CN111983243A - Amino-terminal brain natriuretic peptide precursor determination kit, preparation method and detection method - Google Patents
Amino-terminal brain natriuretic peptide precursor determination kit, preparation method and detection method Download PDFInfo
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- CN111983243A CN111983243A CN202010797622.7A CN202010797622A CN111983243A CN 111983243 A CN111983243 A CN 111983243A CN 202010797622 A CN202010797622 A CN 202010797622A CN 111983243 A CN111983243 A CN 111983243A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
- G01N2333/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Brain natriuretic peptide [BNP, proBNP]; Cardionatrin; Cardiodilatin
Abstract
The invention discloses an amino-terminal brain natriuretic peptide precursor determination kit, which is characterized by comprising the following components: the detection line of the reagent card is coated with an amino terminal brain natriuretic peptide precursor monoclonal antibody, and the quality control line is coated with one of goat anti-chicken IgG antibody, goat anti-rabbit IgG antibody or goat anti-mouse IgG antibody. The kit is characterized in that a reaction buffer solution is adopted to redissolve a reaction substance before use, a sample to be detected is added, and the amino-terminal pro-brain natriuretic peptide in the sample to be detected and the amino-terminal pro-brain natriuretic peptide monoclonal antibody marked by the fluorescent microspheres are enabled to fully react.
Description
Technical Field
The invention relates to the technical field of in-vitro diagnosis and detection, in particular to an amino-terminal brain natriuretic peptide precursor determination kit, a preparation method and a detection method.
Background
The amino-terminal brain natriuretic peptide precursor (NT-proBNP) is released primarily by ventricular cells in response to myocardial stress and filling pressure, acting to balance intravascular volume. After stimulation of the cardiomyocytes, the brain natriuretic peptide is released in the form of prohormone (proBNP) and is subsequently cleaved by an endonuclease into an amino-terminal brain natriuretic peptide precursor (NT-proBNP) containing 76 amino acids and a C-terminal polypeptide BNP containing 32 amino acids. BNP has important biological activity because of having a characteristic amino acid ring, but has short half-life in vivo, namely only 18-22 minutes; NT-proBNP is linear structure, has no biological activity, has long half-life period in vivo, about 120 minutes, has stable content and is not influenced by factors such as body position, daily activity and the like, so that NT-proBNP is easier to detect. Therefore, NT-proBNP is considered to be a better biochemical marker capable of reflecting cardiac functions, and has important value for auxiliary diagnosis and treatment evaluation detection of cardiovascular related diseases such as ventricular pressure increase condition, congestive heart failure and dyspnea complication caused by the congestive heart failure, essential hypertension and left ventricular dysfunction evaluation of the essential hypertension, and the like. Of course, the detection result of NT-proBNP is only used for clinical reference, and can not be used alone as the basis for diagnosis or case exclusion, and the clinical diagnosis and treatment of patients should be combined with the symptoms/signs, medical history, and other laboratory examinations, such as echocardiogram, left heart ejection fraction, etc., to comprehensively consider.
Currently, the detection methods of the amino-terminal pro-brain natriuretic peptide mainly include radioimmunoassay, magnetic particle chemiluminescence immunoassay, enzyme-linked immunoassay, colloidal gold method, fluorescence immunoassay, and the like. However, the current detection methods for amino-terminal pro-brain natriuretic peptide have various problems: the radioimmunoassay adopts isotope labeling, which is extremely harmful to human bodies and brings inconvenience to experiments; the magnetic particle chemiluminescence immunoassay method has the advantages of poor reagent stability, low detection precision and high instrument failure rate; enzyme-linked immunoassay is greatly influenced by other factors due to the instability of enzyme, and has poor repeatability; the colloidal gold method has poor sensitivity and cannot carry out quantitative detection; the fluorescence immunoassay method also has the problems of low accuracy and poor precision.
Myocardial infarction and heart failure are common diseases and complications in cardiology, have the characteristics of acute morbidity and critical illness, and can effectively reduce the mortality of patients and improve the prognosis of the patients by acute and early diagnosis. POCT is a detection mode which utilizes portable equipment to obtain a detection result in a short time, and has the characteristics of quick detection, simple operation, capability of being operated at the bedside and the like. The POCT platform can directly detect the cardiac markers in the whole blood sample at the patient, saves the sample transmission and sample preparation time, simultaneously does not need to queue, and can be used for sampling and detecting immediately, thereby greatly shortening the sample detection time and meeting the requirements of rapid differential diagnosis of the cardiac diseases of the patients in the department of cardiology. Therefore, the POCT detection of the amino-terminal brain natriuretic peptide precursor has important clinical value.
CN102103143 discloses a colloidal gold technology in POCT detection, in which a test strip is simultaneously provided with a test line coated with an N-terminal brain natriuretic peptide precursor polyclonal antibody, a test line coated with a cardiac troponin I monoclonal antibody, and a control line of a rabbit anti-mouse IgG antibody, so that two indexes can be synchronously detected.
CN107389950 discloses a detection kit prepared by using a magnetic separation principle and an acridinium ester chemiluminescence method, wherein a magnetic bead-SA-biotin-NT-proBNP monoclonal antibody solvent is combined with NT-proBNP, and then is subjected to a combination reaction with the acridinium ester-NT-proBNP monoclonal antibody solvent.
CN104865387 discloses an immunofluorescence test paper strip for rapid quantitative detection of NT-proBNP, wherein a conjugate pad is arranged between a sample pad and a detection membrane in the test paper strip, a fluorescent-labeled NT-proBNP antibody conjugate is coated on the conjugate pad, NT-proBNP in a sample and the NT-proBNP antibody conjugate on the conjugate pad are subjected to immunoreaction during detection to form an immune complex, and the immune complex reacts with the NT-proBNP antibody coated on the immune complex to be fixed on a detection line when the immune complex is chromatographed to the detection line. The method is easy to cause the non-uniform activity of the fluorescent coupling compound coated on the unit area of the coupling compound gasket, so that the reagent variation coefficient CV is higher.
Therefore, in the field of POCT detection of NT-proBNP, an amino-terminal brain natriuretic peptide precursor detection kit with high detection accuracy, strong specificity, good reproducibility and quick operation still needs to be developed.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides an amino-terminal brain natriuretic peptide precursor detection kit, a preparation method and a detection method, wherein the detection kit has the advantages of high detection accuracy, strong specificity, good reproducibility and quick operation.
In order to realize the purpose, the invention provides an amino-terminal brain natriuretic peptide precursor detection kit, which adopts the following technical scheme:
an amino-terminal pro-brain natriuretic peptide detection kit, comprising: reagent card, reaction substance and reaction buffer solution.
The reagent card comprises a shell and a test strip, wherein the test strip comprises a sample pad, a nitrocellulose membrane, a water absorption pad and a backing plate, the nitrocellulose membrane is attached to the backing plate, one end of the sample pad and one end of the water absorption pad are respectively lapped at two ends of the nitrocellulose membrane, the nitrocellulose membrane is provided with a detection line and a quality control line which are parallel to each other, and the quality control line is positioned at one end of the nitrocellulose membrane close to the water absorption pad, the detection line is coated with an amino-terminal brain natriuretic peptide precursor monoclonal antibody, the quality control line is coated with one of a goat anti-chicken IgY antibody, a goat anti-rabbit IgG antibody or a goat anti-mouse IgG antibody, the casing includes upper cover and the lower cover of lock each other, the upper cover corresponds nitrocellulose membrane's position is equipped with the display window, corresponds the position of sample pad is equipped with the application of sample hole.
The reaction substance comprises a detection line fluorescent conjugate and a quality control line fluorescent conjugate; the detection line fluorescent conjugate contains an amino-terminal brain natriuretic peptide precursor monoclonal antibody marked by fluorescent microspheres, and the quality control line fluorescent conjugate contains one of chicken IgY antibody, rabbit IgG antibody or mouse IgG antibody marked by fluorescent microspheres.
The reaction buffer comprises buffer solution, surfactant, saccharide, preservative and sodium chloride.
