CN113092763B - Buffer solution and application thereof in neuron specific enolase detection kit - Google Patents

Buffer solution and application thereof in neuron specific enolase detection kit Download PDF

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CN113092763B
CN113092763B CN202110380903.7A CN202110380903A CN113092763B CN 113092763 B CN113092763 B CN 113092763B CN 202110380903 A CN202110380903 A CN 202110380903A CN 113092763 B CN113092763 B CN 113092763B
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buffer solution
parts
buffer
kit
reagent
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CN113092763A (en
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王法龙
刘聪
魏宏娟
李博飞
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Beijing Meilian Taike Biotechnology Co ltd
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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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • 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
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • 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
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/988—Lyases (4.), e.g. aldolases, heparinase, enolases, fumarase

Abstract

The invention belongs to the technical field of immunodiagnosis, and particularly relates to a buffer solution and application thereof in a neuron specific enolase detection kit. The buffer solution comprises the following components: trihydroxymethyl aminomethane, KCl, disodium hydrogen phosphate dodecahydrate, magnesium sulfate, trehalose, and BSA. The buffer solution can be used for preparing a kit for detecting NSE, the kit can quickly and simply detect the content of neuron-specific enolase in human peripheral blood, is simple and convenient to operate, and can provide an in vitro diagnosis kit for auxiliary diagnosis of small cell lung cancer, neuroblastoma and nephroblastoma, and a preparation method and application thereof.

Description

Buffer solution and application thereof in neuron specific enolase detection kit
Technical Field
The invention belongs to the technical field of kit preparation, and particularly relates to a buffer solution and application thereof in a neuron specific enolase detection kit.
Background
Neuron-Specific Enolase (NSE) is an acid protease with a molecular weight of about 78000. The NSE is one of the important markers of the small cell lung cancer, and can be used for differential diagnosis and detection of the treatment effect of the small cell lung cancer after radiotherapy and chemotherapy. The NSE can also be used for differential diagnosis of neuroblastoma and nephroblastoma, and has higher clinical application value for early diagnosis of neuroblastoma.
The detection methods in the prior art are various, including ELISA, FIA, plate type chemiluminescence and other methods, but the sensitivity and stability of NSE in ELISA, FIA, plate type chemiluminescence and other methodologies are still unsatisfactory, and the application of NSE in disease detection is seriously influenced. The detection time of the methodology is generally 30 minutes to 1.5 hours, and the detection time is long, so that the diagnosis and treatment of patients are not facilitated.
For example, the chinese patent application CN 109187971a provides a chemiluminescence immunoassay kit for neuron-specific enolase and a preparation method thereof, belonging to the technical field of in vitro detection. The kit comprises: the kit comprises a streptavidin magnetic particle suspension, a chemiluminescent marker labeled neuron-specific enolase monoclonal antibody and a conjugate marker labeled neuron-specific enolase monoclonal antibody.
The Chinese patent application CN 106645700A is a kit for rapid diagnosis of neuron-specific enolase and a use method thereof, and the kit comprises a neuron-specific enolase standard substance, a reaction buffer solution, a cleaning solution, a liposome compound and a magnetic separation reagent. The liposome compound is a compound which is internally wrapped by bipyridyl ruthenium Ru (bpy)32+ and nano-gold particles, the surface of the liposome compound is coated by an anti-neuron specific enolase antibody, and the nano-gold particles are spherical-like nano-gold with the particle size of 2-50 nm. The magnetic separation reagent is prepared from a biotinylated anti-neuron-specific enolase antibody and streptavidin-coated magnetic particles.
However, in the prior art, the detection process and the sensitivity cannot be obtained at the same time. The invention aims to provide an in vitro diagnostic kit which is simple and convenient to operate and can provide auxiliary diagnosis for small cell lung cancer, neuroblastoma and nephroblastoma, and a preparation method and application thereof, so that the content of neuron-specific enolase in human peripheral blood can be detected quickly and simply.
Disclosure of Invention
In order to overcome the technical problems, the invention provides a buffer solution which can be used for preparing a kit for detecting NSE, the kit can quickly and simply detect the content of neuron-specific enolase in human peripheral blood, is simple and convenient to operate, and can provide an in vitro diagnosis kit for auxiliary diagnosis of small cell lung cancer, neuroblastoma and nephroblastoma, and a preparation method and application thereof.
In order to achieve the above purpose, the technical scheme provided by the invention is as follows:
a buffer comprising the following components: tris (hydroxymethyl) aminomethane (Tris), potassium chloride (KCl), disodium hydrogen phosphate dodecahydrate, magnesium sulfate, trehalose, and Bovine Serum Albumin (BSA).
Preferably, the buffer solution comprises the following components in parts by weight: 4-20 parts of tris (hydroxymethyl) aminomethane, 6-25 parts of KCl, 1-6 parts of disodium hydrogen phosphate dodecahydrate, 0.1-10 parts of magnesium sulfate, 5.0-50 parts of trehalose and 1.0-50 parts of BSA.
