CN116973576A

CN116973576A - D-dimer detection kit and detection method thereof

Info

Publication number: CN116973576A
Application number: CN202310977916.1A
Authority: CN
Inventors: 张翠杰
Original assignee: Beijing Antu Bioengineering Co ltd
Current assignee: Beijing Antu Bioengineering Co ltd
Priority date: 2023-08-04
Filing date: 2023-08-04
Publication date: 2023-10-31

Abstract

The invention relates to the technical field of medical detection, in particular to a D-dimer detection kit and a detection method thereof. The invention provides a D-dimer detection kit, which consists of a reagent R1 and a reagent R2. Wherein the R1 reagent mainly comprises buffer solution, preservative, inorganic salt, surfactant and protein stabilizer; the R2 latex reagent mainly comprises buffer solution, protein protecting agent, sucrose and preservative. Experiments show that the kit is easy to operate and short in analysis time, has high analysis sensitivity, high precision, high test accuracy and wide linear range, and can be combined with an automatic analyzer, thereby being beneficial to wide popularization and use in clinic.

Description

D-dimer detection kit and detection method thereof

Technical Field

The invention relates to the technical field of medical detection, in particular to a D-dimer detection kit and a detection method thereof.

Background

D-dimer (DD) is a specific fibrin degradation product of crosslinked fibrin under the action of a fibrin enzyme. During blood coagulation, fibrinogen is hydrolyzed to soluble fibrin monomers, which are automatically polymerized into network polymers, which are covalently crosslinked between monomers under the action of thrombin and Factor XIIIa to form crosslinked fibrin. At the same time, in vivo cross-linked fibrin is degraded by plasmin into various fibrin degradation products of different molecular weights, including D-dimers, which are specific degradation products thereof.

Activation of the coagulation system results in the formation of thrombin, which, upon binding of the central domain of fibrinogen (region E), releases fibrinopeptides A (FPA) and fibrinopeptides B (FPB) while exposing complementary binding sites to facilitate the autopolymerization of the two. Fibrin units polymerize side-by-side, end-by-end into polymeric chains, producing Fibrin Monomers (FM) and polymers. In Factor XIIIa and Ca ²⁺ Under the action of the above, the glutamine at 398 or 399 position of the gamma chain on the fibrin monomer is crosslinked with the lysine at 406 position of the gamma chain on the other fibrin monomer to form a gamma '-gamma' linkage, so as to generate crosslinked fibrin. Plasmin cleaves the complementary structure of the DE region, degrading fibrinogen (Fg) and Fibrinogen Monomer (FM) to form fibrinogen degradation products (Fg DPs); at the same time, cross-linked fibrin is reduced, and a plurality of degradation products (Fb DPs) of cross-linked fibrin are generated, wherein the degradation products comprise D-dimer and other fragments.

The increased D-dimer content can detect the course of various diseases, such as deep vein thrombosis, disseminated intravascular coagulation, myocardial infarction, severe hepatitis, pulmonary embolism and other diseases; has certain monitoring effect on the possible complications of the parturient with preeclampsia and pregnancy high risk. In addition, the change of the D-dimer level can be used as an index for monitoring thrombolytic therapy and guiding the dosage of thrombolytic drugs. Therefore, the detection of the D-dimer content in blood has important clinical significance in the aspects of early diagnosis of thrombotic diseases, disease course monitoring, treatment monitoring of thrombolytic drugs and the like.

The currently prevailing methods for detecting D-dimers are mainly three: whole blood agglutination method, enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunofluorescence (ELFA), latex turbidimetry. Among them, latex immunoturbidimetry is widely used in clinic with the advantages of high sensitivity, rapid and accurate detection. Latex immunoturbidimetry is widely used in clinic because of its high sensitivity and rapid and accurate detection. The detection of D-dimer by latex immunonephelometry is mainly based on the fact that the detection object D-dimer is combined with an antibody coated on the surface of latex particles to cause the increase of turbidity, thereby realizing the detection of D-dimer. However, the current kit for detecting DD has the problems of low precision, narrow linear range, weak anti-interference capability and poor stability, and limits the application of the kit in clinical detection.

