CN111830264A

CN111830264A - D-dimer detect reagent box

Info

Publication number: CN111830264A
Application number: CN202010883495.2A
Authority: CN
Inventors: 李小英; 刘芳; 王玉玉
Original assignee: Baoding Tianyue Bioengineering Co ltd
Current assignee: Baoding Tianyue Bioengineering Co ltd
Priority date: 2020-08-28
Filing date: 2020-08-28
Publication date: 2020-10-27

Abstract

The invention relates to the technical field of in-vitro diagnosis, in particular to a D-dimer detection kit, which solves the defect that in the detection process of a D-dimer latex immunoturbidimetry in the prior art, the natural reaction time is longer, so that the detection result needs to wait for a longer time, and comprises a first reagent, a second reagent, a supplementary reagent, a diluent and a calibrator; the reagent I comprises buffer solution and sodium azide; the reagent II comprises polystyrene latex microspheres of the cross-linked D dimer monoclonal antibody and a buffer solution; the supplementary reagent comprises acrylic resin and chondroitin sulfate; the diluent is normal saline. The detection support of the latex immunoturbidimetry is carried out through the polystyrene latex microspheres of the cross-linked D-dimer monoclonal antibody, then the buffer solution is utilized for carrying out buffer proportioning reaction, sodium azide is added, the stability of the detection reagent is improved, and the kit can be ensured to stably realize the detection of the D-dimer through the latex immunoturbidimetry.

Description

D-dimer detect reagent box

Technical Field

The invention relates to the technical field of in-vitro diagnosis, in particular to a D-dimer detection kit.

Background

The D-dimer is derived from a plasmin-solubilized cross-linked fibrin clot, and reflects primarily fibrinolytic function. Clinical testing of D-dimers has been used primarily in the diagnosis of Venous Thromboembolism (VTE), Deep Vein Thrombosis (DVT), and Pulmonary Embolism (PE).

Fibrin exists in blood, and is activated and hydrolyzed to generate specific degradation products, which are called fibrin degradation products. D-dimer is the simplest fibrin degradation product, and an elevated level of D-dimer indicates the presence of hypercoagulable state and secondary hyperfibrinolysis in vivo. Therefore, the mass concentration of the D-dimer has important significance for the diagnosis, the curative effect evaluation and the prognosis judgment of the thrombotic diseases.

The conventional detection method of the D-dimer mainly comprises the following steps: latex agglutination, enzyme-linked immunosorbent assay (ELISA), latex-enhanced immunoturbidimetry, immunogold labeling, and the like. Among them, the latex immunoturbidimetry has the advantages of simple and convenient operation, rapidness, accurate quantification, strong specificity, high sensitivity and the like, can meet the requirements of outpatient and emergency treatment, and is increasingly widely applied in clinical application. In the detection process of the D-dimer latex immunoturbidimetry, the natural reaction time is long, so that the detection result needs to wait for a long time, and in the gradual reaction process, the internal pH changes, so that the detection terminal speed is slow, and the whole detection time of the D-dimer is prolonged.

Therefore, we propose a D-dimer detection kit to solve the above problems.

Disclosure of Invention

The invention aims to solve the defects that in the detection process of a D-dimer latex immunoturbidimetry in the prior art, the natural reaction time is long, the detection result needs to wait for a long time, the internal pH changes in the reaction process gradually, the detection terminal rate is slow, and the overall detection time of the D-dimer is prolonged, and provides a D-dimer detection kit.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a D-dimer detection kit, which comprises a first reagent, a second reagent, a supplementary reagent, a diluent and a calibrator;

the reagent I comprises buffer solution and sodium azide;

the reagent II comprises polystyrene latex microspheres of the cross-linked D dimer monoclonal antibody and a buffer solution;

the supplementary reagent comprises acrylic resin and chondroitin sulfate;

the diluent is normal saline.

Preferably, the first reagent comprises the following components in parts by weight: 10-15 parts of buffer solution and 20-30 parts of sodium azide.

Preferably, the second reagent comprises the following components in parts by weight: 40-50 parts of polystyrene latex microspheres of the cross-linked D dimer monoclonal antibody and 10-15 parts of buffer solution.

