阅读说明：本技术 D-二聚体检测试剂和方法、试剂制备方法及聚乙二醇应用 (D-dimer detection reagent and method, preparation method of reagent and application of polyethylene glycol ) 是由 李坷坷 赵玉梅 雷霆 于 2018-12-28 设计创作，主要内容包括：本申请公开了一种D-二聚体检测试剂和方法、试剂制备方法及聚乙二醇的应用。本申请D-二聚体检测试剂,包括聚乙二醇、缓冲液体系和无机盐；聚乙二醇为相对分子量6000-10000的聚乙二醇；无机盐用于调节缓冲液体系离子强度。本申请D-二聚体检测试剂或检测方法,利用低分子量聚乙二醇对血红蛋白与D-二聚体相互作用进行干扰,减小血红蛋白对D-二聚体与特异性抗体反应的影响,起到抗血红蛋白干扰的作用,提高了D-二聚体检测的准确性。并且,由于本申请的D-二聚体检测试剂能够抗血红蛋白干扰,使得D-二聚体检测时,无需采用双波法检测或采用高波长来消除由血红蛋白带来的影响,降低了D-二聚体检测的难度和仪器成本。(The application discloses a D-dimer detection reagent and a method, a preparation method of the reagent and application of polyethylene glycol. The D-dimer detection reagent comprises polyethylene glycol, a buffer solution system and inorganic salt; the polyethylene glycol has a relative molecular weight of 6000-10000; inorganic salts are used to adjust the ionic strength of the buffer system. According to the detection reagent or the detection method for the D-dimer, the interaction of the hemoglobin and the D-dimer is interfered by the low-molecular-weight polyethylene glycol, the influence of the hemoglobin on the reaction of the D-dimer and a specific antibody is reduced, the effect of resisting the interference of the hemoglobin is achieved, and the accuracy of the detection of the D-dimer is improved. In addition, the D-dimer detection reagent can resist hemoglobin interference, so that the influence caused by hemoglobin is eliminated without adopting a double wave method or adopting high wavelength during D-dimer detection, and the difficulty and the instrument cost of D-dimer detection are reduced.)

1. A D-dimer detection reagent, characterized in that: comprises polyethylene glycol, a buffer solution system and inorganic salt;

the polyethylene glycol is low molecular weight polyethylene glycol with the relative molecular weight of 6000-10000;

the inorganic salt is used for adjusting the ionic strength of the buffer solution system;

preferably, the working concentration of the low molecular weight polyethylene glycol is 5-50 g/L;

preferably, the pH of the buffer system is between 5.0 and 8.5.

2. The D-dimer detection reagent according to claim 1, wherein: the buffer liquid is at least one of boric acid buffer solution, citric acid-sodium citrate buffer solution, glycine buffer solution, phosphate buffer solution, Tris-hydrochloric acid buffer solution, MES, HEPES and MOPS buffer solution;

preferably, the inorganic salt is selected from at least one of potassium chloride, lithium chloride, sodium chloride, zinc chloride, magnesium chloride, potassium nitrate, and sodium nitrate; more preferably, the inorganic salt is at least one selected from the group consisting of potassium chloride, sodium chloride, zinc chloride and magnesium chloride.

3. The D-dimer detection reagent according to claim 1 or 2, wherein: also includes a protein protectant;

preferably, the protein protectant is selected from at least one of bovine serum albumin, immunoglobulin, glycine, polysaccharide, and sorbitol.

4. The D-dimer detection reagent according to any one of claims 1 to 3, wherein: also comprises a suspending agent;

preferably, the suspending agent is Tween20 and/or triton-X100.

5. The D-dimer detection reagent according to any one of claims 1 to 4, wherein: also comprises a preservative;

preferably, the preservative is selected from at least one of Proclin300, thimerosal, sodium benzoate, nitrate, nitrite, and sodium azide.

6. The D-dimer detection reagent according to any one of claims 1 to 5, wherein: all reagents are divided into a reagent I and a reagent II;

the reagent I comprises polyethylene glycol, a buffer solution system, inorganic salt, a preservative and a protein protective agent;

the second reagent comprises particles coated with D-dimer specific antibodies, a buffer solution system, a protein protective agent, a preservative and a suspending agent;

preferably, in the reagent one: the pH value of the buffer solution system is 5.0-7.0, the buffer solution system is at least one of boric acid buffer solution, citric acid-sodium citrate buffer solution, glycine buffer solution, phosphate buffer solution, MES, HEPES and MOPS buffer solution, and preferably boric acid buffer solution, phosphate buffer solution, MOPS or MES buffer solution; the preservative is Proclin300, thimerosal or sodium azide; the protein protectant is at least one selected from bovine serum albumin, immunoglobulin and sorbitol;

preferably, in the reagent two: the particles coated with the D-dimer specific antibody are polystyrene microspheres; the pH value of the buffer solution system is 6.5-8.5, and the buffer solution system is at least one of boric acid buffer solution, phosphate buffer solution, Tris-hydrochloric acid buffer solution, HEPES and MOPS buffer solution.

