CN114236122A - Kit and preparation method and application thereof - Google Patents
Kit and preparation method and application thereof Download PDFInfo
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- CN114236122A CN114236122A CN202111391504.7A CN202111391504A CN114236122A CN 114236122 A CN114236122 A CN 114236122A CN 202111391504 A CN202111391504 A CN 202111391504A CN 114236122 A CN114236122 A CN 114236122A
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- G—PHYSICS
- 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
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- G—PHYSICS
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
Abstract
The invention discloses a kit and a preparation method and application thereof, relating to the technical field of medical diagnosis, wherein the kit is used for detecting the content of a tissue plasminogen activator-plasminogen activator inhibitor-1 compound, and comprises the following components: the first working solution comprises a biotinylation coating antibody and a first buffer solution, wherein the biotinylation coating antibody is obtained by coupling biotin and a coating antibody; the second working solution comprises a streptavidin-coated superparamagnetic particle and a second buffer solution; the third working solution comprises a labeled antibody labeled with enzyme and a third buffer solution, wherein the labeled antibody labeled with enzyme is obtained by coupling enzyme activated by carbodiimide and N-hydroxysuccinimide with a labeled antibody; luminescent substrate and cleaning solution. The kit provided by the invention has high accuracy and good stability when detecting the content of the tissue plasminogen activator-plasminogen activator inhibitor-1 compound.
Description
Technical Field
The invention relates to the technical field of medical diagnosis, in particular to a kit and a preparation method and application thereof.
Background
The tissue plasminogen activator inhibitor (PAI-1) is a physiological inhibitor of tissue plasminogen activator (t-PA), and after the two 1:1 form a compound, i.e. t-PAI-C, the activity of t-PA for activating plasminogen is inhibited, thereby inhibiting the degradation of fibrin. When the blood coagulation is activated, fibrin is formed to activate tissue plasminogen activator (t-PA) synthesized in vascular endothelial cells, and the activated t-PA further activates plasminogen to be converted into plasmin which degrades fibrin to generate fibrin degradation products. The PAI-1 concentration in plasma was 5 times higher than that of t-PA, and thus it was considered that t-PA released into blood almost formed a complex with PAI-1. the level of t-PAI-C is positively correlated with the concentration of t-PA and the degree of vascular endothelial injury, and is a direct marker for evaluating the t-PAI-C and the t-PA.
the increase in t-PAI-C is mainly seen in Disseminated Intravascular Coagulation (DIC), vascular endothelial injury, and various arterial venous thrombosis (deep vein thrombosis, acute myocardial infarction, etc.). Studies have shown that t-PAI-C can be used as a risk prediction index of myocardial infarction.
At present, only a few manufacturers at home and abroad in the market have the kit, the price is high and the kit is difficult to obtain, and the number of commercialized kits is small; with clinical popularization, the market demand of the tissue plasminogen activator-plasminogen activator inhibitor-1 complex (t-PAI-C) commercial assay kit is also increasing sharply.
Disclosure of Invention
The invention mainly aims to provide a kit, a preparation method and application thereof, and aims to provide a kit which has high accuracy and good stability and is used for detecting the content of a tissue plasminogen activator-plasminogen activator inhibitor-1 complex.
In order to achieve the above object, the present invention provides a kit for detecting the content of tissue plasminogen activator-plasminogen activator inhibitor-1 complex, the kit comprising:
the first working solution comprises a biotinylation coating antibody and a first buffer solution, wherein the biotinylation coating antibody is obtained by coupling biotin and a coating antibody;
the second working solution comprises a streptavidin-coated superparamagnetic particle and a second buffer solution;
the third working solution comprises a labeled antibody labeled with enzyme and a third buffer solution, wherein the labeled antibody labeled with enzyme is obtained by coupling enzyme activated by carbodiimide and N-hydroxysuccinimide with a labeled antibody;
luminescent substrate and cleaning solution.
Optionally, the enzyme comprises alkaline phosphatase or horseradish peroxidase.
Optionally, the first buffer comprises disodium hydrogen phosphate, NaCl, BSA, CaCl2Triton X-100, Tween-20 and glycine.
Optionally, the second buffer comprises tris, NaCl, Triton X-100, Proclin300, and Tween 20.
Optionally, the third buffer solution further comprises morpholine ethanesulfonic acid, NaCl, ZnCl2、MgCl2Glycine, glycerol, BSA, Proclin300, and Tween 20; and/or the presence of a gas in the gas,
the luminescent substrate comprises 9- (4-chlorphenyl thiophosphoryloxymethylene) -10-methyl dihydro acridine disodium salt; and/or the presence of a gas in the gas,
the cleaning solution comprises trihydroxy aminomethane, NaCl, Tween-20, Triton X-100 and Proclin 300.
Optionally, the biotin comprises biotin-N-succinimidyl ester.
Optionally, the Biotin comprises NHS-LC-Biotin.