The inventor discovers through a large number of research and experimental investigation that a traditional fluorescence immunoassay method enables a sample to be detected and an amino-terminal brain natriuretic peptide precursor monoclonal antibody marked on a fluorescent conjugate pad to generate specific immunological combination to form a fluorescence compound, the fluorescence compound passes through a test band, and an antigen in the fluorescence compound and the amino-terminal brain natriuretic peptide precursor monoclonal antibody coated in the test band generate specific immunological combination to be fixed in the test band for fluorescence intensity detection. According to the traditional fluorescence immunoassay method, the fluorescence conjugate is coated on the combination pad, and in the process from spraying to drying, the fluorescence conjugate cannot be rapidly fixed and kept on the glass fiber due to the fact that wet materials are not directionally diffused, so that the activity of the fluorescence conjugate contained in the unit area of the combination pad is uneven, the content accuracy of the amino terminal brain natriuretic peptide precursor in a sample to be detected is low, and the precision is low.
On the basis of the research findings, the invention improves the traditional technology, one of the amino-terminal brain natriuretic peptide precursor monoclonal antibody containing the fluorescent microsphere mark and chicken IgY antibody, rabbit IgG antibody or mouse IgG antibody marked by the fluorescent microsphere is freeze-dried to obtain a reaction substance, a reaction buffer solution is adopted for redissolving before use, and a sample to be detected is added, so that the amino-terminal brain natriuretic peptide precursor in the sample to be detected and the amino-terminal brain natriuretic peptide precursor monoclonal antibody marked by the fluorescent microsphere are fully reacted, the antigen-antibody binding rate is improved, and the detection accuracy and precision are improved.
Furthermore, the fluorescent microsphere is a time-resolved fluorescent microsphere, rare earth ions are embedded in the time-resolved fluorescent microsphere, and at least one functional group of amino, carboxyl or hydroxyl is modified on the surface of the time-resolved fluorescent microsphere; the rare earth ions are selected from at least one of Sm samarium, Eu europium, Gd gadolinium, Tb terbium and Dy dysprosium in lanthanide elements.
Further, the reaction substance is prepared by freeze-drying a detection line fluorescent conjugate and a quality control line fluorescent conjugate under the protection of a freeze-drying protection solution.
Further, the freeze-drying protective solution contains buffer solution, protective protein and saccharide; the buffer solution is at least one of phosphate, borate or Tris buffer solution, and the concentration is 10mM-50 mM; the protective protein is at least one of bovine serum albumin, ovalbumin or casein, and the concentration is 0.1-3%; the saccharide is at least one of trehalose, sucrose and lactose, and the concentration is 0.1-10%.
Further, in the reaction buffer solution, the buffer solution is at least one selected from phosphate, borate or Tris buffer solution, and the concentration is 10mM-50 mM; the surfactant is at least one of Triton x-100, Tween 20, S16 and S9, and the concentration is 0.1% -1%; the saccharide is selected from one or more of trehalose, sucrose and lactose, and the concentration is 0.1-10%; the concentration of the sodium chloride is 0.8-2%; the preservative is selected from at least one of sorbic acid, benzoic acid or proclin300, and the concentration is 0.01-0.1%.
Further, the kit for determining the amino-terminal pro-brain natriuretic peptide further comprises an ID card, wherein the ID card stores the project name, the batch number and the standard curve data of the product.
The invention also provides a preparation method of the kit for determining the precursor of the amino-terminal brain natriuretic peptide, which adopts the following technical scheme:
the preparation method of the reagent card comprises the following steps:
1) preparation of detection lines and quality control lines: respectively preparing an amino-terminal brain natriuretic peptide precursor monoclonal antibody with the concentration of 0.5-3mg/mL and a goat anti-chicken IgY antibody, a goat anti-rabbit IgG antibody or a goat anti-mouse IgG antibody with the concentration of 0.5-2mg/mL, scribing to a detection line and a quality control line area on a nitrocellulose membrane by using a film spraying machine, wherein the coating amount is 0.5-1.0 mu L/cm, and drying;
2) preparation of sample pad: preparing a sealing solution, adding the sealing solution onto the glass fiber to completely wet the glass fiber, and drying;
3) assembling the reagent card: sticking the nitrocellulose membrane to a backing plate, respectively lapping one end of a sample pad and one end of a water absorption pad on two ends of the nitrocellulose membrane, cutting the nitrocellulose membrane into test strips with specific widths, putting the test strips into a shell to fix the test strips in position, covering the shell, and pressing the test strips.
The preparation method of the reaction substance comprises the following steps:
1) preparing a detection line fluorescent conjugate:
a) placing the fluorescent microspheres in a centrifuge tube, adding an activating solution, centrifuging, removing supernatant, adding the activating solution into the precipitate, and carrying out ultrasonic resuspension; cleaning again;
b) adding crosslinking agents EDC and Sulfo-NHS into the fluorescent microspheres, reacting for 10-30min, centrifuging, removing supernatant, adding a precipitate into a coupling buffer solution, and performing ultrasonic resuspension, wherein the final concentration of EDC is 0.04-0.2%, and the final concentration of Sulfo-NHS is 0.12-0.6%;
c) adding 30-100 μ g of amino terminal brain natriuretic peptide precursor monoclonal antibody into 0.1mL of fluorescent microsphere, performing room temperature rotation reaction, centrifuging, removing supernatant, adding a sealing solution into the precipitate, performing ultrasonic resuspension, and performing room temperature rotation reaction; centrifuging, removing supernatant, adding Washing Buffer into the precipitate, cleaning twice, removing supernatant, and ultrasonically resuspending the precipitate by using a redissolving solution to obtain a detection line fluorescent conjugate;
2) preparing a quality control line fluorescent conjugate:
a) placing the fluorescent microspheres in a centrifuge tube, adding an activating solution, centrifuging, removing supernatant, adding the activating solution into the precipitate, and carrying out ultrasonic resuspension; cleaning again;
b) adding crosslinking agents EDC and Sulfo-NHS into the fluorescent microspheres, reacting for 10-30min, centrifuging, removing supernatant, adding a precipitate into a coupling buffer solution, and performing ultrasonic resuspension, wherein the final concentration of EDC is 0.04-0.2%, and the final concentration of Sulfo-NHS is 0.12-0.6%;
c) adding 30-100 μ g of one of chicken IgY antibody, rabbit IgG antibody or mouse IgG antibody into 0.1mL of fluorescent microsphere, performing room temperature rotary reaction, centrifuging, removing supernatant, adding the precipitate into confining liquid, performing ultrasonic resuspension, and performing room temperature rotary reaction; centrifuging, removing supernatant, adding Washing Buffer into the precipitate, cleaning twice, removing supernatant, and ultrasonically resuspending the precipitate with a redissolving solution to obtain the quality control line fluorescent conjugate;
3) preparing liquid: diluting the detection line fluorescent conjugate by 100-1000 times by using a freeze-drying protective solution, and diluting the quality control line fluorescent conjugate by 500-2000 times to prepare a reaction substance solution;
4) subpackaging: subpackaging the reaction substance solution in plastic test tubes, placing the subpackaged plastic test tubes on a freeze-drying tray, freeze-drying the plastic test tubes into powder, sealing and capping the reaction substance after freeze-drying, and storing at the temperature of 2-8 ℃.
The preparation method of the reaction buffer comprises the following steps:
1) preparing a reaction buffer solution: adding tween 20 with the final concentration of 0.1-1 percent, trehalose with the final concentration of 0.1-10 percent, NaCl with the final concentration of 0.8-2 percent and proclin300 with the final concentration of 0.01-0.1 percent into Tris buffer solution with the concentration of 10-50 mM;
2) subpackaging: and subpackaging the reaction buffer solution into plastic test tubes, covering and sealing the plastic test tubes, and storing the plastic test tubes at the temperature of between 2 and 8 ℃.
