CN114034854A

CN114034854A - Kit for quantitatively detecting cTnT/NT-proBNP/D-dimer and application thereof

Info

Publication number: CN114034854A
Application number: CN202111466897.3A
Authority: CN
Inventors: 石磊; 黎文娟; 邓成刚; ***; 陈魏穗
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: 2022-02-11

Abstract

The embodiment of the application belongs to the technical field of fluorescence immunochromatography, and relates to a kit for quantitatively detecting cTnT/NT-proBNP/D-dimer and application thereof, wherein the kit comprises a detection card, the detection card comprises a card shell and a test paper card, the test paper card comprises a PVC (polyvinyl chloride) bottom plate, and a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper are arranged on the PVC bottom plate; three monoclonal antibodies of anti-cardiac troponin T, anti-N-terminal atrial natriuretic peptide and anti-D dimer respectively marked by rare earth Eu3+ fluorescent microspheres and goat anti-chicken IgY antibody marked by rare earth Eu3+ fluorescent microspheres are adsorbed on the bonding pad; the nitrocellulose membrane is provided with a first detection line, a second detection line, a third detection line and a quality control line, the first detection line is coated with a D-dimer monoclonal antibody, the second detection line is coated with a cTnT monoclonal antibody, the third detection line is coated with an NT-proBNP monoclonal antibody and the quality control line is coated with a chicken IgY antibody, so that the cTnT, the NT-proBNP and the D-dimer can be rapidly and accurately quantitatively detected by one-time sampling.

Description

Kit for quantitatively detecting cTnT/NT-proBNP/D-dimer and application thereof

Technical Field

The application relates to the technical field of fluorescence immunochromatography, in particular to a kit for quantitatively detecting cTnT/NT-proBNP/D-dimer and application thereof.

Background

Chest pain is a common but life-threatening condition, and causes of chest pain are complex and diverse, including Acute Coronary Syndrome (ACS), Acute Aortic Dissection (AAD), and Acute Pulmonary Embolism (APE), among which ACS accounts for the highest proportion of these serious life-threatening diseases. In recent years, the morbidity of the patient is increased year by year, the death risk of the patient is higher and higher, and how to quickly and accurately diagnose and identify ACS and other causes of fatal chest pain becomes the difficulty and the key point of emergency treatment. All patients who see a doctor due to chest pain need to be subjected to electrocardiographic examination, the electrocardiogram is an important means for diagnosing ischemic chest pain, and the first electrocardiogram is completed within 10 minutes after the patients are subjected to a doctor. However, merely performing an electrocardiographic examination at an early stage of chest pain is not sufficient to provide more sufficient and powerful evidence for further diagnosis and treatment of the disease, because the electrocardiographic examination at the early stage of the disease does not show typical AMI in about 50% of cases in which symptoms such as chest pain are finally diagnosed as AMI (acute myocardial infarction), and thus it is of great significance to improve the early rapid diagnosis rate of chest pain diseases.

cTnT: cTn (cardiac troponin) is present in cardiac muscle cells and is composed of three subunits, cTnI (cardiac troponin I), cTnT (cardiac troponin T), and cTnC (cardiac troponin C). The cTnT is an antigen specific to the cardiac muscle cells, after the cardiac muscle cells are damaged, the cTnT appears in blood at the earliest time and has long duration, and the cTnT has high sensitivity and specificity on the cardiac muscle damage. Research shows that the cardiac troponin T (cTnT) can sensitively prompt the state of myocardial injury, has high myocardial specificity, can be used as a cardiac injury marker, can be used as a 'gold standard' for judging the myocardial injury, particularly diagnosing acute myocardial infarction, and can more quickly diagnose and eliminate the AMI (acute myocardial infarction) due to high sensitivity and long window period of the myocardial injury. (ACS is divided into AMI and UAP (unstable angina) according to the severity of coronary lesions.

NT-proBNP: the NP (natriuretic peptide) family is a large class of substances used in vertebrates to regulate circulatory system volume and osmotic pressure. BNP (brain natriuretic peptide) is a major member of the natriuretic peptide family and is secreted mainly by cardiomyocytes. The myocardial cells are stimulated to firstly secrete pre-proBNP and then form proBNP, the proBNP is cracked into BNP (B-type natriuretic peptide containing 32 amino acids) and NT-proBNP (N-terminal B-type natriuretic peptide containing 76 amino acids) with bioactivity such as benefiting natrium, diuresis, blood vessel expansion and the like under the action of endonuclease, and the BNP and the NT-proBNP have certain correlation in blood level. In the 2015 national standard "cardiac marker detection and clinical application in coronary artery disease and heart failure", both of them are used as important cardiac markers for risk stratification of ACS, early detection, diagnosis and prognosis evaluation of heart failure, and play important roles. And the NT-proBNP has more advantages than the BNP as a cardiac marker in the two aspects, which are shown in the following concrete points: 1. the half-life period in vivo is longer (90-120 min is more than 18-20 min); 2. the diagnosis rate for early and mild heart failure is higher; 3. the concentration change level can more accurately reflect the treatment effect; 4. the in vitro stability is better (72h is more than 4 h); 5. the detection reagents all adopt antibodies from the same source, and the correlation of the diagnosis result is better. Therefore, the detection of NT-proBNP is of great importance for the diagnosis of heart diseases. Researches show that the concentration of the NT-proBNP is high in consistency with the severity of heart failure (acute heart failure AHF and chronic heart failure CHF), the NT-proBNP can be used as a strong prediction factor of HF (HF), and when suspected Heart Failure (HF) occurs in a patient, N-terminal atrial natriuretic peptide (NT-proBNP) detection can be firstly carried out, so that chronic obstructive dyspnea and cardiogenic dyspnea can be effectively identified.