7. A D-dimer detection reagent, characterized in that: the reagent is composed of a first reagent and a second reagent;

the reagent I consists of 5-50g/L of low molecular weight polyethylene glycol with the relative molecular weight of 6000-10000, a buffer solution system, inorganic salt, a preservative and a protein protective agent;

the reagent II consists of particles coated with the D-dimer specific antibody, a buffer solution system, a protein protective agent, a preservative and a suspending agent;

preferably, the inorganic salt is selected from at least one of potassium chloride, lithium chloride, sodium chloride, zinc chloride, magnesium chloride, potassium nitrate, and sodium nitrate; more preferably, the inorganic salt is selected from at least one of potassium chloride, sodium chloride, zinc chloride and magnesium chloride;

preferably, the suspending agent is tween20 and/or triton-X100;

preferably, the preservative of the first reagent is at least one selected from Proclin300, thimerosal, sodium benzoate, nitrate, nitrite and sodium azide; more preferably, the preservative of the first reagent is Proclin300, thimerosal or sodium azide;

preferably, the protein protective agent of the first reagent is at least one selected from bovine serum albumin, immunoglobulin, glycine, polysaccharide and sorbitol; more preferably, the protein protectant of the first reagent is at least one selected from the group consisting of bovine serum albumin, immunoglobulin and sorbitol;

preferably, the pH value of the buffer system adopted by the solution of the reagent I is 5.0-7.0, and the buffer liquid system is at least one of boric acid buffer solution, citric acid-sodium citrate buffer solution, glycine buffer solution, phosphate buffer solution, MES, HEPES and MOPS buffer solution; preferably boric acid buffer, phosphate buffer, MOPS or MES buffer;

preferably, the D-dimer specific antibody coated particles in the reagent II are polystyrene microspheres;

preferably, the preservative of the reagent II is at least one selected from Proclin300, thimerosal, sodium benzoate, nitrate, nitrite and sodium azide;

preferably, the protein protective agent of the reagent two is at least one selected from bovine serum albumin, immunoglobulin, glycine, polysaccharide and sorbitol;

preferably, the pH value of the buffer system of the solution of the second reagent is 6.5-8.5, and the buffer liquid system is at least one of boric acid buffer, phosphate buffer, Tris-hydrochloric acid buffer, HEPES and MOPS buffer.

8. The application of low molecular weight polyethylene glycol in preparing a D-dimer detection reagent for reducing hemoglobin interference, wherein the low molecular weight polyethylene glycol is polyethylene glycol with the relative molecular weight of 6000-10000;

preferably, the D-dimer detection reagent is an immune turbidimetric detection reagent;

preferably, the working concentration of the low molecular weight polyethylene glycol in the D-dimer detection reagent is 5-50 g/L.

9. A method for detecting a D-dimer, comprising: comprises the following steps of (a) carrying out,

obtaining a blood sample to be detected which is treated by sodium citrate; then one of the following actions is taken:

operation one: uniformly mixing the blood sample to be detected with the first reagent, and carrying out warm bath to the reaction temperature; then adding a reagent II for reaction; preferably, the reagent II is pre-heated to the reaction temperature;

and operation II: carrying out warm bath on a blood sample to be detected to a reaction temperature, and then adding a D-dimer detection reagent for reaction; preferably, the D-dimer detection reagent is pre-incubated to the reaction temperature;

after the first operation or the second operation, the interference of hemoglobin is eliminated without using dual-wavelength correction or high wavelength, and the content of the D-dimer is calculated according to the change of the light transmittance value of the reaction system directly through single-wavelength detection;

the reagent I comprises polyethylene glycol, a buffer solution system, inorganic salt, a preservative and a protein protective agent;

the second reagent comprises particles coated with D-dimer specific antibodies, a buffer system, a protein protective agent, a preservative and a suspending agent;

the D-dimer detection reagent comprises polyethylene glycol, microparticles coated with D-dimer specific antibodies, a buffer solution system and inorganic salts; preferably, the D-dimer detection reagent further comprises at least one of a protein protecting agent, a suspending agent, and a preservative;

preferably, the polyethylene glycol of the reagent I or the D-dimer detection reagent is low molecular weight polyethylene glycol with the relative molecular weight of 6000-10000;

preferably, the working concentration of the low molecular weight polyethylene glycol is 5-50 g/L;

preferably, the buffer liquid of the reagent I, the reagent II or the D-dimer detection reagent is at least one of boric acid buffer, citric acid-sodium citrate buffer, glycine buffer, phosphate buffer, Tris-hydrochloric acid buffer, MES, HEPES and MOPS buffer;

