CN113092786A - Buffer solution and application thereof in central nervous system specific protein detection kit - Google Patents
Buffer solution and application thereof in central nervous system specific protein detection kit Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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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
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- 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
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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
- 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
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4727—Calcium binding proteins, e.g. calmodulin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2814—Dementia; Cognitive disorders
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette
Abstract
The invention belongs to the technical field of immunodetection, and particularly relates to a buffer solution and application thereof in a central nervous specific protein detection kit. The buffer solution comprises the following components: 4-hydroxyethyl piperazine Ethanesulfonic acid (HEPES), sodium chloride (NaCl), Potassium chloride (KCl), magnesium sulfate (MgSO)4) Bovine Serum Albumin (BSA), trehalose, glycine, Casein (Casein), and glycerol. The buffer solution can be applied to a central nervous specific protein detection kit for detecting brain injury, has higher sensitivity, and can complete detection in a shorter timeAnd (6) measuring.
Description
Technical Field
The invention belongs to the technical field of immunodetection, and particularly relates to a buffer solution and application thereof in a central nervous specific protein detection kit.
Background
Accurate assessment of the severity and prognosis of central nervous system injury following Traumatic Brain Injury (TBI) is important for clinical treatment. At present, no accurate means is available for evaluating the severity of TBI, and prognosis is difficult to judge. Cognitive dysfunction, motor dysfunction, sensory dysfunction, emotional dysfunction and the like caused by central nervous system injury seriously affect the life quality of patients.
S100-beta is an acidic calcium-binding protein with a molecular weight of 21KD, which is mainly produced by astrocytes, forms a disulfide bond via cysteine residues, and is abundantly present in the central nervous system in a dimer-active form. The content of the TBI can reflect the degree of TBI and evaluate the prognosis. Clinically, the brain injury degree can be judged by detecting the expression of the S100-beta protein, and meanwhile, the prognosis condition of a patient can be evaluated.
Currently, the severity of traumatic brain injury and postoperative recovery conditions are clinically evaluated mostly through imaging observation and behavior scoring, and sufficient accuracy and sensitivity are lacked.
However, there are also a variety of methods for using the kit in the detection of brain injury in the prior art, for example, chinese patent application CN109266739A discloses a miRNA marker and a kit for detecting brain injury, where the marker is any one or more of miR-9, miR-124, miR-128a, and miR-128 b; the kit is used for detecting the content of the microRNA marker in cerebrospinal fluid; the marker and the kit can be used for auxiliary diagnosis of brain injury.
Chinese patent application CN107202890A discloses a rapid diagnostic reagent for mild and moderate brain injuries, a kit containing the reagent and a preparation and detection method of the kit, wherein the reagent and the kit comprise monoclonal capture antibodies respectively prepared from S100, GFAP and UCHL 1; the kit also comprises a detection antibody, and polyclonal antibodies respectively prepared from S100, GFAP and UCHL 1.
Chinese patent application CN109856403A discloses an S100-beta protein time-resolved fluorescence immunochromatography detection kit, which adopts lanthanide chelates to mark fluorescent microspheres, and then marks an S100-beta monoclonal antibody.
However, the use of the kit generally has the conditions of poor sensitivity, complex preparation process, long detection time, high cost and the like. The invention aims to solve the problems of poor sensitivity, slow detection time and the like of a brain injury detection kit in the prior art.
Disclosure of Invention
In order to overcome the technical problems, the invention provides a buffer solution which can be applied to a central nervous specific protein detection kit for detecting brain injury, has higher sensitivity and can complete detection in a shorter time.