D-dimer: the D-dimer is one of Fibrin Degradation Products (FDPs), and is specially used for the fragment of fibrin after being decomposed, and the structure of the D-dimer retains the structure of a gamma chain staggered molecule in fibrin monomers. The structural composition of D-dimer has three forms: one consists of two 180kDa D domain molecules, one consists of two 195kDa D domain molecules, and the other consists of two 340kDa D domain molecules and one fibrinogen E domain molecule. This staggered molecular structure is present in fibrin, but not in fibrinogen. Thus, D-dimer can be said to be the only product resulting from the breakdown of fibrin. Elevated D-dimer in blood suggests two implications: firstly, fibrin agglutination occurs in blood; secondly, the anticoagulant mechanism in the body is started, and the existing fibrin is cut and decomposed. Thus, an increase or positive in D-dimer is an important molecular marker for the presence of increased clotting and fibrinolytic activity in vivo. The detection of the D-dimer is helpful for early diagnosis of thromboembolic diseases and disseminated intravascular coagulation; but also has important significance in the occurrence and development processes of a plurality of diseases such as cardiovascular and cerebrovascular diseases (such as myocardial infarction, angina, hypertension, coronary heart disease, cerebral infarction, cerebral hemorrhage and the like): the D-dimer is possibly increased by cardiovascular and cerebrovascular diseases, and the change of the D-dimer is dynamically monitored by selecting an appropriate time in combination with clinical manifestations and other examinations, so that valuable information can be provided for clinical thrombosis prevention, disease outcome assessment and the like, and the method has great significance in the aspects of detection of the D-dimer in early chest pain, prevention, diagnosis and treatment of cardiovascular and cerebrovascular diseases, disease outcome assessment and the like.

At present, detection methods for cTnT, NT-proBNP and D-dimer include an enzyme-linked immunosorbent assay, a fluorescence immunoassay, a chemiluminescence method, an immunoturbidimetry method, a colloidal gold immunochromatography method and the like, but detection products and methods are mainly limited to detection of one or two of the methods, and methods for simultaneously detecting the three methods are extremely few, so that the methods are not favorable for more rapidly, comprehensively and specifically carrying out early warning and diagnosis on occurrence and development of various heart diseases, particularly ACS, and whole course. Therefore, the application provides a rapid detection kit which is simpler and faster and can simultaneously detect the cTnT, the NT-proBNP and the D-dimer chest pain triplet by combining clinical feedback and the defects of the prior art. The method can realize timely and rapid combined detection of cTnT, NT-proBNP and D-dimer of the patient suffering from acute chest pain, and provides a basis for distinguishing whether cardiogenic or non-cardiogenic chest pain occurs to the patient suffering from chest pain, thereby effectively achieving timely targeted treatment and improving the survival rate of the patient.

1. The detection methods for cTnI, NT-proBNP and D-dimer include enzyme-linked immunosorbent assay, fluorescence immunoassay, chemiluminescence assay, immunoturbidimetry assay, colloidal gold immunochromatography and the like, but the detection products and methods are mainly limited to detection of one or two items, and the methods for simultaneous detection of the three items are few.

2. Most myocardial injuries adopt troponin I/T (cardiac troponin I/cardiac troponin T, cTnT/T), creatine kinase isozyme (CK-MB) and Myoglobin (MYO), and the three combined tests are important, but relatively single in terms of diagnosis, have certain limitations for patients with non-cardiogenic chest pain and are particularly challenging for emergency departments.

Disclosure of Invention

The embodiment of the application aims to provide a kit for quantitatively detecting cTnT/NT-proBNP/D-dimer and application thereof, which can be used for rapidly detecting 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 quantitatively detecting cTnT/NT-proBNP/D-dimer and applications thereof, and adopt the following technical solutions:

a kit for quantitatively detecting cTnT/NT-proBNP/D-dimer comprises a detection card, wherein the detection card comprises a card shell and a test paper card, the test paper card comprises a PVC (polyvinyl chloride) bottom plate, and a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper which are sequentially lapped are arranged on the PVC bottom plate;

wherein the binding pad is adsorbed with rare earth Eu3+ fluorescent microsphere labeled anti-cardiac troponin T antibody, rare earth Eu3+ fluorescent microsphere labeled anti-N-terminal atrial natriuretic peptide antibody, rare earth Eu3+ fluorescent microsphere labeled anti-D-dimer antibody and rare earth Eu3+ fluorescent microsphere labeled goat anti-chicken IgY antibody;

the nitrocellulose membrane is sequentially provided with a first detection line, a second detection line, a third detection line and a quality control line, wherein the first detection line is coated with a D-dimer monoclonal antibody, the second detection line is coated with a cTnT monoclonal antibody, the third detection line is coated with an NT-proBNP monoclonal antibody and the quality control line is coated with a chicken IgY antibody.

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

soaking the glass fiber membrane in a treatment solution for soaking treatment;

and drying the glass fiber membrane after the soaking treatment to obtain the sample pad.

Further, the treatment solution comprises 1.0% of Triton X-100, 2.5% of bovine serum albumin and 0.15M of Tris buffer.