preferably, the pH value of the buffer solution system of the first reagent is 5.0-7.0, and the buffer solution system of the first reagent is boric acid buffer solution, phosphate buffer solution, MOPS or MES buffer solution;

preferably, the pH value of the buffer solution system of the reagent II is 6.5-8.5;

preferably, the preservative of the reagent one, the reagent two or the D-dimer detection reagent is selected from at least one of Proclin300, thimerosal, sodium benzoate, nitrate, nitrite and sodium azide;

preferably, the preservative of the first reagent is Proclin300, thimerosal or sodium azide;

preferably, the protein protective agent of the reagent one, the reagent two or the D-dimer detection reagent is selected from at least one of bovine serum albumin, immunoglobulin, glycine, polysaccharide and sorbitol;

preferably, the protein protective agent of the first reagent is at least one of bovine serum albumin, immunoglobulin and sorbitol;

preferably, the reagent two or the D-dimer detection reagent-coated D-dimer specific antibody particles are polystyrene microspheres;

preferably, the suspending agent of the reagent two or the D-dimer detection reagent is Tween20 and/or triton-X100;

preferably, the inorganic salt of the first reagent is at least one selected from potassium chloride, lithium chloride, sodium chloride, zinc chloride, magnesium chloride, potassium nitrate and sodium nitrate; preferably at least one of potassium chloride, sodium chloride, zinc chloride and magnesium chloride.

10. A method for preparing a D-dimer detection reagent, which is characterized by comprising the following steps:

dispersing polyethylene glycol, inorganic salt, a preservative and a protein protective agent into a buffer solution system to prepare a reagent I; dispersing the particles coated with the D-dimer specific antibody, a protein protective agent, a preservative and a suspending agent into a buffer solution system to prepare a reagent II;

or dispersing polyethylene glycol, D-dimer specific antibody coated particles and inorganic salt into a buffer solution system to prepare a D-dimer detection reagent;

preferably, further comprising dispersing at least one of a protein protecting agent, a suspending agent, and a preservative into the D-dimer detection reagent;

preferably, the polyethylene glycol of the reagent I or the D-dimer detection reagent is low molecular weight polyethylene glycol with the relative molecular weight of 6000-10000;

preferably, the working concentration of the low molecular weight polyethylene glycol is 5-50 g/L;

preferably, the buffer liquid of the reagent I, the reagent II or the D-dimer detection reagent is at least one of boric acid buffer, citric acid-sodium citrate buffer, glycine buffer, phosphate buffer, Tris-hydrochloric acid buffer, MES, HEPES and MOPS buffer;

preferably, the pH value of the buffer solution system of the first reagent is 5.0-7.0, and the buffer solution system of the first reagent is boric acid buffer solution, phosphate buffer solution, MOPS or MES buffer solution;

preferably, the pH value of the buffer solution system of the reagent II is 6.5-8.5;

preferably, the preservative of the reagent one, the reagent two or the D-dimer detection reagent is selected from at least one of Proclin300, thimerosal, sodium benzoate, nitrate, nitrite and sodium azide;

preferably, the preservative of the first reagent is Proclin300, thimerosal or sodium azide;

preferably, the protein protective agent of the reagent one, the reagent two or the D-dimer detection reagent is selected from at least one of bovine serum albumin, immunoglobulin, glycine, polysaccharide and sorbitol;

preferably, the protein protective agent of the first reagent is at least one of bovine serum albumin, immunoglobulin and sorbitol;

preferably, the reagent two or the D-dimer detection reagent-coated D-dimer specific antibody particles are polystyrene microspheres;

preferably, the suspending agent of the reagent two or the D-dimer detection reagent is Tween20 and/or triton-X100;

preferably, the inorganic salt of the first reagent is at least one selected from potassium chloride, lithium chloride, sodium chloride, zinc chloride, magnesium chloride, potassium nitrate and sodium nitrate; preferably at least one of potassium chloride, sodium chloride, zinc chloride and magnesium chloride.

Technical Field

The application relates to the field of D-dimer detection reagents, in particular to a detection method and a detection method of D-dimer, a preparation method of the detection reagent and application of polyethylene glycol.

Background

D-dimer (abbreviated DD), the smallest fragment resulting from degradation of fibrin; DD has important significance for diagnosis and treatment of a plurality of diseases as direct evidence for thrombosis formation and thrombus degradation. Under pathological and physiological conditions, when the coagulation system is activated, thrombin acts on fibrinogen to convert it into fibrin monomers (α, β, γ)2, i.e., D-E-D structure, which are regularly cross-linked into soluble fibrin monomer polymers (SFMC). Soluble fibrin monomers are crosslinked with each other in alpha and gamma chains between fibrin monomers under the action of Ca + and XIIIa to form insoluble fibrin monomer aggregates. The formation of crosslinked fibrin sets activates the plasmin dissolution system. The fibrinolytic system is composed of three main components, namely, plasmin (namely plasmin), a plasmin activator and a plasmin inhibitor. In the presence of a fibrin clot, plasminogen is activated and converted to plasmin, from which the fibrinolysis process begins, plasmin degrading the fibrin clot to form soluble fragments of varying molecular weight, such as X 'Y', D, E, etc. Wherein the D-dimer is two fragments connected together by a gamma chain, and comprises products of the specific degradation of cross-linked fibrin such as DD, DXD, DD/E, YXD and the like.