In order to achieve the above purpose, the technical scheme provided by the invention is as follows:
a buffer comprising the following components: 4-hydroxyethyl piperazine Ethanesulfonic acid (HEPES), sodium chloride (NaCl), Potassium chloride (KCl), magnesium sulfate (MgSO)4) Bovine Serum Albumin (BSA), trehalose, glycine, Casein (Casein), glycerol;
preferably, the buffer solution comprises the following components in parts by weight: 1.0-10 parts of HEPES, 5-10 parts of sodium chloride, 0.5-5 parts of potassium chloride, 0.1-10 parts of magnesium sulfate, 4.0-30 parts of BSA, 10-100 parts of trehalose, 4.0-20 parts of glycine, 0.5-25 parts of Casein and 20-200 parts of glycerol.
Preferably, the buffer solution comprises the following components in parts by weight: 3.0-10 parts of HEPES, 9 parts of sodium chloride, 0.5-5 parts of potassium chloride, 0.1-10 parts of magnesium sulfate, 4.0-27 parts of BSA, 10-100 parts of trehalose, 4.0-18 parts of glycine, 0.5-25 parts of Casein and 20-200 parts of glycerol.
Preferably, the preparation method of the buffer solution comprises the following steps:
(1) weighing HEPES, NaCl, KCl and MgSO4The trehalose, the bovine serum albumin, the glycine, the casein and the glycerol are sequentially added into purified water and stirred until the trehalose, the bovine serum albumin, the glycine, the casein and the glycerol are completely dissolved, the pH value is adjusted, and the volume is fixed;
(2) filtering with a filter membrane to obtain the buffer solution.
Preferably, the pH is between 6.5 and 8.2.
Preferably, the filter membrane pore size is 0.22 μm.
The invention also aims to provide application of the buffer solution in preparing a central nervous system specific protein detection reagent.
The invention also aims to provide a central nervous specificity protein detection kit, and the kit 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 S100-beta 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 S100-beta 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 an alkaline phosphatase-labeled S100-beta antibody solution;
the reagent B is a biotin-labeled S100-beta antibody solution;
the reagent C is a streptavidin-labeled magnetic particle solution;
the cleaning solution is as follows: 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 S100-beta antibody conjugate is used as a raw material of a reagent A, and is dissolved by using a buffer solution A to prepare the reagent A.
Preferably, the preparation method of the reagent B comprises the following steps: the biotinylation S100-beta 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 preparation method of the enzyme-labeled S100-beta 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 protein purification analyzer and a Superdex200 preparative grade 2.6/60 gel column; eluting with buffer 2; and preparing the purified enzyme-labeled antibody conjugate.
Preferably, the method for preparing the biotinylated S100- β antibody conjugate comprises the steps of:
preparing a biotin solution by using PBS, shaking, adding the biotin solution into a desalting column after reaction, and adding an isometric PBS buffer solution to start elution; and preparing the purified biotinylated antibody conjugate.
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, reacting, and performing magnetic separation; the separated magnetic particles are resuspended by using a buffer solution 4, and the magnetic particles-streptavidin connectors after magnetic separation are fully mixed and reacted for 1 to 4 hours at room temperature; 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.
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 S100-beta 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 has a great influence on the detection sensitivity of the kit. Hair brushThe sensitivity of the kit provided by the invention to the detection of the central nervous specific protein detection kit reaches 50 picograms/milliliter (50 multiplied by 10)-12g/mL)。
(2) 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.
(3) The kit provided by the invention has higher precision, sensitivity and specificity. Compared with other methodologies, the method has the advantages of high sensitivity, wide linearity, short reaction time and high accuracy.
(4) The antigen buffer solution has better protection effect on S100-beta, can keep activity for 7 days at normal temperature (10-30 ℃) in a liquid state, can keep activity for 6 months at 2-8 ℃ and can keep activity for 12 months at-20 ℃. The activity can be maintained for 3 years under the freeze-drying condition.
Drawings
FIG. 1: a schematic view of an S100-beta 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: 3.0g of HEPES, 9g of sodium chloride, 2g of potassium chloride, 5g of magnesium sulfate, 27g of BSA, 40g of trehalose, 18g of glycine, 15g of Casein and 100ml of glycerol.
