Hemolytic agent and detection kit

Abstract

The invention discloses a hemolytic agent and a detection kit, the hemolytic agent comprises a surfactant and guanidine substances, and the pH value of the hemolytic agent is 2.8-9.5; the concentration of the guanidine substance is 0.2 g / L-12 g / L, and the guanidine substance is a guanidyl compound or guanidine salt. The surfactant can realize red blood cell hemolysis and perform different degrees of treatment on white cell membranes, besides normal contact, guanidine substances are positively charged and can attract hemoglobin with negative charges, and after the guanidine substances are combined with the hemoglobin, disulfide bonds in the hemoglobin can be promoted to be damaged, so that the hemoglobin is disintegrated. Meanwhile, the guanidine substances almost have no destructive effect on the leukocytes, so that the leukocytes still keep respective states in the hemolytic agent disclosed by the invention. In combination with a test case, compared with a traditional hemolytic agent, the hemolytic agent disclosed by the invention can be simultaneously used for detection of hemoglobin and leukocyte classification.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro diagnostic reagents, and in particular to a hemolytic agent and a detection kit. Background Art

[0002] Hemoglobin is a protein responsible for carrying oxygen in living organisms. It has a molecular weight of 64.5KD. The hemoglobin molecule contains 6 tryptophan residues, 12 tyrosine residues and three pairs of disulfide bonds. Human hemoglobin is composed of four subunits, two α subunits and two β subunits. The adult hemoglobin molecule is negatively charged.

[0003] Because when testing hemoglobin, a hemolytic agent is needed to perform a strong hemolysis treatment on red blood cells; when testing white blood cell classification, white blood cells need to be treated very weakly. At present, it is difficult for common blood cell analyzers on the market to strike a balance between hemolysis of red blood cells and treatment of white blood cells when testing hemoglobin and white blood cell classification. Therefore, different hemolytic agents are often used to hemolyze and treat red blood cells and white blood cells respectively. There are relatively few studies on combining hemoglobin hemolytic agents and white blood cell classification hemolytic agents into one hemolytic agent. Summary of the invention

[0004] Based on this, it is necessary to provide a hemolytic agent that can solve the above problems.

[0005] In addition, it is also necessary to provide a detection kit including the above-mentioned hemolytic agent.

[0006] A hemolytic agent, comprising a surfactant and a guanidine substance, wherein the pH of the hemolytic agent is 2.8 to 9.5;

[0007] The concentration of the guanidine substance is 0.2 g / L to 12 g / L, and the guanidine substance is a guanidine compound or a guanidine salt;

[0008] The chemical structure formula of the guanidine compound is as follows:

[0009]

[0010] Among them, R 1 , R 2 , R 3 , R 4 and R 5 Each of the following is independently selected from at least one of H, an alkyl group, an aryl group, a guanidinyl group and a heteroaryl group.

[0011] In one embodiment, R 1 is guanidine, R 2 , R 3 and R 4 Both H, R 5is at least one of an alkyl group, an aryl group and a heteroaryl group.

[0012] In one embodiment, the guanidine compound is metformin, phenformin hydrochloride or buformin.

[0013] In one embodiment, R 2 , R 3 and R 4 Both H, R 1 and R 5 At least one group independently selected from a C6-C20 aryl group and a C5-C20 heteroaryl group.

[0014] In one embodiment, the guanidine compound is

[0015] In one embodiment, the guanidine compound is a surfactant containing a guanidine group.

[0016] In one embodiment, the guanidine compound is dodecylguanidine hydrochloride or dodecylguanidine acetate.

[0017] In one embodiment, the guanidine salt is selected from at least one of guanidine hydrochloride, guanidine nitrate, guanidine carbonate and guanidine sulfate.

[0018] In one embodiment, the surfactant includes a cationic surfactant and a nonionic surfactant, the hemolytic agent further includes a reducing agent and a buffer, and the pH of the hemolytic agent is 3.0 to 7.8;

[0019] The concentration of the cationic surfactant is 0 to 12 g / L, the concentration of the nonionic surfactant is 1 g / L to 7 g / L, and the concentration of the reducing agent is 0.1 g / L to 2.0 g / L;

[0020] The cationic surfactant is at least one selected from octaalkyltrimethylammonium chloride, octaalkyltrimethylammonium bromide, dodecyltrimethylammonium chloride, dodecyltrimethylammonium bromide, dodecyltrimethylammonium chloride, dodecyltrimethylammonium bromide, tetradecyltrimethylammonium chloride, tetradecyltrimethylammonium bromide, octadecyltrimethylammonium chloride and octadecyltrimethylammonium bromide;

[0021] The nonionic surfactant is selected from at least one of fatty polyoxyethylene ether, Tween and Triton;

[0022] The reducing agent is selected from at least one of 2-mercaptoethanol, dithiothreitol and tris(2-formylethyl)phosphine hydrochloride.