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

respectively labeling an anti-cardiac troponin T antibody, an anti-N-terminal atrial natriuretic peptide antibody, an anti-D-dimer antibody and a goat anti-chicken IgY antibody by rare earth Eu3+ fluorescent microspheres to respectively obtain an anti-cardiac troponin T antibody-fluorescent microsphere coupled compound, an anti-N-terminal atrial natriuretic peptide antibody-fluorescent microsphere coupled compound, an anti-D-dimer antibody-fluorescent microsphere coupled compound and a goat anti-chicken IgY antibody-fluorescent microsphere coupled compound;

uniformly mixing the anti-cardiac troponin T antibody-fluorescent microsphere coupled compound, the anti-N-terminal atrial natriuretic peptide antibody-fluorescent microsphere coupled compound, the anti-D-dimer antibody-fluorescent microsphere coupled compound and the goat anti-chicken IgY antibody-fluorescent microsphere coupled compound to obtain a fluorescence labeling mixed solution;

and spraying the fluorescent labeling mixed solution onto a target glass fiber membrane through a non-contact type micro quantitative spray head to obtain the combined pad.

Further, the rare earth Eu3+ fluorescent microspheres for labeling the anti-cardiac troponin T antibody, the anti-N-terminal atrial natriuretic peptide antibody, the anti-D-dimer antibody and the anti-chicken IgY antibody are aldehyde-treated rare earth Eu3+ fluorescent microspheres, wherein the aldehyde-treatment method of the rare earth Eu3+ fluorescent microspheres comprises the following steps:

washing the rare earth fluorescent nano microspheres by using 20mM carbonate buffer solution with pH of 9.5 and adopting a centrifugal method to obtain washed rare earth fluorescent nano microspheres;

resuspending the washed rare earth fluorescent nano-microspheres in a carbonate buffer solution with the concentration of 20mM and the pH value of 9.5, adding aldehyde dextran, uniformly mixing, and carrying out dark reaction in a greenhouse for a certain time to obtain the dark-reacted rare earth fluorescent nano-microspheres;

and (3) centrifugally washing and resuspending the rare earth fluorescent nano-microspheres subjected to dark reaction by using 20mM carbonate buffer solution with pH of 9.5 to obtain aldehyde-based rare earth Eu3+ fluorescent microspheres.

Further, the anti-cardiac troponin T antibody-fluorescent microsphere coupled compound is prepared by the following steps:

mixing monoclonal antibodies of monoclonal cell strains with different antigen epitopes in equal proportion, adding the monoclonal antibodies into carbonate buffer solution, and dialyzing at 4 ℃;

adding the aldehyde rare earth Eu3+ fluorescent microspheres, and reacting at 4 ℃;

and adding sodium borohydride to a final concentration of 5mM, adding a blocking solution to block, washing by a Tris-HCL buffer solution centrifugation method, and suspending in a Tris-HCL buffer solution to obtain the anti-cardiac troponin T antibody-fluorescent microsphere coupled complex.

Further, the method for preparing the target glass fiber membrane comprises the following steps:

soaking the glass fiber membrane by 150mM Tris-HCL treatment solution to obtain the soaked glass fiber membrane;

and drying the soaked glass fiber membrane to obtain the target glass fiber membrane.

Further, the nitrocellulose membrane provided with the first detection line, the second detection line, the third detection line and the quality control line is prepared by the following steps:

adopting cell strains different from the cell strains of the anti-cardiac troponin T antibody, the anti-N-terminal atrial natriuretic peptide antibody and the anti-D-dimer antibody on the combination pad as target cell strains;

extracting a D-dimer monoclonal antibody, a cTnT monoclonal antibody and an NT-proBNP monoclonal antibody from the target cell strain by an ascites production process;

respectively adjusting the concentrations of the D-dimer monoclonal antibody, the cTnT monoclonal antibody, the NT-proBNP monoclonal antibody and the chicken IgY antibody to 1.5mg/ml by using coating diluent, wherein the amount of the membrane solution is 1.5 mu l/cm, and respectively obtaining a first detection solution, a second detection solution, a third detection solution and a quality control solution;

and respectively spraying the first detection solution, the second detection solution, the third detection solution and the quality control solution on the original nitrocellulose membrane in parallel to obtain the nitrocellulose membrane provided with the first detection line, the second detection line, the third detection line and the quality control line.

Further, the interval between the first detection line, the second detection line, the third detection line and the quality control line is 3 mm.

In order to solve the above technical problems, the embodiment of the present application further provides a use method of a kit for quantitatively detecting cTnT/NT-proBNP/D-dimer, which adopts the following technical scheme:

the application of the kit in quantitative detection of cTnT/NT-proBNP/D-dimer comprises the following steps:

placing the IC card at the labeling position of a dry type fluorescence immunoassay analyzer, and reading related information;

adding 100 mu L of serum sample or 150 mu L of whole blood sample into the buffer solution, fully and uniformly mixing to obtain a mixed solution, and vertically dropwise adding 100 mu L of the mixed solution to a sample adding position of the detection card;

and inserting the detection card into a loader of a dry type fluorescence immunoassay analyzer to perform test operation, and obtaining a detection result output by the dry type fluorescence immunoassay analyzer.

