, centrifuged at 4-5℃for 10-15min, the MACS buffer was decanted, and AB serum and intravenous immunoglobulin were added.

5. The method according to claim 1, 3 or 4, wherein the enzyme mixture is obtained by the following steps:

6. The method according to claim 1, wherein in the first step, after adding CD117 immunomagnetic beads and incubating, adding MACS buffer to wash cells, centrifuging at 4-5 ℃ for 10-15min, pouring out MACS buffer, then resuspending the pellet in MACS buffer, and then running immunomagnetic bead cell sorting system to sort out mast cells and other negative cells; adding MC culture medium, counting MC and negative cells, obtaining purity and dead cell number, centrifuging at 4-5deg.C for 10-15min, pouring out supernatant, and re-suspending cells in MC culture medium.

7. The method according to claim 1, wherein in the first step, the cytokines added to the mast cell medium include human SCF and human IL-4 cells, no cytokines are added for the first 24 hours of the culture, 100-120ng/mL human SCF and 10-12ng/mL human IL-4 cells are added after 24 hours, respectively, and the cell density is always 5X 10 during the cell culture ⁵ The cell density is kept to be a fixed value; the culture can be maintained for at least 2 months by changing the liquid 1-2 times per week depending on the cell culture condition.

8. The method according to claim 6, wherein in the first step, the MC medium comprises the following components: 500mL of classical Iscove's medium, 50mL of fetal bovine serum with the concentration of 10-12%, 5-6mL of penicillin/streptomycin liquid, 5-6mL of non-essential amino acid solution and 10-12 mu L of alpha-monothioglycerol with the concentration of 11574mol/L.