Under physiological conditions, 2% -3% of plasma fibrinogen is degraded after being converted into fibrin, and under normal physiological conditions, the content of D-dimer in plasma is very low. Therefore, the high D-dimer content in vivo indicates that the fibrinolytic system in vivo is abnormally activated, and is an important index for the formation of the microthrombus in the blood vessel. D-dimers have therefore become a routine project for clinical coagulation tests, providing a major basis for the differential diagnosis and treatment of numerous diseases.

The current DD detection method mainly comprises a latex agglutination method, an enzyme-linked immunosorbent assay, a colloidal gold immunofiltration method and an immunoturbidimetry. The latex agglomeration method is characterized in that a D-dimer specific antibody is adsorbed by latex particles and forms macroscopic agglomeration with DD in plasma to judge whether the antibody is positive or negative; the method is simple and rapid to operate. However, the detection result is observed by naked eyes, so that the method has strong subjectivity, and false results are easy to appear when the detection result is inaccurate. The enzyme-linked immunosorbent assay is characterized in that a double-antibody sandwich method is adopted, an antibody of DD is coated on a solid phase carrier, after the DD in plasma is combined with the antibody, an enzyme-labeled second labeled DD monoclonal antibody is combined to form an antibody-antigen-enzyme-labeled antibody sandwich composite product, and finally, the DD content in the plasma is quantitatively determined by adding an enzyme substrate and developing. When the method is used for testing the DD, the DD is not interfered by bilirubin, fibrinogen and the like, the test result is accurate, but the DD is not suitable for detecting a small amount of samples, and the test process consumes a long time. The colloidal gold immune penetration method is to coat a DD monoclonal antibody on a porous film, the DD in plasma is combined with the antibody to generate color change, and the red intensity of 540nm in the plasma is tested by an instrument to quantitatively detect the D-dimer content. The immunoturbidimetry is characterized in that a DD monoclonal antibody is coupled to polystyrene particles in advance, aggregation reaction is caused by specific reaction between DD in plasma and the antibody, the turbidity of a reaction system is changed, when the incident wavelength is smaller than the diameter of an aggregate, the absorbance is changed, and the content of a D-dimer in a plasma sample can be quantitatively tested according to the change of the absorbance. The immunoturbidimetry has the characteristics of quick and simple operation, high sensitivity and strong anti-interference capability, can provide reliable test data for clinical detection, and can realize full-automatic test of a large number of samples by means of a full-automatic hemagglutination instrument, so that the immunoturbidimetry becomes the most commonly used methodology for detecting DD of the full-automatic hemagglutination instrument.

Because plasma components are complex, various interference factors such as interference of bilirubin, hemoglobin, rheumatoid factors, HAMA and the like exist when different methods are used for measuring DD content in plasma. Among the above methods, immunoturbidimetry can effectively avoid the interference caused by fibrinogen and bilirubin, and in addition, the interference caused by rheumatoid factor, HAMA and the like to DD test results can be eliminated by introducing an anti-interference agent. However, the interference caused by hemoglobin is currently solved by eliminating the influence caused by hemoglobin by using dual-wave method detection or using high wavelength, which greatly increases the complexity of the optical system of the instrument and the cost of the instrument.

Therefore, there is a need to develop a method for reducing the interference of hemoglobin in a sample with DD detection.

Disclosure of Invention

The application aims to provide a D-dimer detection reagent, a detection method, a preparation method of the detection reagent and new application of polyethylene glycol.

In order to achieve the purpose, the following technical scheme is adopted in the application:

one aspect of the application discloses a D-dimer detection reagent comprising polyethylene glycol, a buffer system, and an inorganic salt; the polyethylene glycol is low molecular weight polyethylene glycol with the relative molecular weight of 6000-10000; inorganic salts are used to adjust the ionic strength of the buffer system.

It should be noted that, during the detection of D-dimer (abbreviated as DD), especially when DD reacts with its specific antibody, if hemoglobin exists in the sample or reaction system, the hemoglobin interacts with DD, thereby affecting the reaction of DD with the specific antibody and further interfering with the test result. The research of the application finds that polyethylene glycol (abbreviated as PEG) can influence the interaction of the hemoglobin and the DD, so that the influence of the hemoglobin on the reaction of the DD and a specific antibody is avoided or reduced, namely the PEG can resist the interference of the hemoglobin; particularly, the anti-interference capability of the low molecular weight polyethylene glycol is better, such as PEG6000, PEG8000, PEG10000 and the like; it will be appreciated that other molecular weight polyethylene glycols may be used if the interference resistance is not critical. Thus, the present application provides, inter alia, a D-dimer detection reagent comprising a low molecular weight polyethylene glycol having a relative molecular weight of 6000-10000. In the D-dimer detection reagent, a buffer solution system and inorganic salt can refer to the existing D-dimer detection reagent, and particularly can refer to the existing D-dimer immunoturbidimetric detection reagent; however, in consideration of the detection effect of the D-dimer detection reagent, the preferred technical scheme of the present application defines the buffer solution system, inorganic salts and other components, and will be described in detail in the following preferred technical scheme.