Disclosure of Invention

In view of the above, the present invention aims to provide a D-dimer detection kit and a detection method thereof.

The invention provides a kit for detecting D-dimer, which comprises a reagent R1 and a reagent R2,

the reagent R1 comprises: buffer solution, inorganic salt, coagulant, surfactant, preservative and protective agent; the surfactant comprises: at least one of a silicone surfactant, a fluorosurfactant, and/or a nonionic surfactant;

further, the silicone surfactant includes, but is not limited to, silwet L-7600; the nonionic surfactants include, but are not limited to, triton X-100.

The reagent R2 comprises: buffer solution, protective agent, stabilizer and latex microsphere coated with D-Dimer antibody;

the latex microsphere is made of polystyrene latex microsphere with the particle size of 300nm.

The invention optimizes the particle size of the emulsion microsphere, and the test result shows that the monoclonal antibody has good affinity, high reactivity and high sensitivity when the particle size of the carboxylated polystyrene emulsion microsphere is 300nm.

Further, in the reagent R1,

the buffer solution is at least one selected from MOPS buffer solution and/or PB buffer solution;

the inorganic salt is at least one selected from NaCl and/or KCl;

the coagulant is at least one selected from PEG6000 and/or PEG 8000;

the surfactant comprises Silwet L-7600 and Triton X-100;

the preservative is ProClin 300;

the protective agent is selected from at least one of BSA, casein and/or ADP;

in the case of the reagent R2,

the buffer solution is at least one selected from MOPS buffer solution, TAPS buffer solution, PB buffer solution, glycine buffer solution and/or HEPES buffer solution;

the protective agent is selected from at least one of BSA, casein and/or ADP;

the stabilizer is at least one selected from sucrose, trehalose, glycerol and/or glucose.

Still further, the method comprises the steps of,

the reagent R1 comprises: PB buffer with pH of 6.5 50mM, 9-15 g/L NaCl, 8-10 g/LPEG6000, 1.0-2.0 g/L Silwet L-7600, 1.0-5.0 g/L Triton X-100, 1.0-2.0 ml/LProClin 300 and 2-5 g/L BSA.

The reagent R2 comprises: 20mM MOPS buffer solution, 2-5 g/L BSA, 60-80 g/L glucose and 1-3 mg/mL polystyrene latex microsphere coated with D-Dimer antibody; or (b)

The reagent R2 comprises: 20mM TAPS buffer solution, 2-5 g/L BSA, 60-80 g/L glucose and 1-3 mg/mL polystyrene latex microsphere coated with D-Dimer antibody; or (b)

The reagent R2 comprises: 20mM HEPES buffer, 2-5 g/L BSA, 60-80 g/L glucose and 1-3 mg/mL polystyrene latex microspheres coated with D-Dimer antibody.

Still further, the method comprises the steps of,

in some embodiments of the invention, the reagent R1 is PB buffer at pH 6.5 50mM, 9g/L NaCl, 8g/L PEG6000, 1.0g/L Silwet L-7600, 1.0g/L Triton X-100, 1.0ml/LProClin 300, and 5g/L BSA; the reagent R2 is: 20mM MOPS buffer, 2g/L BSA, 60g/L glucose and 1.25mg/mL D-Dimer antibody coated polystyrene latex microspheres; or (b)

In other embodiments of the invention, the reagent R1 is PB buffer at pH 6.5 50mM, 12g/L NaCl, 9g/L PEG6000, 1.0g/L Silwet L-7600, 2.0g/L Triton X-100, 1.0ml/LProClin 300, and 2g/L BSA; the reagent R2 comprises: 20mM TAPS buffer, 2g/L BSA, 60g/L glucose and 1.25mg/mL D-Dimer antibody coated polystyrene latex microspheres; or (b)

In other embodiments of the invention, the reagent R1 is PB buffer at pH 6.5 50mM, 15g/L NaCl, 10g/L PEG6000, 2.0g/L Silwet L-7600, 5.0g/L Triton X-100, 1.0ml/LProClin 300, and 2g/L BSA; the reagent R2 comprises: 20mM HEPES buffer, 5g/L BSA, 80g/L glucose and 1.25mg/mL D-Dimer antibody coated polystyrene latex microspheres.