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

the method can be used for rapidly and accurately carrying out chest pain triple time-resolved fluorescence immunofluorescence quantitative detection for quantitatively analyzing three cardiovascular and cerebrovascular disease related indexes of cTnT, NT-proBNP and D-dimer in serum and whole blood samples. Provides a basis for identifying whether the patient with chest pain is cardiogenic or non-cardiogenic, thereby effectively achieving timely and targeted treatment and improving the survival rate of the patient.

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 structural diagram of a detection card of a kit for quantitatively detecting cTnT/NT-proBNP/D-dimer according to the present application;

FIG. 2 is another schematic structural view of a detection card of the kit for quantitatively detecting cTnT/NT-proBNP/D-dimer according to the present application;

FIG. 3 is a schematic diagram showing the results of comparative analysis of the methodology of the kit for quantitative detection of cTnT/NT-proBNP/D-dimer according to the present application;

FIG. 4 is a schematic diagram of another comparative analysis of the methodology of the kit for quantitative detection of cTnT/NT-proBNP/D-dimer according to the present application;

FIG. 5 is a schematic diagram of another comparative analysis of the methodology of the kit for quantitative detection of cTnT/NT-proBNP/D-dimer according to the present application;

FIG. 6 is a schematic diagram of another comparative analysis of the methodology of the kit for quantitative detection of cTnT/NT-proBNP/D-dimer according to the present application;

FIG. 7 is a schematic diagram of another comparative analysis of the methodology of the kit for quantitative detection of cTnT/NT-proBNP/D-dimer according to the present application;

FIG. 8 is a schematic diagram of another comparative analysis of the methodology of the kit for quantitative detection of cTnT/NT-proBNP/D-dimer according to the present application.

Reference numerals: 1. a PVC base plate; 2. a sample pad; 3. a bonding pad; 4. a nitrocellulose membrane; 5. a first detection line, 6, a second detection line; 7. a third detection line; 8. a quality control line; 9. absorbent paper; 10. detecting the card; 11. a sample adding position; 12. a test paper card; 13. a hand-held area.

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.

A kit for quantitatively detecting cTnT/NT-proBNP/D-dimer is shown in figures 1 and 2, and figure 1 is a structural schematic diagram of a detection card of the kit for quantitatively detecting cTnT/NT-proBNP/D-dimer; FIG. 2 is a schematic view of the structure of a kit for quantitative determination of cTnT/NT-proBNP/D-dimer according to the present application after assembly of a test card; the kit comprises a detection card, wherein the detection card comprises a card shell and a test paper card, the test paper card comprises a PVC (polyvinyl chloride) base plate, and a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper which are sequentially overlapped are arranged on the PVC base plate;

The rare earth Eu3+ fluorescent microsphere labeled anti-cardiac troponin T (cTnT) antibody specifically comprises: rare earth Eu3+ fluorescent microsphere labeled anti-cTnT murine monoclonal IgG antibody; the rare earth Eu3+ fluorescent microsphere labeled anti-N-terminal atrial natriuretic peptide (NT-proBNP) antibody specifically comprises: a rare earth Eu3+ fluorescent microsphere labeled anti-NT-proBNP mouse-derived monoclonal IgG antibody; the rare earth Eu3+ fluorescent microsphere labeled anti-D-dimer (D-dimer) antibody specifically comprises the following components: rare earth Eu3+ fluorescent microsphere labeled anti-D-dimer murine monoclonal IgG antibody.

The diameter of the rare earth fluorescent microsphere is 100-250 nm. The rare earth fluorescent microsphere contains one or more rare earth lanthanide elements, is stable under the ground state, and emits fluorescence with the wavelength range of 550-650nm under the action of an excitation light source with the wavelength range of 300-400 nm. Wherein, the diameter of the rare earth fluorescent microsphere of the combined pad is preferably 120-200 nm. Further preferably, the diameter of the rare earth fluorescent microsphere is 150nm, the rare earth fluorescent microsphere contains rare earth lanthanide element Eu3+ (europium), is stable under a ground state, and emits fluorescence with the wavelength of 615nm under the action of an excitation light source of 337 nm.

The antibody marked by the rare earth fluorescent microspheres on the bonding pad is preferably derived from monoclonal cell strains aiming at 2 different antigen epitopes. Specifically, the monoclonal antibodies are purified and mixed monoclonal antibodies, and are respectively derived from monoclonal antibody cell strains aiming at different cardiac troponin T, N terminal atrial natriuretic peptides, D dimers and other antigen epitopes.

The kit has the advantages of simple and convenient operation, rapid reaction, high sensitivity, strong specificity and the like, and can realize one-time sampling and simultaneously, rapidly and accurately carry out quantitative detection on cTnT, NT-proBNP and D-dimer, thereby effectively assisting the clinical diagnosis and treatment of patients with chest pain. The method can rapidly and accurately carry out quantitative analysis on three cardiovascular and cerebrovascular disease related indexes of cTnT, NT-proBNP and D-dimer in serum and whole blood samples simultaneously, and can be used for chest pain triple time-resolved fluorescence immunofluorescence quantitative detection. Provides a basis for identifying whether the patient with chest pain is cardiogenic or non-cardiogenic, thereby effectively achieving timely and targeted treatment and improving the survival rate of the patient.

Secondly, a preparation method of the sample pad comprises the following steps:

the glass fiber membrane was immersed in a treatment solution containing 1.0% Triton X-100, 2.5% BSA, 0.15M Tris buffer, pH7.5, at 4 ℃ for 4 hours, and then placed in an oven and dried at 37 ℃ for 2 hours.