[0023] A detection kit for detecting hemoglobin and / or leukocyte classification, comprising the above-mentioned hemolytic agent.

[0024] The hemolytic agent of the present invention comprises a surfactant and a guanidine substance. The surfactant can realize hemolysis of red blood cells and treat the membrane of white blood cells to different degrees. In addition to normal contact, the guanidine compound is positively charged, which attracts the negatively charged hemoglobin. After the guanidine substance is combined with the hemoglobin, it can promote the destruction of the disulfide bonds inside the hemoglobin and disintegrate the hemoglobin, so that after the above-mentioned hemolytic agent is added to the blood sample, the absorption peak of the hemoglobin is blue-shifted, and finally a peak value appears at about 380. At the same time, the degree of treatment of the guanidine substance on the white blood cells is weak, so the white blood cells still maintain their respective states in the hemolytic agent of the present invention. After the treatment of the surfactant in the hemolytic agent of the present invention, the white blood cells can be characteristically classified in three directions of SSC (side scatter), SFL (fluorescence intensity), and FSC (forward scatter).

[0025] In combination with the test examples, compared with the traditional hemolytic agent, the hemolytic agent of the present invention can be used for the detection of hemoglobin and leukocyte classification at the same time.

[0026] In addition, guanidine substances themselves have bactericidal and bacteriostatic effects, so the shelf life of the hemolytic agent of the present invention can still be extended without adding preservatives.

[0027] Preferably, the hemolytic agent of the present invention further comprises a reducing agent, which can accelerate the destruction of the hemoglobin structure, ie, reduce the disulfide bonds inside the hemoglobin to -SH groups.

[0028] Preferably, in the guanidine compound, R 1 is guanidine, R 2 , R 3 and R 4 Both H, R 5 is at least one of an alkyl group, an aryl group and a heteroaryl group. At this time, the biguanide-based surfactant has a relatively moderate effect on hemolysis of red blood cells and treatment of white blood cells. The red blood cells are completely hemolyzed, while the morphology of white blood cells is basically retained.

[0029] Preferably, in the guanidine compound, R 2 , R 3 and R 4 Both H, R 1 and R 5 At least one of C6-C20 aromatic groups and C5-C20 heteroaromatic groups is independently selected. In this case, the guanidine compound containing an aromatic ring or an aromatic heterocyclic ring has good fat solubility and a strong hemolytic effect on red blood cells, which can reduce the use of other types of surfactants and reduce the material cost of the hemolytic agent.

[0030] Preferably, the guanidine compound is a surfactant containing a guanidine group. In this case, the surfactant containing a guanidine group is itself a cationic surfactant, which has a hemolytic effect on red blood cells and a treating effect on white blood cells, thereby reducing the amount of the cationic surfactant used.

[0031] Preferably, the surfactant includes a cationic surfactant and a nonionic surfactant. The selection of a cationic surfactant and a nonionic surfactant can better achieve hemolysis of red blood cells.

[0032] The hemolytic agent of the present invention adopts a guanidine compound to achieve simultaneous detection of hemoglobin and leukocyte classification, and truly realizes the simultaneous testing of leukocyte classification and hemoglobin concentration. The two hemolytic agents are combined into one, and the hemoglobin concentration can be tested without affecting the leukocyte classification, which can reduce the amount of blood used, reduce the workload of laboratory doctors to a certain extent, reduce the number of operating steps, and can also cooperate with modern medical testing instruments to achieve the effect of in vitro diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0034] in:

[0035] Figure 1 This is a graph showing the correlation test results of the hemolytic agent prepared in Example 1.

[0036] Figure 2 This is a graph showing the correlation test results of the hemolytic agent prepared in Example 2.

[0037] Figure 3 This is a graph showing the correlation test results of the hemolytic agent prepared in Example 3.

[0038] Figure 4 This is a graph showing the correlation test results of the hemolytic agent prepared in Comparative Example 1.

[0039] Figure 5 This is a correlation test result diagram of the hemolytic agent prepared in Comparative Example 2.

