CN114994332A - Application of buffer solution in glial fibrillary acidic protein detection kit - Google Patents
Application of buffer solution in glial fibrillary acidic protein detection kit Download PDFInfo
- Publication number
- CN114994332A CN114994332A CN202210554502.3A CN202210554502A CN114994332A CN 114994332 A CN114994332 A CN 114994332A CN 202210554502 A CN202210554502 A CN 202210554502A CN 114994332 A CN114994332 A CN 114994332A
- Authority
- CN
- China
- Prior art keywords
- buffer
- solution
- buffer solution
- antibody
- serum albumin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- 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
- G01N33/68—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
-
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- 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
- G01N33/58—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)
-
- 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
- G01N2333/435—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
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides an application of a buffer solution in a detection kit for acidic protein in glial fibers, which relates to the technical field of detection, and the buffer solution specifically comprises: buffer 8 and buffer 9; the buffer solution 8 includes: tris (hydroxymethyl) aminomethane, sodium chloride, bovine serum albumin, sucrose, an enzyme stabilizer, a 1M magnesium chloride solution, a 0.1M zinc chloride solution, glycerol, and glycine; the buffer 9 includes: disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate, sodium chloride, bovine serum albumin, sucrose, cellulose salt or cellulose derivative, and gelatin. The buffer solution can be applied to a collagen fiber acidic protein detection kit, the kit is high in sensitivity and accuracy, the result is more objective, and the occurrence of misjudgment and missed judgment in detection can be avoided.
Description
Technical Field
The invention relates to the technical field of detection, and particularly relates to application of a buffer solution in a detection kit for acidic protein in glial fibers.
Background
Traumatic Brain Injury (TBI) is brain injury caused by external forces that disrupt normal brain function, resulting in impaired cognitive or physical performance in humans. Among all types of TBI, the most common sequelae are headache (47.9%) and memory abnormalities (42%) ("british journal of neurosurgery, 2018), with about three adults in need of psychological counseling or neurological treatment. Currently, CT scanning is the only objective, simple and reliable option widely used to help clinicians assess TBI. However, the correctness of the CT result is directly related to the accuracy of the CT device and the level of interpretation of the physician, and is a relatively subjective judgment method compared with other detection methods. CT scans with about 90% mild TBI (sometimes referred to as "concussion") were negative (Toth, 2015). Less than 1% of these patients require neurosurgical intervention (Papa L, 2012). In view of the very low percentage of CT scan positivity and the unnecessary imaging detection of these patients may increase the risk of radiation-induced carcinogenesis, it is of great clinical and strategic importance to find and develop other brain injury diagnostic methods to accurately determine the extent of craniocerebral injury and to assess prognosis.
Glial Fibrillary Acidic Protein (GFAP), a type iii intermediate filament protein, consists of 432 amino acids, is mainly distributed in astrocytes of the central nervous system, and is involved in cytoskeleton formation and maintenance of its tonicity strength. GFAP is a nervous system specific protein. It has important influence on the recovery of nervous system function in brain injury (arm, glial fiber acidic protein foundation and clinical research progress, 2009). In TBI, GFAP enters the blood through the blood brain barrier within 1 hour, resulting in a significant increase in serum GFAP. Has important significance for early diagnosis, differential diagnosis and prognosis judgment of TBI (Liuxia, biological characteristics and clinical research progress of glial fibrillary acidic protein 2015), and is mainly used for auxiliary diagnosis of brain trauma clinically.
So far, the detection method of the glial fibrillary acidic protein specifically discloses a magnetic microsphere electrochemiluminescence immunoassay kit for detecting the glial fibrillary acidic protein and a preparation method thereof as patent CN202011545701.5, and the invention mainly adopts ruthenium pyridine as a chemiluminescent marker, which has obvious advantages and is mainly shown in that: the stability is better, ruthenium is metal ion, the molecular weight is small, and the steric hindrance of the antibody is not influenced. Short production process, good repeatability and wide detection range. The electrochemical luminescence reaction is controllable, and the signal acquisition difficulty is reduced. Patent cn201811391978.x discloses a magnetic particle separation chemiluminescence immunoassay for detecting Glial Fibrillary Acidic Protein (GFAP), the kit composition comprising: the kit comprises a calibrator, a quality control product reagent A, a reagent B, a cleaning solution concentrate and a luminescent substrate solution, wherein the reagent A is a colloidal fiber acidic protein (GFAP) antibody solution containing magnetic particles with certain concentration for marking; the reagent B is a colloidal fiber acidic protein (GFAP) antibody solution containing a certain concentration of alkaline phosphatase label; the invention can greatly improve the signal intensity and the sensitivity of immunoreaction, so that a low-content substance can generate a strong chemiluminescent signal when carrying out immune combination, and a more accurate, precise, convenient, quick and simple method is provided for the detection of human Glial Fibrillary Acidic Protein (GFAP).
The invention aims to provide a buffer solution which is further applied to a glial fibrillary acidic protein detection kit, so that the detection has stronger objectivity and higher sensitivity and accuracy.