The invention also provides a fluorescence immunoassay quantitative detection method for detecting the amino-terminal brain natriuretic peptide precursor, which is characterized in that the kit is adopted and comprises the following steps:
step A: placing the reaction substance and the reaction buffer solution which are stored in a refrigerating way at room temperature to ensure that the reaction substance and the reaction buffer solution are fully balanced;
and B: sucking reaction buffer solution and putting the reaction buffer solution into a reaction substance test tube, and immediately sucking whole blood, serum or plasma and adding the whole blood, serum or plasma into the reaction substance test tube filled with the reaction buffer solution to obtain sample mixed solution;
and C: covering a reaction substance test tube cover, and shaking to fully and uniformly mix the sample mixed solution;
step D: absorbing the sample mixed liquid, adding the sample mixed liquid into a sample adding hole of a reagent card, and reacting;
step E: and inserting the reacted reagent card into a fluorescence immunoassay analyzer, and reading the result.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention subpackages the fluorescent conjugate, namely the fluorescent microsphere marked amino-terminal brain natriuretic peptide precursor monoclonal antibody and the quality control line fluorescent conjugate into independent centrifugal tubes, and then prepares freeze-dried powder in a freeze-drying mode for packaging, thereby maximally preserving the biological activity of the fluorescent microsphere marked antibody, having good uniformity and reducing the difference among batches.
(2) The fluorescence coupling is sprayed on the bonding pad through a film spraying machine and then dried, and in the process from spraying to drying, the wet material is not directionally diffused and can not be rapidly fixed and kept on the glass fiber, so that the activity of the fluorescence coupling compound contained in the unit area of the bonding pad is not uniform, and the fluorescence coupling is an important source for causing higher coefficient of variation CV of the immunochromatography reagent. By adopting the process, the fluorescent conjugate is subpackaged into the independent centrifugal tubes, and then is prepared into the freeze-dried powder in a freeze-drying mode, and the freeze-dried powder is packaged, so that the fluorescent conjugate has the advantages of good uniformity, small environmental influence and easiness in stable storage.
(3) The fluorescent conjugate, namely the fluorescent microsphere marked amino-terminal brain natriuretic peptide precursor monoclonal antibody and the quality control line fluorescent conjugate are subpackaged in independent centrifugal tubes, freeze-dried powder is prepared in a freeze-drying mode and is packaged, and after reaction buffer solution is added, the fluorescent conjugate can be quickly dissolved and can be completely released, so that the biological activity of the fluorescent microsphere marked amino-terminal brain natriuretic peptide precursor monoclonal antibody is greatly maintained, the fluorescent microsphere marked amino-terminal brain natriuretic peptide precursor monoclonal antibody can be fully combined with the amino-terminal brain natriuretic peptide precursor in a sample, and the detection sensitivity and accuracy are improved.
(4) And adding a sample to be detected into the centrifugal tube, performing immunoreaction with a reaction substance, adding the mixed solution into the reagent card sample adding hole, and performing immunoreaction with antibodies on the detection line and the quality control line to form an immune complex. The fluorescent conjugate is fully released and has immunoreaction with the terminal brain natriuretic peptide precursor in the sample, the immunoreaction is more full and complete, and the sensitivity and the repeatability of the detection are obviously improved.
Drawings
In order to more clearly illustrate the embodiments of the present application or technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention, and other drawings can be obtained by those skilled in the art according to the drawings.
FIG. 1 is a schematic diagram of the components of an amino-terminal pro-brain natriuretic peptide detection kit according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of the internal structure of an immunochromatographic reagent card according to an embodiment of the present invention;
FIG. 3 is a standard curve of an amino-terminal brain natriuretic peptide precursor according to an embodiment of the present invention;
FIG. 4 is a graph of the linear range of amino-terminal brain natriuretic peptide precursors in accordance with one embodiment of the present invention;
FIG. 5 is a diagram showing the correlation between the kit and the chemiluminescence kit according to one embodiment of the invention.
Wherein: 1. a reagent card; 2. a reactive material; 3. a display window; 4. a sample application hole; 5. detecting lines; 6. a quality control line; 7. a sample pad; 8. a water absorbent pad; 9. a nitrocellulose membrane; 10. a backing plate.
Detailed Description
In order to make the technical solutions of the present invention better understood, those skilled in the art will now describe the present invention in further detail with reference to the accompanying drawings. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
An amino-terminal brain natriuretic peptide precursor assay kit of an embodiment, comprising: the reagent card comprises a shell and a test strip, wherein the test strip comprises a sample pad, a nitrocellulose membrane, a water absorption pad and a backing plate, the nitrocellulose membrane is attached to the backing plate, one end of the sample pad and one end of the water absorption pad are respectively lapped at two ends of the nitrocellulose membrane, the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line and the quality control line are parallel to each other, the quality control line is positioned at one end, close to the water absorption pad, of the nitrocellulose membrane, the detection line is coated with an amino terminal brain natriuretic peptide precursor monoclonal antibody, the quality control line is coated with one of goat anti-chicken IgY antibody, goat anti-rabbit IgG antibody or goat anti-mouse IgG antibody, the shell comprises an upper cover and a lower cover which can be mutually buckled, a display window is arranged at the position, corresponding to the nitrocellulose membrane, and a sample adding hole is arranged at the position corresponding to the sample pad.
Wherein, the reaction substance comprises a detection line fluorescent conjugate and a quality control line fluorescent conjugate; the detection line fluorescent conjugate contains an amino terminal brain natriuretic peptide precursor monoclonal antibody marked by fluorescent microspheres, and the quality control line fluorescent conjugate contains one of chicken IgY antibody, rabbit IgG antibody or mouse IgG antibody marked by fluorescent microspheres.
Wherein the reaction buffer solution is a buffer solution containing a surfactant, saccharides, a preservative and sodium chloride.
The amino-terminal brain natriuretic peptide precursor detection kit can be used for rapidly detecting acute heart failure, myocardial infarction and cardiogenic dyspnea based on the amino-terminal brain natriuretic peptide precursor as a detection marker. During detection, adding a reaction buffer solution into a reaction substance, immediately sucking 50 mu L of whole blood, serum or plasma into a reaction substance test tube filled with the reaction buffer solution, fully and uniformly mixing, fully combining an amino-terminal brain natriuretic peptide precursor in a sample to be detected with an amino-terminal brain natriuretic peptide precursor monoclonal antibody marked by fluorescent microspheres in the reaction substance to form a compound, adding the compound onto a sample adding hole of a reagent card, combining the compound with the amino-terminal brain natriuretic peptide precursor monoclonal antibody coated on a detection line through chromatography to form an antibody-antigen-antibody sandwich compound, and inserting the reagent card into a fluorescence immunoassay analyzer to detect the content of the amino-terminal brain natriuretic peptide precursor in the sample to be detected. The detection method is convenient and rapid, full in reaction and simple in operation.
Specifically, the fluorescent microsphere is a time-resolved fluorescent microsphere, the time-resolved fluorescent microsphere is embedded with rare earth ions, and meanwhile, one functional group of amino, carboxyl or hydroxyl is modified on the surface of the time-resolved fluorescent microsphere; the rare earth ions are one or more of Sm samarium, Eu europium, Gd gadolinium, Tb terbium and Dy dysprosium in lanthanide elements.
An antibody, also called an immunoglobulin (Ig), is a glycoprotein that specifically binds to an antigen. Antibodies exist as one or more Y-shaped monomers, each of which consists of 4 polypeptide chains, comprising two identical heavy chains and two identical light chains. In general, the active groups that can be used for conjugation in antibody protein molecules are free amino groups (e.g., lysine or terminal amino groups), free carboxyl groups (e.g., aspartic acid residues, glutamic acid residues, and terminal carboxyl groups), thiol groups (e.g., cysteine), hydroxyl groups (e.g., serine or threonine), and the like.