Preferably, the buffer solution comprises the following components in parts by weight: 4.2-15.6 parts of trihydroxymethyl aminomethane, 8.5-24 parts of KCl, 1.2-5.8 parts of disodium hydrogen phosphate dodecahydrate, 0.1-10 parts of magnesium sulfate, 5.0-50 parts of trehalose and 1.0-50 parts of BSA;
preferably, the preparation method of the buffer solution comprises the following steps:
(1) weighing Tris, KCl and NaH2PO4、MgSO4Adding trehalose and bovine serum albumin into purified water, stirring until the trehalose and the bovine serum albumin are completely dissolved, adjusting the pH value, and fixing the volume;
(2) filtering with filter membrane.
Preferably, the pH value is between 6.8 and 8.5.
Preferably, the pore size of the filter is 0.22 μm.
The invention also aims to provide application of the buffer solution in preparing a neuron-specific enolase detection reagent.
The invention also aims to provide a neuron-specific enolase detection kit, which comprises the buffer solution.
Preferably, the kit comprises: detecting reagent strips, quality control products and calibration products.
Preferably, the preparation method of the quality control product comprises the following steps: the NSE recombinant protein is used as a raw material of a calibrator, dissolved by the buffer solution, and fully mixed to prepare the calibrator.
Preferably, the preparation method of the calibrator comprises the following steps: the NSE recombinant protein is used as a raw material of a quality control product, dissolved by the buffer solution, and fully mixed to prepare the product.
Preferably, the detection reagent strip comprises a reagent A, a reagent B, a reagent C, a cleaning solution, a luminescent substrate, a reading hole position, a reaction hole position, a cleaning hole position, an elution sleeve and a suction head.
Wherein the reagent A is NSE antibody solution marked by alkaline phosphatase;
the reagent B is a biotin-labeled NSE antibody solution;
the reagent C is a streptavidin-labeled magnetic particle solution;
the cleaning solution is; tris buffer containing a nonionic surfactant.
Preferably, the cleaning solution comprises the following components: the concentration of Tween-20 is 0.5-5% (w/v), the concentration of TritonX-100 is 0.05-0.5% (v/v), and the rest is water.
The luminogenic substrate is an ALP-catalyzed luminogenic substrate.
Preferably, the preparation method of the reagent A comprises the following steps: the enzyme-labeled NSE antibody conjugate was used as a raw material of the reagent a, and dissolved in the buffer a to prepare the reagent a.
Preferably, the preparation method of the reagent B comprises the following steps: the biotinylated NSE antibody conjugate is used as a raw material of a reagent B, and is dissolved by using a buffer solution B to prepare the reagent B.
Preferably, the preparation method of the reagent C comprises the following steps: the streptavidin magnetic particle conjugate is used as a raw material of the reagent C, and is dissolved by using a buffer solution C to prepare the reagent C.
Preferably, the buffer solution a, the buffer solution B and the buffer solution C all have the following compositions in parts by weight:
3.5-15 parts of trihydroxymethyl aminomethane, 9.0 parts of sodium chloride, 1.0-50 parts of bovine serum albumin, 10-100 parts of bovine serum, 1.0-20 parts of sheep serum and 1.0-20 parts of horse serum.
The pH values of the buffer solution A, the buffer solution B and the buffer solution C are 5.8-7.6.
Preferably, the method for preparing the enzyme-labeled NSE antibody conjugate comprises the following steps:
(1) antibody activation: weighing 2IT, dissolving the 2IT by using a buffer solution 1, adding the buffer solution into an antibody solution for activation, oscillation and reaction, then adding a buffer solution 2, removing excessive 2IT by using a PD10 desalting column after reaction, and preparing an activated antibody solution;
(2) activation of alkaline phosphatase: weighing SMCC, dissolving with DMF, shaking, reacting to obtain SMCC solution, adding ALP into SMCC solution, shaking, reacting, adding buffer solution 2, reacting, removing excessive SMCC with PD10 desalting column to obtain activated ALP solution;
(3) linking of antibody and ALP: adding an ALP solution into the antibody solution, shaking and reacting to obtain an enzyme-labeled antibody conjugate;
(4) termination and purification of enzyme-labeled antibody conjugates: weighing maleimide, dissolving with DMF, and diluting with buffer solution 1 to obtain maleimide solution; carrying out reaction;
dissolving ethanolamine by using a buffer solution 1, shaking, and concentrating the enzyme-labeled antibody conjugate by using an ultrafiltration concentration tube; antibody purification using a purified protein analyzer and a Superdex 200 preparative grade 2.6/60 gel column; eluting with buffer 2; and preparing the purified enzyme-labeled antibody conjugate.
