阅读说明：本技术 一种等张溶血素及其制备方法、处理生物样本方法和检测白细胞膜抗原方法 (Isohemolysin, preparation method thereof, biological sample treatment method and leukocyte membrane antigen detection method ) 是由 俞秋兴 于 2019-09-10 设计创作，主要内容包括：本等张溶血素,包括氧化溶解剂、助溶剂、细胞增溶剂、细胞固定剂、无机盐及水质,氧化溶解剂为亚硝酸钠,且含量为2-20g/L；助溶剂为甘油、二甘醇、丙二醇中的一种或多种,且含量为0.3-3mmol/L；细胞增溶剂为异丁醇、甲醇、乙醇中的一种或多种,且含量为0.1-1mol/L；细胞固定剂为多聚甲醛、甲醛、乙醇、丙酮的一种或多种,且含量为10-40g/L；无机盐为氯化钠、氯化镁和氯化钙,且所述氯化钠的含量为1-10g/L,氯化镁的含量为1-100mmol/L,氯化钙的含量为1-100mmol/L。同时提出了该溶血素制备方法、溶血素处理生物样本方法和通过流式细胞仪检测白细胞膜抗原方法,该溶血素处理样本后白细胞各群体分区明显,不损伤白细胞膜蛋白,既保留原有的生物学活性,又能完全裂解红细胞,结果准确、性能稳定。(The isotonic hemolysin comprises an oxidation dissolving agent, a cosolvent, a cell solubilizer, a cell fixing agent, inorganic salt and water, wherein the oxidation dissolving agent is sodium nitrite, and the content of the oxidation dissolving agent is 2-20 g/L; the cosolvent is one or more of glycerol, diethylene glycol and propylene glycol, and the content is 0.3-3 mmol/L; the cell solubilizer is one or more of isobutanol, methanol and ethanol, and the content of the cell solubilizer is 0.1-1 mol/L; the cell fixing agent is one or more of paraformaldehyde, formaldehyde, ethanol and acetone, and the content is 10-40 g/L; the inorganic salt is sodium chloride, magnesium chloride and calcium chloride, and the content of the sodium chloride is 1-10g/L, the content of the magnesium chloride is 1-100mmol/L, and the content of the calcium chloride is 1-100 mmol/L. The hemolysin preparation method, the hemolysin treatment biological sample method and the method for detecting leukocyte membrane antigen by the flow cytometer are provided at the same time, each group of leukocytes after the hemolysin treatment of the sample are obviously partitioned, leukocyte membrane protein is not damaged, the original biological activity is retained, erythrocytes can be completely cracked, the result is accurate, and the performance is stable.)

1. the isotonic hemolysin is characterized by comprising an oxidation dissolving agent, a cosolvent, a cell solubilizer, a cell fixing agent, inorganic salt and water, wherein the oxidation dissolving agent is sodium nitrite, and the content of the oxidation dissolving agent is 2-20 g/L; the cosolvent is one or more of glycerol, diethylene glycol and propylene glycol, and the content of the cosolvent is 0.3-3 mmol/L; the cell solubilizer is one or more of isobutanol, methanol and ethanol, and the content of the cell solubilizer is 0.1-1 mol/L; the cell fixing agent is one or more of paraformaldehyde, formaldehyde, ethanol and acetone, and the content of the cell fixing agent is 10-40 g/L; the inorganic salt is sodium chloride, magnesium chloride and calcium chloride, the content of the sodium chloride is 1-10g/L, the content of the magnesium chloride is 1-100mmol/L, and the content of the calcium chloride is 1-100 mmol/L.

2. the isotonic hemolysin according to claim 1, further comprising a minor amount of stabilizers and preservatives.

3. The isopinolysin of claim 2, further comprising a PH modifier, wherein said PH modifier is sodium bicarbonate, and wherein said sodium bicarbonate is present in an amount of 0.1 to 1 mmol/L.