Preferably, the supplementary reagent comprises the following components in parts by weight: 20-30 parts of acrylic resin and 10-15 parts of chondroitin sulfate.

Preferably, the buffer solution is Tris, the pH value of the buffer solution is 7.0-9.0, and the concentration of the buffer solution is 250-500 mmol/L.

Preferably, the diluent is 0.5-0.9% of physiological saline by mass fraction.

Preferably, the calibration sample preparation method of the D-dimer detection kit comprises the following steps:

and S1 acquisition: collecting blood;

s2 freezing: freezing the blood plasma at-30 to-18 ℃ within 4 hours after blood collection;

s3 uses: the frozen plasma was thawed within 10min at 37 ℃, mixed well and centrifuged at 10000-15000 Xg for 70min and after centrifugation D-Dimer was determined within 2 hours.

Preferably, in S1, after the blood is collected, the blood tube is directly centrifuged at 1500xg to 2500xg for 15min, and then centrifuged again at 15000xg for 70min to sufficiently centrifuge the high fat plasma.

Preferably, the use method of the D-dimer detection kit comprises the following steps:

s1: taking plasma to be detected, placing the plasma into a first reagent bottle and a second reagent bottle, placing a calibration sample into a third reagent bottle, and adding a diluent into the first reagent bottle, the second reagent bottle and the third reagent bottle, wherein the volume ratio of the diluent to the detected plasma/the calibration sample is (5-9): 1;

s2: adding a first reagent into the first reagent bottle, the second reagent bottle and the third reagent bottle respectively, and incubating for 5-10 min;

s3: adding a second reagent into the first reagent bottle, the second reagent bottle and the third reagent bottle respectively, mixing uniformly, and incubating for 5-10 min;

s4: and adding supplementary reagents into the first reagent bottle, the second reagent bottle and the third reagent bottle respectively, determining the change rate of the light transmittance by adopting a blood coagulation analyzer or a biochemical analyzer so as to make a calibration curve of a calibration sample, and then determining the change rate of the light transmittance of the plasma to be detected, namely obtaining the content of the D-dimer in the plasma to be detected on the calibration curve.

Compared with the prior art, the invention has the beneficial effects that:

1. the detection support of the latex immunoturbidimetry is carried out through the polystyrene latex microspheres of the cross-linked D-dimer monoclonal antibody, then the buffer solution is utilized for carrying out buffer proportioning reaction, and sodium azide is also arranged, so that the stability of the detection reagent is improved, and the kit can be ensured to stably realize the detection of the D-dimer through the latex immunoturbidimetry.

2. According to the invention, the supplementary reagent is arranged in the kit, the acrylic resin is arranged in the supplementary reagent, and after the reagent I and the reagent II are added, the acrylic resin in the supplementary reagent is added to realize the reaction acceleration and coagulation acceleration of the reagent I and the reagent II, so that the plasma to be detected and the polystyrene latex microspheres of the cross-linked D dimer monoclonal antibody are subjected to a quick-reading agglutination reaction to generate agglutination to cause turbidity rise, further, the reduction of light transmittance is realized, and the detection data is quickly obtained.

3. The reagent is also provided with the chondroitin sulfate in the supplementary reagent, the pH value of the kit in the agglutination reaction process can be timely adjusted through the chondroitin sulfate, the kit is ensured to be under the standard of fast agglutination reaction, an environment is provided for the agglutination reaction, fast detection is assisted, and D-dimer detection data can be efficiently obtained.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. When "mass, concentration, temperature, time, or other value or parameter is expressed as a range, preferred range, or as a range defined by a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, a range of 1 to 50 should be understood to include any number, combination of numbers, or subrange selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, and all fractional values between the above integers, e.g., 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, specifically consider "nested sub-ranges" that extend from any endpoint within the range. For example, nested sub-ranges of exemplary ranges 1-50 may include 1-10, 1-20, 1-30, and 1-40 in one direction, or 50-40, 50-30, 50-20, and 50-10 in another direction. "

The present invention is further illustrated below with reference to specific examples in which various processes and methods not described in detail are conventional methods well known in the art. Materials, reagents, devices, instruments, apparatuses and the like used in the following examples are commercially available unless otherwise specified.