Preferably, the method of preparing the biotinylated NSE antibody conjugate comprises the steps of:
preparing a biotin solution by using a PBS buffer solution, oscillating, adding the biotin solution into a desalting column after reaction, and adding the PBS buffer solution with the same volume to start elution; and preparing the purified biotinylated antibody conjugate.
Preferably, the preparation method of the PBS buffer solution comprises the following steps: dissolving 3-10g of disodium hydrogen phosphate dodecahydrate, 0.2-1.0g of sodium dihydrogen phosphate, 5-8g of sodium chloride and 0.01-0.8g of potassium chloride in water, and fixing the volume to 1000 mL.
Preferably, the preparation method of the streptavidin magnetic particle conjugate comprises the following steps:
washing the magnetic particles with a buffer solution 3, then resuspending, adding streptavidin into the magnetic particle solution, and reacting; adding EDC into the magnetic particle solution, performing magnetic separation after reaction, and suspending the separated magnetic particles by using a buffer solution 4; after fully and uniformly mixing, uniformly mixing at room temperature and reacting for 1-4 hours; and then carrying out magnetic separation on the connecting object, and using a buffer solution 5 to resuspend the separated magnetic particles to prepare the streptavidin magnetic particle conjugate.
Preferably, the preparation method of the buffer solution 1 comprises the following steps:
weighing 14.8-15.1g of ethanolamine and 5.8-6.0g of NaCl, adding into purified water, stirring until the ethanolamine and the NaCl are completely dissolved, adjusting the pH value to 7.3-7.6, and fixing the volume to 1000 ml; filtration was performed with a 0.22 μm filter.
Preferably, the preparation method of the buffer solution 2 comprises the following steps:
weighing 75g of glycine, adding the glycine into purified water, stirring until the glycine is completely dissolved, and metering to 1000 ml; filtration was performed with a 0.22 μm filter.
Preferably, the preparation method of the buffer solution 3 comprises the following steps:
weighing 10.0-50.0g of MES, adding the MES into purified water, stirring until the MES is completely dissolved, adjusting the pH value to be 4.0-7.0, and fixing the volume to 1000 ml; filtration was performed with a 0.22 μm filter.
Preferably, the preparation method of the buffer solution 4 comprises the following steps:
weighing 1.0-10.0g of Tris, 9.0g of NaCl and 5.0-30g of bovine serum albumin, adding the materials into purified water, stirring until the materials are completely dissolved, adjusting the pH value to be 6.4-8.5, and fixing the volume to 1000 ml. Filtration was performed with a 0.22 μm filter.
Preferably, the preparation method of the buffer solution 5 comprises the following steps: weighing 1.5-8.0g of Tris and 9.0g of NaCl, adding into purified water, stirring until the Tris and the NaCl are completely dissolved, adjusting the pH value to 7.0-7.8, and fixing the volume to 1000 ml; filtration was performed with a 0.22 μm filter.
Another object of the present invention is to provide a detection method of the kit, comprising the steps of:
s1: immune reaction: adding a sample, a reagent A, a reagent B and a reagent C into the reaction hole in sequence for reaction;
s2: magnetic separation and cleaning: and adding a cleaning solution into the hole site, sucking the product of the immune reaction out of the reaction hole site by using magnetic force, demagnetizing the cleaning hole site, and cleaning.
S3: reading value: adding a luminescent substrate at a measurement hole site; detecting relative luminescence intensity (RLU) with a self-developed instrument;
s4: obtaining an NSE concentration-luminous value standard curve according to the detected value of the calibrator; the curve is fitted by using a four-parameter Logistic equation;
s5: the detection value of the sample can correspond to the unique concentration value obtained on the curve, so that the concentration detection of the unknown sample is realized.
Compared with the prior art, the invention has the technical advantages that:
(1) the buffer solution provided by the invention can be used for preparing a kit for detecting NSE, and the kit can be used for quickly and simply detecting the content of neuron-specific enolase in human peripheral blood.
(2) The kit provided by the invention is used for quantitatively analyzing the level of human peripheral blood neuron specific enolase by adopting a magnetic particle chemiluminescence method, and is used for auxiliary diagnosis of small cell lung cancer, neuroblastoma and nephroblastoma.
(4) The invention is matched with a full-automatic instrument for detection, and accurate results can be obtained in 18 minutes only by adding the blood collection tube with the sample. Is far superior to the reaction time of 30 minutes of the traditional chemiluminescence or the reaction time of 1-2 hours of the enzyme-linked immunosorbent assay.
(5) The invention uses special buffer system and materials to make the detection sensitivity reach 0.5ng/ml (0.5X 10)-9g/mL)。
Drawings
FIG. 1: schematic diagram of NSE detection reagent strip;
FIG. 2: reaction flow chart of the kit in use;
FIG. 3: a reagent kit preparation process flow chart;
the specific meanings of the symbols in the drawings are as follows: 1: sample hole site; 2: a suction head; 3: eluting the sleeve; 4: cleaning fluid; 5: a luminescent substrate; 6: a reagent B; 7: a reagent C; 8: a reagent A; 9: a dilution hole site 1; 10: a dilution hole site 2; 11: reaction hole sites; 12: cleaning the hole site 1; 13: cleaning the hole site 2; 14: cleaning a hole position 3; 15: and (6) measuring and reading holes.