Thirdly, a preparation method of the combined pad comprises the following steps:

the conjugate pad described herein is prepared by the following steps:

the glass fiber membrane was soaked in 150mM Tris-HCl treatment solution (containing 1.0% Triton X-100, 2.5% BSA, pH7.4) at 4 ℃ for 2 hours, and then taken out of the oven at 37 ℃ and dried for 4 hours for use. The preparation method comprises the steps of putting a glass fiber membrane on a Bio-DotXYZ3050 three-dimensional spraying platform, using a Bio-Jet Quanti300 non-contact type micro quantitative nozzle to mix the anti-cardiac troponin T, the anti-N-terminal atrial natriuretic peptide, the anti-D-dimer antibody and the goat anti-chicken IgY antibody marked by rare earth fluorescent microspheres to obtain an anti-cardiac troponin T antibody-fluorescent microsphere coupled compound, an anti-N-terminal atrial natriuretic peptide antibody-fluorescent microsphere coupled compound, an anti-D-dimer antibody-fluorescent microsphere coupled compound and the goat anti-chicken IgY antibody-fluorescent microsphere coupled compound, spraying the four coupled compounds on the glass fiber membrane, and drying the glass fiber membrane for 1 hour at 37 ℃ to obtain the anti-cardiac troponin T antibody-fluorescent microsphere coupled compound, the anti-N-terminal atrial natriuretic peptide antibody-fluorescent microsphere coupled compound, the anti-D-dimer antibody-fluorescent microsphere coupled compound and the goat anti-chicken IgY antibody-fluorescent microsphere coupled compound.

In the application, the three monoclonal antibodies in the T/N-terminal atrial natriuretic peptide precursor/D dimer three-in-one assay kit marked by the rare earth fluorescent microspheres on the combination pad are prepared by the following steps:

step 1: obtaining of monoclonal antibody cell strain: respectively immunizing mice with atrial natriuretic peptide at the tail end of cardiac troponin T, N and pure D dimer, preparing a monoclonal antibody cell strain with high specificity and affinity by adopting a standard monoclonal antibody preparation method, pairing and screening the obtained monoclonal antibody cell strain, and preferably selecting the monoclonal antibody cell strain for the kit according to pairing results and affinity data;

step 2: preparation of monoclonal antibody: preparing and purifying three mouse-derived monoclonal antibodies such as an anti-cardiac troponin T antibody, an anti-N-terminal atrial natriuretic peptide antibody and an anti-D dimer antibody by adopting a standard ascites production process, subpackaging and storing at-20 ℃ for later use;

and step 3: aldehyde group of the rare earth fluorescent microsphere: taking 5mg rare earth fluorescent microspheres, washing the microspheres for 3 times by using 20mM carbonate buffer solution with pH9.5 by using a centrifugal method, wherein the centrifugal speed is 12000rpm, the time is 5 minutes, finally suspending the microspheres in 100 mu l of the carbonate buffer solution, adding 500 mu l of aldehyde-group dextran, uniformly mixing the microspheres, carrying out dark reaction for 4 hours at room temperature, washing and suspending the microspheres in 100 mu l of the carbonate buffer solution by using the same centrifugal method, and placing the microspheres in 4 ℃ for later use;

and 4, step 4: preparing three coupling compounds of rare earth Eu3+ fluorescent microsphere labeled anti-cardiac troponin T antibody, anti-N-terminal atrial natriuretic peptide antibody and anti-D-dimer antibody: three antibody-microsphere conjugate complexes were separately conjugated, as follows: selecting anti-cardiac troponin T monoclonal antibodies from 2 monoclonal cell strains with different antigen epitopes, dialyzing 2mg of the anti-cardiac troponin T monoclonal antibodies with the carbonate buffer solution at 4 ℃ overnight according to the mass ratio of 1:1, mixing the dialyzed anti-cardiac troponin T monoclonal antibodies with the aldehyde-based rare earth fluorescent microspheres, and reacting at 4 ℃ overnight; then, sodium borohydride was added to a final concentration of 5mM, and the reaction was carried out at 4 ℃ for 4 hours; adding equal volume of blocking solution (50mM Tris-HCl, pH7.4, containing 2% BSA, 5% sucrose), and blocking at 4 deg.C overnight; then, the mixture was washed 3 times with 50mM Tris-HCl, pH7.4 buffer by centrifugation, resuspended in 100. mu.l of 50mM Tris-HCl buffer (containing 1.2% NaCL, 0.5% BSA, 0.1% Tween 20), and stored at 4 ℃ in the dark for further use. The same operation is carried out to respectively prepare the anti-N-terminal atrial natriuretic peptide antibody-microsphere coupled compound and the anti-D-dimer antibody-microsphere coupled compound, and the compounds are stored at 4 ℃ in dark for standby.