Preferably, the working concentration of the low molecular weight polyethylene glycol in the D-dimer detection reagent of the present application is 5 to 50 g/L.

It should be noted that the research of the present application finds that the anti-hemoglobin interference capability of the low molecular weight polyethylene glycol is best at the working concentration of 5-50 g/L; it will be appreciated that lower or higher concentrations of polyethylene glycol may also be employed in applications where interference rejection is less desirable.

Preferably, in the D-dimer detection reagent of the present application, the pH of the buffer system is 5.0 to 8.5.

Preferably, in the D-dimer detection reagent of the present application, the buffer liquid is at least one of a boric acid buffer, a citric acid-sodium citrate buffer, a glycine buffer, a phosphate buffer, a Tris-hydrochloric acid buffer, MES, HEPES, and MOPS buffer.

Preferably, in the D-dimer detection reagent of the present application, the inorganic salt is at least one selected from the group consisting of potassium chloride, lithium chloride, sodium chloride, zinc chloride, magnesium chloride, potassium nitrate and sodium nitrate.

More preferably, in the test agent for D-dimer of the present application, the inorganic salt is at least one selected from the group consisting of potassium chloride, sodium chloride, zinc chloride and magnesium chloride.

It should be noted that the inorganic salt serves to provide suitable ionic strength for the reaction of the D-dimer with the specific antibody; therefore, any inorganic salt that can adjust the ionic strength and does not inhibit the reaction of the D-dimer with the specific antibody can be used in the present application, for example, potassium chloride, lithium chloride, sodium chloride, zinc chloride, magnesium chloride, potassium nitrate, sodium nitrate, and the like.

Preferably, the D-dimer detection reagent of the present application further comprises a protein protectant.

It should be noted that the protein protecting agent of the present application is a protein stabilizing agent, and the role thereof is to protect the stability of a protein, and therefore, in principle, any agent capable of stabilizing a protected protein can be used in the present application. However, in a preferred embodiment of the present application, the protein protectant is selected from at least one of bovine serum albumin (abbreviated BSA), immunoglobulin (abbreviated IgG), glycine, polysaccharide, and sorbitol.

Preferably, the D-dimer detection reagent herein further comprises a suspending agent.

It is noted that suspending agents function to facilitate uniform suspension of materials that are insoluble in solvents, and thus suspending agents that facilitate suspension of microparticles or gels may be used herein. However, in a preferred embodiment of the present application, the suspending agent is tween20 and/or triton-X100.

Preferably, the D-dimer detection reagent of the present application further comprises a preservative.

It is noted that the preservative functions to eliminate the problem of changes in the properties of the agent caused by microorganisms, and therefore, conventionally used preservatives can be used in the present application. However, the present application preferably employs at least one of Proclin300, thimerosal, sodium benzoate, nitrate, nitrite, and sodium azide.

In one mode of use of the present application, all reagents in the D-dimer detection reagent are divided into reagent one and reagent two; the first reagent comprises polyethylene glycol, a buffer solution system, inorganic salt, a preservative and a protein protective agent; the second reagent comprises particles coated with D-dimer specific antibodies, a buffer system, a protein protective agent, a preservative and a suspending agent. While not wishing to be bound by theory, applicants have found through extensive experimentation that the low molecular weight polyethylene glycol placed in reagent one contributes to the stability of the long term performance of the reagent system.

Preferably, the buffer system of the first reagent has a pH value of 5.0-7.0, and the buffer liquid system is at least one of boric acid buffer, citric acid-sodium citrate buffer, glycine buffer, phosphate buffer, MES, HEPES and MOPS buffer.

More preferably, the buffer liquid of the first reagent is a boric acid buffer, a phosphate buffer, a MOPS buffer or a MES buffer.

Preferably, the preservative of the first reagent is Proclin300, thimerosal or sodium azide.

Preferably, the protein protectant of the first reagent is at least one selected from the group consisting of bovine serum albumin, immunoglobulin and sorbitol.

Preferably, the D-dimer specific antibody coated microparticles in the second reagent are polystyrene microspheres.

Preferably, the pH value of the buffer system of the reagent two is 6.5-8.5, and the buffer liquid system is at least one of boric acid buffer, phosphate buffer, Tris-hydrochloric acid buffer, HEPES and MOPS buffer.