The invention will now be further illustrated with reference to the accompanying drawings and examples:
Detailed Description
The present invention will be described below with reference to specific examples to make the technical aspects of the present invention easier to understand and grasp, but the present invention is not limited thereto. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.
1. Preparation of buffer
Example 1
The buffer solution comprises the following components in parts by weight: 4.2g of trihydroxymethyl aminomethane, 8.5g of KClI, 5.8g of disodium hydrogen phosphate dodecahydrate, 5g of magnesium sulfate, 10g of trehalose and 15g of BSA.
The preparation method of the buffer solution comprises the following steps:
(1) weighing Tris, KCl and NaH2PO4、MgSO4Adding trehalose and BSA (bovine serum albumin) into purified water, stirring until the trehalose and the BSA are completely dissolved, adjusting the pH value to 7.2, and metering the volume to 1000 ml;
(2) filtering with 0.22 μm filter membrane to obtain buffer solution. (recording as buffer S1)
Example 2
The buffer solution comprises the following components in parts by weight: 15.6g of trihydroxymethyl aminomethane, 24g of KCl, 1.2g of disodium hydrogen phosphate dodecahydrate, 0.1g of magnesium sulfate, 50g of trehalose and 1.0g of BSA.
The preparation method of the buffer solution comprises the following steps:
(1) weighing Tris, KCl and NaH2PO4、MgSO4Adding trehalose and BSA (bovine serum albumin) into purified water, stirring until the trehalose and the BSA are completely dissolved, adjusting the pH value to 6.8, and metering the volume to 1000 ml;
(2) filtering with 0.22 μm filter membrane to obtain buffer solution. (recording as buffer S2)
Example 3
The buffer solution comprises the following components in parts by weight: 20g of tris (hydroxymethyl) aminomethane, KCl6g, 6g of disodium hydrogen phosphate dodecahydrate, 10g of magnesium sulfate, 5.0g of trehalose and 50g of BSA.
Preferably, the preparation method of the buffer solution comprises the following steps:
(1) weighing Tris, KCl and NaH2PO4、MgSO4Sea, seaAdding the trehalose and BSA into purified water, stirring until the trehalose and the BSA are completely dissolved, adjusting the pH value to 8.5, and metering the volume to 1000 ml;
(2) filtering with 0.22 μm filter membrane to obtain buffer solution. (recording as buffer S3)
Comparative example 1
The difference compared to example 1 is only in the pH value.
A buffer solution was prepared in the same manner as in example 1.
The preparation method of the buffer solution comprises the following steps:
(1) weighing Tris, KCl and NaH2PO4、MgSO4Adding trehalose and BSA (bovine serum albumin) into purified water, stirring until the trehalose and the BSA are completely dissolved, adjusting the pH value to 9.5, and metering the volume to 1000 ml;
(2) filtering with 0.22 μm filter membrane to obtain buffer solution. (recording as buffer D1)
Comparative example 2
The only difference compared to example 1 is the use of magnesium chloride instead of magnesium sulfate.
The buffer solution comprises the following components in parts by weight: 4.2g of trihydroxymethyl aminomethane, 8.5g of KClI, 5.8g of disodium hydrogen phosphate dodecahydrate, 5g of magnesium chloride, 10g of trehalose and 15g of BSA.
The preparation method of the buffer solution comprises the following steps:
(1) weighing Tris, KCl and NaH2PO4Adding magnesium chloride, trehalose and BSA (bovine serum albumin) into purified water, stirring until the magnesium chloride, the trehalose and the BSA are completely dissolved, adjusting the pH value to 7.2, and fixing the volume to 1000 ml;
(2) filtering with 0.22 μm filter membrane to obtain buffer solution. (recording as buffer D2)
Comparative example 3
The only difference compared to example 1 is the replacement of trehalose with glucose.
The buffer solution comprises the following components in parts by weight: 4.2g of trihydroxymethyl aminomethane, 8.5g of KClI, 5.8g of disodium hydrogen phosphate dodecahydrate, 5g of magnesium sulfate, 10g of glucose and 15g of BSA.
The preparation method of the buffer solution comprises the following steps:
(1) weighing Tris, KCl and NaH2PO4、MgSO4Adding glucose and BSA (bovine serum albumin) into purified water, stirring until the glucose and the BSA are completely dissolved, adjusting the pH value to 7.2, and metering the volume to 1000 ml;
(2) filtering with 0.22 μm filter membrane to obtain buffer solution. (recording as buffer D3)
Comparative example 4
The only difference compared to example 1 was the use of sheep serum protein instead of BSA.