[0040] Figure 6 This is a graph showing the correlation test results of the hemolytic agent prepared in Comparative Example 3. DETAILED DESCRIPTION

[0041] The following will be combined with the specific embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] It should be noted that all directional indications in the embodiments of the present invention (such as up, down, left, right, front, back, etc.) are only used to explain the relative position relationship, movement status, etc. between the various parts in a certain specific posture. If the specific posture changes, the directional indication will also change accordingly.

[0043] In addition, the descriptions of "first", "second", etc. in the present invention are only used for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the ability of ordinary technicians in the field to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0044] The invention discloses a hemolytic agent according to an embodiment of the invention, comprising a surfactant and a guanidine substance, wherein the pH value of the hemolytic agent is 2.8-9.5.

[0045] The concentration of the guanidine substance is 0.2 g / L to 12 g / L, and the guanidine substance is a guanidine compound or a guanidine salt.

[0046] The general chemical structure of guanidine compounds is as follows:

[0047]

[0048] Among them, R 1 , R 2 , R 3 , R 4 and R 5 Each of the following is independently selected from at least one of H, an alkyl group, an aryl group, a guanidinyl group and a heteroaryl group.

[0049] The hemolytic agent of the present invention comprises a surfactant and a guanidine compound. The surfactant can realize hemolysis of red blood cells and treat the membrane of white blood cells to different degrees. In addition to normal contact, guanidine substances are positively charged and attract each other with negatively charged hemoglobin. After the guanidine substances are combined with hemoglobin, they can promote the destruction of the disulfide bonds inside the hemoglobin and disintegrate the hemoglobin, so that after the above-mentioned hemolytic agent is added to the blood sample, the absorption peak of the hemoglobin is blue-shifted and finally a peak value appears at about 380. At the same time, the degree of treatment of guanidine substances on white blood cells is weak, so the white blood cells still maintain their respective states in the hemolytic agent of the present invention. After being treated with the surfactant in the hemolytic agent of the present invention, the white blood cells can be characteristically classified in three directions: SSC (side scatter), SFL (fluorescence intensity), and FSC (forward scatter).

[0050] In combination with the test examples, compared with the traditional hemolytic agent, the hemolytic agent of the present invention can be used for the detection of hemoglobin and leukocyte classification at the same time.

[0051] In addition, guanidine substances themselves have bactericidal and bacteriostatic effects, so the shelf life of the hemolytic agent of the present invention can still be extended without adding preservatives.

[0052] Preferably, in this embodiment, R 1 is guanidine, R 2 , R 3 and R 4 Both H, R 5 is at least one of an alkyl group, an aryl group and a heteroaryl group.

[0053] At this time, the biguanide-based surfactant has a relatively moderate effect on red blood cell hemolysis and white blood cell treatment. The red blood cell hemolysis is complete, while the white blood cell morphology is basically retained.

[0054] Specifically, in this embodiment, the guanidine compound is metformin, phenformin hydrochloride or buformin.

[0055] Preferably, in this embodiment, R 2 , R 3 and R 4 Both H, R 1 and R 5 At least one group independently selected from a C6-C20 aryl group and a C5-C20 heteroaryl group.

[0056] At this time, the guanidine compound containing an aromatic ring or an aromatic heterocycle has good fat solubility and a stronger hemolytic effect on red blood cells, which can reduce the use of other types of surfactants and reduce the material cost of the hemolytic agent.

[0057] Specifically, in this embodiment, the guanidine compound is:

[0058]

[0059] Preferably, in this embodiment, the guanidine compound is a surfactant containing a guanidine group.

[0060] At this time, the surfactant containing guanidine groups is itself a cationic surfactant, which has a hemolytic effect on red blood cells and a treating effect on white blood cells, thereby reducing the amount of cationic surfactant used.

[0061] Specifically, in this embodiment, the guanidine compound is dodecylguanidine hydrochloride or dodecylguanidine acetate.

[0062] Preferably, in this embodiment, the surfactant includes a cationic surfactant and a nonionic surfactant, the hemolytic agent further includes a reducing agent and a buffer, and the pH of the hemolytic agent is 3.0 to 7.8.

[0063] Selecting cationic surfactants and nonionic surfactants can better achieve red blood cell hemolysis.

[0064] Reducing agents can accelerate the destruction of hemoglobin structure, that is, reducing the disulfide bonds inside hemoglobin to -SH groups.