Fourthly, a preparation method of the nitrocellulose membrane coated with the detection line and the quality control line comprises the following steps:

the nitrocellulose membrane coated with the detection line and the quality control line is prepared by the following steps:

step 1: adopting cell strains different from three monoclonal antibody cell strains of anti-cardiac troponin T, anti-N-terminal atrial natriuretic peptide, anti-D dimer and the like used on the combination pad, preparing and purifying three murine monoclonal antibodies of anti-cardiac troponin I antibody, anti-N-terminal atrial natriuretic peptide antibody, anti-D dimer antibody and the like by adopting a standard ascites production process to obtain monoclonal antibodies matched with the labeled antibodies, subpackaging and storing at-20 ℃ for later use;

step 2: the three mouse-derived monoclonal antibodies and the goat anti-mouse IgG antibody are respectively adjusted to the concentration of 1-3mg/ml (preferably 1.5mg/ml) by using coating diluent, the membrane solution amount is 1-3 mul/cm (preferably 1.5ul/cm), the three mouse-derived monoclonal antibodies and the goat anti-mouse IgG antibody are respectively used as a detection line and a quality control line and are parallelly sprayed on a nitrocellulose membrane for coating, the intervals among the three detection lines and the quality control lines are 3-7mm (preferably 3mm), and then the nitrocellulose membrane is placed in an oven and dried for 2 hours at 37 ℃.

Fifthly, the use method of the kit comprises the following steps:

the application also provides a three-in-one preparation method of cTnT, NT-proBNP and D-dimer realized by the kit, which comprises the following steps:

step 1: balancing the detection reagent and the sample to room temperature, taking out the test paper card, and horizontally placing;

step 2: reading a card: placing an IC card at a labeling position of a dry type fluorescence immunoassay analyzer, and reading related information, wherein the fluorescence immunoassay analyzer is an optical detection system, and the detection ranges are respectively as follows: cTnT: 0.10-50ng/mL, NT-proBNP 30-30000pg/mL, D-dimer: 0.2-10 ug/mL;

and step 3: sample adding: adding 100 mu L of serum sample or 150 mu L of whole blood sample into a tube of buffer solution, fully and uniformly mixing, and vertically dropwise adding 100 mu L of mixed solution to a sample adding position of the test card; taking sample without sucking bubbles;

and 4, step 4: detection can be carried out by adopting two modes of automatic test or instant test, and the automatic test comprises the following steps: inserting the test card into the carrier of the dry-type fluorescence immunoassay analyzer, pressing the test key, automatically scanning, analyzing and detecting the test card by the dry-type fluorescence immunoassay analyzer, and testing immediately: and (3) placing the test card for 15min at room temperature, inserting the test card into a carrier of a dry fluorescence immunoassay analyzer, and clicking for immediate test.

The application provides a cTnT, NT-proBNP, three-in-one assay kit of D-dimer that fluorescence immunochromatography technique that utilizes tombarthite carboxyl latex microballon mark was prepared, be fit for serum and whole blood sample simultaneously, and be fit for single one-person detection clinically, for qualitative colloidal gold reagent, can quantitative determination sample in cardiac troponin T, N-end brain natriuretic peptide precursor and the content of D dimer, have more definite clinical guiding significance, have easy and simple to handle, the reaction is quick, high sensitivity, the specificity is strong, be fit for advantages such as on-the-spot test and economical and practical.

Sixthly, assembling the test paper card:

sequentially adhering a processed sample pad 2, a binding pad 3 adsorbed with a rare earth fluorescence labeled antibody, a nitrocellulose membrane 4 coated with a detection line and a quality control line and a water absorption pad 5 on a PVC (polyvinyl chloride) plate 1, and assembling to obtain a large test paper plate; cutting into 4mm wide according to requirements, and filling the test paper into a plastic card to form the test paper card.

The equipment and raw materials selected in the steps are preferably as follows:

three specific paired antibodies, namely atrial natriuretic peptide at the tail end of cardiac troponin T, N, D dimer and the like; three quality control products of myocardial troponin T, N terminal atrial natriuretic peptide, D dimer and the like: british langway laboratory diagnostics ltd; rare earth fluorescent microspheres: shanghai Yangzi Biotechnology Ltd; cellulose Nitrate (NC) membrane: millipore corporation; bovine Serum Albumin (BSA), polyethylene glycol PEG20000, hydrolyzed casein: sigma products, other common reagents are analytically pure reagents.

Sixthly, analysis performance examples of the anemia three-term time-resolved fluorescence immunochromatography kit of the present application:

1. analysis of the properties: accuracy of

(1) Performance requirements and inspection methods

The kit of this patent determines cTnI: adding a certain volume of traceable standard solution or high-concentration sample (the ratio of the volume of the standard solution to the volume of the human source sample is not more than 1: 9) into a low-concentration serum matrix (or other body fluid components), preparing accuracy reference substances with the concentrations of about 0.3ng/mL and 10ng/mL, repeatedly detecting each concentration for 3 times, averaging, and calculating according to a formula (1), wherein the recovery rate is 85-115%.

Formula (1):

wherein, R: recovery rate; v: volume of standard solution added; v0: a volume of a human sample; c: the average value of the detection concentration of the human sample after the human sample is added into the standard solution; c0: an average value of the detected concentration of the human sample; CS: concentration of standard solution.

The kits described herein determine the NT-proBNP: and (3) measuring NT-proBNP enterprise reference products with the concentrations of about 300pg/mL and 10000pg/mL, detecting according to the steps of the specification, recording the test result (Xi) after repeating the measurement for 3 times, calculating the relative deviation (Bi) according to the formula (2), and judging as qualified if the results of 3 times meet the requirement that the relative deviation does not exceed +/-10%. If the results of more than or equal to 2 times are not met, the product is judged to be unqualified. If the result of 1 test is not satisfactory, the test is repeated 20 times, and the relative deviation (Bi) is calculated according to the formula (2), and if the result of more than or equal to 19 tests is satisfactory, the relative deviation is not more than +/-10 percent.

