CN110501490A - A kind of isotonic hemolysin and preparation method thereof, processing biological sample method and detection leucocyte membranous antigen method - Google Patents
A kind of isotonic hemolysin and preparation method thereof, processing biological sample method and detection leucocyte membranous antigen method Download PDFInfo
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- CN110501490A CN110501490A CN201910866938.4A CN201910866938A CN110501490A CN 110501490 A CN110501490 A CN 110501490A CN 201910866938 A CN201910866938 A CN 201910866938A CN 110501490 A CN110501490 A CN 110501490A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1404—Fluid conditioning in flow cytometers, e.g. flow cells; Supply; Control of flow
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1434—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its optical arrangement
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- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
Abstract
This isotonic hemolysin, including boric acid-sodium tetraborate, cosolvent, propylene glycol, inorganic salts and water quality, buffer include, and the content of boric acid is 0.2-2mol/L, sodium tetraborate content is 0.05-0.5mol/L；Cosolvent is glycerol or formaldehyde, and content is 10-40g/L；Propylene glycol, and its content is 0.2-2mol/L；Inorganic salts include sodium chloride, magnesium chloride and calcium chloride, and the content of sodium chloride is 1-100mmol/L, and the content of magnesium chloride is 1-100mmol/L, and the content of calcium chloride is 1-100mmol/L.The preparation method, hemolysin processing biological sample method and hemolysin for proposing the hemolysin simultaneously pass through the method for flow cytomery leucocyte membranous antigen, this hemolysin, detection accuracy is high, and interference impurity is few, and stability is high, is easy to save.Each group's subregion of leucocyte is obvious after handling sample, and cell fragment is few, does not damage leucocyte memebrane protein, can retain original biological activity and complete splitting erythrocyte.The hemolysin that import original product can be substituted reaches import reagent production domesticization, reduces scientific research and medical treatment cost.
Description
Technical field
The present invention relates to immunological cells analysis fields, and in particular to a kind of isotonic hemolysin and preparation method thereof, processing
Biological sample method and detection leucocyte membranous antigen method.
Background technique
Crowd do it is known, blood cell analysis for carry out scientific research, the prevention of disease, diagnosing and treating have important valence
Value, from the form, size, quantity of original research cell to the antigenic component of nearest cell surface.Traditional cellular immunity and
Non-specific immune function detection technique cannot be from individual cell level to the size of target cell, form, plasma membrane and cell interior knot
Structure carries out multi-parameter, highly sensitive analysis, and flow cytometer can be floated on a liquid with rapid survey, sorting, storage, display
A series of important biophysics of cell dispersion, the characteristic parameter in terms of biochemistry, and can be according to the parameter of pre-selection
Range therefrom sorts out specified cell subsets, is applied to immunology, hematology, oncology, cytogenetics, thin at present
The clinical medicine such as born of the same parents' biology, biochemistry and basic medical research field, lymphocyte and its Asia such as cell surface molecule
Cluster analysis, intracellular cytokine assay, leukaemia immunophenotyping, the analysis of tumor drug resistance GAP-associated protein GAP, autoimmune disease
Related HLA antigenic analysis, the crossmatch in trnasplantion immunity, mitochondrial membrane potential, Apoptosis, intracellular calcium concentration,
Functions of neutrophils analysis etc..
Majority due to playing immune function in vivo is leucocyte, and in human blood content it is most be red blood cell, be
Reducing influences target cell assay, needs to reject the interference effect of red blood cell in blood, to keep away when carrying out erythrocyte hemolysis
Exempt from antileukocytic structure and function, retain the memebrane protein of leukocyte surface, therefore, requirement to haemolysis very it is high, it is existing
Some fluidic cell hemolysins are mostly hypotonic haemolysis element, that is, are lower than the solution of osmotic pressure, and isotonic solution hemolysin is and osmotic pressure phase
Deng solution, have previously been thought that red blood cell in isotonic solution, be able to maintain its normal morphology (not shrinkage, not haemolysis), but some point
Son can also pass freely through cell membrane, such as 1.9% urea liquid, wherein can haemolysis immediately by red blood cell merging.