[0065] The concentration of the cationic surfactant is 0-12 g / L, the concentration of the nonionic surfactant is 1 g / L-7 g / L, and the concentration of the reducing agent is 0.1 g / L-2.0 g / L.

[0066] Specifically, in this embodiment, the cationic surfactant is selected from at least one of octaalkyltrimethylammonium chloride, octaalkyltrimethylammonium bromide, dodecyltrimethylammonium chloride, dodecyltrimethylammonium bromide, dodecyltrimethylammonium chloride, dodecyltrimethylammonium bromide, tetradecyltrimethylammonium chloride, tetradecyltrimethylammonium bromide, octadecyltrimethylammonium chloride and octadecyltrimethylammonium bromide.

[0067] Specifically, in this embodiment, the nonionic surfactant is selected from at least one of fatty polyoxyethylene ether, Tween and Triton.

[0068] Specifically, in this embodiment, the reducing agent is selected from at least one of 2-mercaptoethanol, dithiothreitol and tris(2-formylethyl)phosphine hydrochloride (TCEP).

[0069] Specifically, in this embodiment, the buffer is a glycine-hydrochloric acid buffer system, a TRIS-hydrochloric acid buffer system, a phosphate buffer system or a citric acid buffer system, and the osmotic pressure is 30 to 220 (preferably 60 to 110).

[0070] The hemolytic agent of the present invention adopts a guanidine compound to achieve simultaneous detection of hemoglobin and leukocyte classification, and truly realizes the simultaneous testing of leukocyte classification and hemoglobin concentration. The two hemolytic agents are combined into one, and the hemoglobin concentration can be tested without affecting the leukocyte classification, which can reduce the amount of blood used, reduce the workload of laboratory doctors to a certain extent, reduce the number of operating steps, and can also cooperate with modern medical testing instruments to achieve the effect of in vitro diagnosis.

[0071] In addition, the hemolytic agent of the present invention does not use harmful cyanide or SLS which is difficult to degrade, and tests the hemoglobin content from a new perspective.

[0072] The above-mentioned hemolytic agent can be used to detect hemoglobin and / or leukocyte classification.

[0073] Specifically, the present invention also discloses a detection kit for detecting hemoglobin and / or leukocyte classification according to an embodiment, comprising the above-mentioned hemolytic agent.

[0074] The following are specific embodiments.

[0075] In the specific embodiment, octaalkyltrimethylammonium chloride is purchased from McLean Company as model T819345, tetradecyltrimethylammonium chloride is purchased from McLean Company as model S817662, Tween 20 is purchased from Sinopharm Group as model 30189328, mercaptoethanol is purchased from Aladdin Reagent Company as model M301573, Triton X-100 is purchased from Sinopharm Group as model 30188928, metformin is purchased from McLean Company as model M813341, dithiothreitol is purchased from McLean Company as model D806827, AEO-3 is purchased from McLean Company as model A871881, dodecylguanidine hydrochloride is purchased from Rowan Reagent Company as model R139359, and tris(hydroxymethyl)aminomethane is purchased from Aladdin Reagent Company as model T110599.

[0076] Example 1

[0077] Prepare the hemolytic agent according to the following formula.

[0078] Octaalkyltrimethylammonium chloride 7g / L;

[0079] Tween 20 1.2 g / L;

[0080] Guanidine compounds 1.3 g / L;

[0081]

[0082] In this embodiment, the guanidine compound is Custom synthesis.

[0083] Example 2

[0084] Prepare the hemolytic agent according to the following formula.

[0085]

[0086] Example 3

[0087] Prepare the hemolytic agent according to the following formula.

[0088]

[0089]

[0090] Comparative Example 1

[0091] Prepare the hemolytic agent according to the following formula.

[0092]

[0093] Comparative Example 2

[0094]

[0095] Comparative Example 3

[0096]

[0097] Test Example 1

[0098] 1) Hemoglobin concentration test

[0099] Eight bottles of fresh anticoagulated blood were mixed with the hemolytic agents prepared in Examples 1 to 3 and Comparative Examples 1 to 3 at a volume ratio of 1:33, and then HGB test was performed using a blood cell analyzer from Coman. The test results are shown in Table 1 below.

[0100] Table 1

[0101]

[0102] Combined with Table 1, it can be seen that the HGB test results of the formula containing guanidine compounds are close to the standard values, indicating that the use of guanidine compounds can effectively enhance the hemolysis of red blood cells and improve the accuracy of HGB detection.

