CN114167052A

CN114167052A - Kit for quantitative detection of NT-proBNP/ST2 by time-resolved fluorescence immunochromatography and application thereof

Info

Publication number: CN114167052A
Application number: CN202111467425.XA
Authority: CN
Inventors: 石磊; 谢丽娟; 陈魏穗; 黎文娟; 邓成刚
Original assignee: Guangzhou Datai Bioengineering Technology Co ltd
Current assignee: Guangzhou Datai Bioengineering Technology Co ltd
Priority date: 2021-12-03
Filing date: 2021-12-03
Publication date: 2022-03-11

Abstract

The embodiment of the application belongs to the technical field of detection, and relates to a time-resolved fluorescence immunochromatography kit for quantitatively detecting NT-proBNP/ST2 and application thereof, wherein the kit comprises a detection card, the detection card comprises a card shell and a test strip, the test strip comprises a PVC (polyvinyl chloride) bottom plate, and a treatment pad, a combination pad, a nitrocellulose membrane and absorbent paper which are sequentially overlapped are arranged on the PVC bottom plate; the detection kit comprises a nitrocellulose membrane, a binding pad and a nitrocellulose membrane, wherein the nitrocellulose membrane is fixedly provided with a fluorescence-labeled NT-proBNP-L antibody, a fluorescence-labeled ST2-L antibody and a fluorescence-labeled goat anti-chicken IgY antibody, and on the basis of the direction from the binding pad to the absorbent paper, the nitrocellulose membrane is sequentially provided with a first detection line, a second detection line and a quality control line, the NT-proBNP-C antibody is coated at the first detection line, the ST2-C antibody is coated at the second detection line, and the chicken IgY antibody is coated at the quality control line. The method can be used for rapidly detecting the values of the amino terminal pro-brain natriuretic peptide and the growth stimulation expression gene 2 protein in the heart failure condition.

Description

Kit for quantitative detection of NT-proBNP/ST2 by time-resolved fluorescence immunochromatography and application thereof

Technical Field

The application relates to the technical field of time-resolved fluorescence immunoassay, in particular to a kit for quantitatively detecting NT-proBNP/ST2 by using a time-resolved fluorescence immunochromatography method and application thereof.

Background

NT-proBNP (amino terminal pro-brain natriuretic peptide) is a biomarker widely applied in clinic at present, has a half-life of 120 minutes, is not easy to degrade in plasma, and has certain value in the aspects of heart failure screening, diagnosis, identification and cardiovascular event prediction. Compared with BNP, NT-proBNP has longer half-life and better stability, so the NT-proBNP has higher sensitivity for detecting early or mild heart failure, and the time for sending blood samples to a laboratory is more sufficient, thus being more suitable for clinical application. ST2, growth-stimulating expression gene 2 protein, is a novel soluble biomarker, and the ST2 gene product has three distinct clonal types: soluble ST2(sST2), transmembrane receptor type ST2L, polymorphic ST 2V. sST2 has no transmembrane sequence and can be secreted to the outside of cells, and ST2L contains a transmembrane fragment and a Toll/IL-1R receptor (TIR) intracellular domain and is transmembrane ST 2. ST2, the sole receptor for interleukin-33, is involved in cardioprotection, reflects ventricular remodeling and cardiac fibrosis associated with heart failure, and is closely related to symptoms such as inflammation and oxidative stress. ST2 has a better prognostic power and allows the risk to be detected very early. The abnormal rising of the levels of NT-proBNP and ST2 in the serum of the patients with heart failure, and the combined diagnosis of the two indexes, especially the two indexes, has obvious value on the disease progression and treatment of the patients. However, there are currently few studies on the combined prediction of readmission and mortality rates of heart failure patients with both indices.

NT-proBNP is used as an important index of myocardial injury, and currently, the researched methodology mainly comprises: colloidal gold, enzyme-linked immunosorbent assay, chemiluminescence assay, radioimmunoassay, fluorescence chromatography and the like. ST2 is a novel heart failure detection marker, and the methodologies currently studied are: enzyme-linked immunosorbent assay, chemiluminescence assay, fluorescence chromatography, etc.

(1) Colloidal gold method: the colloidal gold technology is an application form formed by combining a gold labeling technology and an antigen-antibody reaction, and has the advantages of convenience, rapidness, strong stability, no need of special equipment and reagents, visual result judgment and the like. However, the colloidal gold product has large batch difference and low sensitivity, and can only give qualitative or semi-quantitative results.