In the kit, the surfactant in the reagent R1 is a combination of a high-efficiency organosilicon surfactant Silwet L-7600 and a nonionic surfactant 100 (polyethylene glycol octyl phenyl ether) (Triton X-100). The organosilicon surfactant not only has higher surface activity (such as lower cmc and gamma cmc) than the common surfactant, but also has the performance which is not possessed by many common surfactants. The surfactant plays a role of a dispersing agent in the reagent R1, so that the antigen site is exposed more fully, and the mutual attraction and combination between the D-dimer of the object to be detected and the latex particles of the D-dimer antibody are promoted. The problem of poor repeatability of the reagent at a low value is effectively solved, the accuracy of reagent detection is improved, and the anti-interference capability of the reagent is obviously enhanced.

In the present invention, the surfactant in R1 can also effectively solve the problem of reagent repeatability. In some embodiments, the surfactant of the R1 reagent is preferably a combination of both the high potency silicone surfactant Silwet L-7600 and the conventional surfactant 100 (polyethylene glycol octyl phenyl ether) (Triton X-100). On the one hand, the buffer solution of the R1 reagent and the surfactant can be matched to effectively solve the problem of reagent repeatability. In some specific experimental groups, the buffer for the R1 reagent is preferably 50mM PB buffer at pH 6.5. On the other hand, the anti-interference capability of the reagent is further enhanced by adding sodium chloride in the presence of the surfactant and other components in R1.

The kit is used for measuring the D-dimer based on an immunoturbidimetry, and the detection principle is as follows: after the reagent R2 and the D-dimer in the sample are combined to generate agglutination reaction, an antigen-antibody immune complex is formed, turbidity is generated, the turbidity is related to the concentration of the D-dimer in the sample, the absorbance value at the moment is measured, and the content of the D-dimer is calculated according to a calibration curve. Typically, the particle size of the polystyrene latex microspheres affects the sensitivity of the reagent. The particle size of the polystyrene latex microsphere is 270 nm-400 nm; in some embodiments, the polystyrene latex microspheres have a particle size of 300nm.

The invention provides a method for detecting D-dimer, which utilizes the kit disclosed by the invention to detect a sample.

Further, the detecting includes the steps of: after the sample is sequentially mixed with the reagent R1 and the reagent R2, the difference between the initial absorbance and the measured absorbance is obtained, and the concentration of the D-dimer is obtained according to a standard curve.

Still further, the method comprises the steps of,

the volume ratio of the sample, the reagent R1 and the reagent R2 is 150:50:6.

The initial absorbance is measured at 5min after mixing of reagent R2;

the absorbance was measured at 10min after mixing of reagent R2.

The sample is blood plasma, and the blood plasma is treated by sodium citrate.

Specifically, firstly, uniformly mixing a reagent R1 with a sodium citrate anticoagulation plasma sample to be detected for 5 minutes, and then adding a reagent R2 to start a reaction, wherein the volume ratio of the reagent R1 to the reagent R2 is calculated as sample=150:50:6; the initial absorbance A1 is measured by comparing with the blank at 5 minutes, the absorbance A2 is measured by comparing with the blank at 10 minutes, the difference delta A between A2 and A1 is calculated, and the D-dimer content in the plasma sample to be measured is obtained according to a standard curve.

The invention provides a D-dimer detection kit, which consists of a reagent R1 and a reagent R2. Wherein the R1 reagent mainly comprises buffer solution, preservative, inorganic salt, surfactant and protein stabilizer; the R2 latex reagent mainly comprises buffer solution, protein protecting agent, sucrose and preservative. Experiments show that the kit is easy to operate and short in analysis time, has high analysis sensitivity, high precision, high test accuracy and wide linear range, and can be combined with an automatic analyzer, thereby being beneficial to wide popularization and use in clinic.

Drawings

FIG. 1 shows a correlation curve of experimental group 1 in a correlation experiment;

fig. 2 shows the linear range curve of experimental group 1 in a linear experiment.

Detailed Description

The invention provides a D-dimer detection kit and a detection method thereof, and a person skilled in the art can properly improve the process parameters by referring to the content of the text. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred experimental sets, it will be apparent to those skilled in the relevant art that the methods and applications herein can be modified or adapted and practiced without departing from the spirit and scope of the invention.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

The invention is further illustrated in the following in connection with the experimental group:

experimental group D-dimer detection kit and detection method thereof

1. Preparation of reagent R1: the kit provided by the invention is used for determining the D-dimer based on a latex immunoturbidimetry and consists of a reagent R1 and a reagent R2, wherein the surfactant in the reagent R1 is a combination of Silwet L-7600 and Triton X-100. The reagent R2 is a polystyrene latex microsphere compound coated with the D-dimer antibody.

TABLE 1 composition of reagent R1

The preparation procedure of reagent R1 is (preparation 1L): preparing PB buffer solution by using purified water according to the component concentrations of the experimental groups 1-3 in the table 1 and the component content of the reagent R1, and adjusting the pH to obtain R1 buffer solution; then adding corresponding amounts of sodium chloride, polyethylene glycol 6000, silwet L-7600, triton X-100, bovine serum albumin and ProClin300, mixing well, and adjusting pH with sodium hydroxide/hydrochloric acid to obtain reagent R1.

2. Preparation of reagent R2:

TABLE 2 composition of reagent R2

Component (A)	Experiment group 1	Experiment group 2	Experiment group 3
				20mM buffer	MOPS	TAPS	HEPES
Bovine serum albumin (BSA, g/L)	2	2	5
				Glucose (g/L)	60	60	80
Latex microsphere coated DD antibody (g/L)	1.25	1.25	1.25

1. Preparation of buffer for reagent R2: preparing a buffer solution by using purified water according to the component concentration of the reagent R2 in the embodiment shown in the table 2, adjusting the pH, and adding corresponding amounts of bovine serum albumin and glucose as the buffer solution of the reagent R2;

2. preparation of reagent R2:

step 1: carboxylated polystyrene latex microspheres with the particle size of 300nm are added into 10mM MES buffer solution to the concentration of 8.3mg/ml;

step 2: 4mg/ml EDC ((1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride)) was added to each of the latex microspheres, and the mixture was stirred at room temperature for 0.5h;

step 3: dissolving and diluting the DD monoclonal antibody to 0.24mg/ml by using PB buffer solution with the pH of 8.0, then respectively adding the antibody diluted solution into the activated polystyrene latex microspheres, and stirring at room temperature for reaction for 3 hours;

step 4: adding a commercial blocking agent 110010 to terminate the reaction to a final concentration of 0.05%, and stirring overnight at room temperature; centrifuging the obtained reaction liquid, and discarding supernatant to obtain precipitate;

step 5: dispersing the precipitate with a buffer solution of the reagent R2, and carrying out ultrasonic treatment for 3 minutes to obtain a uniformly distributed R2 latex reagent;

step 6: and (3) placing the R2 in a water bath kettle at 42 ℃ for ageing treatment for 16-18 hours to obtain the final R2 reagent.

3. Preparation of the comparative 1-3 kit

Reagent R1 was prepared as in one of experimental groups 1 to 3.

Preparation of reagent R2: the particle sizes of the polystyrene latex microspheres used in the step 1 of the experimental group are respectively 145nm, 190nm and 218nm, and the other steps are consistent with the experimental group.

4. Preparation of comparative sets 4-9 kits

The kit can be used for measuring the D-dimer by a latex immunoturbidimetry method, and comprises a reagent R1 and a reagent R2.

TABLE 3 composition of reagent R1

The preparation procedure of reagent R1 is (preparation 1L): preparing a buffer solution by using purified water according to the component concentrations of comparative groups 4 to 9 in Table 3 and the component content of the reagent R1, and adjusting the pH to obtain an R1 buffer solution; then adding corresponding amounts of sodium chloride, polyethylene glycol 6000, silwet L-7600, triton X-100, bovine serum albumin and ProClin300, mixing well, and adjusting pH with sodium hydroxide/hydrochloric acid to obtain reagent R1.

The preparation of the reagent R2 is consistent with the preparation method of the reagent R2 in the experimental group.

5. Results

The performance evaluation result levels of the kits in the experimental groups 1-3 are basically consistent, and the relevant performances such as relevance, linear range, precision, anti-interference performance, stability and the like are verified by taking the kits prepared in the experimental groups 1-3 as examples.

The testing method comprises the following steps: endpoint method, incremental reaction. At 37 ℃, firstly, uniformly mixing the reagent R1 with the DD plasma sample for 5 minutes, and then adding the reagent R2 to start the reaction, wherein the volume ratio of the reagent R1 to the reagent R2 is that of the sample=150:50:6; initial absorbance A1 was measured at 5 minutes relative to the blank, and absorbance A2 was measured at 10 minutes relative to the blank, and the difference Δa between A2 and A1 was calculated.

1. Determination of a Standard Curve

The delta A values of a series of standard plasma samples of DD with standard concentration of 0-17.6 mug/mL were measured one by using the test group 1-3 kit and the control DD kit (SEKISUID-dimer kit, cat# 346903), and the partial concentration detection results are shown in Table 4. And standard curves of all the kits can be respectively fitted according to a series of DD concentration and measured delta A values, and the standard curves of the experimental groups 1-3 are SPLINE fitting functions.

Table 4 shows the correspondence between DD concentration and measured Δa value, and it is clear from table 4 that the absorbance change is more pronounced and the sensitivity is higher in the experimental groups 1 to 3 than in the comparative groups 1 to 3 under the same conditions.

TABLE 4 delta A test results for standard plasma samples

As can be seen from Table 4, the comparative groups 1 to 3 have lower reactivity and poorer precision of the test samples (Table 7) than the experimental groups 1 to 3, which indicates that the carboxylated polystyrene latex microspheres of the invention have proper particle size selection, and the used monoclonal antibodies have good affinity, and the reactivity and the sensitivity can reach the level of the comparative kit.

2. Correlation experiments

A comparative example (SEKISUID-dimer kit, cat# 346903) was prepared by using a commercially available D-dimer kit with excellent accuracy, and a test group kit was used as a test group to conduct a comparative experiment, and 40 samples were examined, the results of which are shown in Table 5.

Taking a test result of a comparison kit on the market as an abscissa independent variable, taking a test result of the kit of the invention as an ordinate dependent variable, and making a linear regression curve to obtain a regression equation of an experiment group 1 as Y=1.031X-0.0524, wherein a linear correlation coefficient R= 0.9971; experimental group 2 regression equation y=1.0251x+0.0767, linear correlation coefficient r=0.9990; experimental group 3 regression equation y=1.0263x+0.0392, linear correlation coefficient r=0.9970; the linear relation is good, and the test result can be effectively used as clinical test. The correlation curves of experimental group 1 are shown in figure 1.

TABLE 5 correlation experiment detection results (Unit: μg/mL)

3. Precision detection

Two plasma samples with high and low DD concentration are respectively taken, each plasma sample is continuously tested for 20 times, the variation coefficient of each plasma sample is calculated, the precision results of an experimental group are shown in a table 6, and the precision results of a comparison group 1-9 are shown in a table 7. As can be seen from the experimental results of experimental groups 1 to 3 and comparative groups 1 to 3 in tables 6 and 7, the use of 300nm microspheres has higher sensitivity in the low value region and the test sample has better reproducibility performance than the use of 145nm, 190nm and 218nm microspheres for preparing reagent 2.

As can be seen from the experimental results of experimental groups 1 to 3 and comparative groups 4 to 7 in tables 6 and 7, the addition of the common surfactant to the R1 reagent can improve the precision of the reagent, the addition of the silicon surfactant can remarkably improve the precision of the reagent, and the addition of the common surfactant and the special silicon surfactant has a synergistic effect in improving the precision. And it can be seen from the experimental results of experimental groups 1 to 3 and comparative groups 8 to 9 in tables 6 and 7 that the buffer type affects the precision of the reagent, preferably 50mM PB.

TABLE 6 results of the precision tests of Experimental groups 1 to 3

TABLE 7 comparative examples 1 to 9 precision test results

4. Tamper resistant detection

The serum of the clinical normal patient is divided into two parts, one part is added with the interfering substance with the highest concentration, the other part is added with the same amount of solvent, 3 gradient equal differential dilutions are carried out on the samples added with the interfering substance and the samples without the interfering substance, each sample is detected three times, and the deviation of the measured value is calculated. The test groups were evaluated for their ability to resist interference and the results are shown in Table 8. And the anti-interference ability of the comparison groups 4 to 7 was evaluated, and the results are shown in Table 9.

TABLE 8 anti-interference test results for experimental groups 1-3

TABLE 9 comparison of the results of anti-interference tests for groups 4-7

The results show that the anti-interference performance of the kit disclosed by the invention on bilirubin, hemoglobin, fat emulsion, vitamin C and heparin sodium interfering substances with three concentration gradients is within +/-5%, and the kit has stronger anti-interference performance. In contrast groups 4 to 7, the anti-hemoglobin and chyle interference abilities were all more than 10%, and the anti-interference ability was poor. The experimental results show that the addition of the surfactants Silwet L-7600 and Triton X-100 can obviously improve the anti-interference capability of the reagent, and the anti-interference capability of the reagent is optimal under the addition of sodium chloride, namely, the compounding of the surfactant Silwet L-7600 and Triton X-100 and the co-use of the surfactant Silwet L-7600 and the Triton X-100 can more obviously improve the anti-interference capability of the reagent.

5. Linear experiments

The samples were mixed (equal ratio) with high and low values to 7 diluted concentration samples, each sample was averaged 2 times. Mixing the normal samples into low-value samples of 0.32 mug/mL, and adding DD high-value samples of 17 mug/mL into the mixed normal samples to obtain linear high values; the high and low value samples were mixed proportionally into 7 equally diluted samples. The results of the linear measurement are shown in Table 10. Calculating a linear regression correlation coefficient R by taking the theoretical DD concentration as an abscissa independent variable X and taking an actual test value as an ordinate dependent variable Y, wherein the result shows that the linear regression equation of the experimental group 1 is Y= 0.9955X-0.0419, and the correlation coefficient R=0.9998; the linear regression equation of the experimental group 2 is Y=1.0093X-0.01, and the correlation coefficient R=0.9999; the linear regression equation of the experimental group 3 is Y=1.0109X-0.0207, and the correlation coefficient R=0.9998; the invention has better correlation in the linear range of 0.3 mu g/mL-17 mu g/mL. The linear range curve of experimental group 1 is shown in figure 2.

TABLE 10 Linear Range analysis results

6. Stability test

The kit of the invention was subjected to bottle opening stability and long-term stability tests. The kit is calibrated on a testing instrument, stored for 30 days at the temperature of 2-8 ℃ after bottle opening, and the plasma samples with the concentration of 4.0 mug/mL and 8.0 mug/mL are subjected to bottle opening stability test after 30 days, and the deviation value of the test result after bottle opening for 30 days is calculated, and the result is shown in Table 11.

TABLE 11 results of stability to opening of test groups 1 to 3

The kit is taken and calibrated on a testing instrument, sealed and stored for 18 months at the temperature of 2-8 ℃, plasma samples with the concentration of 4.0 mug/mL and 8.0 mug/mL are respectively subjected to long-term stability testing for 3 months, 6 months, 9 months, 12 months and 18 months, and the deviation value of the testing result after the bottle is opened for 18 months is calculated, and the result is shown in Table 12.

TABLE 12 Long-term stability measurement results (Unit: μg/mL) for experimental groups 1-3

The results in tables 11 and 12 show that after 30 days of bottle opening and 18 months of sealed storage at 2-8 ℃, the test deviation of the plasma samples of 4.0 mug/mL and 8.0 mug/mL of the kit is within 3%, and the stability is good. The preparation reagent R2 has better stability.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

A kit for detecting D-dimer is characterized by comprising a reagent R1 and a reagent R2,

the reagent R1 comprises: buffer solution, inorganic salt, coagulant, surfactant, preservative and protective agent; the surfactant comprises: at least one of a silicone surfactant, a fluorosurfactant, and/or a nonionic surfactant;

the reagent R2 comprises: buffer solution, protective agent, stabilizer and latex microsphere coated with D-Dimer antibody;

the latex microsphere is made of polystyrene latex microsphere with the particle size of 300nm.
2. The kit according to claim 1, wherein in the reagent R1,

the buffer solution is at least one selected from MOPS buffer solution and/or PB buffer solution;

the inorganic salt is at least one selected from NaCl and/or KCl;

the coagulant is at least one selected from PEG6000 and/or PEG 8000;

the surfactant comprises Silwet L-7600 and Triton X-100;

the preservative is ProClin 300;

the protective agent is selected from at least one of BSA, casein and/or ADP.
3. The kit according to claim 1, wherein in the reagent R2,

the buffer solution is at least one selected from MOPS buffer solution, TAPS buffer solution, PB buffer solution, glycine buffer solution and/or HEPES buffer solution;

the protective agent is selected from at least one of BSA, casein and/or ADP;

the stabilizer is at least one selected from sucrose, trehalose, glycerol and/or glucose.
4. The kit according to claim 1 or 2, wherein,

the reagent R1 comprises: PB buffer with pH of 6.5 50mM, 9-15 g/L NaCl, 8-10 g/LPEG6000, 1.0-2.0 g/L Silwet L-7600, 1.0-5.0 g/L Triton X-100, 1.0-2.0 ml/LProClin 300 and 2-5 g/L BSA.
5. A kit according to claim 1 or 3, wherein,

the reagent R2 comprises: 20mM MOPS buffer solution, 2-5 g/L BSA, 60-80 g/L glucose and 1-3 mg/mL polystyrene latex microsphere coated with D-Dimer antibody; or (b)

The reagent R2 comprises: 20mM TAPS buffer solution, 2-5 g/L BSA, 60-80 g/L glucose and 1-3 mg/mL polystyrene latex microsphere coated with D-Dimer antibody; or (b)

The reagent R2 comprises: 20mM HEPES buffer, 2-5 g/L BSA, 60-80 g/L glucose and 1-3 mg/mL polystyrene latex microspheres coated with D-Dimer antibody.
A method for detecting a D-dimer, wherein a sample is detected by the kit according to any one of claims 1 to 5.
7. The method of detection according to claim 6, wherein the detecting comprises the steps of: after the sample is sequentially mixed with the reagent R1 and the reagent R2, the difference between the initial absorbance and the measured absorbance is obtained, and the concentration of the D-dimer is obtained according to a standard curve.
8. The method according to claim 6 or 7, wherein the volume ratio of the sample, the reagent R1 and the reagent R2 is 150:50:6.
9. The method for detecting according to claim 6 or 7, wherein,

the initial absorbance is measured at 5min after mixing of reagent R2;

the absorbance was measured at 10min after mixing of reagent R2.
10. The method according to any one of claims 6 to 9, wherein the sample is plasma, and the plasma is treated with sodium citrate.