The preparation method of the buffer solution comprises the following steps:
(1) weighing HEPES, NaCl, KCl and MgSO4The trehalose, the bovine serum albumin, the glycine, the casein and the glycerol are sequentially added into purified water and stirred until the trehalose, the bovine serum albumin, the glycine, the casein and the glycerol are completely dissolved, the pH value is adjusted to 6.5, and the volume is fixed 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: 1.0g of HEPES, 5g of sodium chloride, 0.5g of potassium chloride, 10g of magnesium sulfate, 30g of BSA, 10g of trehalose, 20g of glycine, 0.5g of Casein and 20ml of glycerol.
The preparation method of the buffer solution comprises the following steps:
(1) weighing HEPES, NaCl, KCl and MgSO4The trehalose, the bovine serum albumin, the glycine, the casein and the glycerol are sequentially added into purified water and stirred until the trehalose, the bovine serum albumin, the glycine, the casein and the glycerol are completely dissolved, the pH value is adjusted to 8.2, and the volume is fixed 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: 10g of HEPES, 10g of sodium chloride, 5g of potassium chloride, 0.1g of magnesium sulfate, 4.0g of BSA, 100g of trehalose, 4.0g of glycine, 25g of Casein and 200ml of glycerol.
The preparation method of the buffer solution comprises the following steps:
(1) weighing HEPES, NaCl, KCl and MgSO4Trehalose, bovine serum albumin, glycine, casein and glycerol are sequentially added into purified water and stirred until the trehalose, bovine serum albumin, glycine, casein and glycerol are completely dissolved, the pH value is adjusted to 7.0, and the volume is fixed to 1000ml；
(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 HEPES, NaCl, KCl and MgSO4The trehalose, the bovine serum albumin, the glycine, the casein and the glycerol are sequentially added into purified water and stirred until the trehalose, the bovine serum albumin, the glycine, the casein and the glycerol are completely dissolved, the pH value is adjusted to 5.0, and the volume is fixed to 1000 ml;
(2) filtering with 0.22 μm filter membrane to obtain buffer solution. (recording as buffer D1)
Comparative example 2
Compared with example 1, the difference is only the amount of trehalose and glycine.
The buffer solution comprises the following components in parts by weight: 3.0g of HEPES, 9g of sodium chloride, 2g of potassium chloride, 5g of magnesium sulfate, 27g of BSA, 8g of trehalose, 50g of glycine, 15g of Casein and 100ml of glycerol.
The procedure for preparing the buffer was the same as in example 1. (recording as buffer D2)
Comparative example 3
The only difference compared to example 1 was the replacement of HEPES with PIPES (1, 4-piperazine disulfonic acid).
The buffer solution comprises the following components in parts by weight: 3.0g of PIPES, 9g of sodium chloride, 2g of potassium chloride, 5g of magnesium sulfate, 27g of BSA, 40g of trehalose, 18g of glycine, 15g of Casein and 100ml of glycerol.
The preparation method of the buffer solution comprises the following steps:
(1) weighing PIPES, NaCl, KCl and MgSO4The trehalose, the bovine serum albumin, the glycine, the casein and the glycerol are sequentially added into purified water and stirred until the trehalose, the bovine serum albumin, the glycine, the casein and the glycerol are completely dissolved, the pH value is adjusted to 6.5, and the volume is fixed 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 is the replacement of glycerol with propylene glycol.
The buffer solution comprises the following components in parts by weight: 3.0g of HEPES, 9g of sodium chloride, 2g of potassium chloride, 5g of magnesium sulfate, 27g of BSA, 40g of trehalose, 18g of glycine, 15g of Casein and 100ml of propylene glycol.