(2) Enzyme linked immunosorbent assay: the enzyme-linked immunosorbent assay is an application technology for detecting samples based on effective combination of antigen and antibody and under the conditions of immunoreaction specificity and enzyme high-efficiency catalysis. This approach is simple to operate, but relatively poor in specificity.

(3) A radioimmunoassay: the sensitivity is most outstanding, the radioactive homologen is adopted to label the antigen or the antibody, and the antigen or the antigen is combined with the corresponding antibody, and then the radioactivity of the antigen-antibody combination is detected to obtain the result. However, the use of radioimmunoassay can cause some damage to the human body.

(4) Fluorescence chromatography: based on the immunological principle and chromatographic technology, the technology detects the movement of sample on fiber membrane, displays special color via capillary action in short time and analyzes the color. However, the requirements for the apparatus are relatively high, and the spectrophotometer that can be used for detection is expensive in the liquid phase, and as a result, cannot be stored for a long time.

(5) A chemiluminescence method: the chemiluminescence immunoassay technology has the characteristics of high sensitivity, convenient operation, simple preparation of markers, strong stability, high automation degree, environmental pollution and the like, and can obtain a test result in time. The method has low detection precision and high cost of instruments and equipment.

Disclosure of Invention

The embodiment of the application aims to provide a kit for quantitatively detecting NT-proBNP/ST2 by using a time-resolved fluorescence immunochromatography method and application thereof, which can quickly detect the values of amino terminal pro-BNP and growth stimulation expression gene 2 protein in heart failure.

In order to solve the above technical problems, the embodiments of the present application provide a kit for quantitative detection of NT-PROBNP/ST2 by time-resolved fluorescence immunochromatography and applications thereof, and the following technical solutions are adopted:

a kit for quantitatively detecting NT-proBNP/ST2 by a time-resolved fluorescence immunochromatography method comprises a detection card, wherein the detection card comprises a card shell and a test strip, the test strip comprises a PVC (polyvinyl chloride) bottom plate, and a treatment pad, a combination pad, a nitrocellulose membrane and absorbent paper which are sequentially overlapped are arranged on the PVC bottom plate;

the detection kit comprises a nitrocellulose membrane, a binding pad and a nitrocellulose membrane, wherein the nitrocellulose membrane is fixedly provided with a fluorescence-labeled NT-proBNP-L antibody, a fluorescence-labeled ST2-L antibody and a fluorescence-labeled goat anti-chicken IgY antibody, and on the basis of the direction from the binding pad to the absorbent paper, the nitrocellulose membrane is sequentially provided with a first detection line, a second detection line and a quality control line, the NT-proBNP-C antibody is coated at the first detection line, the ST2-C antibody is coated at the second detection line, and the chicken IgY antibody is coated at the quality control line.

Further, the processing pad is prepared by the following steps:

preparing a treatment solution, soaking the sample pad in the treatment solution, and drying the soaked sample pad to obtain the treatment pad, wherein the treatment solution comprises bovine serum albumin, Tween-20 and borax borate buffer solution.

Furthermore, the bovine serum albumin is 10g, the Tween-20 is 5ml, the concentration of the borax borate buffer solution is 0.2M, the pH value is 8, and the dosage is 1000 ml.

Further, the bonding pad is prepared by the following steps:

adding the weighed fluorescent microspheres into a buffer solution, centrifuging, and retaining the precipitate;

dissolving the precipitate into another buffer solution, carrying out ultrasonic crushing, and adding a target antibody;

adding a marking confining liquid, centrifuging, retaining a precipitate, adding a marking preservation liquid, and performing ultrasonic crushing to obtain a target solution;

wherein, when the added target antibody is NT-proBNP-L antibody, the obtained target solution is a first target solution, wherein the first target solution comprises the NT-proBNP-L antibody which is fluorescently labeled;

when the target antibody added is an ST2-L antibody, obtaining the target solution as a second target solution, wherein the second target solution comprises a fluorescence-labeled ST2-L antibody;

when the added target antibody is a goat anti-chicken IgY antibody, the obtained target solution is a third target solution, and the third target solution comprises a fluorescence-labeled goat anti-chicken IgY antibody;

respectively scribing the first target solution, the second target solution and the third target solution on a preset sample pad to fix the fluorescence-labeled NT-proBNP-L antibody, the fluorescence-labeled ST2-L antibody and the fluorescence-labeled goat anti-chicken IgY antibody on the preset sample pad to obtain the binding pad.