In the present application, the first reagent is mixed with the sample in advance to process the sample; the second reagent serves to deposit the particles coated with the D-dimer specific antibody. For example, in one mode of use of the present application, reagent one and reagent two are used as immunoturbidimetric detection reagents.

When the reagent I and the reagent II are used as an immunoturbidimetric assay reagent, corresponding blocking agents can be added into the reagent I in order to eliminate the influence of D-dimer assay partial cover caused by nonspecific binding of autoimmune proteins such as rheumatoid factor and HAMA. The key point of the present application is to eliminate the interference of hemoglobin by adding a specific amount of low molecular weight polyethylene glycol, and therefore, the blocking agent can refer to conventionally used blocking agents against interference of rheumatoid factor, HAMA, and the like, and is not particularly limited herein.

The other side of the application discloses a D-dimer detection reagent, which consists of a reagent I and a reagent II; the reagent I consists of 5-50g/L of low molecular weight polyethylene glycol with the relative molecular weight of 6000-10000, a buffer solution system, inorganic salt, a preservative and a protein protective agent; and the reagent II consists of particles coated with the D-dimer specific antibody, a buffer solution system, a protein protective agent, a preservative and a suspending agent.

In the application, the specific content of the low-molecular-weight polyethylene glycol is added into the reagent I, so that the D-dimer detection reagent can reduce the interference of hemoglobin on D-dimer detection. In the prior art, the components of D-dimer reagent, in particular the D-dimer specific antibody coating particles, buffer solution systems, inorganic salts, preservatives, protein protective agents, suspending agents and the like in the D-dimer immunoturbidimetric detection reagent can also be used in the reagent of the application if the components can be applied through experiments.

The application also discloses application of the low molecular weight polyethylene glycol in preparing a D-dimer detection reagent for reducing hemoglobin interference, wherein the low molecular weight polyethylene glycol is polyethylene glycol with the relative molecular weight of 6000-10000.

It is important to note that the key to the present application is the discovery that polyethylene glycol (abbreviated PEG) can affect the interaction of hemoglobin with D-dimer, thereby avoiding or reducing the effect of hemoglobin on the reaction of D-dimer with specific antibody; therefore, the application proposes a new application of polyethylene glycol, namely, the application in preparing a D-dimer detection reagent for reducing hemoglobin interference.

Preferably, in the application of the present application, the D-dimer detection reagent is an immunoturbidimetric detection reagent.

Preferably, in the application of the present application, the working concentration of the low molecular weight polyethylene glycol in the D-dimer detection reagent is 5-50 g/L.

In another aspect of the present application, there is disclosed a method for detecting a D-dimer, comprising the steps of,

obtaining a blood sample to be detected which is treated by sodium citrate; then one of the following actions is taken:

operation one: uniformly mixing the blood sample to be detected with the first reagent, and carrying out warm bath to the reaction temperature; then adding a reagent II which is subjected to warm bath to the reaction temperature, and uniformly mixing for reaction;

and operation II: carrying out warm bath on a blood sample to be detected and a D-dimer detection reagent to reaction temperature respectively, and then mixing uniformly for reaction;

after the first operation or the second operation, the interference of hemoglobin is eliminated without using dual-wavelength correction or high wavelength, and the content of the D-dimer is calculated according to the change of the light transmittance value of the reaction system directly through single-wavelength detection;

wherein, the reagent I comprises polyethylene glycol, a buffer solution system, inorganic salt, a preservative and a protein protective agent; the reagent II comprises particles coated with D-dimer specific antibodies, a buffer solution system, a protein protective agent, a preservative and a suspending agent; the D-dimer detection reagent contains polyethylene glycol, particles coating D-dimer specific antibodies, a buffer system and inorganic salts.

Preferably, the D-dimer detection reagent further comprises at least one of a protein protecting agent, a suspending agent and a preservative.

Preferably, the polyethylene glycol in the reagent one or the D-dimer test reagent is a low molecular weight polyethylene glycol having a relative molecular weight of 6000-10000.

Preferably, the working concentration of the low molecular weight polyethylene glycol is 5-50 g/L.

It should be noted that the key point of the present application is to add low molecular weight polyethylene glycol into the reagent-I or D-dimer detection reagent, so that the detection method of the present application can eliminate the interference of hemoglobin on the detection result without using dual-wavelength calibration or high wavelength, simplify the test operation, avoid using expensive instruments and equipment with complex optical systems, and reduce the detection cost. It is understood that the key to the present application is the addition of low molecular weight polyethylene glycol to the reagent one or the D-dimer detection reagent; as for the other components, for example, particles coated with D-dimer specific antibodies, buffer systems, inorganic salts, preservatives, protein protecting agents, suspending agents and the like can be referred to the existing D-dimer detecting reagents, and particularly, the existing D-dimer immunoturbidimetric detecting reagents can be referred to.