Formula (2): bi ═ x (Xi-T)/T × 100%, where Bi represents the relative deviation; xi represents the concentration per measurement; t represents the labeled concentration.

The kit described in the present application measures D-Dimer: adding a certain volume of traceable D-Dimer standard solution or high-concentration sample (the volume ratio of the standard solution to the human source sample is not more than 1: 9) into a low-concentration serum matrix (or other body fluid components), preparing accuracy reference substances with the concentration of about 0.5 mu g/mL and 5 mu g/mL, repeatedly detecting each concentration for 3 times, taking an average value, and calculating according to a formula (1), wherein the recovery rate is 85-115%.

(2) And (4) testing results:

table 1 accuracy test results of cTnT detection by kit

TABLE 2 accuracy test results of NT-proBNP detection by kit

TABLE 3 accuracy test results of D-dimer detection with kit

2. Analysis of the properties: linearity

(1) Performance requirements and test methods:

selecting the test paper card and a dry type fluorescence immunochromatographic analyzer (model: AFS-1000), and setting the parameters of the fluorescence immunoassay analyzer: after the technological parameters of the test paper card are set on the fluorescence immunoassay analyzer, the assembled test paper card is taken out and respectively pressed

cTnI: diluting high-value samples close to the upper limit of a linear interval into 50ng/mL, 10ng/mL, 2ng/mL, 0.3ng/mL and 0.1ng/mL according to a certain proportion, operating on a fluorescence immunoassay quantitative analyzer according to a reagent specification, repeatedly detecting samples with each concentration for 3 times, calculating the average value of the samples, performing straight line fitting on the average value of the measured concentration and theoretical concentration or the dilution proportion by using a least square method, calculating a linear correlation coefficient r according to a formula (3), and ensuring that the linear correlation coefficient (r) is not less than 0.9900 in the range of [0.1, 50] ng/mL.

NT-proBNP: diluting high-value samples close to the upper limit of a linear interval into 30000pg/mL, 10000pg/mL, 2000pg/mL, 300pg/mL and 30pg/mL according to a certain proportion, operating the samples with low concentration on a fluorescence immunoassay quantitative analyzer according to a reagent instruction, repeatedly detecting the samples with each concentration for 3 times, calculating the average value of the samples, performing straight line fitting on the average value of the measured concentration and theoretical concentration or the dilution proportion by using a least square method, calculating a linear correlation coefficient r according to a formula (3), and ensuring that the linear correlation coefficient (r) is not lower than 0.9900 within the range of [30, 30000] pg/mL.

D-Dimer: diluting high-value samples close to the upper limit of a linear region into 10 mu g/mL, 5.0 mu g/mL, 2.0 mu g/mL, 0.5 mu g/mL and 0.2 mu g/mL according to a certain proportion, operating on a fluorescence immunoassay quantitative analyzer according to a reagent instruction, repeatedly detecting samples with each concentration for 3 times, calculating the average value of the samples, performing straight line fitting on the average value of the measured concentration and theoretical concentration or dilution ratio by using a least square method, calculating a linear correlation coefficient r according to a formula (3), and enabling the linear correlation coefficient (r) to be not less than 0.9900 in a range of [0.2, 10] mu g/mL.

Formula (3):

wherein n represents the number of assay samples; xi represents the concentration of the measurement sample; yi represents the mean value of the measured values corresponding to the concentration of the measured sample, which are measured in 3 times of repeated measurement; r represents a linear correlation coefficient.

(2) The result of the detection

TABLE 4 test results of kit for linearity

3. Analysis of the properties: repeatability of

(1) Performance requirements and inspection methods

cTnI: randomly extracting the same batch of reagents, detecting cTnI enterprise reference products with the concentrations of about 0.3ng/mL and 10ng/mL, repeating the steps for 10 times respectively, calculating the average value M and the standard deviation SD of 10 measurement results respectively, and obtaining the variation Coefficient (CV) according to a formula (4), wherein the variation Coefficient (CV) in the batch is less than or equal to 15%.

NT-proBNP: randomly extracting the same batch of reagent, detecting NT-proBNP enterprise reference products with the concentration of about 300pg/mL and 10000pg/mL, repeating the steps for 10 times respectively, calculating the average value M and the standard deviation SD of 10 measurement results respectively, and obtaining the Coefficient of Variation (CV) according to a formula (4), wherein the Coefficient of Variation (CV) in batch is less than or equal to 15%.

D-Dimer: randomly extracting the same batch of reagents, detecting D-Dimer enterprise reference products with the concentrations of about 0.5 mu g/mL and 5.0 mu g/mL, repeating the steps for 10 times respectively, calculating the average value M and the standard deviation SD of 10 measurement results, obtaining the Coefficient of Variation (CV) according to a formula (4), wherein the Coefficient of Variation (CV) of the result obtained by repeated tests on normal quality control plasma is not more than 15%, and the Coefficient of Variation (CV) of the result obtained by repeated tests on high-value quality control abnormal plasma is not more than 10%.

Equation 4: CV is SD/M × 100%, where CV represents a coefficient of variation; SD represents the standard deviation of 10 measurements; m represents the average of 10 measurements.