Further, the labeled blocking solution comprises bovine serum albumin and purified water.

Further, the marker preservation solution comprises bovine serum albumin, sucrose, casein sodium salt, trehalose, Proclin300 and Tris buffer solution.

Further, the concentration of the Tris buffer solution is 0.2M, and the pH value is 8.

In order to solve the above technical problems, the embodiments of the present application further provide an application of a time-resolved fluorescence immunochromatographic kit for quantitatively detecting NT-proBNP/ST2, which adopts the following technical scheme:

the application of the kit in quantitative detection of NT-proBNP/ST2 comprises the steps of adding a sample to be detected into a sample adding hole of the detection card, placing the detection card after sample addition into a card slot of an instrument for detection, and obtaining a detection result output by the instrument.

Further, the sample to be detected is whole blood or plasma.

Further, the whole blood or the plasma is collected through an anticoagulant tube containing an anticoagulant, wherein the anticoagulant is heparin lithium, EDTA-K2 or sodium citrate.

Compared with the prior art, the embodiment of the application mainly has the following beneficial effects:

the time-resolved immunochromatography technology is a novel nonradioactive microanalysis technology established by taking lanthanide with unique fluorescence characteristics and a chelating agent thereof as tracers. The technology not only retains the advantages of simple operation, rapid detection and strong portability of the colloidal gold immunochromatography technology, but also realizes the accurate quantification of the detection result by the fluorescent tracing enhancement technology. Compared with the performance of the traditional rapid detection technology, the technology has higher sensitivity, wider detection range and low price. The method simultaneously and quantitatively detects the contents of NT-proBNP and ST2 in plasma by using a double-antibody sandwich method in a fluorescence immunochromatography technology, and establishes an effective NT-proBNP/ST2 fluorescence quantitative immunochromatography detection kit by means of optimizing process conditions, performance evaluation, clinical application and the like. The method can be applied to the reference basis for detecting the heart failure.

Drawings

In order to more clearly illustrate the solution of the present application, the drawings needed for describing the embodiments of the present application will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present application, and that other drawings can be obtained by those skilled in the art without inventive effort.

FIG. 1 is a schematic diagram of the structure of the test card of the kit for time-resolved fluorescence immunochromatography for quantitative detection of NT-proBNP/ST2 according to the present application;

FIG. 2 is another schematic diagram of the structure of the test card of the kit for time-resolved fluorescence immunochromatography for quantitative detection of NT-PROBNP/ST2 according to the present application;

FIG. 3 is a schematic representation of the results of a correlation analysis of a time-resolved fluoroimmunoassay kit for quantitative detection of NT-PROBNP/ST2 according to the present application with a comparison manufacturer;

FIG. 4 is a schematic representation of the results of another correlation analysis of the time-resolved fluoroimmunoassay kit for quantitative detection of NT-PROBNP/ST2 according to the present application with a comparison manufacturer.

Reference numerals: 1. a PVC base plate; 2. a treatment pad; 3. a bonding pad; 4. a nitrocellulose membrane; 5. a first detection line, 6, a second detection line; 7. a quality control line; 8. absorbent paper; 9. a handheld area; 10. taking a reading; 11. and (4) sample adding holes.

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 application belongs; the terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "including" and "having," and any variations thereof, in the description and claims of this application and the description of the above figures are intended to cover non-exclusive inclusions. The terms "first," "second," and the like in the description and claims of this application or in the above-described drawings are used for distinguishing between different objects and not for describing a particular order.

Reference herein to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in at least one embodiment of the application. The appearances of the phrase in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments. It is explicitly and implicitly understood by one skilled in the art that the embodiments described herein can be combined with other embodiments.

The following examples are presented to facilitate a better understanding of the present application and are not intended to limit the present application. The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

In order to make the technical solutions better understood by those skilled in the art, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings.