Example one

The invention provides a D-dimer detection kit, which comprises a first reagent, a second reagent, a supplementary reagent, a diluent and a calibration sample;

the first reagent comprises the following components in parts by weight: 30 parts of buffer solution and 3 parts of sodium azide;

the reagent II comprises the following components in parts by weight: 20 parts of polystyrene latex microspheres of the cross-linked D dimer monoclonal antibody and 30 parts of buffer solution;

the supplementary reagent comprises the following components in parts by weight: 20 parts of acrylic resin and 10 parts of chondroitin sulfate;

the diluent is 0.5 percent of normal saline by mass fraction.

Wherein the buffer solution is Tris, the pH value is 7.0, and the concentration is 250 mmol/L.

In an embodiment, a calibration sample preparation method of the D-dimer detection kit is further provided, which includes the following steps:

and S1 acquisition: collecting blood;

s2 freezing: freezing the plasma at a temperature of-30 ℃ within 4 hours after blood collection;

s3 uses: the frozen plasma was thawed within 10min at 37 ℃, mixed well and centrifuged at 10000xg for 70min and after centrifugation was complete D-Dimer was determined within 2 hours.

Specifically, the use method of the D-dimer detection kit comprises the following steps:

s1: taking plasma to be detected and placing the plasma into a first reagent bottle and a second reagent bottle, placing a calibration sample into a third reagent bottle, and adding diluent into the first reagent bottle, the second reagent bottle and the third reagent bottle, wherein the volume ratio of the diluent to the detected plasma/the calibration sample is 5: 1;

s2: adding a first reagent into the first reagent bottle, the second reagent bottle and the third reagent bottle respectively, and incubating for 5 min;

s3: adding a second reagent into the first reagent bottle, the second reagent bottle and the third reagent bottle respectively, mixing uniformly and incubating for 5 min;

Example two

the first reagent comprises the following components in parts by weight: 40 parts of buffer solution and 4 parts of sodium azide;

the reagent II comprises the following components in parts by weight: 22 parts of polystyrene latex microspheres of the cross-linked D dimer monoclonal antibody and 40 parts of buffer solution;

the supplementary reagent comprises the following components in parts by weight: 24 parts of acrylic resin and 12 parts of chondroitin sulfate;

the diluent is 0.6 percent of normal saline by mass fraction.

Wherein the buffer solution is Tris, the pH value is 8.0, and the concentration is 300 mmol/L.

and S1 acquisition: collecting blood;

s2 freezing: freezing the plasma at a temperature of-28 ℃ within 4 hours after blood collection;

s3 uses: the frozen plasma was thawed within 10min at 37 ℃, mixed well, and centrifuged at 12000xg for 70min, and after centrifugation was complete, D-Dimer was determined within 2 hours.

s1: taking plasma to be detected and placing the plasma into a first reagent bottle and a second reagent bottle, placing a calibration sample into a third reagent bottle, and adding a diluent into the first reagent bottle, the second reagent bottle and the third reagent bottle, wherein the volume ratio of the diluent to the detected plasma/the calibration sample is 6: 1;

s2: adding a first reagent into the first reagent bottle, the second reagent bottle and the third reagent bottle respectively, and incubating for 7 min;

s3: adding a second reagent into the first reagent bottle, the second reagent bottle and the third reagent bottle respectively, mixing uniformly and incubating for 7 min;

EXAMPLE III

the first reagent comprises the following components in parts by weight: 50 parts of buffer solution and 4 parts of sodium azide;

the reagent II comprises the following components in parts by weight: 24 parts of polystyrene latex microspheres of the cross-linked D dimer monoclonal antibody and 50 parts of buffer solution;

the supplementary reagent comprises the following components in parts by weight: 28 parts of acrylic resin and 14 parts of chondroitin sulfate;

the diluent is 0.7 percent of normal saline by mass fraction.