The buffer solution comprises the following components in parts by weight: 4.2g of trihydroxymethyl aminomethane, 8.5g of KClI, 5.8g of disodium hydrogen phosphate dodecahydrate, 5g of magnesium sulfate, 10g of trehalose and 15g of sheep serum protein.
The preparation method of the buffer solution comprises the following steps:
(1) weighing Tris, KCl and NaH2PO4、MgSO4Adding trehalose and sheep serum protein into purified water, stirring until the trehalose and the sheep serum protein are completely dissolved, adjusting the pH value to 7.2, and fixing the volume to 1000 ml;
(2) filtering with 0.22 μm filter membrane to obtain buffer solution. (recording as buffer D4)
2. Preparation of neuron specific enolase detection kit
2.1 buffer solution
In addition to the buffers prepared in examples 1-3 and comparative examples 1-4, the buffers used in the preparation of the neuron-specific enolase assay kit included the following buffers:
(1) buffer solution 1
Weighing 14.8g of ethanolamine and 5.8g of NaCl, adding the ethanolamine and the NaCl into purified water, stirring until the ethanolamine and the NaCl are completely dissolved, adjusting the pH value to be 7.3-7.6, and fixing the volume to 1000 ml; filtration was performed with a 0.22 μm filter.
(2) Buffer solution 2
Weighing 75g of glycine, adding the glycine into purified water, stirring until the glycine is completely dissolved, and metering to 1000 ml; filtration was performed with a 0.22 μm filter.
(3) Buffer solution 3
Weighing 20.0g of MES, adding the MES into purified water, stirring until the MES is completely dissolved, adjusting the pH value to be 4.0-7.0, and fixing the volume to 1000 ml; filtration was performed with a 0.22 μm filter.
(4) Buffer 4
Weighing 5.0g of Tris, 9.0g of NaCl and 20g of bovine serum albumin, adding the Tris into purified water, stirring until the Tris is completely dissolved, adjusting the pH value to be 6.4-8.5, and fixing the volume to 1000 ml; filtration was performed with a 0.22 μm filter.
(5) Buffer solution 5
Weighing 6.0g of Tris and 9.0g of NaCl, adding into purified water, stirring until the Tris and the NaCl are completely dissolved, adjusting the pH value to be 7.0-7.8, and fixing the volume to 1000 ml; filtration was performed with a 0.22 μm filter.
(6) Buffer 6
Weighing 7g of Tris, 9.0g of NaCl, 30g of bovine serum albumin, 50mL of bovine serum, 15mL of sheep serum and 12mL of horse serum, adding the mixture into purified water, stirring until the mixture is completely dissolved, adjusting the pH value to be between 5.8 and 7.6, and fixing the volume to 1000 mL; filtration was performed with a 0.22 μm filter.
2.2 kit Components
The NSE kit consists of a detection reagent strip, a calibrator, a quality control material and a two-dimensional code; the test reagent strip is an integral body formed by a series of solutions and accessories, a sample can be independently tested, and a calibrator is prepared by NSE antigen containing two concentrations and buffer solution and is used for calibrating a standard curve; the quality control product is prepared from NSE antigen containing two concentrations and buffer solution; the standard curve of the current batch is recorded in the two-dimensional code.
TABLE 1 major Components of the kit
Figure BDA0003012933500000091
Figure BDA0003012933500000101
2.3 reagent strip Components
The detection reagent strip consists of a reagent A, a reagent B, a reagent C, a cleaning solution, a luminescent substrate, a reading hole, an elution sleeve and a suction head (the schematic diagram of the reagent strip is shown in figure 1).
The reagent A is NSE antibody solution marked by alkaline phosphatase, and the concentration is 1 mug/mL; the reagent B is a biotin-labeled NSE antibody solution with the concentration of 1 mu g/mL; the reagent C is a streptavidin-labeled magnetic particle solution, and the concentration is 0.4 mg/mL; the cleaning solution is used for cleaning the reaction process; the luminescent substrate is an ALP catalyzed luminescent substrate; assay wells were used for final assay readings.
TABLE 2 main Components of the test strips
Position of Detecting reagent strip components Loading/quantity
1 Sample hole site Is free of
2 Suction head 1 is provided with
3 Elution sleeve 1 is provided with
4 Cleaning liquid 2.0mL
5 Luminescent substrate 180μL
6 Reagent B 80μL
7 Reagent C 60μL
8 Reagent A 80μL
9 Dilution hole site 1 Is free of
10 Dilution hole site 2 Is free of
11 Location of reaction hole Is free of
12 Cleaning hole site 1 Is free of
13 Cleaning hole site 2 Is free of
14 Cleaning hole site 3 Is free of
15 Measuring and reading hole 1 is provided with
2.4 preparation of the kit:
2.4.1 preparation of calibrator and quality control Material
NSE recombinant protein is used as a raw material of a calibrator, and is dissolved by a buffer solution (any one of buffer solutions S1-S3 or buffer solutions D1-D4 provided by the invention), and the buffer solutions are fully mixed to prepare 2 calibrators with the concentrations of 1ng/mL and 150 ng/mL.