The kit for quantitatively detecting NT-proBNP/ST2 by using a time-resolved fluorescence immunochromatography method comprises a detection card, wherein the detection card comprises a card shell and a test strip, the test strip comprises a PVC (polyvinyl chloride) bottom plate, and a treatment pad, a combination pad, a nitrocellulose membrane and absorbent paper which are sequentially overlapped are arranged on the PVC bottom plate;

the detection kit comprises a nitrocellulose membrane, a binding pad and a nitrocellulose membrane, wherein the nitrocellulose membrane is fixedly provided with a fluorescence-labeled NT-proBNP-L antibody, a fluorescence-labeled ST2-L antibody and a fluorescence-labeled goat anti-chicken IgY antibody, and on the basis of the direction from the binding pad to the absorbent paper, the nitrocellulose membrane is sequentially provided with a first detection line, a second detection line and a quality control line, the NT-proBNP-C antibody is coated at the first detection line, the ST2-C antibody is coated at the second detection line, and the chicken IgY antibody is coated at the quality control line. Specifically, as shown in fig. 1, fig. 1 is a schematic structural diagram of a detection card of the time-resolved fluorescence immunochromatography kit for quantitatively detecting NT-proBNP/ST2 according to the present application. PVC soleplate; 2. a sample pad; 3. conjugate pads (fluorescently labeled NT-proBNP-L antibody, ST2-L antibody and goat anti-chicken IgY antibody); 4. a nitrocellulose membrane; 5. the first detection line is coated with NT-proBNP-C antibody; 6. the second detection line is coated with ST2-C antibody; 7. the quality control line is coated with chicken IgY antibody; 8. absorbent paper. Fig. 2 is a schematic structural diagram of the detection card.

The composition, packaging and quantity (25 reactions/boxes) of the kit for quantitative detection of NT-proBNP/ST2 by time-resolved fluorescence immunochromatography are shown in Table 1:

TABLE 1 composition, packaging and quantity of NT-proBNP/ST2 kits

1. The preparation method of the treatment pad comprises the following steps:

the preparation method of the treatment pad comprises the following steps:

preparing a treatment solution, infiltrating the treatment solution into a sample pad by using a paper-impregnating pad pressing machine, and drying the infiltrated sample pad to obtain the treatment pad.

The specific steps of drying the soaked sample pad comprise: and drying the soaked sample pad at the temperature of 18-26 ℃ and the relative humidity of less than or equal to 30% overnight (16-24) for h to obtain the dried sample pad. And (4) placing the dried sample pad into a self-sealing bag, sealing the self-sealing bag, and placing a proper amount of drying agent. Before the intermediate is assembled, cutting off 5mm from each of the upper and lower long edges of the dried sample pad at the temperature of 18-28 ℃ and the relative humidity of less than or equal to 30%, and then cutting into the size of (23 +/-1) mmx (300 +/-10) mm to obtain the processing pad for later use. Calculating the material balance of the processing pad (usage amount + residual amount + loss amount)/collar usage amount; material balance requirement limit: 95 to 105 percent.

The treatment fluid comprises bovine serum albumin, Tween-20 and borax borate buffer solution. 10g of bovine serum albumin, 5ml of Tween-20, 0.2M of borax borate buffer solution, 8 of pH value and 1000ml of dosage. After BSA (bovine serum albumin) is dissolved by borax borate buffer solution, Tween-20 is added, and stirring is continued until all BSA (bovine serum albumin) is dissolved; (2-8) DEG C, the validity period is 3 months, and the specific numerical value proportion is shown in table 2:

TABLE 2 sample pad treatment solution preparation

2. A method of making a conjugate pad comprising T1 wire, T2 wire, and C wire.

The preparation method of the combined pad comprises the following steps:

the method comprises the following specific steps: weighing 100 mu L of fluorescent microspheres, adding the fluorescent microspheres into 1mL of marking buffer MES, and slowly reversing the mixture from top to bottom and uniformly mixing the mixture for 10-20 times; rotating to react for 30 min;

centrifuging at 4 deg.C at 10000rpm for 30min, discarding supernatant, and keeping precipitate;

re-dissolving MES buffer solution, and ultrasonically treating with ultrasonic cell crusher for 3-5 times;

adding 50 μ g of target antibody, then reversing the upper part and the lower part, uniformly mixing for 20 times, placing on a rotary mixing instrument, and carrying out rotary reaction for 30 min;

sucking 100 mu L of labeled confining liquid into the centrifuge tube, and completely scattering the labeled confining liquid by an ultrasonic cell crusher according to a mode of performing ultrasonic treatment at 90W for 2s and intermittent for 5s and repeating for 3-5 times; rotating and reacting for 2 h;