Wherein the buffer solution is Tris, the pH value is 8.0, and the concentration is 400 mmol/L.

and S1 acquisition: collecting blood;

s2 freezing: freezing the plasma at-20 ℃ within 4 hours after blood collection;

s3 uses: the frozen plasma was thawed within 10min at 37 ℃, mixed well, and centrifuged at 14000xg for 70min and after centrifugation was complete, D-Dimer was determined within 2 hours.

s1: taking plasma to be detected and placing the plasma into a first reagent bottle and a second reagent bottle, placing a calibration sample into a third reagent bottle, and adding diluent into the first reagent bottle, the second reagent bottle and the third reagent bottle, wherein the volume ratio of the diluent to the detected plasma/the calibration sample is 8: 1;

s2: adding a first reagent into the first reagent bottle, the second reagent bottle and the third reagent bottle respectively, and incubating for 8 min;

s3: adding a second reagent into the first reagent bottle, the second reagent bottle and the third reagent bottle respectively, mixing uniformly and incubating for 8 min;

Example four

the first reagent comprises the following components in parts by weight: 55 parts of buffer solution and 5 parts of sodium azide;

the reagent II comprises the following components in parts by weight: 25 parts of polystyrene latex microspheres of the cross-linked D dimer monoclonal antibody and 55 parts of buffer solution;

the supplementary reagent comprises the following components in parts by weight: 30 parts of acrylic resin and 15 parts of chondroitin sulfate;

the diluent is 0.9 percent of normal saline by mass fraction.

Wherein the buffer solution is Tris, the pH value is 9.0, and the concentration is 500 mmol/L.

and S1 acquisition: collecting blood;

s2 freezing: freezing the plasma at a temperature of-18 ℃ within 4 hours after blood collection;

s3 uses: the frozen plasma was thawed within 10min at 37 ℃, mixed well, and centrifuged at 15000xg for 70min, and after centrifugation was complete, D-Dimer was determined within 2 hours.

s1: taking plasma to be detected and placing the plasma into a first reagent bottle and a second reagent bottle, placing a calibration sample into a third reagent bottle, and adding a diluent into the first reagent bottle, the second reagent bottle and the third reagent bottle, wherein the volume ratio of the diluent to the detected plasma/the calibration sample is 9: 1;

s2: adding a first reagent into the first reagent bottle, the second reagent bottle and the third reagent bottle respectively, and incubating for 10 min;

s3: adding a second reagent into the first reagent bottle, the second reagent bottle and the third reagent bottle respectively, mixing uniformly and incubating for 10 min;

Comparative example 1

A D-dimer detection kit comprises a first reagent, a second reagent, a supplementary reagent, a diluent and a calibration sample;

the supplementary reagent comprises the following components in parts by weight: 30 parts of acrylic resin;

the diluent is 0.9 percent of normal saline by mass fraction.

Specifically, the preparation method of the calibration sample of the D-dimer detection kit comprises the following steps:

and S1 acquisition: collecting blood;

Comparative example No. two

the supplementary reagent comprises the following components in parts by weight: 15 parts of chondroitin sulfate;

the diluent is 0.9 percent of normal saline by mass fraction.

and S1 acquisition: collecting blood;

Comparative example No. three

the diluent is 0.9 percent of normal saline by mass fraction.

and S1 acquisition: collecting blood;

s4: and (3) determining the change rate of the light transmittance by adopting a blood coagulation analyzer or a biochemical analyzer to make a calibration curve of the calibration sample, and then determining the change rate of the light transmittance of the plasma to be detected, namely obtaining the content of the D-dimer in the plasma to be detected on the calibration curve.

EXAMPLE five

The test was repeated 3 times using the test kit of example four, and the relative deviation was calculated according to the formula (1), and the results are shown in table 1:

in the formula:

b-relative deviation;

xi-the ith measurement;

t-reference substance index value or fixed value for each concentration sample.

TABLE 1

	For the first time	For the second time	The third time
				Relative deviation (%)	7	7	8

EXAMPLE six

The detection kit in the fourth embodiment is used for respectively measuring 3 samples with high (> 50%), medium (30% -50%) and low (< 30%) concentration levels for 10 times, and the average value of the measured values is calculated

And standard deviation(s), coefficient of variation (CV%) was calculated according to equation (2), and the results are shown in table 2:

TABLE 2

According to tables 1 and 2, the data obtained correspond to: the relative deviation is within 15.0%; the repeatability test uses samples with high, medium and low concentration levels, the Coefficient of Variation (CV) of the high-concentration sample is less than or equal to 10%, the Coefficient of Variation (CV) of the medium-concentration sample is less than or equal to 10%, and the Coefficient of Variation (CV) of the low-concentration sample is less than or equal to 15%, so the detection kit is qualified.