In one embodiment, the time-resolved fluorescent microsphere is different from a common fluorescence immunoassay, and the tracer is a rare earth element instead of fluorescein; preferably, the rare earth element is trivalent lanthanide Eu europium; each microsphere can be wrapped with thousands of fluorescent molecules, so that the marking efficiency of fluorescence is greatly improved, and the analysis sensitivity is effectively improved; meanwhile, the surface of the fluorescent microsphere is modified with amino, carboxyl or hydroxyl and other functional groups with proper density for covalent coupling with the amino-terminal brain natriuretic peptide precursor monoclonal antibody, so that the stability of the marker is improved.
The antibody coupling method generally used includes the glutaraldehyde method, the carbodiimide method (EDC), and the like. EDC [ 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide ] is generally believed to have a mechanism in which the carboxyl group of a protein residue is reacted with EDC first to form O-acylisourea (O-acylisourea), which is a reactive intermediate, and EDC is reacted with a substance having a carboxyl group to form an active intermediate, which is extremely unstable, whose active ester is easily hydrolyzed, and whose reaction rate with an amino group is slow. NHS is usually added for use together to improve the solubility and stability of the reactive intermediate. The invention adopts sulfo-NHS which has higher linking reaction efficiency than NHS and has negative charge, thus being not easy to cause the polymerization of linking substrates (such as fluorescent microspheres).
In one embodiment, the reaction substance is prepared by freeze-drying a detection line fluorescent conjugate and a quality control line fluorescent conjugate under the protection of a freeze-drying protective solution, namely, an amino-terminal brain natriuretic peptide precursor monoclonal antibody marked by fluorescent microspheres and chicken IgY antibody, rabbit IgG antibody or mouse IgG antibody marked by the fluorescent microspheres are diluted by the freeze-drying protective solution to prepare a reaction substance solution, and then the reaction substance solution is subpackaged in plastic test tubes for freeze-drying. The freeze drying process can greatly maintain the biological activity of the antibody marked by the fluorescent microspheres.
In one embodiment, the freeze-drying protective solution contains a buffer solution of protective protein and saccharide; the buffer solution is one or more of phosphate, borate or Tris buffer solution, the concentration is 10mM-50mM, the buffer solution provides the pH of a stable solution in freeze drying, and the physical aggregation or chemical denaturation of the protein caused by large pH change is prevented; the protective protein is one or more of bovine serum albumin, ovalbumin or casein, the concentration is 0.1% -3%, the protective protein is a macromolecular substance, and provides a stable supporting framework for active components in freeze drying, so that low-temperature protection and dehydration protection are realized; the saccharide is one or more of trehalose, sucrose and lactose, the concentration is 0.1% -10%, and the saccharide can prevent active components from denaturation and has the functions of low-temperature protection and dehydration protection.
In one embodiment, the buffer in the reaction buffer is one or more of phosphate, borate or Tris buffer, the concentration is 10mM-50mM, and the buffer provides the pH of a stable solution after being redissolved so as to prevent the fluorescent microspheres from polymerizing; the surfactant is one or more of Triton x-100, Tween 20, S16 and S9, the concentration is 0.1% -1%, and the surfactant is quickly dissolved, so that the active components are quickly dispersed; the saccharide is one or more of trehalose, sucrose and lactose, the concentration is 0.1-10%, and the saccharide has a protection effect and improves the viscosity of the solution; the concentration of the NaCl is 0.8-2%, and the NaCl improves the ionic strength in the solution and is beneficial to the immune reaction of antigen and antibody; the preservative is one or more of sorbic acid, benzoic acid or proclin300, the concentration of the preservative is 0.01-0.1%, and the preservative can inhibit the growth of bacteria in a solution.
In one embodiment, the kit further comprises an ID card storing the item name, batch number and standard curve data of the product. When the dry-type fluorescence immunoassay instrument is used with a matched dry-type fluorescence immunoassay instrument, the ID card is inserted, and then the relevant information is read.
Example 1
Preparation of reagent cards
(1) Preparation of detection lines and quality control lines: respectively preparing an amino terminal brain natriuretic peptide precursor monoclonal antibody with the concentration of 2.0mg/mL and a goat anti-chicken IgY antibody with the concentration of 1mg/mL, marking to a detection line and a quality control line area on a nitrocellulose membrane by using a film spraying machine, coating the coating amount of 1.0 mu L/cm, placing the marked nitrocellulose membrane in a blast drying oven, and drying for 18 hours at 50 ℃;
(2) preparation of sample pad: preparing a sealing solution, adding the sealing solution onto the glass fiber to completely wet the glass fiber, placing the glass fiber in a blast drying oven, and drying the glass fiber for 18 hours at 50 ℃;
(3) assembling the reagent card: sticking the nitrocellulose membrane to a backing plate, respectively lapping one end of a sample pad and one end of a water absorption pad on two ends of the nitrocellulose membrane, cutting the nitrocellulose membrane into test strips with specific widths, putting the test strips into a shell to be fixed at a position, covering a cover, pressing the test strips, sealing the test strips into an aluminum foil bag, adding a silica gel drying agent, and sealing.
Preparation of fluorescent conjugate for detection line (amino terminal brain natriuretic peptide precursor monoclonal antibody)
a) Placing 0.1mLEu europium time-resolved fluorescent microsphere-carboxyl group in a centrifuge tube, adding an activating solution, centrifuging at the rotation speed of 13000rpm for 20min, removing supernatant, adding the activating solution into precipitate, and carrying out ultrasonic resuspension; cleaning again;
b) adding crosslinking agents EDC and Sulfo-NHS according to 0.1mLEu europium time resolution fluorescent microsphere-carboxyl groups, wherein the final concentration of EDC is 0.12%, the final concentration of Sulfo-NHS is 0.36%, reacting for 30min, centrifuging for 20min at the rotation speed of 13000rpm, removing supernatant, adding a coupling buffer solution into a precipitate, and carrying out ultrasonic resuspension;
c) adding 80 mu g of amino-terminal brain natriuretic peptide precursor monoclonal antibody into the europium time-resolved fluorescent microsphere-carboxyl group of 0.1mLEu, carrying out rotary reaction for 120min at room temperature, then centrifuging for 20min at the rotation speed of 13000rpm, removing supernatant, adding a sealing solution into a precipitate, carrying out ultrasonic resuspension, and carrying out rotary reaction for 100min at room temperature; centrifuging at 13000rpm for 20min, removing supernatant, adding Washing Buffer into the precipitate, cleaning twice, removing supernatant, and ultrasonically resuspending the precipitate with a redissolution; obtaining a detection line fluorescent conjugate;
preparation of quality control line (chicken IgY antibody) fluorescent conjugate
a) Placing 0.1mLEu europium time-resolved fluorescent microsphere-carboxyl group in a centrifuge tube, adding an activating solution, centrifuging at the rotation speed of 13000rpm for 20min, removing supernatant, adding the activating solution into precipitate, and carrying out ultrasonic resuspension; cleaning again;
b) adding crosslinking agents EDC and Sulfo-NHS according to 0.1mLEu europium time resolution fluorescent microsphere-carboxyl groups, wherein the final concentration of EDC is 0.12%, the final concentration of Sulfo-NHS is 0.36%, reacting for 30min, centrifuging for 20min at the rotation speed of 13000rpm, removing supernatant, adding a coupling buffer solution into a precipitate, and carrying out ultrasonic resuspension;
c) adding 100ug of chicken IgY antibody into 0.1mLEu europium time-resolved fluorescent microsphere-carboxyl group, rotating for reaction at room temperature for 120min, then centrifuging at the revolution of 13000rpm for 20min, removing supernatant, adding sealing liquid into precipitate, carrying out ultrasonic resuspension at the concentration of 0.5%, and rotating for reaction at room temperature for 100 min; centrifuging at 13000rpm for 20min, removing supernatant, adding Washing Buffer into the precipitate, cleaning twice, removing supernatant, and ultrasonically resuspending the precipitate with a redissolution; obtaining a quality control line fluorescent conjugate;
preparation of the reaction Mass
(1) Preparing a freeze-drying protective solution: adding 1% casein into 50mM Tris buffer solution, adding 3% trehalose, adding 3% sucrose, fully dissolving and mixing uniformly.