centrifuging at 4 ℃ for 30min at 10000rpm, discarding the supernatant, taking the precipitate, absorbing 1mL of marked preservative solution, adding into a centrifuge tube, and completely scattering the solution by an ultrasonic cell crusher according to a mode of performing ultrasonic treatment at 90W for 2s and intermittent operation for 5s for 10 times;

and transferring the sample into a 10mL centrifuge tube, sucking 4mL of the label preservation solution, washing 2mL of the centrifuge tube for label washing, transferring the washed sample into the 10mL centrifuge tube, and uniformly mixing the sample and the centrifuge tube in a vortex manner to obtain the target solution.

The marking confining liquid comprises bovine serum albumin and purified water, and the specific numerical ratio is shown in table 3. The marked preservation solution comprises bovine serum albumin, sucrose, casein sodium salt, trehalose, Proclin300 and a Tris buffer solution, wherein the concentration of the Tris buffer solution is 0.2M, the pH value is 8, and the specific numerical proportion is shown in Table 4.

TABLE 3 preparation of marking blocking solution (10mL)

TABLE 4 preparation of labeled preservative solution (100mL)

And secondly, the application of the time-resolved fluorescence immunochromatography kit for quantitatively detecting NT-proBNP/ST2 comprises a detection card, wherein a sample to be detected is added into a sample adding hole of the detection card, and the detection card after sample addition is placed into a card slot of an instrument for detection to obtain a detection result output by the instrument.

Specifically, 1) opening the instrument and inserting a chip with the same reagent batch number;

2) sucking 100 mu L of sample by using a pipette, adding the sample into the diluent, and turning upside down and uniformly mixing;

3) opening the aluminum foil bag, taking out the detection card, and horizontally placing on a desktop;

4) sucking 100 mu L of the diluted and uniformly mixed sample by using a pipettor, and adding the sample into a sample adding hole of the detection card;

5) selecting a sample type 'plasma, serum or whole blood' on a matched instrument;

6) and (3) immediate test: after reacting for 15min at room temperature, placing the detection card into the instrument card slot, selecting an 'instant test' mode, and clicking 'test'; standard test: placing the detection card into a card slot of an instrument, selecting a standard test mode, clicking a test mode, automatically timing by the instrument, and automatically testing and displaying a result after timing is finished;

7) clicking 'printing' can print a detection result report.

Wherein, the sample to be detected is whole blood or plasma, and the step of collecting the whole blood or the plasma comprises the following steps: whole blood or plasma of the subject is collected using an anticoagulation tube. The anticoagulant contained in the anticoagulation tube is heparin lithium, EDTA-K2 or sodium citrate. Samples with hemolysis, lipemia, jaundice are not recommended. Specifically, the whole blood collection is to collect the venous blood of a detected person by using an anticoagulation tube containing lithium heparin, EDTA-K2 or sodium citrate anticoagulant, and the collected blood sample is shaken uniformly for later use; blood plasma collection venous blood of a subject is collected by an anticoagulation tube containing heparin lithium, EDTA-K2 or sodium citrate anticoagulant, and blood plasma should be separated as soon as possible after blood collection so as to avoid hemolysis.

The different blood samples should be used immediately after collection. If the reagent cannot be used for detection in time, the blood plasma/whole blood can be stored for 4 hours at room temperature. Or the blood plasma can be stored for 2 days at the temperature of 2-8 ℃, and the whole blood can be stored for 2 days at the temperature of 2-8 ℃. Alternatively, plasma can be stored for 90 days at-20 + -5 deg.C, and whole blood samples cannot be stored frozen. The refrigerated and frozen blood samples are fully dissolved and uniformly mixed before detection, and can be tested after being restored to room temperature without repeated freezing and thawing.

Third, example 1: sample detection limit detection experiment of kit

a) NT-proBNP: detecting 5 low-value samples with approximate concentration detection limit (5pg/ml) for 5 times, adding 100uL of sample to be detected into 300uL of sample diluent, uniformly mixing, adding 100uL of sample into a sample adding hole, detecting by using a fluorescence detector after 15min, and repeating for 25 times. The number of detection results smaller than the blank limit (1pg/mL) among the 25 detection values is less than or equal to 3 and all of the 25 detection values are not higher than 5 pg/mL. The result shows that the detection limit of the kit is not more than 5.00 pg/mL.