The detection kits of the first to fourth examples and the first to third comparative examples were used for detection, and the results are as follows:

table 3: example one to example four data

	Example one	Example two	EXAMPLE III	Example four
					Detecting total duration (min)	25	22	19	17
pH of the solution after reaction	6	5	5	5

Table 4: comparative examples one to three

Referring to tables 3 and 4, it can be seen that in the first to fourth examples, as the amount of each component increases, the effect of the detection kit becomes stronger, so that the total detection time is gradually reduced, and the pH of the solution after the reaction is gradually reduced.

In example four and comparative examples one to three, all other values being equal:

in the first comparative example, chondroitin sulfate is not added into the supplementary reagent, which is obviously seen, the pH value of the solution after reaction is 8, while in the fourth example, the pH value is only 5, the difference between the two is great, the chondroitin sulfate has great influence on the pH value environment of the detection reagent solution, and simultaneously, due to the change of the pH value of the reaction environment, the suitable reaction environment is lost, so that the total detection time is prolonged;

in the second comparative example, the chondroitin sulfate is added into the supplementary reagent, but no acrylic resin is found, so that the total detection time in the comparative example is obviously increased to 42min, while the fourth comparative example is only 17min, so that the chondroitin sulfate can effectively improve the detection rate of the D-dimer detection kit;

in the third comparative example, no supplementary reagent was added, the total duration of the detection was 48min, and the final pH of the solution after the reaction was 8, which was large both in the total duration of the detection and the final pH, and the pH value affected the detection reaction environment at the same time, resulting in a larger total duration than in the second comparative example.

In conclusion, the acrylic resin can effectively accelerate the reaction coagulation promotion during the detection of the detection kit, the coagulation can be quickly realized, the change of the light transmittance is obtained, the pH can be timely adjusted for the environment of the coagulation reaction by setting the chondroitin sulfate, the environment is provided for the coagulation reaction, the quick detection is assisted, and the D-dimer detection data can be efficiently obtained.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Claims

1. A D-dimer detection kit is characterized by comprising a first reagent, a second reagent, a supplementary reagent, a diluent and a calibrator;

the reagent I comprises buffer solution and sodium azide;

the supplementary reagent comprises acrylic resin and chondroitin sulfate;

the diluent is normal saline.

2. The D-dimer detection kit according to claim 1, wherein the first reagent comprises the following components in parts by weight: 30-55 parts of buffer solution and 3-5 parts of sodium azide.

3. The D-dimer detection kit according to claim 2, wherein the reagent two comprises the following components in parts by weight: 20-25 parts of polystyrene latex microspheres of the cross-linked D dimer monoclonal antibody and 30-55 parts of buffer solution.

4. The D-dimer detection kit according to claim 3, wherein the supplementary reagents comprise, in parts by weight: 20-30 parts of acrylic resin and 10-15 parts of chondroitin sulfate.

5. The D-dimer detection kit according to any one of claims 1 to 4, wherein the buffer is Tris, the pH value is 7.0 to 9.0, and the concentration is 250 to 500 mmol/L.

6. The D-dimer detection kit according to claim 5, wherein the diluent is 0.5-0.9% by weight of normal saline.

7. A preparation method of a calibration sample of a D-dimer detection kit is characterized by comprising the following steps:

and S1 acquisition: collecting blood;

8. The method of claim 7, wherein the blood tube is centrifuged at 1500 Xg-2500 Xg for 15min and then at 15000Xg for 70min to centrifuge the high lipid plasma fully in S1.

9. The use method of the D-dimer detection kit is characterized by comprising the following steps:

s1: taking plasma to be detected, placing the plasma into a first reagent bottle and a second reagent bottle, placing a calibration sample into a third reagent bottle, and adding a diluent into the first reagent bottle, the second reagent bottle and the third reagent bottle, wherein the volume ratio of the diluent to the detected plasma/calibrator is (5-9): 1;