The preparation method of the buffer solution comprises the following steps:
(1) weighing HEPES, NaCl, KCl and MgSO4The trehalose, the bovine serum albumin, the glycine, the casein and the propylene glycol are sequentially added into purified water and stirred until the trehalose, the bovine serum albumin, the glycine, the casein and the propylene glycol are completely dissolved, the pH value is adjusted to 6.5, and the volume is fixed to 1000 ml;
(2) filtering with 0.22 μm filter membrane to obtain buffer solution. (recording as buffer D4)
2. Preparation of central nervous specific protein 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 central nervous specific protein assay kit included the following:
(1) buffer solution 1
15g of ethanolamine and 6.0g of NaCl are weighed and added into purified water to be stirred until the ethanolamine and the NaCl are completely dissolved, the PH value is adjusted to be between 7.3 and 7.6, the volume is adjusted to be 1000ml, and the mixture is filtered by a filter membrane with the diameter of 0.22 mu m.
(2) Buffer solution 2
75g of glycine was weighed into purified water and stirred until completely dissolved, and then the volume was set to 1000ml, and the solution was filtered through a 0.22 μm filter.
(3) Buffer solution 3
30.0g of MES was weighed out and added to purified water and stirred until completely dissolved, the pH was adjusted to 4.0-7.0 and the volume was adjusted to 1000ml, and then filtered through a 0.22 μm filter membrane.
(4) Buffer 4
Weighing 6.0g of Tris, 9.0g of NaCl and 15g of bovine serum albumin, adding into purified water, stirring until the Tris is completely dissolved, adjusting the pH value to be between 6.4 and 8.5, metering to 1000ml, and filtering by using a filter membrane of 0.22 mu m.
(5) Buffer solution 5
Weighing 4.0g of Tris and 9.0g of NaCl into purified water, stirring until the Tris and the NaCl are completely dissolved, adjusting the pH value to be between 7.0 and 7.8, metering to 1000ml, and filtering by using a filter membrane with the diameter of 0.22 mu m.
(6) Buffer 6
Weighing 7g of Tris, 9.0g of NaCl, 30g of bovine serum albumin, 50mL of bovine serum, 10mL of sheep serum and 10mL 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, fixing the volume to 1000mL, and filtering with a 0.22 mu m filter membrane.
2.2 kit Components
The S100-beta kit consists of a detection reagent strip, a calibrator, a quality control material and a two-dimensional code; the detection reagent strip is an integral body formed by a series of solutions and accessories, a sample can be independently detected, and a calibrator is prepared by S100-beta antigen with two concentrations and buffer solution and is used for calibrating a standard curve; the quality control product is prepared from S100-beta antigen with 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
| The main components of the kit | Loading capacity |
| |
10 strips |
| Quality control product | 200μL×1 |
| |
200μL×1 |
| Calibration article 2 | 200μL×1 |
| Box label two- |
1 is provided with |
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 an alkaline phosphatase-labeled S100-beta antibody solution, and the concentration is 0.8 mu g/mL; the reagent B is a biotin-labeled S100-beta antibody solution, and the concentration is 1.2 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
2.4 preparation of the kit:
2.4.1 preparation of calibrator and quality control Material
The S100-beta recombinant protein is used as a raw material of a calibrator, and is dissolved by a buffer solution (any one of the buffer solutions S1-S3 or D1-D4 provided by the invention), and the solution is fully mixed to prepare 2 calibrators with the concentrations of 1ng/mL and 100 ng/mL.
The S100-beta 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 S100-beta 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 ethanol, amine). 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 Superdex200 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 S100-beta antibody conjugate is used as a raw material of a reagent A, and the reagent A is prepared by fully and uniformly mixing the raw material with a buffer solution 6.