TABLE 5 NT-proBNP detection limit test results

b) ST 2: detecting 5 low-value samples with approximate detection limit (3ng/ml) of concentration for 5 times, adding 300uL of sample diluent into 100uL of samples to be detected, uniformly mixing, adding 100uL of sample diluent into a sample adding hole, detecting by using a fluorescence detector after 15min, and repeating for 25 times. The number of detection results smaller than the blank limit (1ng/mL) among the 25 detection values is less than or equal to 3, and all of the 25 detection values are not higher than 3 ng/mL. The result shows that the detection limit of the kit is not more than 3.00 ng/mL.

TABLE 6 ST2 detection Limit test results

Example 2: sample linear detection experiment of kit of the application

a) The BNP linear reference substances are mixed according to a certain proportion, the mixed concentration is respectively 5pg/mL, 100pg/mL, 1000pg/mL, 3000pg/mL and 5000pg/mL, 100uL of the reference substances are added into 300uL of sample diluent, 100uL of the reference substances are added into a sample adding hole after uniform mixing, each reference substance is repeatedly measured for 3 times, a fluorescence detector is used for detection after 15min, the average value (Yi) of the feedback concentration is calculated, Xi is used as an independent variable, Yi is used as a dependent variable to calculate a linear regression equation, and a linear correlation coefficient r is calculated. The results show that the linear correlation coefficient r is greater than 0.9900.

TABLE 7 NT-proBNP Linear test results

b) Mixing ST2 linear reference substances according to a certain proportion, wherein the mixed concentrations are respectively 3.125ng/mL, 20ng/mL, 50ng/mL, 100pg/mL and 200pg/mL, taking 100uL of the reference substance, adding 300uL of sample diluent, uniformly mixing, taking 100uL of the sample diluent, adding into a sample adding hole, repeatedly measuring each reference substance for 3 times, detecting by using a fluorescence detector after 15min, calculating a feedback concentration average value (Yi), taking Xi as an independent variable, taking Yi as a dependent variable to calculate a linear regression equation, and calculating a linear correlation coefficient r. The results show that the linear correlation coefficient r is greater than 0.9900.

TABLE 8 ST2 Linear test results

Example 3: accuracy detection of the kit of the present application

Measuring NT-proBNP enterprise reference substances with the concentration of about 100pg/mL and 3000pg/mL, adding 100uL of the reference substances into 300uL of sample diluent, uniformly mixing, adding 100uL of the reference substances into a sample adding hole of a reagent card, repeatedly measuring for 3 times, horizontally placing the reagent card for 15min, inserting the reagent card into a fluorescence quantitative detector, reading the T/C value of a fluorescence signal, and calculating the corresponding sample concentration according to a standard curve. As can be seen from the test data in Table 7, the relative deviation of the samples is not more than + -10%.

TABLE 9 NT-proBNP accuracy test results

b) Measuring ST2 enterprise reference products with the concentration of about 500ng/mL and 200pg/mL, adding 100uL of the reference product into 300uL of sample diluent, uniformly mixing, adding 100uL of the reference product into a sample adding hole of a reagent card, repeatedly measuring for 3 times, horizontally placing the reagent card for 15min, inserting the reagent card into a fluorescence quantitative detector, reading the T/C value of a fluorescence signal, and calculating the corresponding sample concentration according to a standard curve. As can be seen from the test data in Table 7, the relative deviation of the samples is not more than + -10%.

TABLE 10 ST2 accuracy test results

Example 4: comparison of the comparative relevance of the kits of the present application to the kits of comparative manufacturers

The time-resolved fluorescence immunochromatographic kit of the reagent NT-proBNP/ST2 and the NT-proBNP determination kit produced by BAO-Chuang biotechnology limited of Guangzhou of a comparative manufacturer are respectively used for testing linear values, each point is tested for 9 times, and the correlation is analyzed, wherein the correlation coefficient is not lower than 0.9900. The correlation of the NT-proBNP reagent of the present application with the comparison manufacturer is shown in FIG. 3.

The time-resolved fluorescence immunochromatographic kit of the reagent NT-proBNP/ST2 and the ST2 determination kit produced by BAO-Chuang biotechnology limited of Guangzhou of a comparative manufacturer are respectively used for testing linear values, each point is tested for 9 times, and the correlation is analyzed, wherein the correlation coefficient is not lower than 0.9900. The correlation of the reagent ST2 of the present application with the comparison manufacturer is shown in FIG. 4.