The formula and instrument of hemolysin have certain relationship, and different instruments uses different hemolysins, same hemolysin
The effect shown on different instruments is different, and main cause is that the WBC Appearance after hemolysin cracking is variant, this difference combines
The light path design of FSC/SSC will lead to the difference that lymph, monokaryon, granulocyte divide group.
In view of this, the present inventor furthers investigate in view of the above-mentioned drawbacks in the prior art, there is this case generation then.
Summary of the invention
In order to solve the above technical problems, being mentioned simultaneously we have proposed a kind of isotonic hemolysin hemolysin and preparation method thereof
Hemolysin processing biological sample method is gone out and hemolysin passes through the method for flow cytomery leucocyte membranous antigen, this hair
Bright hemolysin, detection accuracy is high, and interference impurity is few, can substitute the hemolysin of import similar product, reduce Hospital medical
Cost mitigates Medical Expense for Patients.
In order to achieve the above objectives, technical scheme is as follows:
A kind of isotonic hemolysin, including buffer, cell cosolvent, cell solubilizing agent, inorganic salts and water quality, the buffering
Agent includes boric acid and sodium tetraborate, and the content of boric acid is 0.2-2mol/L, and sodium tetraborate content is 0.05-0.5mol/L；Institute
The one or two that cell cosolvent is glycerol, formaldehyde are stated, and cell hydrotropy agent content is 0.3-3mol/L, content of formaldehyde is
10-40g/L；The cell solubilizing agent is propylene glycol, and its content is 0.2-2mol/L；The inorganic salts include sodium chloride, chlorine
Change magnesium and calcium chloride, and the content of sodium chloride is 1-100mmol/L, the content of magnesium chloride is 1-100mmol/L, and calcium chloride contains
Amount is 1-100mmol/L, with the conductivity and osmotic pressure for adjusting hemolysin.
Boric acid and sodium tetraborate mixed liquor therein, simple boric acid hemolyzing effect is poor under the conditions of isotonic, and sodium tetraborate is not
Has haemocylolysis, the two has good hemolyzing effect after mixing in proportion, and has certain buffer function.In borate
Under the adjusting of buffer, guarantee that hemolysin under the variation of temperature, there is a stable pH value and a longer stationary phase.It is sweet
The quick lysed erythrocyte of the ingredients such as oil energy, has protective effect to leucocyte, and glycerol can also reduce the activity of water, prevents degradation from protecting
Soluble albumen increases stability, volatilization prevention.There are also the effect of fixed cell, formalins to save to most of antigens for formaldehyde
Preferably, penetration into tissue is good, and tissue contracts are smaller, but formaldehyde effumability and with certain toxicity.
Preferably, the hemolysin further include micro cell preservative be one of Proclin300, Sodium azide or
Two kinds；And its content is 0.1-0.5ml/L, the advantage is that stabilization, at low cost, long action time.
Preferably, the buffer be boric acid-sodium tetraborate, phosphate, Tris-HCL buffer it is one or more, and
Its content is 0.25-2.5mol/L.
Preferably, the cell solubilizing agent is one or more, and its content of propylene glycol, ethyl alcohol, isobutanol, diethylene glycol (DEG)
For 0.2-2mol/L.
A kind of preparation method of isotonic hemolysin, comprising the following steps:
1) by it is above-mentioned containing measure boric acid, sodium chloride, calcium chloride, magnesium chloride be added aqueous medium sufficiently dissolve, add formaldehyde,
Glycerol, propylene glycol, Proclin 300 obtain boric acid mixed liquor (A liquid).