[0103] 2) Correlation analysis

[0104] Thirty fresh anticoagulated blood samples were mixed with the hemolytic agents prepared in Examples 1 to 3 and Comparative Examples 1 to 3 at a volume ratio of 1:33, and then HGB test was performed using a blood cell analyzer from Coman. The test results are as follows: Figure 1 to Figure 6 shown.

[0105] Combination Figure 1 to Figure 6It can be seen that for the formula containing guanidine compounds, the correlation R-square between the HGB test results and the standard value is greater than 0.99, while for the formula without guanidine compounds, the correlation is poor (R-square is less than 0.8), indicating that the use of guanidine compounds can effectively improve the correlation of HGB test results.

[0106] 3) Hemolytic test for leukocyte classification

[0107] The hemolytic agents prepared in Examples 1 to 3 and Comparative Examples 1 to 3 were mixed at a volume ratio of 1:113, and then a WBC test was performed using a blood cell analyzer from Coman. The test results are shown in Table 2 below.

[0108] Table 2

[0109]

[0110]

[0111] Combined with Table 2, it can be seen that for the formulation containing guanidine compounds, the WBC test results are close to the standard values ​​and the test deviation is small; for the formulation not containing guanidine compounds, the white blood cell classification counts vary greatly; this indicates that the use of guanidine compounds has a moderate effect on white blood cell treatment and can better maintain the state of white blood cells, ensuring that white blood cells can be correctly classified during detection, thereby improving the accuracy of WBC detection.

[0112] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A hemolytic agent, characterized in that It includes a surfactant and a guanidine substance, and the pH of the hemolytic agent is 2.8 to 9.5; The concentration of the guanidine substance is 0.2 g / L to 12 g / L, and the guanidine substance is a guanidine compound or a guanidine salt; The chemical structure formula of the guanidine compound is as follows: Wherein, R1, R2, R3, R4 and R5 are independently selected from at least one of H, alkyl, aryl, guanidinyl and heteroaryl.

2. The hemolytic agent according to claim 1, characterized in that R1 is a guanidine group, R2, R3 and R4 are all H, and R5 is at least one of an alkyl group, an aryl group and a heteroaryl group.

3. The hemolytic agent according to claim 2, characterized in that The guanidine compound is metformin, phenformin hydrochloride or buformin.

4. The hemolytic agent according to claim 1, characterized in that R2, R3 and R4 are all H, and R1 and R5 are each independently selected from at least one of a C6-C20 aryl group and a C5-C20 heteroaryl group.

5. The hemolytic agent according to claim 4, characterized in that The guanidine compound is 6. The hemolytic agent according to claim 1, characterized in that The guanidine compound is a surfactant containing a guanidine group.

7. The hemolytic agent according to claim 6, characterized in that The guanidine compound is dodecylguanidine hydrochloride or dodecylguanidine acetate.

8. The hemolytic agent according to claim 1, characterized in that The guanidine salt is selected from at least one of guanidine hydrochloride, guanidine nitrate, guanidine carbonate and guanidine sulfate.

9. The hemolytic agent according to any one of claims 1 to 8, characterized in that The surfactant includes a cationic surfactant and a nonionic surfactant, the hemolytic agent also includes a reducing agent and a buffer, and the pH of the hemolytic agent is 3.0 to 7.8; The concentration of the cationic surfactant is 0 to 12 g / L, the concentration of the nonionic surfactant is 1 g / L to 7 g / L, and the concentration of the reducing agent is 0.1 g / L to 2.0 g / L; The cationic surfactant is at least one selected from octaalkyltrimethylammonium chloride, octaalkyltrimethylammonium bromide, dodecyltrimethylammonium chloride, dodecyltrimethylammonium bromide, dodecyltrimethylammonium chloride, dodecyltrimethylammonium bromide, tetradecyltrimethylammonium chloride, tetradecyltrimethylammonium bromide, octadecyltrimethylammonium chloride and octadecyltrimethylammonium bromide; The nonionic surfactant is selected from at least one of fatty polyoxyethylene ether, Tween and Triton; The reducing agent is selected from at least one of 2-mercaptoethanol, dithiothreitol and tris(2-formylethyl)phosphine hydrochloride.

10. A detection kit for detecting hemoglobin and / or leukocyte classification, characterized in that: The method comprises the hemolytic agent according to any one of claims 1 to 9.

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