Currently, the single NT-proBNP detection mostly adopts a colloidal gold method and a chemiluminescence method. The single term ST2 is primarily chemiluminescent. The emission intensity of chemiluminescence methods depends on various environmental factors, the curves of emission intensity and time in different environmental systems have large differences, and instruments are expensive and need to be used by professional operators. The methodological time-resolved immunofluorescence is improved on the basis of colloidal gold, the billows elements are used as tracers, two parameters of wavelength and time are detected simultaneously for signal resolution, the interference of non-specific fluorescence can be effectively eliminated, and the method is high in sensitivity, wide in analysis range, stable in labeled conjugate, long in effective service life, rapid in measurement, easy to automate and free of radioactive pollution.

In addition, ST2 is used as a novel heart failure detection index, the application combines NT-proBNP/ST2 detection, and the NT-proBNP and ST2 in a sample are detected by combining the high sensitivity characteristic of a time-resolved fluorescence technology, so that the kit can be used as a novel heart failure diagnosis means.

It should be understood that, although the steps in the flowcharts of the figures are shown in order as indicated by the arrows, the steps are not necessarily performed in order as indicated by the arrows. The steps are not performed in the exact order shown and may be performed in other orders unless explicitly stated herein. Moreover, at least a portion of the steps in the flow chart of the figure may include multiple sub-steps or multiple stages, which are not necessarily performed at the same time, but may be performed at different times, which are not necessarily performed in sequence, but may be performed alternately or alternately with other steps or at least a portion of the sub-steps or stages of other steps.

It is to be understood that the above-described embodiments are merely illustrative of some, but not restrictive, of the broad invention, and that the appended drawings illustrate preferred embodiments of the invention and do not limit the scope of the invention. This application is capable of embodiments in many different forms and is provided for the purpose of enabling a thorough understanding of the disclosure of the application. Although the present application has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that the present application may be practiced without modification or with equivalents of some of the features described in the foregoing embodiments. All equivalent structures made by using the contents of the specification and the drawings of the present application are directly or indirectly applied to other related technical fields and are within the protection scope of the present application.

Claims

1. A kit for quantitatively detecting NT-proBNP/ST2 by a time-resolved fluorescence immunochromatography method is characterized by comprising a detection card, wherein the detection card comprises a card shell and a test strip, the test strip comprises a PVC (polyvinyl chloride) bottom plate, and a treatment pad, a combination pad, a nitrocellulose membrane and absorbent paper which are sequentially overlapped are arranged on the PVC bottom plate;

2. The kit for quantitative determination of NT-proBNP/ST2 according to claim 1, wherein the treatment pad is prepared by the following steps:

3. The kit for quantitative determination of NT-proBNP/ST2 of claim 2, wherein the amount of bovine serum albumin is 10g, the amount of Tween-20 is 5ml, the concentration of borax borate buffer is 0.2M, the pH value is 8, and the amount is 1000 ml.

4. The kit for quantitative determination of NT-proBNP/ST2 according to claim 1, wherein the conjugate pad is prepared by the following steps:

5. The kit for quantitative determination of NT-proBNP/ST2 according to claim 4, wherein the labeled blocking solution comprises bovine serum albumin and purified water.

6. The kit for quantitative determination of NT-proBNP/ST2 according to claim 4, wherein the label-preserving solution comprises bovine serum albumin, sucrose, sodium caseinate, trehalose, Proclin300 and Tris buffer.

7. The kit for the quantitative determination of NT-proBNP/ST2 according to claim 6, wherein the Tris buffer has a concentration of 0.2M and a pH value of 8.

8. Use of the kit according to any one of claims 1 to 7 for quantitative detection of NT-proBNP/ST2, wherein a sample to be tested is added to the sample application hole of the test card, and the test card after application is placed in the card slot of the apparatus for detection, so as to obtain the detection result output by the apparatus.

9. The use of the kit according to claim 8 for the quantitative detection of NT-proBNP/ST2, wherein the sample to be tested is whole blood or plasma.

10. Use of the kit according to claim 9 for the quantitative detection of NT-proBNP/ST2, wherein the whole blood or plasma is collected through an anticoagulant tube containing an anticoagulant selected from the group consisting of lithium heparin, EDTA-K2, and sodium citrate.