2) aqueous medium is added by the above-mentioned sodium tetraborate containing measurement, sodium chloride, calcium chloride, magnesium chloride sufficiently to dissolve, adds
Formaldehyde, glycerol, propylene glycol, Proclin 300 obtain sodium tetraborate mixed liquor (B liquid).
3) A liquid and B liquid are mixed well in proportion, pH value 7.0-8.0 is adjusted to obtain hemolysin using filter.
A kind of isotonic hemolysin processing biological sample method, comprising the following steps: institute is added into biological sample in room temperature
The hemolysin stated, mixed processing 10-15 minutes；The biological sample includes one kind of whole blood, peripheral blood, anti-coagulants venous whole
Or it is a variety of；The volume ratio of the hemolysin and biological sample is 0.5-1.5: 20.
A kind of method that isotonic hemolysin passes through flow cytomery leucocyte membranous antigen, comprising the following steps:
1) fluorescent labeled antibody of detection cell surface molecule is added into sample to be tested in room temperature, mixes, is protected from light incubation
10-15min；
2) hemolysin described in claim is added, mixed processing is protected from light and is incubated for 10min；
3) treated sample to be tested centrifuge is precipitated into sample 5-10min with the centrifugal speed of 300-500g；
4) after the sample after centrifugal treating being removed supernatant, PBS buffer solution is added, mixes sample, it is to be measured；
5) sample to be tested is through flow cytometry analysis, by cell volume size and granularity be divided into lymphocyte populations,
Monocyte group, neutrophil leucocyte group.
Hemolysin of the invention can pass through forward angle (SSC) on flow cytometer and side according to cell size and granularity
Leukocyte population is divided into lymphocyte, monocyte, neutrophil leucocyte and fragment to angle scattering light (FSC).Handle sample
Each group's subregion of leucocyte is obvious afterwards, and cell fragment is few, does not damage leucocyte memebrane protein, and it is living can to retain original biology
Property, and can complete splitting erythrocyte.Processing result is accurate, performance is stable, can substitute the hemolysin of import original product, reach into
Opening reagent production domesticization, reduces scientific research and medical treatment cost.Hemolysin of the invention has well on the mainstreams analyzer such as BD series
Divide group's map, and stability is high, is easy to save.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with
It obtains other drawings based on these drawings.
Fig. 1 is a kind of isotonic hemolysin disclosed in the embodiment of the present invention in BD company FACS Calibuar fluidic cell
Instrument hemolyzing effect figure (one)；
Fig. 2 is a kind of isotonic hemolysin disclosed in the embodiment of the present invention in BD company FACS Calibuar fluidic cell
Instrument hemolyzing effect figure (two)；
Fig. 3 is a kind of isotonic hemolysin FACS Calibuar flow cytomery T leaching disclosed in the embodiment of the present invention
Bar Immunophenotyping analysis chart (one)；
Fig. 4 is a kind of isotonic hemolysin FACSCalibuar flow cytomery T leaching disclosed in the embodiment of the present invention
Bar Immunophenotyping analysis chart (two)；
Fig. 5 is a kind of isotonic hemolysin FACS Calibuar flow cytomery T leaching disclosed in the embodiment of the present invention
Bar Immunophenotyping analysis chart (three)；
Fig. 6 is a kind of isotonic hemolysin FACSCalibuar flow cytomery T leaching disclosed in the embodiment of the present invention
Bar Immunophenotyping analysis chart (four).
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, the test method that actual conditions are preferably not specified in following examples usually according to normal condition lift implementation
Example is to preferably be illustrated to the contents of the present invention, it is clear that described embodiment is only that present invention a part is real
Example is applied, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not making creation
Property labour under the premise of every other embodiment obtained, shall fall within the protection scope of the present invention.
Below with reference to embodiment and specific embodiment, the present invention is described in further detail.
As shown in figures 1 to 6, a kind of isotonic hemolysin, including buffer, cell cosolvent, cell solubilizing agent, inorganic salts and
Water quality, the buffer includes boric acid and sodium tetraborate, and the content of boric acid is 0.2-2mol/L, and sodium tetraborate content is
0.05-0.5mol/L；The cell cosolvent is the one or two of glycerol, formaldehyde, and cell hydrotropy agent content is 0.3-
3mol/L, content of formaldehyde 10-40g/L, cell glycerol can reduce the activity of water in hemolysin, prevent degradation protection soluble
Albumen and leucocyte increase stability, volatilization prevention；The cell solubilizing agent is propylene glycol, and its content is 0.2-2mol/L；Institute
Stating inorganic salts includes sodium chloride, magnesium chloride and calcium chloride, and the content of sodium chloride is 1-100mmol/L, and the content of magnesium chloride is
The content of 1-100mmol/L, calcium chloride are 1-100mmol/L, with the conductivity and osmotic pressure for adjusting hemolysin.
Boric acid and sodium tetraborate mixed liquor therein, simple boric acid hemolyzing effect is poor under the conditions of isotonic, and sodium tetraborate is not
Has haemocylolysis, the two has good hemolyzing effect after mixing in proportion, and has certain buffer function.In borate
Under the adjusting of buffer, guarantee that hemolysin under the variation of temperature, there is a stable pH value and a longer stationary phase.It is sweet
The quick lysed erythrocyte of the ingredients such as oil energy, has protective effect to leucocyte, and glycerol can also reduce the activity of water, prevents degradation from protecting
Soluble albumen increases stability, volatilization prevention.There are also the effect of fixed cell, formalins to save to most of antigens for formaldehyde
Preferably, penetration into tissue is good, and tissue contracts are smaller, but formaldehyde effumability and with certain toxicity.
Wherein, it is one of Proclin 300, Sodium azide or two that the hemolysin, which further includes micro cell preservative,
Kind；And its content is 0.1-0.5ml/L, the advantage is that stabilization, at low cost, long action time.The buffer is boric acid-four
Boratex, phosphate, Tris-HCL buffer one or more, and its content 0.25-2.5mol/L.The cell solubilizing agent
For propylene glycol, ethyl alcohol, isobutanol, diethylene glycol (DEG) it is one or more, and its content be 0.2-2mol/L.
The preparation method of isotonic hemolysin in this example, comprising the following steps:
S1, it is sufficiently dissolved by the above-mentioned boric acid containing measurement, sodium chloride, calcium chloride, magnesium chloride addition aqueous medium, adds first
Aldehyde, glycerol, propylene glycol, Proclin 300 obtain boric acid mixed liquor (A liquid)；
S2, it sufficiently dissolves, adds by the above-mentioned sodium tetraborate containing measurement, sodium chloride, calcium chloride, magnesium chloride addition aqueous medium
Formaldehyde, glycerol, propylene glycol, Proclin 300 obtain sodium tetraborate mixed liquor (B liquid)；
S3, A liquid and B liquid are mixed well in proportion, pH value 7.0-8.0 is adjusted to obtain hemolysin using filter.
Isotonic hemolysin in this example handles biological sample method, comprising the following steps: adds in room temperature into biological sample
Hemolysin described in entering, mixed processing 10-15 minutes；The biological sample includes whole blood, peripheral blood, anti-coagulants venous whole
It is one or more；Volume ratio 0.5-1.5: 20 of the hemolysin and biological sample.
The method that the medium vadose solution sanguinin of this example passes through flow cytomery leucocyte membranous antigen, comprising the following steps:
S1, the fluorescent labeled antibody that detection cell surface molecule is added into sample to be tested in room temperature, mix, are protected from light incubation
10-15min；
S2, hemolysin described in claim is added, mixed processing is protected from light and is incubated for 10min；
S3, treated sample to be tested centrifuge is precipitated into sample 5-10min with the centrifugal speed of 300-500g；
S4, after the sample after centrifugal treating is removed supernatant, PBS buffer solution is added, mixes sample, it is to be measured；
S5, sample to be tested through flow cytometry analysis, by cell volume size and granularity be divided into lymphocyte populations,
Monocyte group, neutrophil leucocyte group.
Embodiment:
| _ | A liquid | B liquid | Unit |
| Boric acid | 0.2-2 | mol/L | |
| Boratex | 0.05-0.5 | mol/L | |
| Glycerol | 0.3-3 | 0.3-3 | mol/L |
| Formaldehyde | 10-40 | 10-40 | g/L |
| Propylene glycol | 0.2-2 | 0.2-2 | mol/L |
| Magnesium chloride | 1-100 | 1-100 | mmol/L |
| Calcium chloride | 1-100 | 1-100 | mmol/L |
| Sodium chloride | 1-100 | 1-100 | mmol/L |
| Proclin300 | 0.1-0.5 | 0.1-0.5 | ml/L |
Example one: measuring A liquid 900ml, B liquid 100ml, is uniformly mixed, and adjusts pH value 7.1, is flowed with BD FACS Calibuar
Formula cell instrument detects 15 parts of blood samples, every group blood examination 2 times, record experimental result, and the FACSTM with BD company
The actually detected result of LysinqSolution carries out correlation analysis.
Example two: measuring A liquid 800ml, B liquid 200ml, is uniformly mixed, and adjusts pH value 7.6, is flowed with BD FACS Calibuar
Formula cell instrument detects 15 parts of blood samples, every group blood examination 2 times, record experimental result, and the FACSTM with BD company
The actually detected result of LysinqSolution carries out correlation analysis.
Example three: measuring A liquid 700ml, B liquid 300ml, is uniformly mixed, and adjusts pH value 7.9, is flowed with BD FACS Calibuar
Formula cell instrument detects 15 parts of blood samples, every group blood examination 2 times, record experimental result, and the FACSTM Lysing with BD company
The actually detected result of Solution carries out correlation analysis.
Experimental subjects and reagent: the peripheral blood sample EDTA of 15 parts of health and sub-health population, heparin, sodium citrate are anti-
Solidifying, (18~25 DEG C) of room temperature save, and detection is completed within 8h.Accuracy estimating uses FACS Calibuar flow cytometer,
Matched reagent is four color reagent CD45-PerCP-Cy5.5/CD4-FITC/ of T lymph subsets counts
CD8-APC/CD3-PE, mating hemolysin are the FACSTM LysingSolution of BD company.
Each component is accurately weighed according to recipe requirements, uses pure water constant volume after successively dissolving, with 0.22um filtering with microporous membrane,
Hemolysin base fluid A and B of the present invention are obtained, A liquid and B liquid are mixed according to different ratios respectively, obtain different PH
Three parts of hemolysins.
Experimental method: take 10 μ l CD45-PerCP-Cy5.5/CD4-FITC/CD8-APC/CD3-PE antibody that streaming is added
In loading pipe, then with method 100 μ l blood samples of addition are reversely loaded, room temperature, which is protected from light, after mixing is incubated for 10-15min；Respectively plus certainly
Hemolysin processed and mating 1000 μ l of hemolysin FACSTM Lysing Solution (1X), room temperature is protected from light haemolysis after concussion mixes
10min；Treated sample to be tested centrifuge is precipitated into sample 5-10min with the centrifugal speed of 300-500g；At centrifugation
After sample after reason removes supernatant, PBS buffer solution is added, mixes well and completes upper machine testing in rear 1h.
Degree and effect are as follows after hemolysin haemolysis of the invention:
1, haemolysis tube exterior: supernatant is limpid bright, and erythrocyte splitting is abundant；
2, tube bottom after centrifugation: haemolysis pipe is centrifuged after ten minutes through 300g, and the red precipitate that bottom of the tube is visible by naked eyes is red
Cell fragment is few.