The invention further provides a preparation method of the kit, which comprises the following steps:
coupling biotin with a coating antibody, removing redundant biotin, and diluting with a first buffer solution to obtain a first working solution;
taking a superparamagnetic particle stock solution coated with streptavidin, and diluting the superparamagnetic particle stock solution with a second buffer solution to obtain a second working solution;
activating enzyme with carbodiimide and N-hydroxysuccinimide, coupling the activated enzyme with a labeled antibody for 0.5-5 h, adding enzyme confining liquid, sealing for 5-30 min, purifying to obtain the labeled antibody labeled with the enzyme, and adding a third buffer solution into the labeled antibody labeled with the enzyme to obtain a third working solution.
Optionally, the enzyme blocking solution comprises hydroxylamine hydrochloride.
The invention further provides a method for detecting the content of the tissue plasminogen activator-plasminogen activator inhibitor-1 complex, which adopts the kit and comprises the following steps:
uniformly mixing a sample to be detected with the first working solution and the third working solution, and incubating to obtain a mixed solution;
adding a second working solution into the mixed solution, and uniformly mixing to obtain a reaction solution;
cleaning the reaction solution with a cleaning solution, and adding a luminescent substrate to obtain a solution to be detected;
detecting the luminous intensity of the liquid to be detected;
calculating the content of tissue plasminogen activator-plasminogen activator inhibitor-1 complex according to the luminous intensity.
According to the technical scheme provided by the invention, when a sample contains a tissue plasminogen activator-plasminogen activator inhibitor-1 complex, the biotinylation coating antibody, the enzyme-labeled antibody and the tissue plasminogen activator-plasminogen activator inhibitor-1 complex are subjected to immunoreaction to form a biotinylation coating antibody-tissue plasminogen activator-plasminogen activator inhibitor-1 complex-enzyme-labeled antibody sandwich immune complex; by adding magnetic particles coated with streptavidin as a carrier, the streptavidin on the superparamagnetic particles is specifically combined with biotin on a sandwich immune complex, the sandwich immune complex is adsorbed by a magnetic field, unreacted substances are washed away by cleaning liquid, and the superparamagnetic particles coated with the streptavidin, biotinylated coated antibody, tissue plasminogen activator, plasminogen activator inhibitor-1 complex and enzyme-labeled antibody sandwich immune complex are obtained; the alkaline phosphatase on the sandwich immune complex acts on the luminescent substrate by adding chemiluminescence substrate liquid to emit light, and the concentration of the tissue plasminogen activator-plasminogen activator inhibitor-1 complex in the sample is calculated by detecting the luminescent signal. The kit provided by the invention can rapidly detect the content of the tissue plasminogen activator-plasminogen activator inhibitor-1 compound, and has high accuracy and good stability.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other related drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a graph showing the correlation between the concentration of a test antigen and the signal value in the kit of example 1 of the present invention;
FIG. 2 is a graph showing a comparison of the measurement value of the kit with the measurement value of the Sysmex in example 1 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. 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.
In addition, technical solutions between various embodiments may be combined with each other, but must be realized by a person skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination should not be considered to exist, and is not within the protection scope of the present invention.
The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
At present, only a few manufacturers at home and abroad in the market have the kit, the price is high and the kit is difficult to obtain, and the number of commercialized kits is small; with clinical popularization, the market demand of the tissue plasminogen activator-plasminogen activator inhibitor-1 complex (t-PAI-C) commercial assay kit is also increasing sharply.
In view of this, the present invention provides a kit, and aims to provide a kit for detecting the content of tissue plasminogen activator-plasminogen activator inhibitor-1 complex, which is accurate, high in stability and good in stability.
In an embodiment of the kit provided by the invention, the kit is used for detecting the content of a tissue plasminogen activator-plasminogen activator inhibitor-1 complex, and comprises a first working solution, a second working solution, a third working solution, a luminescent substrate and a cleaning solution, wherein the first working solution comprises a biotinylated coating antibody and a first buffer solution, and the biotinylated coating antibody is obtained by coupling biotin and the coated antibody; the second working solution comprises superparamagnetic particles coated with streptavidin and a second buffer solution; the third working solution comprises a labeled antibody labeled with enzyme and a third buffer solution, wherein the labeled antibody labeled with enzyme is obtained by coupling enzyme activated by carbodiimide and N-hydroxysuccinimide with the labeled antibody.
According to the technical scheme provided by the invention, when a sample contains a tissue plasminogen activator-plasminogen activator inhibitor-1 complex, the biotinylation coating antibody, the enzyme-labeled antibody and the tissue plasminogen activator-plasminogen activator inhibitor-1 complex are subjected to immunoreaction to form a biotinylation coating antibody-tissue plasminogen activator-plasminogen activator inhibitor-1 complex-enzyme-labeled antibody sandwich immune complex; by adding magnetic particles coated with streptavidin as a carrier, the streptavidin on the superparamagnetic particles is specifically combined with biotin on a sandwich immune complex, the sandwich immune complex is adsorbed by a magnetic field, unreacted substances are washed away by cleaning liquid, and the superparamagnetic particle-biotinylated coated antibody-tissue plasminogen activator-plasminogen activator inhibitor-1 complex-enzyme-labeled antibody sandwich immune complex is formed; the alkaline phosphatase on the sandwich immune complex acts on the luminescent substrate by adding chemiluminescence substrate liquid to emit light, and the concentration of the tissue plasminogen activator-plasminogen activator inhibitor-1 complex in the sample is calculated by detecting the luminescent signal. The kit provided by the invention can rapidly detect the content of the tissue plasminogen activator-plasminogen activator inhibitor-1 compound, and has high accuracy and good stability.
According to the invention, EDC (carbodiimide) and NHS (N-hydroxysuccinimide) are used to activate the carboxyl of the hydrophobic end of the enzyme and then react with primary amine on the labeled antibody, so that the enzyme can label the labeled antibody; wherein the biotinylated coated antibody is biotinylated by reacting biotin, activated under conditions, with a primary amine of the coated antibody to form a stable peptide bond. The reaction steps of the kit in the scheme of the invention are that a calibrator, a quality control product or a clinical sample is firstly subjected to incubation reaction with a biotinylated coated antibody and a labeled antibody labeled with enzyme, then the superparamagnetic particles of streptavidin are added for incubation reaction, and then a substrate is added after cleaning by a cleaning solution and a luminescence value is read.
The invention does not limit the types of the coating antibody and the labeled antibody, and preferably, in the embodiment of the invention, the coating antibody and the labeled antibody are both selected from Shenzhen elegant Shiji science and technology Limited (the company: network http:// www.yarewell.com /), the coating antibody is selected from a monoclonal antibody with the product number of YM0213, and the labeled antibody is selected from a monoclonal antibody with the product number of YM0212, and by adopting the two, the coating antibody and the labeled antibody can generate immunoreaction with the tissue plasminogen activator-plasminogen activator inhibitor-1 complex, and have strong specificity.
The kind of the enzyme in the labeled antibody labeled with the enzyme is not limited in the present invention, and preferably, the enzyme includes alkaline phosphatase (AP enzyme) or horseradish peroxidase (HRP enzyme). The alkaline phosphatase is one of the most commonly used marking enzymes of the current immunodiagnostic reagent products, and has high stability and high accuracy; the horseradish peroxidase is high-purity peroxidase and has an important influence on the quality of the kit.
Preferably, the pH of the first buffer is 7.2; the pH value of the second buffer solution is 7.2; the pH of the third buffer was 6.0. It is understood that the above three pH values may be satisfied by only one or by both, and as a preferred embodiment of the present invention, the above three pH values are satisfied by both, and the accuracy of the obtained kit is the highest.
Preferably, the first buffer comprises: disodium hydrogen phosphate, NaCl, BSA, CaCl2Triton X-100, Tween-20 and glycine; wherein, BSA is bovine serum albumin, Triton X-100 is polyethylene glycol octyl phenyl ether, and Tween-20 is polyoxyethylene sorbitol ester belonging to polysorbate family. More preferably, the concentration of each component in the first buffer is: 50mM disodium hydrogen phosphate, 0.9% NaCl (w/w), 1% BSA (w/w), 0.5mM CaCl20.05% Triton X-100(w/w), 0.05% Tween-20(w/w), 1% glycine (w/w). The reagent kit has high accuracy and good stability.
The second buffer comprises Tris, NaCl, Triton X-100, Proclin300 and Tween20, the active ingredients of Proclin300 are mainly 2-methyl-4-isothiazolin-3-one (MCI) and 5-chloro-2-methyl-4-isothiazolin-3-one (CMCI), which rapidly penetrate the cell membrane and inhibit specific enzymes essential for cellular respiration, thus immediately inhibiting cellular activity upon contact with the microbial organism. More preferably, the concentration of each component in the second buffer is: 50mM Tris, 0.9% NaCl (w/w), 0.05% Tween-20(w/w), 0.05% Triton X-100(w/w) and 0.05% Proclin300(w/w), and the reagent kit has high accuracy and good stability.
The third buffer solution comprises morpholine ethanesulfonic acidAcid (MES), NaCl, ZnCl2、MgCl2Glycine, glycerol, BSA, Proclin300 and Tween 20. More preferably, in the third buffer, the concentration of each component is: 0.05M MES, 0.15M NaCl, 0.1% Tween-20(w/w), 0.05% Proclin300(w/w), 1% BSA (w/w), 1% glycine (w/w), 10% glycerol (w/w), 5mM MgCl2，0.1mM ZnCl2The reagent kit has high accuracy and good stability in the proportion.
The present invention is not limited to the kind of Biotin, and preferably, the Biotin includes Biotin-N-succinimidyl ester (Biotin-NHS), and more preferably, the Biotin includes NHS-LC-Biotin, which has CAS number 72040-63-2, is a biotinylation reagent long chain, is an amine-reactive biotinylation reagent, and is formed by linking a valeric acid side chain of D-Biotin to NHS ester group through a spacer. The NHS ester group at the end of NHS-LC-Biotin is covalently combined with amine group on protein or other molecules to form stable amido bond, and NHS group is released. The 6-aminocaproic acid spacer of NHS-LC-Biotin greatly increased the length between the covalently modified molecule and the bicyclic Biotin ring, resulting in better binding potential to avidin or streptomycin probes.
In the embodiment of the invention, the superparamagnetic particles coated with streptavidin are Dynabeads magnetic beads, wherein the diameter of the superparamagnetic particles can be 0.5-10 mu m, and the concentration of the superparamagnetic particle working solution coated with streptavidin can be 0.05-5.0 mg/mL. In the specific embodiment of the invention, the concentration of the working solution of the superparamagnetic particles coated with streptavidin is 0.5mg/mL, and the diameter of the working solution is 2.8 μm.
The invention is not limited to the kind of the luminescent substrate, preferably, the luminescent substrate comprises 9- (4-chlorphenyl thiophosphoryloxymethylene) -10-methyl dihydro acridine disodium salt (APS-5), which is a hypersensitive AP enzyme substrate and is purchased from New enzyme Biotechnology Limited in Han, Wuhan, when the APS-5 is taken as an alkaline phosphatase detection substrate, the detection sensitivity can reach 10 to 19 times of molar concentration, the color development time can reach a peak value in about 10 seconds, and the fluorescence duration is long.
In addition, preferably, the cleaning solution comprises trihydroxy aminomethane, NaCl, Tween-20, 0.05% Triton X-100 and Proclin300, more preferably, the cleaning solution is Tris buffer solution, and the concentration of each component is as follows: 50mM Tris, 0.9% NaCl (w/w), 0.05% Tween-20(w/w), 0.05% Triton X-100(w/w), 0.05% Proclin300(w/w), pH 7.2. The kit is more accurate due to the components and the proportion.
The invention further provides a preparation method of the kit, which comprises the following steps:
and S10, coupling biotin and the coated antibody, removing redundant biotin, and diluting with a first buffer solution to obtain a first working solution.
In the specific operation, the following method can be adopted:
taking 60 mu g of coated antibody, diluting the antibody to a final concentration of 2mg/mL by using biotinylation activation buffer solution (the biotinylation activation buffer solution is 50mM phosphate buffer solution, contains 50mM disodium hydrogen phosphate and 0.9% of NaCl by mass fraction, and has a pH value of 8.0), weighing 0.3mg of biotin, adding 300 mu L of DMSO (dimethyl sulfoxide) to prepare 10mg/mL biotin solution, adding 4-10 mu L of biotin solution to the coated antibody solution, incubating for 0.5-3 h at 22 ℃ in a dark place, purifying by using a desalting column to remove unreacted excessive biotin, and diluting the buffer solution to 50-500 mL by using biotinylation antibody to obtain a first working solution.
In the process, the coating antibody is coupled with biotin, and the specific reaction is as follows:
and S20, taking the superparamagnetic particle stock solution coated with streptavidin, and diluting the superparamagnetic particle stock solution with a second buffer solution to obtain a second working solution.
In the embodiment of the invention, the streptavidin-coated superparamagnetic particle working solution with the concentration of 0.5mg/mL and the diameter of 2.8 μm is stored in a second buffer solution, wherein the second buffer solution is TBS buffer solution, and the composition comprises 50mM Tris, 0.9% NaCl (w/w), 0.05% Tween-20(w/w), 0.05% Triton X-100(w/w), 0.05% Proclin300(w/w) and the pH value is 7.2.
In the specific operation, the following method can be adopted in the step:
and (3) taking the superparamagnetic particle stock solution coated with the streptavidin, washing the superparamagnetic particle stock solution coated with the streptavidin for 3 times by using a second buffer solution, and diluting the superparamagnetic particle stock solution coated with the streptavidin to 0.5mg/mL to obtain a second working solution.
S30, activating the enzyme with carbodiimide and N-hydroxysuccinimide, coupling the activated enzyme with a labeled antibody for 0.5-5 h, adding an enzyme blocking solution, sealing for 5-30 min, purifying to obtain the labeled antibody labeled with the enzyme, and adding a third buffer solution into the labeled antibody labeled with the enzyme to obtain a third working solution.
In the specific operation, the following method can be adopted in the step:
taking 100 mu g of enzyme, diluting the enzyme into 1mg/mL (the activation buffer is MES buffer, the components are 0.1M MES and 0.9% of NaCl by mass, pH is 6.0), respectively weighing 0.4mg of carbodiimide (EDC) and 0.6mg of N-hydroxysuccinimide (NHS), adding 0.1mL of the activation buffer, performing vortex dissolution, taking 10ul of the solution, adding the solution into the enzyme solution diluted by the activation buffer, and reacting at room temperature for 15-45 min; purifying with desalting column to obtain activated enzyme solution; diluting the labeled antibody to 1mg/mL (the crosslinking buffer is PBS (phosphate buffer solution) with the components of 0.1M disodium hydrogen phosphate, 0.15mM NaCl, pH7.2), adding 30-200 mu g of labeled antibody solution into the activated enzyme solution, and reacting at room temperature for 1-4 h; and adding 2 mu L of enzyme blocking solution after the reaction is finished, reacting for 10min, purifying by using a desalting column to obtain a labeled antibody mother solution labeled with the enzyme, and diluting by 200-5000 times by using a third buffer solution to obtain a third working solution.
In the step, the carboxyl of the hydrophobic end of the enzyme is activated by EDC (carbodiimide) and NHS (N-hydroxysuccinimide) and then can react with primary amine on the labeled antibody to realize the labeling of the labeled antibody by the enzyme, and the specific reaction is as follows:
preferably, the enzyme blocking solution comprises hydroxylamine hydrochloride. More preferably, the enzyme blocking solution is a PBS buffer solution, which comprises: 0.1M disodium hydrogenphosphate, 0.15mM NaCl, 50mM hydroxylamine hydrochloride, pH7.2, the hydroxylamine hydrochloride hydrolyzed NHS on alkaline phosphatase that did not react with the labeled antibody, and the reaction was terminated early, reducing nonspecific binding.
It is understood that the sequence of steps S10, S20 and S30 is not limited in the present invention.
According to the preparation method of the kit, the first working solution, the second working solution, the third working solution, the luminescent substrate and the cleaning solution are assembled into the kit, the preparation of the biotinylated coated antibody and the labeled antibody labeled with the enzyme can be completed within 5 hours, the time is short compared with other coupling processes, the performance is excellent, the kit can be applied to a chemiluminescence immunoassay detection kit, and the clinical relevance of the kit to the existing kit is good.
The preparation method of the kit provided by the invention at least has all the beneficial effects of the kit, and is not repeated herein.
The invention further provides a method for detecting the content of the tissue plasminogen activator-plasminogen activator inhibitor-1 complex, which adopts the kit and comprises the following steps:
uniformly mixing a sample to be detected with the first working solution and the third working solution, and incubating to obtain a mixed solution;
adding a second working solution into the mixed solution, and uniformly mixing to obtain a reaction solution;
cleaning the reaction solution with a cleaning solution, and adding a luminescent substrate to obtain a solution to be detected;
detecting the luminous intensity of the liquid to be detected;
calculating the content of tissue plasminogen activator-plasminogen activator inhibitor-1 complex according to the luminous intensity.
Specifically, when the kit is used for detecting the content of the tissue plasminogen activator-plasminogen activator inhibitor-1 complex, the following method is adopted:
(1) taking 50 mu L of first working solution and 50 mu L of third working solution to perform specific reaction with 10 mu L of sample to be detected to obtain the double-antibody sandwich immune complex;
(2) adding a second working solution to combine the immune complex with the double antibody sandwich with the magnetic beads;
(3) adding magnetic field to fix magnetic beads, washing with cleaning solution, adding chemiluminescent substrate, performing enzymatic reaction with the double antibody sandwich immune complex, and measuring luminescent signal to obtain tissue plasminogen activator-plasminogen activator inhibitor-1 complex concentration;
the reaction temperature of the first step is 37 ℃, and the reaction time is 10 min; the second step is that the reaction temperature is 37 ℃ and the reaction time is 5 min; the third step is at 37 deg.C for 10 s.
The method for detecting the content of the tissue plasminogen activator-plasminogen activator inhibitor-1 complex has the advantages of simple and rapid operation, short time consumption, high accuracy and good stability during detection, has good clinical relevance with the existing kit, at least has all the beneficial effects of the kit, and is not repeated herein.
The technical solutions of the present invention are further described in detail below with reference to specific examples and drawings, it should be understood that the following examples are merely illustrative of the present invention and are not intended to limit the present invention.
EXAMPLE 1 kit
A first working fluid: biotinylated coating antibody (coating antibody: monoclonal antibody with stock number YM0213, Shenzhen Yashi century science and technology Co., Ltd.), first buffer solution [50mM disodium hydrogen phosphate, 0.9% NaCl (w/w), 1% BSA (w/w), 0.5mM CaCl20.05% Triton X-100(w/w), 0.05% Tween-20(w/w), 1% glycine (w/w)]Wherein the pH value of the first buffer solution is 7.2;
a second working liquid: superparamagnetic particles coated with streptavidin, a second buffer [50mM Tris, 0.9% NaCl (w/w), 0.05% Tween-20(w/w), 0.05% Triton X-100(w/w), 0.05% Proclin300(w/w) ], wherein the pH value of the second buffer is 7.2;
a third working liquid: sign boardLabeled antibody (labeled antibody: monoclonal antibody having stock number YM0212, Shenzhen, Shiji science and technology Co., Ltd.), third buffer [0.05M MES, 0.15M NaCl, 0.1% Tween-20(w/w), 0.05% Proclin300(w/w), 1% BSA (w/w), 1% glycine (w/w), 10% glycerol (w/w), 5mM MgCl2，0.1mM ZnCl2]Wherein the pH value of the third buffer solution is 6.0;
luminescent substrate: APS-5;
cleaning solution: 50mM Tris, 0.9% NaCl (w/w), 0.05% Tween-20(w/w), 0.05% Triton X-100(w/w), 0.05% Proclin300(w/w), pH 7.2.
EXAMPLE 2 preparation of the kit
A kit for the preparation of a substance according to example 1, comprising the steps of:
1. preparation of various buffers
A second buffer solution: 50mM Tris, 0.9% NaCl (w/w), 0.05% Tween-20(w/w), 0.05% Triton X-100(w/w), 0.05% Proclin300(w/w), pH 7.2;
a third buffer solution: 0.05M MES, 0.15M NaCl, 0.1% Tween-20(w/w), 0.05% Proclin300(w/w), 1% BSA (w/w), 1% glycine (w/w), 10% glycerol (w/w), 5mM MgCl2，0.1mM ZnCl2The pH value is 6.0;
a first buffer solution: 50mM disodium hydrogen phosphate, 0.9% NaCl (w/w), 1% BSA (w/w), 0.5mM CaCl20.05% Triton X-100(w/w), 0.05% Tween-20(w/w), 1% glycine (w/w), pH 7.2;
biotinylation activation buffer: 50mM phosphate buffer containing 50mM disodium hydrogen phosphate, and 0.9% NaCl (w/w), pH 8.0;
enzyme blocking solution: 0.1M disodium hydrogen phosphate, 0.15mM NaCl, 50mM hydroxylamine hydrochloride, pH 7.2.
2. Preparation of the second working fluid
Transferring 10mg of superparamagnetic particles (the diameter is 1-3 mu m) with surface active groups being streptavidin, placing the superparamagnetic particles on a magnetic separator for magnetic separation to remove supernatant, adding 1mL of second buffer solution, carrying out vortex mixing uniformly, then separating the supernatant, repeating the cleaning step for 3 times, adding 20mL of second buffer solution to obtain second working solution, and storing the second working solution at the temperature of 2-8 ℃ for later use.
3. Preparation of the third working fluid
Activating alkaline phosphatase with EDC and NHS, desalting and purifying with a desalting column, adding the labeled antibody according to the molar ratio of 1:1.4 of AP enzyme to the labeled antibody (a monoclonal antibody with the product number of YM0212, Shenzhen, elegant, century science and technology Limited) in a molar ratio, shaking and uniformly mixing for reaction at 25 ℃ in a dark place for 2h, adding an enzyme confining liquid, sealing for 10min, purifying with the desalting column, finally adding a third buffer solution, uniformly mixing to obtain a second working solution, and storing at 2-8 ℃ for later use.
4. Preparation of the first working fluid
Taking 60 mu g of coating antibody (coating antibody: monoclonal antibody with the product number of YM0213, Shenzhen is elegant to century science and technology Limited), diluting the concentration of the coating antibody to 1mg/mL by using biotinylation activation buffer solution, weighing 0.3mg of biotin, adding 300 mu L of biotinylation activation buffer solution to prepare 10mg/mL biotin solution, adding 4-10 mu L of biotin solution to the coating antibody solution, incubating for 1h at 22 ℃ in the dark, purifying by using a desalting column to remove unreacted biotin, finally diluting by using first buffer solution to obtain first working solution, and storing for later use at 2-8 ℃.
EXAMPLE 3 detection of the content of tissue plasminogen activator-plasminogen activator inhibitor-1 Complex
(1) Taking trisodium citrate anticoagulation plasma as a sample to be detected, taking a full-automatic chemiluminescence apparatus as a detection tool, and taking 50 mu L of first working solution, 50 mu L of third working solution and 10 mu L of sample to be detected to perform a specific reaction to obtain a mixed solution;
(2) adding a second working solution to combine the immune complex with the double antibody sandwich with the magnetic beads to obtain a reaction solution;
(3) adding magnetic field fixed magnetic beads, washing with a cleaning solution, adding a chemiluminescent substrate, and carrying out enzymatic reaction with the immune complex sandwiched by the double antibodies to obtain a solution to be detected; measuring the luminous intensity of the liquid to be measured, and calculating according to the luminous intensity to obtain the concentration of the tissue plasminogen activator-plasminogen activator inhibitor-1 compound;
the reaction temperature of the first step is 37 ℃, and the reaction time is 10 min; the second step is that the reaction temperature is 37 ℃ and the reaction time is 5 min; the third step is at 37 deg.C for 10 s.
1. Linear evaluation
The tissue plasminogen activator-plasminogen activator inhibitor-1 complex solutions in the tissue plasminogen activator-plasminogen activator inhibitor-1 complex calibrator of the kit of example 1 were subjected to linear analyses at concentrations of 0ng/mL, 20ng/mL, 40ng/mL, 60ng/mL, 80ng/mL, 100ng/mL, and 120ng/mL, respectively, to obtain Table 1 and FIG. 1, respectively.
Table 1 results of linear range detection of the kit of example 1
The linear correlation coefficient r is calculated to be 0.9937, the linear range of the kit is 0-120 ng/mL, specifically, in fig. 1, the abscissa is the sample concentration value (ng/mL), the ordinate is the luminescence intensity (RLU), and the linear equation is y 42787x-1119.8, which is good in linearity.
2. Clinical sample test alignment
28 clinical samples were taken and tested simultaneously with the Simon's kit of example 1. The results are shown in Table 2 below. The regression equation was used to determine the content of tissue plasminogen activator-plasminogen activator inhibitor-1 complex (measured on the Hexemeikang test) as the abscissa X and the concentration as ng/mL, and the regression equation was used to determine the content of tissue plasminogen activator-plasminogen activator inhibitor-1 complex (measured on the invention) as the ordinate y and the concentration as ng/mL, respectively, as determined on the kit of example 1, to obtain FIG. 2 and Table 2.
Table 2 example 1 test comparison of kit and schirmenzen kit
Referring to table 2 and fig. 2, the correlation equation between them is: and y is 1.0681x-1.4169, the correlation coefficient is 0.9855, the K value is-1.4169, and the correlation between the y and the K is better.
3. Accelerated stability testing
The kit of example 1 was placed in an oven at 37 ℃ for accelerated aging, the reagents were taken out on days 0, 2, 5, 7 and 9, respectively, 5 samples were tested, and deviations of the luminescence values on days 2, 5, 7 and 9 from the non-accelerated luminescence value (day 0) were calculated, and the results are shown in tables 3 and 4 below. The deviation of the luminous value of the kit from the non-accelerated kit in the 2 nd, 5 th, 7 th and 9 th days of the accelerated aging test at 37 ℃ is within 10 percent, and the stability of the kit is good.
TABLE 3 example 1 mean luminescence values of test kit accelerated by day 0/2/5/7/9
Table 4 example 1 relative deviation (%) -of kit accelerated day 2/5/7/9 from accelerated day 0
In conclusion, the kit provided by the invention has high accuracy and good stability when detecting the content of the tissue plasminogen activator-plasminogen activator inhibitor-1 complex, and can be widely applied to the detection of the content of the tissue plasminogen activator-plasminogen activator inhibitor-1 complex.
The above is only a preferred embodiment of the present invention, and it is not intended to limit the scope of the invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present invention shall be included in the scope of the present invention.
Claims (10)
1. A kit for detecting the amount of tissue plasminogen activator-plasminogen activator inhibitor-1 complex, comprising:
the first working solution comprises a biotinylation coating antibody and a first buffer solution, wherein the biotinylation coating antibody is obtained by coupling biotin and a coating antibody;
the second working solution comprises a streptavidin-coated superparamagnetic particle and a second buffer solution;
the third working solution comprises a labeled antibody labeled with enzyme and a third buffer solution, wherein the labeled antibody labeled with enzyme is obtained by coupling enzyme activated by carbodiimide and N-hydroxysuccinimide with a labeled antibody;
luminescent substrate and cleaning solution.
2. The kit of claim 1, wherein the enzyme comprises alkaline phosphatase or horseradish peroxidase.
3. The kit of claim 1, wherein the first buffer comprises disodium phosphate, NaCl, BSA, CaCl2Triton X-100, Tween-20 and glycine.
4. The kit of claim 1, wherein the second buffer comprises tris, NaCl, Triton X-100, Proclin300, and Tween 20.
5. The kit of claim 1, wherein the third buffer further comprises morpholine ethanesulfonic acid, NaCl, ZnCl2、MgCl2Glycine, glycerol, BSA, Proclin300, and Tween 20; and/or the presence of a gas in the gas,
the luminescent substrate comprises 9- (4-chlorphenyl thiophosphoryloxymethylene) -10-methyl dihydro acridine disodium salt; and/or the presence of a gas in the gas,
the cleaning solution comprises trihydroxy aminomethane, NaCl, Tween-20, Triton X-100 and Proclin 300.
6. The kit of claim 1, wherein the biotin comprises biotin-N-succinimidyl ester.
7. The kit of claim 6, wherein the Biotin comprises NHS-LC-Biotin.
8. A method of preparing a kit according to any one of claims 1 to 7, comprising the steps of:
coupling biotin with a coating antibody, removing redundant biotin, and diluting with a first buffer solution to obtain a first working solution;
taking a superparamagnetic particle stock solution coated with streptavidin, and diluting the superparamagnetic particle stock solution with a second buffer solution to obtain a second working solution;
activating enzyme with carbodiimide and N-hydroxysuccinimide, coupling the activated enzyme with a labeled antibody for 0.5-5 h, adding enzyme confining liquid, sealing for 5-30 min, purifying to obtain the labeled antibody labeled with the enzyme, and adding a third buffer solution into the labeled antibody labeled with the enzyme to obtain a third working solution.
9. The method of claim 8, wherein the enzyme blocking solution comprises hydroxylamine hydrochloride.
10. Method for detecting the content of tissue plasminogen activator-plasminogen activator inhibitor-1 complex, characterized in that, with a kit according to any of claims 1 to 7, the method for detecting the content of tissue plasminogen activator-plasminogen activator inhibitor-1 complex comprises the following steps:
uniformly mixing a sample to be detected with the first working solution and the third working solution, and incubating to obtain a mixed solution;
adding a second working solution into the mixed solution, and uniformly mixing to obtain a reaction solution;
cleaning the reaction solution with a cleaning solution, and adding a luminescent substrate to obtain a solution to be detected;
detecting the luminous intensity of the liquid to be detected;
calculating the content of tissue plasminogen activator-plasminogen activator inhibitor-1 complex according to the luminous intensity.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114736948A (en) * | 2022-06-10 | 2022-07-12 | 深圳市帝迈生物技术有限公司 | Alpha 2-antifibrinolytic enzyme activity determination kit |
| CN115166244A (en) * | 2022-06-28 | 2022-10-11 | 北京美联泰科生物技术有限公司 | Application of buffer solution in PGP9.5 detection kit |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH022936A (en) * | 1988-06-14 | 1990-01-08 | Snow Brand Milk Prod Co Ltd | Method for measuring tissue type plasminogen activator and its measuring reagent kit |
| CN1076784A (en) * | 1993-03-10 | 1993-09-29 | 北京放射医学研究所 | Magnetic corpuscular immune separating agent and preparation |
| JP2561753B2 (en) * | 1989-10-02 | 1996-12-11 | 帝人株式会社 | Immunological assay kit for human tissue plasminogen activator / human plasminogen activator complex and assay method |
| CN104849467A (en) * | 2014-02-15 | 2015-08-19 | 北京勤邦生物技术有限公司 | Fluorescent microsphere immunochromatography test paper strip for detecting clenbuterol hydrochloride residue, preparation method and applications thereof |
| CN106841613A (en) * | 2017-01-18 | 2017-06-13 | 上海良润生物医药科技有限公司 | A kind of method and system for detecting excretion body |
| CN113419061A (en) * | 2020-06-19 | 2021-09-21 | 南京金斯瑞生物科技有限公司 | Magnetic particle chemiluminescence kit for detecting SARS-CoV-2 virus neutralizing antibody and application thereof |
| CN113533753A (en) * | 2021-07-14 | 2021-10-22 | 江苏拜明生物技术有限公司 | Kit and detection method for detecting tissue type plasminogen activator-inhibitor 1 complex in single plasma |
-
2021
- 2021-11-22 CN CN202111391504.7A patent/CN114236122A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH022936A (en) * | 1988-06-14 | 1990-01-08 | Snow Brand Milk Prod Co Ltd | Method for measuring tissue type plasminogen activator and its measuring reagent kit |
| JP2561753B2 (en) * | 1989-10-02 | 1996-12-11 | 帝人株式会社 | Immunological assay kit for human tissue plasminogen activator / human plasminogen activator complex and assay method |
| CN1076784A (en) * | 1993-03-10 | 1993-09-29 | 北京放射医学研究所 | Magnetic corpuscular immune separating agent and preparation |
| CN104849467A (en) * | 2014-02-15 | 2015-08-19 | 北京勤邦生物技术有限公司 | Fluorescent microsphere immunochromatography test paper strip for detecting clenbuterol hydrochloride residue, preparation method and applications thereof |
| CN106841613A (en) * | 2017-01-18 | 2017-06-13 | 上海良润生物医药科技有限公司 | A kind of method and system for detecting excretion body |
| CN113419061A (en) * | 2020-06-19 | 2021-09-21 | 南京金斯瑞生物科技有限公司 | Magnetic particle chemiluminescence kit for detecting SARS-CoV-2 virus neutralizing antibody and application thereof |
| CN113533753A (en) * | 2021-07-14 | 2021-10-22 | 江苏拜明生物技术有限公司 | Kit and detection method for detecting tissue type plasminogen activator-inhibitor 1 complex in single plasma |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114736948A (en) * | 2022-06-10 | 2022-07-12 | 深圳市帝迈生物技术有限公司 | Alpha 2-antifibrinolytic enzyme activity determination kit |
| CN114736948B (en) * | 2022-06-10 | 2022-11-08 | 深圳市帝迈生物技术有限公司 | Alpha 2-antifibrinolytic enzyme activity determination kit |
| CN115166244A (en) * | 2022-06-28 | 2022-10-11 | 北京美联泰科生物技术有限公司 | Application of buffer solution in PGP9.5 detection kit |
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