4. a process for the preparation of an isopinolysin according to claims 1-3, comprising the steps of:

1) Sodium nitrite, sodium chloride, calcium chloride and magnesium chloride are measured according to the content and added into a water medium to be fully dissolved to obtain a mixed solution.

2) adding formaldehyde, glycerol and isobutanol solution into the mixed buffer solution obtained in the step 1), adding an aqueous medium to 1 liter, adjusting the pH value of sodium bicarbonate to 7.4-7.6, and filtering to obtain the hemolysin.

5. The method for treating a biological sample with an isotonic hemolysin according to claims 1 to 3, wherein said hemolysin is added to a biological sample at room temperature and mixed for 10 to 15 minutes; the biological sample is anticoagulant venous whole blood; the volume ratio of the hemolysin to the biological sample is 1: 40-3: 40.

6. the method of detecting a leukocyte membrane antigen by flow cytometry of an isophtholysin according to claims 1-3, comprising the steps of:

1) Adding a fluorescence labeling antibody for detecting cell surface molecules into a sample to be detected at room temperature, uniformly mixing, and incubating for 10-15min in a dark place;

2) Adding the isohemolysin, mixing, and incubating in dark for 10 min;

3) Precipitating the treated sample to be detected for 5-10min at the centrifugal speed of 300-500g by using a centrifugal machine;

4) Removing supernatant from the centrifuged sample, adding PBS buffer solution, mixing the sample uniformly, and detecting;

5) the sample to be detected is analyzed by a flow cytometry and is divided into a lymphocyte population, a monocyte population and a neutrophil population according to the cell volume and granularity.

Technical Field

The invention relates to the field of immunological cell analysis, in particular to an isoproxysin, a preparation method thereof, a method for treating a biological sample and a method for detecting a leukocyte membrane antigen.

Background

Blood cell analysis is of great value for the development of scientific research, prevention, diagnosis and treatment of disease, ranging from the initial study of cell morphology, size, number to the recent antigenic components on cell surfaces. The traditional cell immunity and nonspecific immunity detection technology can not carry out multi-parameter and high-sensitivity analysis on the size, the shape, the plasma membrane and the internal structure of a target cell from a single cell level, a flow cytometer can quickly measure, sort, store and display a series of important characteristic parameters of dispersed cells suspended in liquid in biophysical and biochemical aspects, and can sort out specified cell subsets according to preselected parameter ranges, and the traditional cell immunity and nonspecific immunity detection technology is currently applied to the fields of clinical medicine and basic medicine research such as immunology, hematology, oncology, cytogenetics, cytobiology, biochemistry and the like, such as lymphocyte and the subset analysis of cell surface molecules, intracellular cytokine analysis, leukemia immunity typing, tumor drug resistance related protein analysis, autoimmune disease related HLA antigen analysis, cross-matching type in transplantation immunity, cross-matching type analysis, cell-specific immune function analysis, cell, Mitochondrial membrane potential, apoptosis, intracellular calcium ion concentration, neutrophil function analysis, and the like.

Because the majority of the immune function in vivo is leucocytes, and the most abundant erythrocyte in human blood, in order to reduce the influence on the analysis of target cells, the interference effect of eliminating the erythrocyte in the blood is needed, when red blood cell hemolysis is carried out, the structure and function of white blood cells are prevented from being damaged, membrane proteins on the surfaces of the white blood cells are reserved, therefore, the requirement for hemolysis is very high, most of the existing flow cytohemolysins are hypolysins, i.e., a solution below osmotic pressure, and isotonic hemolysin, which is a solution equal to osmotic pressure, it has previously been believed that erythrocytes in isotonic solutions retain their normal form (no shrinkage, no hemolysis), but some molecules can freely pass through cell membranes to cause hemolysis of red blood cells, and the hemolysis reaction is realized by changing the physical structure of the red blood cells by utilizing the osmotic pressure difference between the inside and the outside of the cells to cause the rupture of the cell membranes. Some chemicals such as drugs, bacterial toxins, snake venom, etc. bind to ferrous hemoglobin in erythrocytes and become methemoglobin, which changes the structure of erythrocytes and leads to hemolytic reactions.