The NSE recombinant protein is used as a raw material of a quality control product, dissolved by a buffer solution (the buffer solution of the calibrator) and fully mixed to prepare the quality control product. The concentration was 20 ng/mL.
2.4.2 preparation of reagent A
(1) Preparation of enzyme-labeled NSE antibody conjugate
Weighing 6mg of 2-iminosulfane hydrochloride (2IT), dissolving the 2-iminosulfane hydrochloride (2IT) in a buffer solution 1 to 13.76mg/mL, adding the 2IT solution into an antibody solution according to a proportion (adding 1mg of the antibody into 15 mu L of the 2IT solution), activating by shaking, uniformly mixing, reacting at room temperature for 30 minutes, stopping activation, adding the buffer solution 2 into the antibody solution according to a proportion that 1mg of the antibody is added into 15 mu L of the buffer solution 2, reacting at room temperature for 10 minutes, removing excessive 2IT by using a PD10 desalting column, and collecting the activated antibody.
Weighing 3mg of SMCC, dissolving the SMCC in DMF to 6.69mg/mlL, adding an SMCC solution (30 mu L of SMCC solution is added into 1mg of ALP) into an ALP solution according to a proportion, shaking and uniformly mixing, reacting at room temperature for 30 minutes to terminate activation, adding a buffer solution 2 into the ALP solution according to a proportion that 1mg of ALP is added into 30 mu L of buffer solution 2, and reacting at room temperature for 10 minutes; excess SMCC was removed using a PD10 desalting column and the ALP was collected after activation.
The ALP solution (1.5 mg of ALP added to 1.0mg of antibody) was added to the antibody solution in a ratio, and after shaking and mixing, the mixture was reacted at 6 ℃ for 16 hours.
6mg of maleimide was weighed, dissolved in DMF to 9.7mg/mL, diluted with buffer 1 at a ratio of 1/10 to give a 0.97mg/mL maleimide solution, and added at a ratio of 1mg of antibody to 10. mu.L of 0.97mg/mL maleimide solution, and reacted at room temperature for 15 minutes. mu.L ethanolamine was measured accurately and dissolved to 100mM with buffer 1 (i.e., 994. mu.L buffer 1 was added to 6. mu.L ethanolamine). The solution was added in a ratio of 1mg antibody to 30. mu.L 100mM ethanolamine solution, and mixed by shaking. The antibody conjugate to be purified was concentrated to 2mg/mL using an ultrafiltration concentration tube. Antibody purification was performed using a purified protein analyzer and Superdex 200 preparative 2.6/60 gel column, buffer 2 as eluent. The purified liquid is an enzyme-labeled antibody conjugate.
(2) Preparation of reagent A
The enzyme-labeled NSE antibody conjugate is used as a raw material of the reagent A, and the reagent A is prepared by fully and uniformly mixing the enzyme-labeled NSE antibody conjugate with the buffer solution 6.
2.4.3 preparation of reagent B
(1) Preparation of biotinylated NSE antibody conjugates
A10 mM biotin solution was prepared in 0.02M PBS, and a 10mM biotin solution (26.68. mu.L of 10mM biotin was added to 1.0mg of antibody) was added to the antibody solution in a ratio, and after shaking to mix well, the mixture was reacted at room temperature for 1 hour.
The antibody conjugate to be desalted is added to a desalting column. When the antibody conjugate completely entered the column, the column volume was made up using 0.02M PBS. When the liquid in the column completely enters the column material, an equal volume of 0.02M PBS buffer is added to start elution. Equal volumes of protein eluate were collected and tested for concentration. The purified liquid was a biotinylated antibody conjugate.
(2) Preparation of reagent B: the biotinylated NSE antibody conjugate is used as a raw material of a reagent B, and the reagent B is prepared by fully and uniformly mixing the biotinylated NSE antibody conjugate with a buffer solution 6.
2.4.4 preparation of reagent C
(1) Preparation of streptavidin magnetic particle conjugate
After washing the magnetic particles with buffer 3, the particles were resuspended to 10 mg/mL. Streptavidin is added into the magnetic particle solution according to the mass ratio of 100:10 of the magnetic particles to the streptavidin. After thorough mixing, the reaction mixture was mixed well at room temperature for 10 minutes. EDC is added to the magnetic particle solution in a ratio of the magnetic particles to (3-dimethylaminopropyl) ethylcarbodiimide hydrochloride (EDC) mass ratio of 100: 50. After thorough mixing, the reaction mixture was mixed at room temperature for 1 hour.
The linker is magnetically separated. The separated magnetic microparticles were resuspended to 10mg/mL using buffer 4. After mixing well, mix well for anti-2 hours at room temperature. The conjugate was then magnetically separated, and the separated magnetic microparticles were resuspended to 10mg/mL using buffer 5 and stored at 6 ℃.
(2) Preparation of reagent C
The streptavidin magnetic particle conjugate is used as a raw material of the reagent C, and the reagent C is prepared by fully and uniformly mixing the streptavidin magnetic particle conjugate with the buffer solution 6.
3. Examples of effects
3.1 detection principle of the kit of the invention
The kit adopts a double-antibody sandwich method to determine the content of NSE. The NSE in the sample is combined with the NSE enzyme labeled antibody in the reagent A and the NSE biotinylated antibody in the reagent B to form a sandwich structure. Adding a reagent C into the sandwich structure, and combining the biotin in the sandwich structure and the streptavidin in the reagent C to form an antigen-antibody-magnetic particle composite. After washing, a luminescent substrate is added into the compound, the luminescent substrate is catalyzed and cracked by enzyme in the compound to form an unstable excited state intermediate, and when the excited state intermediate returns to a ground state, a photon is emitted. The number of photons generated is positively correlated with the concentration of NSE in the sample.
The detection is carried out by adopting a full-automatic chemiluminescence immunoassay analyzer self-developed by Beijing Meiliantaceae biotechnology limited company. The amount of sample required for the reaction was 30. mu.L, and the automatic assay procedure was:
A. immune reaction: and sequentially adding 30 mu L of sample, 50 mu L of reagent A, 50 mu L of reagent B and 50 mu L of reagent C into the reaction hole, and reacting at 37 ℃ for 20 min.
B. Magnetic separation and cleaning: adding 300 mu L of cleaning solution into the No. 1 cleaning hole, sucking the mixture containing the magnetic particles out of the reaction hole by magnetic force, and demagnetizing the No. 1 cleaning hole. Cleaning for 2 min; and (3) respectively carrying out 1-time magnetic separation and cleaning at No. 2 and No. 3 cleaning hole sites.
C. Reading value: adding 150uL of luminescent substrate at the reading hole position, sucking the mixture containing magnetic particles out of the No. 3 cleaning hole position by magnetic force, and demagnetizing at the reading hole position; relative luminescence intensity (RLU) was measured after luminescence of the ALP-catalyzed luminescent substrate.
D. And obtaining an NSE concentration-luminous value standard curve according to the detected value of the calibrator. The curve was fitted using a four parameter Logistic equation.
E. The detection value of the sample can correspond to the unique concentration value obtained on the curve, so that the concentration detection of the unknown sample is realized.
3.2 detection of indicators
3.2.1 accuracy
Neuron-specific enolase (NSE) solution (A) at a concentration of about 20ng/mL (tolerance. + -. 10%) was added to sample B at a concentration ranging from 0ng/mL to 0.5ng/mL, the volume ratio between the added NSE antigen and sample B was 1:9, and the recovery rate R was calculated according to equation (1) and should be in the range of 85% to 115%.
Figure BDA0003012933500000131
In the formula:
r-recovery rate;
v is the volume of the sample A liquid;
V0-volume of serum sample B fluid;
c is the average value of 3 measurements after the serum sample B liquid is added into the liquid A;
C0-mean of 3 measurements of serum sample B fluid;
CS-concentration of sample a liquid.
3.2.2 sensitivity
Repeating the test for 20 times to obtain concentration values of 20 test results, and calculating average value
Figure BDA0003012933500000141
And standard deviation of (SD). Mean value of
Figure BDA0003012933500000142
The blank limit is obtained, and the result is less than or equal to 0.3 ng/mL.
3.2.3 Linear Range
Mixing a high value sample close to the upper limit of the linear region and a low value sample close to the lower limit of the linear region or a zero concentration sample to obtain not less than 5 dilution concentrations, wherein the low value concentration sample is close to the lower limit of the linear region. The test was repeated 3 times for each concentration of the sample to obtain the luminescence value, the measurement result of each sample was recorded, and the average value (y) of the 3 measurements of each sample was calculatedi). In diluted concentration (x)i) As independent variable, the mean value (y) of the results is determinedi) Linear regression equations were solved for the dependent variables. And (3) calculating a correlation coefficient (r) of the linear regression according to the formula (2), wherein the correlation coefficient r is not less than 0.990 within a linear interval of 0.5-300 ng/mL.
Figure BDA0003012933500000143
In the formula:
r- — -correlation coefficient;
xi-dilution ratio;
yi-mean value of individual sample measurements;
Figure BDA0003012933500000144
-mean value of dilution ratio;
Figure BDA0003012933500000145
sample measurement Total mean.
3.2.4 repeatability
The quality control product is tested repeatedly for 10 times by the same batch number kit, and the average value of 10 test results is calculated
Figure BDA0003012933500000146
And standard deviation SD. The Coefficient of Variation (CV) was calculated according to equation (3) and the result CV was less than or equal to 8%.
Figure BDA0003012933500000147
In the formula: s-standard deviation of sample test values;
Figure BDA0003012933500000148
-average of sample test values.
3.2.5 batch to batch difference
The quality control materials are tested repeatedly for 10 times by using the kits with 3 batch numbers respectively, and the average value of the test results of 30 times is calculated
Figure BDA0003012933500000149
And standard deviation SD, and obtaining Coefficient of Variation (CV) according to formula (3), wherein the result CV is less than or equal to 12%.
3.2.6 specificity experiments
A nonspecific neuronal enolase was added to the sample without any analyte at a concentration of 500. mu.g/L. Taking the average value 3 times, calculating the result according to the formula (4), and determining the result to be not higher than 0.5 ng/mL.
The effect of the buffers of examples 1-3 and comparative examples 1-4 when applied to the kit was examined in the same manner as in 3.2.1-3.2.6, and the results are shown in the following table:
TABLE 3 Effect data
Test group Linear interval r Accuracy R Repetitive CV Sensitivity of the probe Specificity of Inter-batch difference CV
Buffer S1 0.9983 92.6％ 5.51％ 0.24ng/mL 0.28ng/mL 9.88％
Buffer S2 0.9921 88.7％ 6.94％ 0.27ng/mL 0.17ng/mL 11.04％
Buffer S3 0.9954 102.5％ 7.32％ 0.19ng/mL 0.33ng/mL 8.62％
Buffer D1 0.9816 132.7％ 10.22％ 4.15ng/mL 12.53ng/mL 21.37％
Buffer D2 0.9915 74.1％ 8.55％ 0.57ng/mL 3.28ng/mL 11.24％
Buffer D3 0.9939 89.5％ 8.06％ 0.49ng/mL 1.61ng/mL 15.61％
Buffer D4 0.9906 86.3％ 8.34％ 0.35ng/mL 0.63ng/mL 12.09％
Therefore, the kit provided by the invention has higher precision, sensitivity and specificity. And the composition of the buffer solution and the dosage of the effective components thereof have great influence on the effect.
The above detailed description is specific to one possible embodiment of the present invention, and the embodiment is not intended to limit the scope of the present invention, and all equivalent implementations or modifications without departing from the scope of the present invention should be included in the technical scope of the present invention.

Claims (9)

1. A buffer solution for preparing a neuron-specific enolase detection reagent is prepared from the following components: trihydroxymethyl aminomethane, KCl, disodium hydrogen phosphate dodecahydrate, magnesium sulfate, trehalose, BSA and purified water;
the buffer solution comprises the following components in parts by weight: 4-20 parts of trihydroxymethyl aminomethane, 6-25 parts of KCl, 1-6 parts of disodium hydrogen phosphate dodecahydrate, 0.1-10 parts of magnesium sulfate, 5.0-50 parts of trehalose and 1.0-50 parts of BSA.
2. The buffer of claim 1, wherein the buffer comprises the following components in parts by weight: 4.2-15.6 parts of trihydroxymethyl aminomethane, 8.5-24 parts of KCl, 1.2-5.8 parts of disodium hydrogen phosphate dodecahydrate, 0.1-10 parts of magnesium sulfate, 5.0-50 parts of trehalose and 1.0-50 parts of BSA.
3. The method of preparing a buffer according to any of claims 1-2, comprising the steps of:
(1) weighing Tris, KCl and NaH2PO4、MgSO4Adding trehalose and bovine serum albumin into purified water, stirring until the trehalose and the bovine serum albumin are completely dissolved, adjusting the pH value, and fixing the volume;
(2) filtering with filter membrane.
4. The method of claim 3, wherein the pH is between 6.8 and 8.5.
5. The method of preparing a buffer according to claim 3, wherein the pore size of the filter is 0.22 μm.
6. Use of a buffer according to any of claims 1-2 or a buffer prepared by a method according to any of claims 4-5 for the preparation of a reagent for the detection of neuron-specific enolase.
7. A kit for detecting neuron-specific enolase, wherein the kit comprises the buffer solution according to any one of claims 1 to 2 or the buffer solution prepared by the method for preparing the buffer solution according to any one of claims 3 to 5.
8. The neuron-specific enolase detection kit of claim 7, wherein the kit comprises: detecting reagent strips, quality control products and calibration products.
9. The neuron-specific enolase detection kit according to claim 8, wherein the quality control substance is prepared by a method comprising the following steps: using NSE recombinant protein as a raw material of a calibrator, dissolving the NSE recombinant protein with the buffer solution, and fully mixing to prepare the calibrator;
the preparation method of the calibrator comprises the following steps: the NSE recombinant protein is used as a raw material of a quality control product, dissolved by the buffer solution, and fully mixed to prepare the product.
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