(2) Preparing liquid: diluting the detection line fluorescent conjugate by 300 times and diluting the quality control line fluorescent conjugate by 1000 times by using a freeze-drying protective solution to prepare a reaction substance solution;
(3) subpackaging: 0.1mL of the reaction substance solution is subpackaged into plastic test tubes, placed on a freeze-drying tray, freeze-dried into powder, and immediately sealed and capped after freeze-drying, and stored at 2-8 ℃ in a dark place.
Preparation of reaction buffer
(1) Preparation of reaction buffer: adding 0.5% Tween 20 into 50mM Tris buffer solution, adding 2% trehalose, adding 0.9% NaCL, adding 0.05% proclin300, fully dissolving and mixing uniformly;
(2) subpackaging: and subpackaging the reaction buffer solution into test tubes, covering and sealing the test tubes, and storing the test tubes at the temperature of between 2 and 8 ℃.
Kit assembly and assembly of reaction substances and reaction buffers
Assembly of reaction mass and reaction buffer: packing 30 reaction substances and a package of drying agent into an aluminum foil bag, packing the aluminum foil bag and a piece of reaction buffer solution, and storing at 2-8 ℃.
And (3) final assembly of the kit: and packaging a plurality of reagent cards and one ID card.
The fluorescence immunoassay quantitative detection method for detecting the amino-terminal brain natriuretic peptide precursor comprises the following steps:
step A: placing the reaction substance and the reaction buffer solution which are stored in a refrigerating way at room temperature for 30 minutes to ensure that the reaction substance and the reaction buffer solution are fully balanced at the room temperature;
and B: sucking 100 mu L of reaction buffer solution, putting the reaction buffer solution into a reaction substance test tube, and immediately sucking 50 mu L of whole blood or serum or plasma, and adding the whole blood or serum or plasma into the reaction substance test tube filled with the reaction buffer solution to obtain a sample mixed solution;
and C: covering a reaction substance test tube cover, and shaking to fully and uniformly mix the sample mixed solution;
step D: sucking 80 mu L of sample mixed liquid, adding the sample mixed liquid into a sample adding hole of the reagent card, and reacting for 20 minutes;
step E: and inserting the reacted reagent card into a fluorescence immunoassay analyzer, and reading the result.
Example 2
Preparation of reagent cards
(1) Preparation of detection lines and quality control lines: respectively preparing 3.0mg/mL of amino-terminal brain natriuretic peptide precursor monoclonal antibody and 2mg/mL of goat anti-rabbit IgG antibody, marking to a detection line and a quality control line area on a nitrocellulose membrane by using a film spraying machine, wherein the coating amount is 0.8 muL/cm, placing the marked nitrocellulose membrane in a blast drying oven, and drying for 20 hours at 45 ℃;
(2) preparation of sample pad: preparing a sealing solution, adding the sealing solution onto the glass fiber to completely wet the glass fiber, placing the glass fiber in a blast drying oven, and drying the glass fiber for 20 hours at the temperature of 45 ℃;
(3) assembling the reagent card: sticking the nitrocellulose membrane to a backing plate, respectively lapping one end of a sample pad and one end of a water absorption pad on two ends of the nitrocellulose membrane, cutting the nitrocellulose membrane into test strips with specific widths, putting the test strips into a shell to be fixed at a position, covering a cover, pressing the test strips, sealing the test strips into an aluminum foil bag, adding a silica gel drying agent, and sealing.
Preparation of fluorescent conjugate for detection line (amino terminal brain natriuretic peptide precursor monoclonal antibody)
a) Placing 0.1ml of dysprosium time-resolved fluorescent microspheres into a centrifuge tube, adding an activating solution, centrifuging for 15min at the rotation speed of 10000rpm, removing supernatant, adding the activating solution into precipitates, and carrying out ultrasonic resuspension; cleaning again;
b) adding crosslinking agents EDC and Sulfo-NHS into the fluorescent microspheres according to 0.1ml of dysprosium time resolution, reacting for 30min, centrifuging for 15min at the rotation speed of 10000rpm, removing supernatant, adding the precipitate into coupling buffer solution, and carrying out ultrasonic resuspension;
c) adding 100 mu g of amino-terminal brain natriuretic peptide precursor monoclonal antibody into 0.1ml of dysprosium time-resolved fluorescent microspheres, carrying out rotary reaction at room temperature for 120min, then centrifuging at the revolution of 10000rpm for 15min, removing supernatant, adding a sealing solution into the precipitate, carrying out ultrasonic resuspension, and carrying out rotary reaction at room temperature for 100min, wherein the concentration of the sealing solution is 0.1%; centrifuging at 10000rpm for 15min, removing supernatant, adding Washing Buffer into the precipitate, cleaning twice, removing supernatant, and ultrasonically resuspending the precipitate with a redissolution; obtaining a detection line fluorescent conjugate;
preparation of quality control line (rabbit IgG antibody) fluorescent conjugate
a) Placing 0.1mL Dy dysprosium time-resolved fluorescent microsphere into a centrifuge tube, adding an activating solution, centrifuging for 15min at the rotation speed of 10000rpm, removing supernatant, adding the activating solution into a precipitate, and carrying out ultrasonic resuspension; cleaning again;
b) adding crosslinking agents EDC and Sulfo-NHS into the fluorescent microspheres according to 0.1ml of dysprosium time resolution, reacting for 30min, centrifuging for 15min at the rotation speed of 10000rpm, removing supernatant, adding the precipitate into coupling buffer solution, and carrying out ultrasonic resuspension;
c) adding 100 mu g of rabbit IgG antibody into 0.1mL of Dy dysprosium time-resolved fluorescent microspheres, carrying out room-temperature rotation reaction for 120min, then centrifuging for 15min at the rotation speed of 10000rpm, removing supernatant, adding a sealing solution into the precipitate, carrying out ultrasonic resuspension, and carrying out room-temperature rotation reaction for 100 min; centrifuging at 10000rpm for 15min, removing supernatant, adding Washing Buffer into the precipitate, cleaning twice, removing supernatant, and ultrasonically resuspending the precipitate with a redissolution; obtaining a quality control line fluorescent conjugate;
preparation of the reaction Mass
(1) Preparing a freeze-drying protective solution: 3% bovine serum albumin was added to 30mM phosphate buffer, 10% sucrose was added, and the mixture was dissolved thoroughly and mixed.
(2) Preparing liquid: diluting the detection line fluorescent conjugate by 1000 times and diluting the quality control line fluorescent conjugate by 2000 times by using a freeze-drying protective solution to prepare a reaction substance solution;
(3) subpackaging: 0.1mL of the reaction substance solution is subpackaged into plastic test tubes, the subpackaged plastic test tubes are placed on a freeze-drying tray, freeze-dried into powder, the reaction substance is sealed and pressed immediately after freeze-drying, and the reaction substance is stored at the temperature of 2-8 ℃ in a dark place.
Preparation of reaction buffer
(1) Preparation of reaction buffer: adding 1% triton x-100 into 30mM phosphate buffer solution, adding 0.5% lactose, adding 2% trehalose, adding 2% NaCl, adding 0.1% proclin300, dissolving completely, and mixing;
(2) subpackaging: and subpackaging the reaction buffer solution into plastic test tubes, covering and sealing the plastic test tubes, and storing the plastic test tubes at the temperature of between 2 and 8 ℃.
The fluorescence immunoassay quantitative detection method for detecting the amino-terminal brain natriuretic peptide precursor comprises the following steps:
step A: placing the reaction substance and the reaction buffer solution which are stored in a refrigerating way at room temperature for 30 minutes to ensure that the reaction substance and the reaction buffer solution are fully balanced at the room temperature;
and B: sucking 100 mu L of reaction buffer solution, putting the reaction buffer solution into a reaction substance test tube, and immediately sucking 50 mu L of whole blood or serum or plasma, and adding the whole blood or serum or plasma into the reaction substance test tube filled with the reaction buffer solution to obtain a sample mixed solution;
and C: covering a reaction substance test tube cover, and shaking to fully and uniformly mix the sample mixed solution;
step D: sucking 80 mu L of sample mixed liquid, adding the sample mixed liquid into a sample adding hole of the reagent card, and reacting for 15 minutes;
step E: and inserting the reacted reagent card into a fluorescence immunoassay analyzer, and reading the result.