the hemolysin formula and the instrument have a certain relationship, different hemolysins are used in different instruments, the effect of the same hemolysin displayed in different instruments is different, the main reason is that the leucocyte morphology after hemolysin cracking is different, and the difference of lymphocyte, monocyte and granulocyte grouping can be caused by combining the optical path design of FSC/SSC. Many hemolysins on the market at present are only suitable for a certain type of flow cytometry, and can not achieve the effect of a hemolysin general multi-type flow cytometer.

Therefore, how to develop an isoproteolysin, a preparation method thereof, a method for processing a biological sample and a method for detecting a leukocyte antigen, which can overcome the above defects, are technical problems to be solved urgently by those skilled in the art.

disclosure of Invention

In order to solve the technical problems, the invention provides an isotonic hemolysin preparation, the hemolysin can change the structure of red blood cells without destroying the shape, structure and function of white blood cells, has high detection accuracy and less interference impurities, can replace the hemolysin of imported like products, reduces the medical cost of hospitals, and lightens the medical expenses of patients.

In order to achieve the purpose, the technical scheme of the invention is as follows:

an isotonic hemolysin comprises an oxidation dissolving agent, a cosolvent, a cell solubilizer, a cell fixing agent, inorganic salt and water, wherein the oxidation dissolving agent is sodium nitrite, the content of the oxidation dissolving agent is 2-20g/L, and the sodium nitrite can oxidize red blood cells, so that ferrous hemoglobin in blood can be changed into methemoglobin, the red blood cells can be quickly cracked with the help of the cosolvent, and the fragments are few; the cosolvent is one or more of glycerol, diethylene glycol and propylene glycol, the content of the cosolvent is 0.3-3mmol/L, the activity of water can be reduced, soluble proteins and leukocytes can be prevented from being degraded and protected, the stability is improved, and volatilization is prevented; the cell solubilizer is one or more of isobutanol, methanol and ethanol, the content of the cell solubilizer is 0.1-1mol/L, and the cell solubilizer has high adsorption and permeation capacity on the biological membrane, so that the solubilization and the permeability of the biological membrane are changed; the cell fixing agent is one or more of paraformaldehyde, formaldehyde, ethanol and acetone, the content of the cell fixing agent is 10-40g/L, and the cell fixing agent is good for most antigens, good in tissue penetrability and small in tissue shrinkage; the inorganic salt is sodium chloride, magnesium chloride and calcium chloride, the content of the sodium chloride is 1-10g/L, the content of the magnesium chloride is 1-100mmol/L, and the content of the calcium chloride is 1-100mmol/L, and the inorganic salt is used for adjusting the conductivity and osmotic pressure in the hemolysin.

Preferably, the hemolysin further contains a trace amount of a stabilizer and a preservative.

Preferably, the hemolysin also contains a pH value regulator, wherein the pH value regulator is sodium bicarbonate, and the content of the sodium bicarbonate is 0.1-1 mmol/L.

a method for preparing isohemolysin comprises the following steps:

1) Sodium nitrite, sodium chloride, calcium chloride and magnesium chloride are measured according to the content and added into a water medium to be fully dissolved to obtain a mixed solution.

2) Adding formaldehyde, glycerol and isobutanol solution into the mixed buffer solution obtained in the step 1), adding an aqueous medium to 1 liter, adjusting the pH value of sodium bicarbonate to 7.4-7.6, and filtering to obtain the hemolysin.

a method for processing a biological sample by using isoproxysin comprises the steps of adding the isoproxysin into the biological sample at room temperature, and mixing for 10-15 minutes; the biological sample is anticoagulant venous whole blood; the volume ratio of the hemolysin to the biological sample is 1: 40-3: 40.