Still another aspect of the present application discloses a method for preparing a D-dimer detection reagent,

dispersing polyethylene glycol, inorganic salt, a preservative and a protein protective agent into a buffer solution system to prepare a reagent I; dispersing the particles coated with the D-dimer specific antibody, a protein protective agent, a preservative and a suspending agent into a buffer solution system to prepare a reagent II;

alternatively, polyethylene glycol, D-dimer specific antibody-coated microparticles, and an inorganic salt are dispersed in a buffer system to prepare a D-dimer detection reagent.

Preferably, the preparation method of the present application further comprises dispersing at least one of a protein protecting agent, a suspending agent and a preservative in the D-dimer detection reagent.

Preferably, in the preparation method of the present application, the polyethylene glycol of the reagent I or the D-dimer detection reagent is low molecular weight polyethylene glycol with a relative molecular weight of 6000-10000.

Preferably, the working concentration of the low molecular weight polyethylene glycol is 5-50 g/L.

The key point of the preparation method of the D-dimer detection reagent is that low molecular weight polyethylene glycol is added into the reagent I or the D-dimer detection reagent, so that the D-dimer detection reagent prepared by the preparation method can reduce the interference of hemoglobin on D-dimer detection. As for other components involved in the production method, for example, microparticles coated with D-dimer specific antibodies, buffer systems, inorganic salts, preservatives, protein protecting agents, suspending agents and the like, reference may be made to existing D-dimer detection reagents, particularly to existing D-dimer immunoturbidimetric detection reagents.

Due to the adoption of the technical scheme, the beneficial effects of the application are as follows:

according to the D-dimer detection reagent or the detection method, the interaction of the hemoglobin and the D-dimer is interfered by the low-molecular-weight polyethylene glycol, so that the influence of the hemoglobin on the reaction of the D-dimer and the specific antibody is reduced, the effect of resisting the interference of the hemoglobin is achieved, and the accuracy of D-dimer detection is improved. In addition, the D-dimer detection reagent can resist hemoglobin interference, so that the influence caused by hemoglobin is eliminated without adopting a double wave method or adopting high wavelength during D-dimer detection, and the difficulty and the instrument cost of D-dimer detection are reduced.

Drawings

FIG. 1 is a schematic illustration of an immunoturbidimetric assay in an example of the present application;

FIG. 2 is a graph showing the results of D-dimer detection in clinical samples according to example one of the present application;

FIG. 3 is a graph showing the results of D-dimer detection in clinical samples according to example two of the present application;

FIG. 4 is a graph showing the results of D-dimer detection in clinical specimens according to example three of the present application;

FIG. 5 is a graph showing the results of D-dimer detection in clinical specimens according to example four of the present application;

FIG. 6 is a graph showing the results of D-dimer detection in clinical samples according to example five of the present application;

FIG. 7 is a graph showing the results of D-dimer detection in clinical samples according to example six of the present application;

FIG. 8 is a graph showing the results of D-dimer detection in clinical specimens according to example seven of the present application.

Detailed Description

The existing D-dimer detection method usually adopts dual-wave method detection or high wavelength to eliminate the influence caused by hemoglobin when facing the hemoglobin interference, which can greatly increase the complexity of an optical system of the instrument and the cost of the instrument.

The application creatively discovers that the low-molecular-weight polyethylene glycol with specific concentration has better anti-interference effect on the hemoglobin. In the D-dimer detection process, if the D-dimer interacts with hemoglobin, the D-dimer cannot normally react with a D-dimer specific antibody, so that the accuracy of a detection result is influenced; the application finds that the low molecular weight polyethylene glycol can influence the interaction between the hemoglobin and the D-dimer, and particularly the low molecular weight polyethylene glycol at a specific concentration has a better effect, so that the effect of resisting the interference of the hemoglobin can be achieved.

Based on the above research findings, the present application has developed a D-dimer detection reagent and a D-dimer detection method.

Wherein the D-dimer detection reagent comprises low molecular weight polyethylene glycol with the relative molecular weight of 6000-10000, a buffer solution system and inorganic salt. By utilizing the anti-hemoglobin interference effect of the low-molecular-weight polyethylene glycol, the D-dimer detection reagent can accurately detect the D-dimer, and by adopting the D-dimer detection reagent, the influence of hemoglobin can be eliminated without using a complex optical system.

In one implementation of the present application, the D-dimer detection reagent consists of reagent one and reagent two; the reagent I consists of 5-50g/L of low molecular weight polyethylene glycol with the relative molecular weight of 6000-10000, a buffer solution system, inorganic salt, a preservative and a protein protective agent; and the reagent II consists of particles coated with the D-dimer specific antibody, a buffer solution system, a protein protective agent, a preservative and a suspending agent. Similarly, the D-dimer detection reagent of the application also utilizes the anti-hemoglobin interference effect of the low molecular weight polyethylene glycol, so that the D-dimer can be accurately detected without using a complex optical system to eliminate the influence of hemoglobin.

The D-dimer detection method comprises the step of eliminating the interference of hemoglobin on a detection result in a mode of adding an anti-interference agent into a reagent during the detection of the D-dimer without using dual-wavelength correction or high wavelength. The hemoglobin interference resisting effect of the low molecular weight polyethylene glycol is utilized, so that the detection method is simpler and more convenient, and the instrument cost is lower.

Meanwhile, the application also provides a new application of the low molecular weight polyethylene glycol, namely the application of the low molecular weight polyethylene glycol in preparing a D-dimer detection reagent for reducing hemoglobin interference. The application provides a new thought and method for eliminating hemoglobin interference in D-dimer detection, and the problem of hemoglobin interference can be effectively solved without adopting an expensive and complex optical system.

The D-dimer detection reagent and method of the present application are described in immunoturbidimetric assay in detail as follows:

in the case of the D-dimer test, after the anticoagulant-treated sample is added to the cuvette, the sample is transferred to a constant temperature 37 ℃ incubation position for incubation, and then reagent one is added to the cuvetteAnd after being uniformly mixed, preheating to provide a proper reaction environment for antibody-antigen reaction, namely a reaction environment at 37 ℃, and then adding a second reagent. And the change of the transmittance value of the reaction system is detected at 575 nm. Calculating absorbance values corresponding to samples with different concentrations according to the variation of the light transmittance value within 20-150 seconds, and the test diagram is shown in FIG. 1, wherein T in FIG. 1₁At the time point, the light intensity irradiated on the reaction cup is A0, and the light intensity transmitted after passing through the reaction cup is A1; at T₂At the time point, the light intensity irradiated on the reaction cup is A0, the reaction system has reacted at the time point, the light intensity of the light transmittance is A2, and the difference between A2 and A1 is used as the test signal value.

In addition, the detection method can also be combined with a prisheng C3510 full-automatic hemagglutination instrument to realize automatic test, and the sample test process is as follows:

1. and (3) calibrating the prisheng full-automatic hemagglutination instrument by using a standard substance containing a certain amount of D-dimer antigen, and detecting the qualified sample by using a corresponding quality control substance detection and measurement system.

2. The sample determination process mainly comprises the following steps: firstly, adding a plasma sample adopting a sodium citrate anticoagulant into a reaction cup of a prisheng full-automatic hemagglutination instrument, carrying out warm bath for 60 seconds at 37 ℃, then adding a reagent I, uniformly mixing, carrying out warm bath for 90 seconds, then adding a reagent II, and uniformly stirring. The D-dimer in the plasma and the antibody coated on the polyethylene particles have specific reaction and cause aggregation reaction, and the change of the light transmittance value of the reaction system after the latex is added is detected by a pril full-automatic hemagglutination instrument. The amount of D-dimer in the plasma sample was calculated from the reaction curve.

In the above case of performing the detection by dividing the reagent of the present application into the first reagent and the second reagent, if the reagent is not divided into the first reagent and the second reagent, but a single mixed reagent, that is, polyethylene glycol, a buffer system, an inorganic salt and particles coated with a D-dimer specific antibody are contained in a mixed D-dimer detection reagent, the mixed D-dimer detection reagent preferably further contains at least one of a protein protecting agent, a suspending agent and a preservative, and the D-dimer detection reagent more preferably further contains an additive that is resistant to interference from other substances, for example, a blocking agent that is resistant to interference from rheumatoid factor, HAMA and the like, and the detection method of the single mixed detection reagent is as follows:

firstly, a plasma sample adopting a sodium citrate anticoagulant is added into a reaction cup of a pricele full-automatic hemagglutination instrument, the temperature bath is carried out for 60 seconds at 37 ℃, then a single mixed D-dimer detection reagent is added and stirred uniformly, the D-dimer in the plasma and an antibody coated on particles (such as polyethylene particles) have specific reaction and cause aggregation reaction, and the change of the light transmittance value of a reaction system after the detection reagent is added is detected by the aid of the pricele full-automatic hemagglutination instrument. The detection principle is similar to that of the reagent I and the reagent II, and the description is omitted here.

It should be noted that, the reagents of the present application are divided into reagent one and reagent two, or a single mixed D-dimer detection reagent, and D-dimer detection can be performed by a prisheng fully automatic hemagglutination instrument, and only the detection device needs to be adjusted accordingly.

The terms related to this application are explained below:

MES refers to 2- (N-morpholinyl) ethanesulfonic acid buffer;

MOPS refers to 3- (N-morpholine) propanesulfonic acid buffer;

HEPES means 4-hydroxyethylpiperazine ethanesulfonic acid;

triton-X100 refers to polyethylene glycol octyl phenyl ether.

The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.

The instruments, equipment and solutions used in the examples are all conventional unless otherwise indicated.