Example 3
Preparation of reagent cards
(1) Preparation of detection lines and quality control lines: respectively preparing an amino-terminal brain natriuretic peptide precursor monoclonal antibody with the concentration of 0.5mg/mL and a goat anti-mouse IgG antibody with the concentration of 0.5mg/mL, marking to a detection line and a quality control line area on a nitrocellulose membrane by using a film spraying machine, coating the coating amount of 0.5 mu L/cm, placing the marked nitrocellulose membrane in a blast drying oven, and drying for 24 hours at 37 ℃;
(2) preparation of sample pad: preparing a sealing solution, adding the sealing solution onto the glass fiber to completely wet the glass fiber, placing the glass fiber in a blast drying oven, and drying the glass fiber for 24 hours at 37 ℃;
(3) assembling the reagent card: sticking the nitrocellulose membrane to a backing plate, respectively lapping one end of a sample pad and one end of a water absorption pad on two ends of the nitrocellulose membrane, cutting the nitrocellulose membrane into test strips with specific widths, putting the test strips into a shell to be fixed at a position, covering a cover, pressing the test strips, sealing the test strips into an aluminum foil bag, adding a silica gel drying agent, and sealing.
Preparation of fluorescent conjugate for detection line (amino terminal brain natriuretic peptide precursor monoclonal antibody)
a) Placing 0.1mL of Sm samarium time-resolved fluorescent microspheres into a centrifuge tube, adding an activating solution, centrifuging for 10min at the rotation speed of 13000rpm, removing supernatant, adding the activating solution into precipitates, and carrying out ultrasonic resuspension; cleaning again;
b) adding cross-linking agents EDC and Sulfo-NHS into 0.1mL of Sm samarium time-resolved fluorescent microspheres, wherein the final concentration of EDC is 0.04%, and the final concentration of Sulfo-NHS is 0.12%, reacting for 10min, centrifuging for 10min at the rotation speed of 13000rpm, removing supernatant, adding a coupling buffer solution into precipitates, and carrying out ultrasonic resuspension;
c) adding 30 mu g of amino terminal brain natriuretic peptide precursor monoclonal antibody into 0.1mL of Sm samarium time-resolved fluorescent microspheres, carrying out rotary reaction for 120min at room temperature, then centrifuging for 10min at the rotation speed of 13000rpm, removing supernatant, adding confining liquid into precipitates, carrying out ultrasonic resuspension, and carrying out rotary reaction for 100min at room temperature, wherein the concentration of the confining liquid is 0.3%; centrifuging at 13000rpm for 10min, removing supernatant, adding Washing Buffer into the precipitate, cleaning twice, removing supernatant, and ultrasonically resuspending the precipitate with a redissolution; obtaining a detection line fluorescent conjugate;
preparation of quality control line (mouse IgG antibody) fluorescent conjugate
a) Placing 0.1mL of Sm samarium time-resolved fluorescent microspheres into a centrifuge tube, adding an activating solution, centrifuging for 20min at the rotation speed of 13000rpm, removing supernatant, adding the activating solution into precipitates, and carrying out ultrasonic resuspension; cleaning again;
b) adding cross-linking agents EDC and Sulfo-NHS into 0.1mL of Sm samarium time-resolved fluorescent microspheres, wherein the final concentration of EDC is 0.04%, and the final concentration of Sulfo-NHS is 0.12%, reacting for 10min, centrifuging for 10min at the rotation speed of 13000rpm, removing supernatant, adding a coupling buffer solution into precipitates, and carrying out ultrasonic resuspension;
c) adding 30 mu g of mouse IgG antibody into 0.1mL of Sm samarium time-resolved fluorescent microspheres, carrying out room-temperature rotary reaction for 120min, then centrifuging for 10min at the speed of 13000rpm, removing supernatant, adding a sealing solution into a precipitate, carrying out ultrasonic resuspension, and carrying out room-temperature rotary reaction for 100 min; centrifuging at 13000rpm for 10min, removing supernatant, adding Washing Buffer into the precipitate, cleaning twice, removing supernatant, and ultrasonically resuspending the precipitate with a redissolution; obtaining a quality control line fluorescent conjugate;
preparation of the reaction Mass
(1) Preparing a freeze-drying protective solution: 0.1% ovalbumin was added to 10mM borate buffer, 0.1% sucrose was added, and the mixture was thoroughly dissolved and mixed.
(2) Preparing liquid: diluting the detection line fluorescent conjugate by 100 times by using a freeze-drying protective solution, and diluting the quality control line fluorescent conjugate by 500 times to prepare a reaction substance solution;
(3) subpackaging: 0.1mL of the reaction substance solution is subpackaged into plastic test tubes, placed on a freeze-drying tray, freeze-dried into powder, and immediately sealed and capped after freeze-drying, and stored at 2-8 ℃ in a dark place.
Preparation of reaction buffer
(1) Preparation of reaction buffer: adding 0.1% Tween S9 into 10mM borate buffer solution, adding 0.1% sucrose, adding 0.8% NaCL, adding 0.01% proclin300, fully dissolving and mixing uniformly;
(2) subpackaging: and subpackaging the reaction buffer solution into plastic test tubes, covering and sealing the plastic test tubes, and storing the plastic test tubes at the temperature of 2-8 ℃.
The fluorescence immunoassay quantitative detection method for detecting the amino-terminal brain natriuretic peptide precursor comprises the following steps:
step A: placing the reaction substance and the reaction buffer solution which are stored in a refrigerating way at room temperature for 30 minutes to ensure that the reaction substance and the reaction buffer solution are fully balanced at the room temperature;
and B: sucking 100 mu L of reaction buffer solution, putting the reaction buffer solution into a reaction substance test tube, and immediately sucking 50 mu L of whole blood or serum or plasma, and adding the whole blood or serum or plasma into the reaction substance test tube filled with the reaction buffer solution to obtain a sample mixed solution;
and C: covering a reaction substance test tube cover, and shaking to fully and uniformly mix the sample mixed solution;
step D: sucking 80 mu L of sample mixed solution, adding the sample mixed solution into a sample hole of a reaction plate, and reacting for 8 minutes;
step E: and inserting the reacted reagent card into a fluorescence immunoassay analyzer, and reading the result.
Test example 1 Standard Curve preparation
Samples with concentrations of 0, 30, 125, 1250, 5000, 15000, and 30000 (unit: pg/mL) were prepared, the kit and the detection method of example 1 were used to perform detection, samples with each concentration were repeated 3 times, the average value was calculated, the fluorescence signal was read in the fluorescence immunoassay analyzer, and a fitted curve was drawn with the concentration as ordinate and the fluorescence signal average as abscissa, to obtain a standard curve with good linearity, r being 0.9998, as shown in fig. 3.
Test example 2 repeatability test
Samples with concentrations of 125, 5000, and 15000 (pg/mL) were prepared, and the concentration was measured by the kit and the measurement method of example 2, 10 replicates were performed for each sample concentration, the concentration was read in a fluorescence immunoassay analyzer, and the average value (M) and Standard Deviation (SD) of 10 measurement results were calculated, and the coefficient of variation CV (CV ═ SD/mx 100%) was obtained according to the formula. The results are shown in Table 1.
TABLE 1
As shown in table 1, CV values obtained from samples of different concentrations were all 10% or less, and the amino-terminal pro-brain natriuretic peptide detection kit was excellent in reproducibility.
Test example 3 accuracy test
Samples with the concentration of 300 and 5000 (unit: pg/mL) are prepared, the kit and the detection method of the embodiment 3 are adopted for detection, the detection is carried out, 10 times of detection are carried out for each sample, 3 times of detection are carried out for each sample, and the relative deviation (B) is calculated according to a formula. The results are shown in Table 2.
Bi＝(Xi-T)/T×100％
In the formula: bi-relative deviation; xi-measured concentration; t-calibration concentration
TABLE 2
As shown in Table 2, the relative deviations are within. + -. 10%, so the accuracy of the kit for detecting the amino-terminal pro-brain natriuretic peptide is good.
Test example 4 interference test
Preparing hemoglobin mother liquor (120g/L), triglyceride mother liquor (240g/L) and bilirubin mother liquor (5 mg/mL). Fresh pooled clinical serum samples from healthy individuals were selected and appropriate amounts of the amino-terminal pro-brain natriuretic peptide calibrator were added to achieve concentrations of the amino-terminal pro-brain natriuretic peptide in the samples of approximately 125pg/mL and 1250pg/mL, respectively. Divided into 2 parts, one part is used as a control sample, and the other part is used as an experimental sample. Control sample: no interfering substances are added. Experimental samples: pure interfering substances were added to the control samples: hemoglobin mother liquor (120g/L), triglyceride mother liquor (240g/L), and bilirubin mother liquor (5 mg/mL).
The kit and the detection method in example 1 are adopted for detection, each sample is repeatedly detected for 3 times, and the average value is taken as the final detection result. The relative deviation of the experimental sample from the control sample was calculated. If the relative deviation is within. + -. 10%, it is considered that the detection result of the reagent is not interfered by the added interfering substance. The results are shown in Table 3.
The relative deviation B was calculated according to the following formula, with the test mean result of the experimental sample denoted as M and the test mean result of the control sample denoted as T.
In the formula: b-relative deviation; m is the mean value of the concentration of the experimental sample; t-mean concentration of control samples.
TABLE 3
The result shows that the hemoglobin is less than 5mg/mL, the triglyceride is less than 10mg/mL, the bilirubin is less than 0.2mg/mL, the relative deviation is within +/-10 percent, and the kit has good anti-interference effect.
Test example 5 specificity test
Preparation of ANP mother liquor (20ng/mL), CNP mother liquor (20ng/mL), angiotensin II mother liquor (12ng/mL) and L-norepinephrine mother liquor (14ng/mL) by using normal saline (0.9% NaCl solution). Fresh mixed clinical serum samples of healthy individuals are selected, and a suitable amount of high concentration NT-proBNP calibrator is added to achieve NT-proBNP concentrations in the samples of about 125pg/mL and 1250pg/mL, respectively. Divided into 2 parts, one part is used as a control sample, and the other part is used as an experimental sample. Control sample: no cross-reactive species were added. Experimental samples: cross-reactive substances of ANP, CNP, angiotensin ii, pure L-norepinephrine were added to the control samples.
The kit and the detection method in example 1 are adopted for detection, each sample is repeatedly detected for 3 times, and the average value is taken as the final detection result. The relative deviation of the experimental sample from the control sample was calculated. The relative deviation B was calculated according to the following formula, with the test mean result of the experimental sample denoted as M and the test mean result of the control sample denoted as T. The results are shown in Table 4.
In the formula: b-relative deviation; m is the mean value of the concentration of the experimental sample; t-mean concentration of control samples.
TABLE 4
The results show that the relative deviation of the experimental samples of ANP (1000pg/mL), CNP (1000pg/mL), angiotensin II (600pg/mL) and L-noradrenaline (700pg/mL) is within + -10%. The kit has good anti-interference effect.
Test example 6 stability test
Samples with concentrations of 0, 30, 125, 1250, 5000, 15000, and 30000 (unit: pg/mL) were prepared, the kit and the detection method of example 1 were used for detection, each sample was repeated 3 times, the mean value of the measured concentrations and the theoretical concentration or dilution ratio were fitted with a straight line by the least square method, and the correlation coefficient r of linear regression was calculated according to the formula. The results are shown in Table 5, and the correlation line graph is shown in FIG. 4.
Xi-theoretical concentration or dilution ratio; yi-the actual measured average.
| | Test | 1 | |
|
Mean value of |
| 30000 | 30173.7 | 30925.4 | 28151.4 | 29750.2 | |
| 15000 | 14828.3 | 14575.4 | 14974.4 | 14792.7 | |
| 5000 | 4998.3 | 5415.5 | 5277.9 | 5230.6 | |
| 1250 | 1291.6 | 1151 | 1204.8 | 1215.8 | |
| 125 | 126.1 | 112.2 | 129 | 122.4 | |
| 30 | 32.5 | 30.5 | 28 | 30.3 |
TABLE 5
As shown in table 5 and fig. 4, the correlation coefficient r was 0.999, and the stability of the kit of the present invention was good.
Test example 7 clinical sample correlation test
40 human serum samples were simultaneously tested and compared using the kit of example 1 and an imported kit (Ortho Clinical Diagnostics, kit for N-terminal brain natriuretic peptide assay (chemiluminescence method) national institutes of 20162404805). The data were analyzed for correlation, the results of which are shown in table 7:
TABLE 7
The linear regression equation was calculated using the measurement result (xi) of the imported kit as the independent variable and the measurement result (yi) of the kit of the present invention as the dependent variable, and the correlation linear graph is shown in FIG. 5. The correlation equation is: y is 0.9973x +69.086, and the correlation coefficient r is 0.9831. The statistical processing result shows that the kit has good correlation with the clinical sample measurement value of the imported kit, and meets the clinical test requirements.
Comparative example
Preparation of comparative example: diluting the fluorescent conjugate by 15 times and 50 times by using a freeze-drying protective solution, spraying the diluted fluorescent conjugate onto a bonding pad by using a film spraying machine, and placing the bonding pad in a blast drying oven for drying at 37 ℃ for 18-24 hours. Preparing an immunochromatography reagent card by a conventional method, sequentially sticking a sample pad, a bonding pad, a nitrocellulose membrane and a water absorption pad on a PVC (polyvinyl chloride) base plate, cutting a test strip, and loading the test strip into the card.
Comparative example a fluorescent conjugate was coated on a conjugate pad, the remaining structure and procedure were the same as in example 1, and the sample addition amount and interpretation time were the same as in example 1.
And (3) respectively adopting a comparative example kit and an example 1 kit to carry out repeatability test: samples with concentrations of 125, 5000, and 15000 (pg/mL) were prepared, tested, and the concentration was read in a fluoroimmunoassay analyzer 10 times, and the average value (M) and Standard Deviation (SD) of the 10 measurements were calculated to obtain the coefficient of variation CV (CV: SD/M × 100%) according to the formula. The results are shown in Table 8:
TABLE 8
The results show that the coefficient of variation CV of example 1 is below 10%, and the repeatability is better than that of the comparative example.
Those of ordinary skill in the art will understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to these examples; within the idea of the invention, also technical features in the above embodiments or in different embodiments may be combined and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity. Therefore, any omissions, modifications, substitutions, improvements and the like that may be made without departing from the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (10)
1. An amino-terminal pro-brain natriuretic peptide assay kit, comprising: the reagent card comprises a shell and a test strip, the test strip comprises a sample pad, a nitrocellulose membrane, a water absorption pad and a backing plate, the nitrocellulose membrane is attached to the backing plate, one end of the sample pad and one end of the water absorption pad are respectively lapped at two ends of the nitrocellulose membrane, the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line and the quality control line are parallel to each other, the quality control line is positioned at one end, close to the water absorption pad, of the nitrocellulose membrane, the detection line is coated with an amino terminal natriuretic peptide precursor monoclonal antibody, the quality control line is coated with one of goat anti-chicken IgY antibody, goat anti-rabbit IgG antibody or goat anti mouse IgG antibody, the shell comprises an upper cover and a lower cover which can be mutually buckled, and a display window is arranged at the position of the upper cover corresponding to the nitrocellulose membrane, a sample adding hole is formed in the position corresponding to the sample pad;
the reaction substance comprises a detection line fluorescent conjugate and a quality control line fluorescent conjugate; the detection line fluorescent conjugate contains an amino terminal brain natriuretic peptide precursor monoclonal antibody marked by fluorescent microspheres, and the quality control line fluorescent conjugate contains one of chicken IgY antibody, rabbit IgG antibody or mouse IgG antibody marked by fluorescent microspheres;
the reaction buffer comprises buffer solution, surfactant, saccharide, preservative and sodium chloride.
2. The kit for determining the amino-terminal brain natriuretic peptide precursor according to claim 1, wherein the fluorescent microsphere is a time-resolved fluorescent microsphere, the time-resolved fluorescent microsphere is embedded with rare earth ions, and at least one functional group selected from amino, carboxyl or hydroxyl is modified on the surface of the time-resolved fluorescent microsphere; the rare earth ions are selected from at least one of Sm samarium, Eu europium, Gd gadolinium, Tb terbium and Dy dysprosium in lanthanide elements.
3. The kit for determining the amino-terminal brain natriuretic peptide precursor according to claim 1, wherein the reaction substance is prepared by freeze drying a detection line fluorescent conjugate and a quality control line fluorescent conjugate under the protection of a freeze-drying protective solution.
4. The kit for determining an amino-terminal brain natriuretic peptide precursor according to claim 3, wherein the lyoprotectant comprises a buffer, a protectant protein, and a saccharide; the buffer solution is at least one of phosphate, borate or Tris buffer solution, and the concentration is 10mM-50 mM; the protective protein is at least one of bovine serum albumin, ovalbumin or casein, and the concentration is 0.1-3%; the saccharide is at least one of trehalose, sucrose and lactose, and the concentration is 0.1-10%.
5. The kit for determining an amino-terminal brain natriuretic peptide precursor according to claim 1, wherein the buffer in the reaction buffer is at least one selected from the group consisting of phosphate, borate, and Tris buffer at a concentration of 10mM to 50 mM; the surfactant is at least one of Triton x-100, Tween 20, S16 and S9, and the concentration is 0.1% -1%; the saccharide is selected from one or more of trehalose, sucrose and lactose, and the concentration is 0.1-10%; the concentration of the sodium chloride is 0.8-2%; the preservative is selected from at least one of sorbic acid, benzoic acid or proclin300, and the concentration is 0.01-0.1%.
6. The kit for determining amino-terminal brain natriuretic peptide precursor of claim 1, wherein the kit further comprises an ID card storing a project name, a lot number, and standard curve data of a product.
7. The kit for determining an amino-terminal brain natriuretic peptide precursor according to claim 1, wherein the preparation method of the reagent card comprises:
1) preparation of detection lines and quality control lines: respectively preparing an amino-terminal brain natriuretic peptide precursor monoclonal antibody with the concentration of 0.5-3mg/mL and a goat anti-chicken IgY antibody, a goat anti-rabbit IgG antibody or a goat anti-mouse IgG antibody with the concentration of 0.5-2mg/mL, scribing to a detection line and a quality control line area on a nitrocellulose membrane by using a film spraying machine, wherein the coating amount is 0.5-1.0 mu L/cm, and drying;
2) preparation of sample pad: preparing a sealing solution, adding the sealing solution onto the glass fiber to completely wet the glass fiber, and drying;
3) assembling the reagent card: sticking the nitrocellulose membrane to a backing plate, respectively lapping one end of a sample pad and one end of a water absorption pad on two ends of the nitrocellulose membrane, cutting the nitrocellulose membrane into test strips with specific widths, putting the test strips into a shell to fix the test strips in position, covering the shell, and pressing the test strips.
8. The kit for assaying precursor of amino-terminal brain natriuretic peptide according to claim 3, wherein the method for preparing the reaction substance comprises the steps of:
1) preparing a detection line fluorescent conjugate:
a) placing the fluorescent microspheres in a centrifuge tube, adding an activating solution, centrifuging, removing supernatant, adding the activating solution into the precipitate, and carrying out ultrasonic resuspension; cleaning again;
b) adding crosslinking agents EDC and Sulfo-NHS into the fluorescent microspheres, reacting for 10-30min, centrifuging, removing supernatant, adding a precipitate into a coupling buffer solution, and performing ultrasonic resuspension, wherein the final concentration of EDC is 0.04-0.2%, and the final concentration of Sulfo-NHS is 0.12-0.6%;
c) adding 30-100 μ g of amino terminal brain natriuretic peptide precursor monoclonal antibody into 0.1mL of fluorescent microsphere, performing room temperature rotation reaction, centrifuging, removing supernatant, adding a sealing solution into the precipitate, performing ultrasonic resuspension, and performing room temperature rotation reaction; centrifuging, removing supernatant, adding Washing Buffer into the precipitate, cleaning twice, removing supernatant, and ultrasonically resuspending the precipitate by using a redissolving solution to obtain a detection line fluorescent conjugate;
2) preparing a quality control line fluorescent conjugate:
a) placing the fluorescent microspheres in a centrifuge tube, adding an activating solution, centrifuging, removing supernatant, adding the activating solution into the precipitate, and carrying out ultrasonic resuspension; cleaning again;
b) adding crosslinking agents EDC and Sulfo-NHS into the fluorescent microspheres, reacting for 10-30min, centrifuging, removing supernatant, adding a precipitate into a coupling buffer solution, and performing ultrasonic resuspension, wherein the final concentration of EDC is 0.04-0.2%, and the final concentration of Sulfo-NHS is 0.12-0.6%;
c) adding 30-100 μ g of one of chicken IgY antibody, rabbit IgG antibody or mouse IgG antibody into 0.1mL of fluorescent microsphere, performing room temperature rotary reaction, centrifuging, removing supernatant, adding the precipitate into confining liquid, performing ultrasonic resuspension, and performing room temperature rotary reaction; centrifuging, removing supernatant, adding Washing Buffer into the precipitate, cleaning twice, removing supernatant, and ultrasonically resuspending the precipitate with a redissolving solution to obtain the quality control line fluorescent conjugate;
3) preparing liquid: diluting the detection line fluorescent conjugate by 100-1000 times by using a freeze-drying protective solution, and diluting the quality control line fluorescent conjugate by 500-2000 times to prepare a reaction substance solution;
4) subpackaging: subpackaging the reaction substance solution in plastic test tubes, placing the subpackaged plastic test tubes on a freeze-drying tray, freeze-drying the plastic test tubes into powder, sealing and capping the reaction substance after freeze-drying, and storing at the temperature of 2-8 ℃.
9. The kit for determining an amino-terminal brain natriuretic peptide precursor according to claim 5, wherein the method for preparing the reaction buffer comprises the steps of:
1) preparing a reaction buffer solution: adding tween 20 with the final concentration of 0.1-1 percent, trehalose with the final concentration of 0.1-10 percent, NaCl with the final concentration of 0.8-2 percent and proclin300 with the final concentration of 0.01-0.1 percent into Tris buffer solution with the concentration of 10-50 mM;
2) subpackaging: and subpackaging the reaction buffer solution into plastic test tubes, covering and sealing the plastic test tubes, and storing the plastic test tubes at the temperature of between 2 and 8 ℃.
10. A fluorescence immunoassay quantitative detection method for detecting an amino-terminal brain natriuretic peptide precursor, characterized in that the kit of any one of claims 1 to 9 is used, comprising the steps of:
step A: placing the reaction substance and the reaction buffer solution which are stored in a refrigerating way at room temperature to ensure that the reaction substance and the reaction buffer solution are fully balanced;
and B: sucking reaction buffer solution and putting the reaction buffer solution into a reaction substance test tube, and immediately sucking whole blood, serum or plasma and adding the whole blood, serum or plasma into the reaction substance test tube filled with the reaction buffer solution to obtain sample mixed solution;
and C: covering a reaction substance test tube cover, and shaking to fully and uniformly mix the sample mixed solution;
step D: absorbing the sample mixed liquid, adding the sample mixed liquid into a sample adding hole of a reagent card, and reacting;
step E: and inserting the reacted reagent card into a fluorescence immunoassay analyzer, and reading the result.
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