A method for detecting an antigen of a leukocyte membrane by using an isohemolysin through a flow cytometer comprises the following steps:

1) Adding a fluorescence labeling antibody for detecting cell surface molecules into a sample to be detected at room temperature, uniformly mixing, and incubating for 10-15min in a dark place;

2) Adding the isohemolysin, mixing, and incubating in dark for 10 min;

3) precipitating the treated sample to be detected for 5-10min at the centrifugal speed of 300-500g by using a centrifugal machine;

4) Removing supernatant from the centrifuged sample, adding PBS buffer solution, mixing the sample uniformly, and detecting;

5) The sample to be detected is analyzed by a flow cytometry and is divided into a lymphocyte population, a monocyte population and a neutrophil population according to the cell volume and granularity.

through the technical scheme, the hemolysin is one of common auxiliary analysis reagents for detecting leukocyte antigens through flow cytometry, is also a key reagent for analyzing cellular immune functions and the like, divides leukocyte populations into lymphocytes, monocytes, neutrophils and fragments through forward angle (SSC) and side angle scattering light (FSC) on a flow cytometer according to cell size and granularity, has obvious leukocyte population partition after sample treatment, less cell fragments, does not damage leukocyte membrane protein, can retain original biological activity, can completely crack the hemolysin of erythrocytes, has accurate result and stable performance, can replace the hemolysin of imported original products, achieves the domestication of imported reagents, reduces scientific research and medical cost, reduces the medical expense of patients, has high detection accuracy and less interfering impurities, and can replace the hemolysin of COULTER series products, The BD series and other mainstream analyzers have good grouping maps, high stability and easy storage. The hemolysin of the invention has a slightly inferior color compared with other hemolysins, the color is brown when the hemolysis is started, and the hemolysis can be performed without destroying the shape, structure and function of leucocyte

Drawings

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

FIG. 1 is a chart showing the hemolysis effect of an isohemolysin used in an FC500 flow cytometer (I) according to the present invention;

FIG. 2 is a diagram showing the hemolysis effect of an isopinolysin disclosed in the present invention in an FC500 flow cytometer;

FIG. 3 is a chart showing the hemolysis effect of an isopinolysin disclosed in the embodiments of the present invention in an FC500 flow cytometer;

FIG. 4 is a chart (IV) showing the hemolysis effect of an isohemolysin used in an FC500 flow cytometer, according to the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and the test methods in the preferred embodiments without specific reference to specific conditions are generally performed according to conventional conditions, and the described embodiments are only a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The present invention will be described in further detail with reference to examples and specific embodiments.

as shown in fig. 1, fig. 2, fig. 3 and fig. 4, an isotonic hemolysin comprises an oxidative lytic agent, a cosolvent, a cell solubilizer, a cell fixative, a PH regulator, inorganic salts and water, wherein the oxidative lytic agent is sodium nitrite, the content of the oxidative lytic agent is 2-20g/L, the sodium nitrite can oxidize red blood cells, so that ferrous hemoglobin in blood can be changed into methemoglobin, the red blood cells can be rapidly cracked with the help of the cosolvent, and the fragments are few; the cosolvent is one or more of glycerol, diethylene glycol and propylene glycol, the content of the cosolvent is 0.3-3mmol/L, the activity of water can be reduced, soluble proteins and leukocytes can be prevented from being degraded and protected, the stability is improved, and volatilization is prevented; the cell solubilizer is one or more of isobutanol, methanol and ethanol, the content of the cell solubilizer is 0.1-1mol/L, and the cell solubilizer has high adsorption and permeation capacity on the biological membrane, so that the solubilization and the permeability of the biological membrane are changed; the cell fixing agent is one or more of paraformaldehyde, formaldehyde, ethanol and acetone, the content of the cell fixing agent is 10-40g/L, and the cell fixing agent is good for most antigens, good in tissue penetrability and small in tissue shrinkage; the pH value regulator is sodium bicarbonate, and the content of the sodium bicarbonate is 0.1-1 mmol/L; the inorganic salt is sodium chloride, magnesium chloride and calcium chloride, the content of the sodium chloride is 1-10g/L, the content of the magnesium chloride is 1-100mmol/L, and the content of the calcium chloride is 1-100mmol/L, and the inorganic salt is used for adjusting the conductivity and osmotic pressure in the hemolysin. The hemolysin also contains a trace amount of stabilizer and preservative.