(2) The result of the detection

TABLE 5 test repeatability of the kit

4. Analysis of the properties: difference between batches

(1) Performance requirements and inspection methods

cTnI: randomly extracting three batches of reagents, detecting cTnI enterprise reference products with the concentrations of 0.3ng/mL and 10ng/mL, repeating the steps for 10 times respectively, calculating the average value M and the standard deviation SD of 30 measurement results of the reference products with different concentrations respectively, and obtaining the Coefficient of Variation (CV) according to a formula (5), wherein the Coefficient of Variation (CV) among batches is less than or equal to 15%.

NT-proBNP: randomly extracting three batches of reagents, detecting NT-proBNP enterprise reference products with the concentrations of about 300pg/mL, 2000pg/mL and 10000pg/mL, repeating the detection for 10 times respectively, calculating the average value M and the standard deviation SD of 30 measurement results of the reference products with different concentrations respectively, and obtaining the Coefficient of Variation (CV) according to a formula (5), wherein the batch Coefficient of Variation (CV) is less than or equal to 15%.

D-Dimer: randomly extracting the same batch of reagents, detecting D-Dimer enterprise reference products with the concentrations of about 0.5 mu g/mL and 5.0 mu g/mL, repeating the steps for 10 times respectively, calculating the average value M and the standard deviation SD of 30 measurement results of the reference products with different concentrations respectively, and obtaining the Coefficient of Variation (CV) according to a formula (5), wherein the Coefficient of Variation (CV) among batches is less than or equal to 15%.

Formula (5) CV is SD/M × 100%, where CV represents a coefficient of variation; SD represents the standard deviation of 30 measurements; m represents the average of 30 measurements.

(2) The result of the detection

TABLE 6 repeatability test results of cTnI detection by kit

TABLE 7 repeatability test results of NT-proBNP detection by kit

TABLE 8 repeatability test results of NT-proBNP detection by kit

Seventhly, carrying out methodology comparison:

after testing a portion of the serum or whole blood sample using the corresponding test kit (electrochemiluminescence) produced by Roche diagnostics, the above samples were tested using the kit of the present application, and the test results are shown in Table 9.

Table 9 compares the test results with the values of Roche assay kit

100 Roche samples are detected by using the kit, and a scatter diagram is made of concentration values detected by the Roche kit and detection signal values of the kit. And (3) drawing a scatter diagram by taking the detection value of the Roche experiment system as a Y axis and the test value of the comparison system of the application as an X axis, wherein the results are shown in figures 3 to 8, and performing correlation analysis to find that the coincidence rate is better, and the R value of the overall coincidence rate can reach more than 0.9975. Therefore, the kit has better correlation with the recognized Roche hypersensitivity kit at home and abroad, and can more accurately screen the sick individuals from the healthy population.

The application provides a cTnT, NT-proBNP, three-in-one assay kit of D-dimer that fluorescence immunochromatography technique that utilizes rare earth element as marker substance was prepared, be fit for serum and whole blood sample, and be fit for single one-out-of-the-job detection clinically, for qualitative colloidal gold reagent, can the content of three kinds of material of cTnT, NT-proBNP, D-dimer in the quantitative detection sample simultaneously, have more definite clinical guiding meaning, have easy and simple to handle, the reaction is quick, high sensitivity, the specificity is strong, be fit for advantages such as on-the-spot detection and economical and practical.

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 cTnT/NT-proBNP/D-dimer is characterized by comprising a detection card, wherein the detection card comprises a card shell and a test paper card, the test paper card comprises a PVC (polyvinyl chloride) bottom plate, and a sample 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 quantitatively detecting cTnT/NT-proBNP/D-dimer according to claim 1, wherein the sample pad is prepared by the following steps:

soaking the glass fiber membrane in a treatment solution for soaking treatment;

3. The kit for quantitative determination of cTnT/NT-proBNP/D-dimer according to claim 2, wherein the processing solution comprises 1.0% Triton X-100, 2.5% bovine serum albumin and 0.15M Tris buffer.

4. The kit for quantitatively detecting cTnT/NT-proBNP/D-dimer according to claim 1, wherein the conjugate pad is prepared by the following steps:

5. The kit for quantitatively detecting cTnT/NT-proBNP/D-dimer according to claim 4, wherein the rare earth Eu3+ fluorescent microspheres for labeling the anti-cardiac troponin T antibody, the anti-N-terminal atrial natriuretic peptide antibody, the anti-D-dimer antibody and the goat anti-chicken IgY antibody are aldehyde-modified rare earth Eu3+ fluorescent microspheres, wherein the aldehyde-modified method of the rare earth Eu3+ fluorescent microspheres comprises:

6. The kit for quantitatively detecting cTnT/NT-proBNP/D-dimer according to claim 5, wherein the anti-cardiac troponin T antibody-fluorescent microsphere coupled complex is prepared by the following steps:

7. The kit for quantitatively detecting cTnT/NT-proBNP/D-dimer according to claim 4, wherein the target glass fiber membrane is prepared by the following steps:

8. The kit for quantitatively detecting cTnT/NT-proBNP/D-dimer according to claim 1, wherein the nitrocellulose membrane provided with the first detection line, the second detection line, the third detection line and the quality control line is prepared by the following steps:

9. The kit for quantitatively detecting cTnT/NT-proBNP/D-dimer according to claim 8, wherein the interval between the first detection line, the second detection line, the third detection line and the quality control line is 3 mm.

10. Use of a kit according to any one of claims 1 to 9 for the quantitative detection of cTnT/NT-proBNP/D-dimer, comprising the steps of: