CN113866426A

CN113866426A - Kit for detecting D-dimer and preparation method thereof

Info

Publication number: CN113866426A
Application number: CN202111129914.4A
Authority: CN
Inventors: 陈超; 成晶; 马亚飞
Original assignee: Shanghai I Reader Biological Technology Co ltd
Current assignee: Shanghai I Reader Biological Technology Co ltd
Priority date: 2021-09-26
Filing date: 2021-09-26
Publication date: 2021-12-31

Abstract

The invention discloses a kit for detecting D-dimer and a preparation method thereof, and relates to the technical field of immunological detection. Through carrying out anti erythrocyte to the sample pad and handling, can make the sample pad wrap up anti erythrocyte's antibody, make the sample pad like this and can realize the absorption of erythrocyte through antigen-antibody epitope discernment to the emergence of the rupture phenomenon of erythrocyte has effectively been reduced to a great extent. The inventor adds a layer of reaction pad between the combination pad and the antibody membrane to form a bridging structure of the combination pad, the reaction pad and the antibody membrane, so that the reaction time of the antigen and the antibody is prolonged, the baseline of a signal scanning curve is ensured to be straight when the D-dimer is detected in a sample, and the effect of high precision is achieved. In addition, a visual indicator line (I line) area is added on the basis of the traditional fluorescence chromatography structure, so that whether the D-dimer detection reagent strip is used or not can be quickly and conveniently distinguished.

Description

Kit for detecting D-dimer and preparation method thereof

Technical Field

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

Background

D-dimers are products of the co-action of the human intrinsic coagulation and fibrinolytic systems. Under physiological or various pathological conditions, namely when the body generates blood coagulation, the blood coagulation system is activated, and prothrombin is converted into thrombin; the fibrinogen is cracked to form fibrin monomer under the action of thrombin; the formed fibrin monomer is crosslinked on the blood vessel wall under the action of an activated factor FXIIIa to form crosslinked fibrin; the formation of crosslinked Fibrin promotes the activation of fibrinolytic system, plasminogen is converted into plasmin, the crosslinked Fibrin on the vessel wall is decomposed into Fibrin Degradation Products (FDP) with different sizes under the action of the formed plasmin, and D-dimer is the smallest fragment in the degradation products.

D-dimer levels have been shown to be elevated in various thrombotic disorders. Such as venous thromboembolism, severe infection, acute myocardial infarction, cerebral apoplexy, gestational hypertension, severe liver disease, etc. The main value of D-dimer testing is to exclude Venous Thrombosis (VTE), especially in low-grade suspicious patients, and the likelihood of VTE being present may be substantially excluded when the D-dimer test is negative. Recent studies with the novel coronavirus COVID-19 found that dimer levels in the dead patients were rising, breaking through normal values on days 7-9, and thereafter maintained at higher levels compared to normal D-dimer results in the surviving patients. Therefore, the detection of the D-dimer has great clinical application value.

The type of anticoagulant used in the blood immunoassay project is ethylenediamine tetraacetic acid (EDTA) salt in general, and the type of citrate anticoagulated plasma sample is commonly used when D-dimer is clinically determined at present; when the D-dimer is detected, a tube of blood needs to be independently pumped for a patient, and simultaneously, the pumped blood needs to be centrifuged to obtain plasma for detection; the workload of blood collection of hospital staff is increased, and certain damage is caused to the body of a patient.

At present, the D-dimer detection method mainly comprises the following steps: immunoturbidimetry, enzyme linked immunosorbent assay, immunocolloidal gold assay, fluorescence immunochromatography, chemiluminescence assay, and the like. The detection range of the immunoturbidimetry is generally narrow due to the HOOK effect, and higher-concentration D-dimer can be detected only through an additional dilution effect; in addition, the immunoturbidimetry cannot detect a whole blood sample due to the principle of turbidity detection, and the sample type is limited to citrate plasma. The enzyme-linked immunosorbent assay and the chemiluminescence assay have high requirements on storage environment and detection environment due to protease in the reagent, are generally 2-8 ℃, have multi-step elution and relatively long detection time, and are difficult to meet the requirement on detection turnaround time (TAT) of a result obtained by 20 minutes from D-dimer in chest pain center at present. The immune colloidal gold method is easily affected by blood fat and blood cells due to the principle of detecting color, and has poor accuracy of detecting a whole blood sample.

In the existing fluorescence immunochromatography method, when a whole blood sample is detected, a sample pad is not processed generally, and red blood cells are intercepted through the porous physical adsorption property of the sample pad, but the method has the problems of poor repeatability of a detection result and inaccurate detection result. In addition, the existing fluorescence immunochromatography has the defects of low detection sensitivity and accuracy, low uniformity and detection signal response value, poor chromatographic efficiency, easy base line warping of a signal scanning curve obtained by using a reagent strip for detection, low precision and the like, such as those described in patent CN 212031502U; meanwhile, the current D-dimer fluorescence chromatography method for detection has the defect that the used reagent strip cannot be distinguished from the unused reagent strip as described in the patent CN 210090472U.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a kit for detecting D-dimer and a preparation method thereof so as to solve the technical problems.

The inventor finds that when the D-dimer is measured, the problems of poor accuracy and poor repeatability of fluorescence immunochromatography detection are caused because the porous physical adsorption structure of the sample pad easily destroys the hydrophilic and hydrophobic properties of the surface of red blood cells, so that the red blood cells are easy to break, and the risk of interfering the detection result is increased. Thereby causing the fluorescence detection instrument equipment to be unable to accurately detect the fluorescence intensity on the nitrocellulose membrane. In view of the above, the inventors propose a novel kit for detecting D-dimer, which can realize high-accuracy detection of D-dimer and has good reproducibility.

The invention is realized by the following steps:

the invention provides a kit for detecting D-dimer, which comprises a bottom plate, a sample pad, a combination pad, a reaction pad, an antibody membrane and a water absorption pad, wherein the sample pad is coated with an antibody for resisting red blood cells, the combination pad is coated with a conjugate of fluorescent microspheres and an X anti-D-dimer monoclonal antibody A, the antibody membrane is coated with an X anti-D-dimer monoclonal antibody B, X anti-D-dimer polyclonal antibody and a photoactivator which are sequentially used as a detection line, a quality control line and an I line, and the sample pad, the combination pad, the reaction pad, the antibody membrane and the water absorption pad are sequentially overlapped and arranged on the bottom plate; x is from rat, rabbit, sheep or pig.

The inventor researches and discovers that the sample pad can be coated with an anti-erythrocyte antibody by carrying out anti-erythrocyte treatment on the sample pad, so that the sample pad can realize the adsorption of erythrocytes through antigen-antibody epitope recognition, and the occurrence of erythrocyte rupture phenomenon is effectively reduced to a great extent. Provides a favorable basis for immunofluorescence detection after subsequent chromatography. By processing the sample pad, the sample pad has the function of filtering red blood cells in the whole blood sample, and avoids the interference of a large number of red blood cells on the subsequent immunoreaction and detection, thereby realizing the detection of D-dimer in the whole blood sample type.

The inventor adds a layer of reaction pad between the combination pad and the antibody membrane to form a bridging structure of the combination pad, the reaction pad and the antibody membrane, so that the reaction time of the antigen and the antibody is prolonged, the baseline of a signal scanning curve is ensured to be straight when the D-dimer is detected in a sample, and the effect of high precision is achieved.

In addition, a visual indicator line (I line) area is added on the basis of the traditional fluorescence chromatography structure, so that whether the D-dimer detection reagent strip is used or not can be quickly and conveniently distinguished.

It should be noted that X anti-D-dimer monoclonal antibody A and X anti-D-dimer monoclonal antibody B are two kinds of monoclonal antibodies capable of specifically recognizing D-dimer. All IgG, antigen recognition epitope is different.

In a preferred embodiment of the present invention, the sample pad is coated with a rabbit anti-human erythrocyte polyclonal antibody. In other embodiments, the sample pad may be coated with polyclonal antibodies against human erythrocytes from other sources, such as pigs, monkeys, horses, or sheep.

In one embodiment, the rabbit anti-human red blood cell polyclonal antibody on the sample pad is prepared by coating a rabbit anti-human red blood cell polyclonal antibody solution, and the coating concentration of the rabbit anti-human red blood cell polyclonal antibody solution is 0.01-1 mg/mL; in an alternative embodiment, the coating concentration is 0.05-0.5 mg/mL. The inventor finds that the coating concentration can meet the requirement of efficient interception of red blood cells by the sample pad.

In a preferred embodiment of the present invention, the monoclonal antibody B against X-D-dimer on the detection line is prepared by coating the solution of monoclonal antibody B against X-D-dimer, and the coating concentration of the solution of monoclonal antibody B against X-D-dimer is 0.5-4 mg/mL. The inventors found that, at the above coating concentration, detection of a sample with higher accuracy and an extremely low detection limit can be satisfied.

The solvent in the solution of the X anti-D-dimer monoclonal antibody B is PBS, and the molar concentration of the PBS is 0.005-0.3M. The pH of the PBS was 7 to 7.5.

In a preferred embodiment of the present invention, the polyclonal antibody against X-D-dimer on the quality control line is prepared by coating the polyclonal antibody solution against X-D-dimer on the quality control line, and the coating concentration of the polyclonal antibody solution against X-D-dimer on the quality control line is 0.5-4 mg/mL. The subsequent fluorescent quantitative detection requirement can be met under the coating concentration.

The solvent in the X anti-D-dimer polyclonal antibody solution is PBS, and the molar concentration of the PBS is 0.005-0.3M. The pH of PBS was 7-7.5.

In a preferred embodiment of the present invention, the photoactivator on line I is coated with the photoactivator solution, and the coating concentration of the photoactivator solution on line I is 1-8 mg/mL.

In other embodiments, the photoactivator is a xanthene derivative dye, an azo dye, a biological stain, and a carotenoid.

The xanthene derivative dye is selected from a fluorene dye, a fluorone dye or a rhodole dye; the fluorene dye is selected from pyronin dye and rhodamine dye; the pyronin dye is selected from pyronin Y and pyronin B; rhodamine-based dyes and their derivatives (e.g., including but not limited to red Rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), and the like or analogs thereof); the fluorone dye is selected from fluorescein and fluorescein derivatives, such as including but not limited to Fluorescein Isothiocyanate (FITC) hydroxy Fluorescein (FAM), tetrachlorofluorescein (TET), etc. or analogs thereof, and the fluorescein derivative is selected from phloxine B, rose bengal and merbromin.

The azo dye is selected from methyl violet, neutral red, para red, amaranth, light red, allura red AC, tartrazine, orange G, ponceau, methyl red or ammonium diuranate-ammonium rhodanate.

The biological stain is selected from safranin O, basic fuchsin, acid fuchsin, iodized 3,3' -dihexylcarbocyanine, carminic acid or indocyanine green.

The carotenoid is selected from crocetin, alpha-crocin (8, 8-di-apo-8, 8-carotenoid acid), zeaxanthin, lycopene, alpha-carotene, beta-carotene, erythrosine or fucoxanthin.

The photoactivator is ponceau. The inventors found that the test strip before the sample was tested showed red color (ponceau) on the side of the nitrocellulose membrane adjacent to the absorbent pad after ponceau was coated on the nitrocellulose membrane, and that ponceau disappeared visually as ponceau was gradually chromatographed on the absorbent pad after the sample was applied on the sample pad. Therefore, the test paper strip can be used for identifying whether the test paper strip is detected or not. The coating concentration can meet the detection requirement of the conventional D-dimer.

In an alternative embodiment, the solvent of the photoactivator solution is a PBS solution of 3% to 30% sucrose by volume, the molar concentration of the PBS solution being 0.005 to 0.05M.

In a preferred embodiment of the present invention, the reaction pad is made of at least one material selected from the group consisting of:

filter paper, cotton fibers, polyester fibers, glass fibers, and polyethersulfone.

In other embodiments, the reaction pad may be a structure for slowing down the flow rate of the liquid and prolonging the specific reaction time of the antigen-antibody microsphere, and the functional pad capable of delaying the liquid from entering the antibody membrane from the bonding pad is within the protection scope of the reaction pad of the present invention.

The invention also provides a preparation method of the kit for detecting the D-dimer, which comprises the following steps:

and respectively placing the sample pad, the combination pad, the reaction pad, the antibody membrane and the water absorption pad on the bottom plate to assemble the test strip.

In a preferred embodiment of the present invention, the preparation method further comprises preparing a sample pad, which comprises: and paving a rabbit anti-human erythrocyte polyclonal antibody solution on the sample pad to be coated with the antibody, and drying to obtain the sample pad coated with the antibody.

In an alternative embodiment, the drying is in a vacuum-like environment or a vacuum environment; the pH of the rabbit anti-human erythrocyte polyclonal antibody solution is 5.0-6.5.

In a preferred embodiment of the present invention, the above-mentioned preparation method further includes preparing a bonding pad, which includes: mixing and incubating the activated fluorescent microspheres and the X anti-D-dimer monoclonal antibody A to form a conjugate of the fluorescent microspheres and the X anti-D-dimer monoclonal antibody A, then diluting the conjugate with a PBS solution containing BSA, flatly paving the conjugate on a binding pad to be coated, and drying. The drying is carried out in a vacuum-like environment.

In an alternative embodiment, the mixing mass ratio of the fluorescent microspheres to the X anti-D-dimer monoclonal antibody A is 5-20: 1;

when the conjugate is diluted, the PBS solution is 0.01-0.05M PBS solution containing 0.5-0.6% BSA by mass, and the pH of the PBS solution is 7-7.5.

In a preferred embodiment of the present invention, the above preparation method further comprises coating the antibody on the antibody membrane to be coated; the method comprises the steps of coating a detection line coating liquid, a quality control line coating liquid and an I line coating liquid on a nitrocellulose membrane respectively through a membrane scribing instrument, and then drying.

In one embodiment, the distance between the reaction pad and the sample pad is 0.5-4mm, preferably the distance between the reaction pad and the sample pad is 1.5-2.5 mm.

The invention has the following beneficial effects:

the kit for detecting the D-dimer provided by the invention can coat the anti-erythrocyte antibody on the sample pad by carrying out anti-erythrocyte treatment on the sample pad, so that the sample pad can realize the adsorption of erythrocytes by identifying the epitope of the antigen antibody, thereby effectively reducing the occurrence of the rupture phenomenon of the erythrocytes to the greatest extent. Provides a favorable basis for immunofluorescence detection after subsequent chromatography. By processing the sample pad, the sample pad has the function of filtering red blood cells in the whole blood sample, and avoids the interference of a large number of red blood cells on the subsequent immunoreaction and detection, thereby realizing the detection of D-dimer in the whole blood sample type.

A layer of reaction pad is added between the combination pad and the antibody membrane, so that a bridging structure of the combination pad, the reaction pad and the antibody membrane is formed, the reaction time of the antigen and the antibody is prolonged, the signal scanning curve baseline is ensured to be straight when the D-dimer is detected in a sample, and the effect of high precision is achieved.

The preparation method of the kit provided by the invention is simple and feasible, and is easy to popularize and apply.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a front view of an immunochromatographic reagent strip of the present invention;

FIG. 2 is a side view of FIG. 1;

FIG. 3A is a signal scan of the kit of example 1 for detecting D-dimer at a concentration of 3.2. mu.g/mL;

FIG. 3B is a signal scan of the kit of comparative example 1 for detecting D-dimer at a concentration of 3.2. mu.g/mL;

FIG. 4 is a graph showing the results of the test of EDTA whole blood samples using the kits of example 1 and comparative example 2;

FIG. 5A is a graph of the linear correlation between the D-Dimer in sodium citrate plasma samples as determined by the reference method using the kit of example 1 and the Siemens D-Dimer assay kit;

FIG. 5B is a graph showing the linear correlation between the EDTA whole blood sample and the sodium citrate plasma sample when D-dimer is detected by using the kit of example 1;

FIG. 6 is a graph showing the results of D-dimer detection using the kit of example 1;

FIG. 7 is a graph showing the results of detecting D-dimer with the kit of comparative example 3.

Reference numerals: 1-PVC base plate; 2-sample pad; 3-antibody microsphere conjugate pad; 4-a reaction pad; 5-antibody membrane; 6-absorbent paper; a 51-T detection line; 52-C quality control line; the 53-I line indicates the area.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

Referring to fig. 1 and 2, this embodiment provides a detection kit for detecting D-dimer, which includes a PVC base plate 1, a sample pad 2, an antibody microsphere binding pad 3, a reaction pad 4, an antibody membrane 5, and a water-absorbent paper 6 (i.e., a water-absorbent pad). The sample pad 2 is coated with rabbit anti-human erythrocyte polyclonal antibody solution, and the antibody microsphere combination pad 3 is coated with conjugate of fluorescent microsphere and mouse anti-D-dimer monoclonal antibody. The antibody film 5 is coated with a T detection line 51, a C quality control line 52 and an I line indication area 53 in sequence.

The preparation method of the detection kit comprises the following steps:

(1) preparation of anti-Red Blood Cell (RBC) treated sample pad 1.

Preparing a rabbit anti-human erythrocyte polyclonal antibody solution: diluting Shanghai Jiening rabbit anti-human erythrocyte polyclonal antibody to 0.1mg/mL by using pH6.5, 0.1M PB diluent, and storing at 2-8 ℃ for later use;

sample pad treatment: uniformly spreading the rabbit anti-human erythrocyte polyclonal antibody solution on a sample pad, and drying in a vacuum-like environment for 2 hours;

(2) preparation of antibody film 5.

Preparing a T-line coating liquid: diluting a Roche mouse anti-D-Dimer monoclonal antibody to 2mg/mL by using PBS (phosphate buffer solution) with the pH of 7.3 and the concentration of 0.01M, and keeping the temperature at 2-8 ℃ for later use;

c, preparing a coating solution for the yarn C: a polyclonal antibody of the Changsha Boyou mouse anti-D-Dimer antibody is diluted to 1mg/mL by PBS (pH7.3, 0.01M) diluent and is reserved at 2-8 ℃.

I, preparing a coating liquid for the yarn: the Lichun red was diluted to 5mg/mL with 5% (w/v) sucrose in 0.01M PBS, pH7.3, and kept at 2-8 ℃.

Coating the wires: coating the T-line coating liquid, the C-line coating liquid and the I-line coating liquid onto the NC film by a film-scribing metal spraying instrument, and drying the coated NC film for 2h in a vacuum-like environment.

(3) Preparation of antibody microsphere binding pad 3: selecting carboxyl of Merck 300nm fluorescent microspheres, activating by EDC/NHS, adding another mouse anti-D-Dimer monoclonal antibody of Roche according to the mass ratio of 10:1, wherein amino on the antibody can be coupled with the activated carboxyl on the microspheres to form a coupled labeled compound of the fluorescent microspheres and the antibody; the antibody microsphere complex was diluted 50-fold with 0.6% BSA in PBS, pH7.3, 0.01M, and spread evenly on the conjugate pad, and dried in a vacuum-like environment for 2 h.

(4) Assembling the reagent strip: the sample pad, the antibody microsphere binding pad, the reaction pad, the antibody membrane and the absorbent paper are sequentially adhered and assembled to the PVC base plate as shown in fig. 1 and 2. And cutting the assembled sample into reagent strips with the width of 3.2mm by using a strip cutting machine, filling the reagent strips into the reagent strips, pressing the reagent strips into shells, and preparing the detection card (namely the detection kit).

The material of the reaction pad in this embodiment is cotton fiber, and in other embodiments, the reaction pad includes but is not limited to: filter paper, cotton fibers, polyester fibers, glass fibers, polyethersulfone, or combinations thereof. The reaction pad can be used for slowing down the flow rate of liquid and prolonging the specific reaction time of the antigen-antibody microspheres.

Comparative example 1

In this example, the microsphere binding pad was directly connected to the antibody membrane without the reaction pad, and the rest was the same as the kit in example 1.

Comparative example 2

The sample pad was not treated with anti-RBC, and the rest was the same as the kit of example 1.

Comparative example 3

The antibody membrane 5 was prepared without coating the I-line, and the rest was the same as the kit in example 1.

Experimental example 1

This example used the test strips of example 1 and comparative example 1, respectively, to detect D-Dimer (D-Dimer):

the detection method comprises the following steps: taking out the detection card, diluting a sample in Shanghai Hospital, adding the sample from a sample adding port, carrying out chromatography on the diluted liquid sample to an antibody microsphere combination pad, re-melting antibody microspheres in the antibody microsphere combination pad from the combination pad, carrying out chromatography on a substance to be detected in the sample and the antibody microspheres to a reaction pad together, carrying out sufficient specific reaction and combination in the reaction pad, carrying out chromatography on the formed antigen-antibody microsphere complex to an antibody membrane under the capillary action, and detecting the fluorescence intensity enriched in the T line and the C line region by using a fluorescence quantitative detection system.

Samples containing different concentrations of D-Dimer were tested using the kits of example 1 and comparative example 1, and the results are shown in Table 1.

TABLE 1 data sheet for quantitative determination of each concentration of control group and experimental group of the present invention

	Concentration (μ g/mL)	12.8	3.2	0.8	0.2	0.05	0
								Comparative example 1	Signal value	2277	992	302	120	62	43
Example 1	Signal value	3552	1861	682	191	65	16

(1) Verification of repeatability effects

The test was repeated 10 times (test time 10 minutes) for each concentration using the reagent strip of example 1 and the kit of comparative example 1, and the coefficients of variation were determined and compared, and the test results are shown in tables 2 to 4.

TABLE 20.2 comparison of results for control and experimental groups at μ g/ml

TABLE comparison of results for control and experimental groups at 30.8. mu.g/ml

TABLE 43.2 comparison of results for control and experimental groups at μ g/ml

A signal scan when a D-Dimer was detected at a concentration of 3.2. mu.g/mL using the kits of Experimental example 1 and comparative example 1 is shown in FIG. 3. As can be seen in fig. 3, comparative example 1 has significant warpage in the baseline AB and CD segments (fig. 3B); whereas the baseline AB and CD segments were relatively flat in example 1 (fig. 3A), the kits of the invention were more accurate.

From the above results, it can be seen that the kit provided by the present invention is significantly superior to the kit of comparative example 1 in terms of signal value, reproducibility, or precision. The detection precision of the kit is obviously improved by arranging the reaction pad, the detection repeatability is good, and the signal value is high.

Experimental example 2

This experimental example used the kits of example 1 and comparative example 2 to separately detect the type of whole blood sample.

The detection method comprises the following steps: and taking out the detection card, diluting the whole blood sample in Shanghai Hospital, adding the sample from the sample adding port, and observing the conditions of the kit before and after reaction.

The results of the EDTA whole blood sample assay using the kits of example 1 and comparative example 2 are shown in FIG. 4, where FIG. 4a) is a graph showing the whole blood sample not assayed in example 1, FIG. 4b) is a graph showing the whole blood sample assayed in example 1, and FIG. 4c) is a graph showing the whole blood sample assayed in comparative example 2.

As can be seen from the figure, the antibody membrane 5 reddened after the test kit of comparative example 2 tested the EDTA whole blood sample, indicating that the erythrocytes were not completely intercepted in front of the antibody membrane 5, and that some erythrocytes or their release after rupture were present in the antibody membrane 5; in example 1, however, no significant redness of the antibody membrane 5 occurred, indicating that the red blood cells were substantially intercepted in front of the antibody membrane 5.

It should be noted that, because the reaction pad exists in the kit, if there are many red blood cells, the reaction pad also has a certain effect of intercepting a small amount of red blood cells.

Experimental example 3

This example used the kit of example 1 to detect D-Dimer in samples of different sample types.

The detection method comprises the following steps: 57 sodium citrate plasma samples and homologous EDTA whole blood samples in Shanghai Hospital were selected, and the linear correlation analysis was performed on the sodium citrate plasma samples and the homologous EDTA whole blood samples by using the existing Siemens D-Dimer detection kit as a reference method control group and the results of the kit prepared in example 1 as a test group. The detection method of the test group is the same as that of experimental example 1, and is not described in detail.

The results of detecting D-Dimer in samples of different sample types using the kit of example 1 are shown in FIG. 5, wherein FIG. 5A is a linear correlation between the kit of example 1 and the D-Dimer in the sodium citrate plasma sample when the D-Dimer is detected by the Siemens D-Dimer detection kit as a reference method; FIG. 5B is a linear correlation between the kit of example 1 and the D-Dimer in EDTA whole blood samples and sodium citrate plasma samples. The result shows that the test strip provided by the invention has good detection linearity and clinical relevance.

Experimental example 4

D-Dimer was detected using the kits of example 1 and comparative example 3, respectively.

The detection method comprises the following steps: and taking out the detection card, diluting the whole blood sample, adding the sample from the sample adding port, and observing the conditions of the kit before and after reaction.

The results of detecting D-Dimer using the kits of example 1 and comparative example 3 are shown in FIGS. 6 and 7.

Wherein FIG. 6a) is a schematic view of the kit in example 1 when it is not reacted, and FIG. 6b) is a schematic view of the kit after the reaction in example 1; FIG. 7a) is a schematic view of the kit in the case where comparative example 3 is not reacted, and FIG. 7b) is a schematic view of the kit in the case where comparative example 3 is reacted. As can be seen from the figure, the difference is difficult to be observed by naked eyes before and after the reaction in the comparative example 3, while a red band which can be seen by the naked eyes exists before the reaction in the example 1, and after the reaction is finished, the red band disappears, so that the kits before and after the reaction can be easily distinguished.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The kit for detecting the D-dimer is characterized by comprising a bottom plate, a sample pad, a combination pad, a reaction pad, an antibody membrane and a water absorption pad, wherein the sample pad is coated with an antibody for resisting red blood cells, the combination pad is coated with a conjugate of fluorescent microspheres and an X anti-D-dimer monoclonal antibody A, the antibody membrane is coated with an X anti-D-dimer monoclonal antibody B, X anti-D-dimer polyclonal antibody and a photoactivator which are sequentially used as a detection line, a quality control line and an I line, and the sample pad, the combination pad, the reaction pad, the antibody membrane and the water absorption pad are sequentially overlapped and arranged on the bottom plate; and the X is a mouse source, a rabbit source, a sheep source or a pig source.

2. The kit for detecting D-dimer according to claim 1, wherein said sample pad is coated with a rabbit anti-human red blood cell polyclonal antibody;

preferably, the rabbit anti-human erythrocyte polyclonal antibody on the sample pad is prepared by coating a rabbit anti-human erythrocyte polyclonal antibody solution, and the concentration of the rabbit anti-human erythrocyte polyclonal antibody solution is 0.01-1 mg/mL; preferably, the coating concentration is 0.05-0.5 mg/mL.

3. The kit for detecting D-dimer according to claim 1, wherein the monoclonal antibody B against D-dimer in the detection line is prepared by coating a solution of monoclonal antibody B against D-dimer in X at a concentration of 0.5 to 4 mg/mL;

the solvent in the solution of the X anti-D-dimer monoclonal antibody B is PBS, and the molar concentration of the PBS is 0.005-0.3M.

4. The kit for detecting D-dimer according to claim 3, wherein the polyclonal antibody of X anti-D-dimer on the quality control line is prepared by coating the polyclonal antibody solution of X anti-D-dimer with a coating concentration of 0.5-4 mg/mL;

the solvent in the X anti-D-dimer polyclonal antibody solution is PBS, and the molar concentration of the PBS is 0.005-0.3M.

5. The kit for detecting D-dimer according to claim 1, wherein said photoactivator is prepared by coating said photoactivator solution on line I at a concentration of 1-8 mg/mL; preferably, the photoactivator is a xanthene derivative dye, an azo dye, a biological stain, and a carotenoid; preferably, the azo dye is selected from methyl violet, neutral red, para red, amaranth, carmoisine, allura red AC, tartrazine, orange G, ponceau, methyl red or ammonium diuranate-ammonium rhodanate;

preferably, the solvent of the photoactivator solution is PBS solution with 3-30% of sucrose by mass volume, and the molar concentration of the PBS solution is 0.005-0.05M.

6. The kit for detecting D-dimer according to claim 1, wherein said reaction pad is made of any one of the following materials:

7. A method for preparing a kit for detecting D-dimer according to any one of claims 1 to 6, comprising the steps of:

8. The method of claim 7, further comprising preparing a sample pad comprising: paving a rabbit anti-human erythrocyte polyclonal antibody solution on the sample pad to be coated with the antibody, and drying to prepare the sample pad coated with the antibody;

preferably, the drying is under a vacuum-like environment or a vacuum environment; the pH value of the rabbit anti-human erythrocyte polyclonal antibody solution is 5.0-6.5.

9. The method of manufacturing of claim 7, further comprising the preparation of a conjugate pad comprising: mixing and incubating the activated fluorescent microspheres and the X anti-D-dimer monoclonal antibody A to form a conjugate of the fluorescent microspheres and the X anti-D-dimer monoclonal antibody A, then diluting the conjugate with a PBS solution containing BSA, flatly paving the conjugate on a binding pad to be coated, and drying;

preferably, the mixing mass ratio of the fluorescent microspheres to the X anti-D-dimer monoclonal antibody A is 5-20: 1;

upon dilution of the conjugate, the PBS solution was 0.01-0.05M PBS containing 0.5-0.6% BSA, and the pH of the PBS solution was 7-7.5.

10. The production method according to claim 9, further comprising coating an antibody on an antibody membrane to be coated; the method comprises the steps of coating a detection line coating liquid, a quality control line coating liquid and an I line coating liquid on a nitrocellulose membrane respectively through a membrane scribing instrument, and then drying.