2.4.3 preparation of reagent B
(1) Preparation of biotinylated S100-beta antibody conjugates
A10 mM biotin solution (26.68. mu.L of 10mM biotin was added to 1.0mg of antibody) was added to the antibody solution in proportion, and after shaking and mixing, 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 biotinylation S100-beta antibody conjugate is used as a raw material of a reagent B, and the reagent B is prepared by fully and uniformly mixing the raw material 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 S100-beta. The S100-beta in the sample is combined with the S100-beta enzyme labeled antibody in the reagent A and the S100-beta 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 S100-beta 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 S100-beta 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
A solution (A) of a central nervous system-specific protein (S100-. beta.) at a concentration of about 20ng/mL (tolerance. + -. 10%) was added to a sample B at a concentration ranging from 0ng/mL to 0.1ng/mL, and the volume ratio of the added S100-. beta.antigen to the sample B was 1:9, and the recovery ratio R was calculated according to the formula (1) and was within the range of 85% to 115%.
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
And Standard Deviation (SD). Mean value of
The blank limit is obtained, and the result is less than or equal to 0.05 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) Evaluating lines for dependent variablesA linear regression equation. 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 0.1-150 ng/mL.
In the formula:
r- — -correlation coefficient;
xi-dilution ratio;
yi-mean value of individual sample measurements;
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
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%.
In the formula: s-standard deviation of sample test values;
3.2.5 batch to batch difference
Repeating the test of the quality control product 10 times by using the kits with 3 batch numbers respectively, and calculating 30 times of testsAverage of results
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 sample of S100-alpha was added to a sample without any analyte at a concentration of 200 ng/mL. The average value is measured for 3 times, and the measurement result is not higher than 0.1 ng/mL.
The effect of the buffers of examples 1-3 and comparative examples 1-3 when applied to the kit was examined according to the method of 3.2.1-3.2.6, and the results are shown in Table 3 below:
TABLE 3 Effect data
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 (10)
1. A buffer comprising the following components: HEPES, sodium chloride, potassium chloride, magnesium sulfate, BSA, trehalose, glycine, Casein, glycerol.
2. The buffer of claim 1, wherein the buffer comprises the following components in parts by weight: 1.0-10 parts of HEPES, 5-10 parts of sodium chloride, 0.5-5 parts of potassium chloride, 0.1-10 parts of magnesium sulfate, 4.0-30 parts of BSA, 10-100 parts of trehalose, 4.0-20 parts of glycine, 0.5-25 parts of Casein and 20-200 parts of glycerol.
3. The buffer of claim 1, wherein the buffer comprises the following components in parts by weight: 3.0-10 parts of HEPES, 9 parts of sodium chloride, 0.5-5 parts of potassium chloride, 0.1-10 parts of magnesium sulfate, 4.0-27 parts of BSA, 10-100 parts of trehalose, 4.0-18 parts of glycine, 0.5-25 parts of Casein and 20-200 parts of glycerol.
4. The method of preparing a buffer according to any of claims 1 to 3, comprising the steps of:
(1) weighing HEPES, NaCl, KCl and MgSO4The trehalose, the bovine serum albumin, the glycine, the casein and the glycerol are sequentially added into purified water and stirred until the trehalose, the bovine serum albumin, the glycine, the casein and the glycerol are completely dissolved, the pH value is adjusted, and the volume is fixed;
(2) filtering with a filter membrane to obtain the buffer solution.
5. The method of claim 4, wherein the pH is between 6.5 and 8.2.
6. The method of preparing the buffer according to claim 4, wherein the filter has a pore size of 0.22 μm.
7. Use of a buffer according to any of claims 1 to 3 or a buffer prepared by a method according to any of claims 4 to 6 for the preparation of a reagent for the detection of a central nervous specific protein.
8. A kit for detecting a central nervous specific protein, comprising the buffer according to any one of claims 1 to 3 or the buffer prepared by the method according to any one of claims 4 to 6.
9. The central nervous specific protein detection kit according to claim 8, wherein the kit comprises: detecting reagent strips, quality control products and calibration products.
10. The detection kit for a central nervous specificity protein according to claim 9, wherein the preparation method of the calibrator comprises: the S100-beta recombinant protein is used as a raw material of a calibrator, dissolved by the buffer solution, and fully mixed to prepare the calibrator;
the preparation method of the quality control product comprises the following steps: the S100-beta 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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