3, FSC/SSC scatter plots: each series of cell divide group obvious, distinct, cell aggregation degree it is high, without intersection (see figure
Hemolyzing effect figure shown in 1-2).
4, cell surface marker: each system's cell membrane surface T cell differentiation antigen is had no significant effect (see Fig. 3-6 phenotypic analysis figure).
Hemolysin of the present invention is in BD company FACS Calibuar flow cytometer hemolyzing effect figure such as Fig. 1-2；Invention haemolysis
Plain FACS Calibuar flow cytomery T lymphocyte Immunophenotype analysis figure such as Fig. 3-6.
Invention hemolysin and the FACSTM Lysing Solution hemolysin of BD company detect T lymphocyte immunophenotype
As a result correlation analysis (%) is as follows:
Hemolysin of the invention can pass through forward angle (SSC) on flow cytometer and side according to cell size and granularity
Leukocyte population is divided into lymphocyte, monocyte, neutrophil leucocyte and fragment to angle scattering light (FSC).Handle sample
Each group's subregion of leucocyte is obvious afterwards, and cell fragment is few, does not damage leucocyte memebrane protein, and it is living can to retain original biology
Property, and can complete splitting erythrocyte.Processing result is accurate, performance is stable, can substitute the hemolysin of import original product, reach into
Opening reagent production domesticization, reduces scientific research and medical treatment cost.
The formula and instrument of hemolysin have certain relationship, and different instruments uses different hemolysins, same hemolysin
The effect shown on different instruments is different, and main cause is that the WBC Appearance after hemolysin cracking is variant, this difference combines
The light path design of FSC/SSC will lead to the difference that lymph, monokaryon, granulocyte divide group.Hemolysin of the invention is main in BD series etc.
There is good point of group's map in flow point analyzer, and stability is high, is easy to save.
Hemolysin in this case is one of the common assistant analysis reagent of flow cytometry detection leukocyte antigen, and
The key reagents of the analyses such as cellular immune function.Boric acid and sodium tetraborate mixed liquor in this technology, simple boron under the conditions of isotonic
Sour hemolyzing effect is poor, and sodium tetraborate does not have haemocylolysis, and the two has good hemolyzing effect after mixing in proportion, and has
Certain buffer function.Under the adjusting of borate buffer solution, guarantee that hemolysin under the variation of temperature, there is a stable PH
Value and a longer stationary phase.The quick lysed erythrocyte of the ingredients such as the glycerol in hemolysin energy, has protective effect to leucocyte,
Glycerol can also reduce the activity of water, and the albumen for preventing degradation protection soluble increases stability, volatilization prevention.In the hemolysin
Cell solubilizing agent has very high absorption and penetrating power to biomembrane, and biomembrane solubilising and infiltration is caused to sexually revise.In hemolysin
Inorganic salts sodium chloride, magnesium chloride, calcium chloride are used to adjust the conductivity and osmotic pressure in hemolysin.
Pass through the forward angle (SSC) and side scatter light (FSC) on flow cytometer according to cell size and granularity
Leukocyte population is divided into lymphocyte, monocyte, neutrophil leucocyte and fragment.After hemolysin processing sample of the invention
Each group's subregion of leucocyte is obvious, and cell fragment is few, does not damage leucocyte memebrane protein, can retain original biological activity,
Again can hemolysin described in splitting erythrocyte completely, as a result accurately, performance stablize, the hemolysin of import original product can be substituted,
Reach import reagent production domesticization, reduces scientific research and medical treatment cost.To reach novel in design, formula collocation is reasonable and applies
The good purpose of effect.
Hemolysin of the present invention belongs to isotonicity haemolysis, and these two types of hemolysin haemolysis principles are in accordance with intraor extracellular osmotic pressure
Difference and lead to membranolysis, and utilize red blood cell and the brittle difference of leukocyte infiltration, before protecting WBC Appearance
It puts, makes erythrocyte splitting to greatest extent.There is good point of group on the mainstreams analyzers such as COULTER series, BD series
Map, and stability is high, is easy to save.