On the basis of the above-mentioned isopinolysin, the invention also discloses a preparation method of isopinolysin, comprising the following steps:

S1, adding sodium nitrite, sodium chloride, calcium chloride and magnesium chloride according to the content, and fully dissolving to obtain a mixed solution.

S2, adding formaldehyde, glycerol and isobutanol solution into the mixed buffer solution obtained in the step S1, adding an aqueous medium to 1 liter, adjusting the pH value to 7.4-7.6 by using sodium bicarbonate, and filtering to obtain the hemolysin.

The method for treating a biological sample with the above-described isopinolysin is as follows: adding the hemolysin into a biological sample at room temperature, and mixing for 10-15 minutes; the biological sample is anticoagulant venous whole blood; the volume ratio of the hemolysin to the biological sample is 1: 40-3: 40.

Meanwhile, the method for detecting the leukocyte membrane antigen by using the isohemolysin through a flow cytometer comprises the following steps:

S1, adding a fluorescence labeled antibody for detecting cell surface molecules into a sample to be detected at room temperature, uniformly mixing, and incubating for 10-15min in a dark place;

S2, adding the isohemolysin, mixing, and incubating for 10min in a dark place;

s3, using a centrifuge to precipitate the processed sample to be detected for 5-10min at the centrifugation speed of 300-500 g;

s4, removing supernatant from the centrifuged sample, adding PBS buffer solution, and uniformly mixing the sample to be detected;

And S5, analyzing the sample to be detected by a flow cytometer, and sequentially dividing the sample into a lymphocyte population, a monocyte population and a neutrophil population according to the cell volume and the granularity.

subjects and reagents: peripheral blood samples of 15 healthy and sub-healthy people are anticoagulated by EDTA, heparin and sodium citrate, stored at room temperature (18-25 ℃) and detected within 8 hours. Accuracy evaluation adopts a Cytomics FC500 flow cytometer, matched reagents of the flow cytometer are a T lymph subgroup detection four-color reagent CD45-PerCP-Cy5.5/CD4-FITC/CD8-APC/CD3-PE and matched hemolysin OptiLyse C, and the precision experiment uses Immuno-Trol cells produced by Beckman Coulter company for quality control of blood and is stored at 4 ℃.

The experimental method comprises the following steps: adding 10 mu lCD45-PerCP-Cy5.5/CD4-FITC/CD8-APC/CD3-PE antibody into a flow-type sample loading tube, adding 100 mu l of blood sample specimen by a reverse sample loading method, uniformly mixing, and incubating for 10-15min at room temperature in a dark place; adding 1000 μ l of home-made hemolysin and matched hemolysin OptiLyse C respectively, shaking, mixing, and hemolysis for 10min at room temperature in dark; precipitating the treated sample to be detected for 5-10min at the centrifugal speed of 300-500g by using a centrifugal machine; and removing supernatant from the centrifuged sample, adding PBS buffer solution, fully and uniformly mixing, and finishing the detection on the machine within 1 h.

the hemolysin of the present invention has the following degree and effect:

a, appearance of the hemolysis tube: the supernatant is clear and bright, and the red blood cells are fully cracked;

b, centrifuging to obtain a tube bottom: after centrifugation at 300g for 10min, the vessels had no visible red precipitate at the bottom and little red cell debris.

c, FSC/SSC scatter plot: the cells of each series are obviously grouped, have clear limits, high cell aggregation degree and no cross (see a hemolysis effect chart).

d, cell surface labeling: there was no significant effect on the cell membrane surface CD antigen of each line (correlation analysis table).

and e, the hemolysin can be universally used for main flow cytometers such as BD series, COULTER series and the like.