What has been described above is only a preferred embodiment of the present invention, it is noted that for those of ordinary skill in the art
For, without departing from the concept of the premise of the invention, various modifications and improvements can be made, these belong to the present invention
Protection scope.
Claims (7)
1. a kind of isotonic hemolysin, which is characterized in that including buffer, cell cosolvent, cell solubilizing agent, inorganic salts and water
Matter, the buffer includes boric acid and sodium tetraborate, and the content of boric acid is 0.2-2mol/L, and sodium tetraborate content is 0.05-
0.5mol/L；The cell cosolvent is the one or two of glycerol, formaldehyde, and cell hydrotropy agent content is 0.3-3mol/L,
Content of formaldehyde is 10-40g/L；The cell solubilizing agent is propylene glycol, and its content is 0.2-2mol/L；The inorganic salts include
Sodium chloride, magnesium chloride and calcium chloride, and the content of sodium chloride is 1-100mmol/L, the content of magnesium chloride is 1-100mmol/L,
The content of calcium chloride is 1-100mmol/L.
2. a kind of isotonic hemolysin according to claim 1, which is characterized in that the hemolysin further includes micro cell
Preservative is one or both of Proclin 300, Sodium azide；And its content is 0.1-0.5ml/L.
3. a kind of isotonic hemolysin according to claim 2, which is characterized in that the buffer is boric acid-tetraboric acid
Sodium, phosphate, Tris-HCL buffer it is one or more, and its content be 0.25-2.5mol/L.
4. a kind of isotonic hemolysin according to claim 3, which is characterized in that the cell solubilizing agent is propylene glycol, second
Alcohol, isobutanol, diethylene glycol (DEG) it is one or more, and its content be 0.2-2mol/L.
5. a kind of preparation method of hemolysin according to any one of claims 1-4, which comprises the following steps:
1) it sufficiently dissolves by above-mentioned containing measuring boric acid, sodium chloride, calcium chloride, magnesium chloride aqueous medium is added, adds formaldehyde, sweet
Oil, propylene glycol, Proclin 300 obtain boric acid mixed liquor (A liquid)；
2) by it is above-mentioned containing measure sodium tetraborate, sodium chloride, calcium chloride, magnesium chloride be added aqueous medium sufficiently dissolve, add formaldehyde,
Glycerol, propylene glycol, Proclin 300 obtain sodium tetraborate mixed liquor (B liquid)；
3) A liquid and B liquid are mixed well in proportion, pH value 7.0-8.0 is adjusted to obtain hemolysin using filter.
6. a kind of method of hemolysin processing biological sample according to any one of claims 1-4, which is characterized in that in room temperature
Hemolysin described in being added into biological sample, mixed processing 10-15 minutes；The biological sample includes whole blood, peripheral blood, resists
Coagulate the one or more of agent venous whole；The volume ratio of the hemolysin and biological sample is 0.5-1.5: 20.
7. a kind of hemolysin hemolysin according to any one of claims 1-4 passes through flow cytomery leucocyte membranous antigen
Method, which comprises the following steps:
1) fluorescent labeled antibody of detection cell surface molecule is added into sample to be tested in room temperature, mixes, is protected from light and is incubated for 10-
15min；
2) hemolysin described in claim is added, mixed processing is protected from light and is incubated for 10min；
3) treated sample to be tested centrifuge is precipitated into sample 5-10min with the centrifugal speed of 300-500g；
4) after the sample after centrifugal treating being removed supernatant, PBS buffer solution is added, mixes sample, it is to be measured；
5) sample to be tested is divided into lymphocyte populations, monokaryon by cell volume size and granularity through flow cytometry analysis
Cell mass, neutrophil leucocyte group.
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