Disclosure of Invention
The invention provides the application of the buffer solution in the detection kit for the acidic protein in the glial fibrillary fibers aiming at the problems in the prior art, the buffer solution can be applied in the kit, the kit has high sensitivity and accuracy and objective results, and the occurrence of misjudgment and missed judgment in detection can be avoided.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention provides a buffer solution, which comprises: buffer 8 and buffer 9; the buffer solution 8 includes: tris (hydroxymethyl) aminomethane, sodium chloride, bovine serum albumin, sucrose, an enzyme stabilizer, a 1M magnesium chloride solution, a 0.1M zinc chloride solution, glycerol, and glycine; the buffer 9 includes: disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate, sodium chloride, bovine serum albumin, sucrose, cellulose salt or cellulose derivative, gelatin;
the cellulose derivative comprises one or more of methyl cellulose, croscarmellose sodium, carboxymethyl cellulose and hydroxyethyl cellulose.
Further, the cellulose salt is sodium carboxymethylcellulose.
Further, the buffer 8 includes: 12.0-15.0g/L of trihydroxymethyl aminomethane, 9.0g/L of sodium chloride, 1.0-50g/L of bovine serum albumin, 10.0-35g/L of cane sugar, 30-120mL/L of enzyme stabilizer, 0.5-5mL/L of 1M magnesium chloride solution, 0.5-5mL/L of 0.1M zinc chloride solution, 5.0-20.0g/L of glycerol and 5.0-40g/L of glycine.
Further, the buffer 9 includes: 5.6-5.9g/L disodium hydrogen phosphate dodecahydrate, 0.55-0.6g/L sodium dihydrogen phosphate, 9g/L sodium chloride, 1-5g/L bovine serum albumin, 80-140g/L sucrose, 1-5g/L cellulose salt or cellulose derivative, and 5-50g/L gelatin.
Furthermore, the glycerin in the buffer solution 8, the cellulose salt or cellulose derivative and the gelatin in the buffer solution 9 have a synergistic effect under a specific proportioning condition, so that the uniformity and the dispersibility of the magnetic particles in the buffer solution 9 during reaction can be further improved, and the method is favorable for the immune reaction.
Preferably, the weight ratio of the glycerol in the buffer solution 8 to the cellulose salt or cellulose derivative and the gelatin in the buffer solution 9 is 5-20: 1-5: 5-50.
The invention also provides application of the buffer solution in a glial fibrillary acidic protein detection kit.
Further, the kit comprises: detecting a reagent strip, a calibrator and a quality control product; the test reagent strip comprises: reagent A and reagent B;
the production of the reagent A comprises the following steps: uniformly mixing the enzyme-labeled GFAP antibody conjugate with a buffer solution 8 to obtain the enzyme-labeled GFAP antibody conjugate; the buffer solution 8 includes: tris (hydroxymethyl) aminomethane, sodium chloride, bovine serum albumin, sucrose, an enzyme stabilizer, a magnesium chloride solution, a zinc chloride solution, glycerol, glycine and water;
the production of the reagent B comprises: mixing the buffer solution 9 with the GFAP antibody magnetic particle conjugate to obtain the GFAP antibody magnetic particle conjugate; the buffer solution 9 comprises disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate, sodium chloride, bovine serum albumin, sucrose, cellulose salt or cellulose derivative, and gelatin.
Further, the production of the calibration material and the quality control material is as follows: and mixing and dissolving the buffer solution 7 and the GFAP recombinant protein, and diluting to prepare the recombinant protein.
Further, the preparation of the enzyme-labeled GFAP antibody conjugate specifically comprises the following steps:
(1) activation of the antibody: dissolving 2-iminothiolane hydrochloride by using a buffer solution, adding a GFAP antibody solution for activation, uniformly mixing and reacting; after the activation is stopped, adding a buffer solution 2, reacting, and collecting the activated GFAP antibody;
(2) activation of alkaline phosphatase: dissolving (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester in dimethylformamide, adding into alkaline phosphatase solution, and reacting after mixing; after stopping activation, adding a buffer solution 2, reacting, and collecting the activated alkaline phosphatase;
(3) linking of antibody and ALP: uniformly mixing the GFAP antibody obtained in the step I and the alkaline phosphatase obtained in the step II, and reacting;
(4) termination and purification of antibody conjugates: dissolving maleimide with dimethylformamide, diluting with buffer solution 1 to obtain maleimide solution, mixing the maleimide solution with the mixture obtained in the step (3), and reacting to obtain a mixed solution; dissolving ethanolamine by using buffer solution 1, adding the dissolved ethanolamine into the mixed solution, uniformly mixing to obtain a GFAP antibody conjugate to be purified, and concentrating and purifying the GFAP antibody conjugate to obtain an enzyme-labeled GFAP antibody conjugate;
the preparation method of the GFAP antibody magnetic particle conjugate comprises the following steps:
and cleaning the magnetic particles by using a buffer solution 4, then carrying out heavy suspension, adding a GFAP antibody, then adding the buffer solution 4, adding a buffer solution 5 after reaction, carrying out reaction, adding a buffer solution 6, carrying out cleaning, carrying out heavy suspension, carrying out reaction, cleaning by using a buffer solution 8, and carrying out heavy suspension to obtain the GFAP antibody magnetic particle conjugate.
Further, the buffer 7 includes tris, bovine serum albumin, and glycine.
Further, the buffer solution 1 comprises ethanolamine and sodium chloride; the buffer solution 2 comprises glycine; the buffer 4 comprises sodium tetraborate decahydrate; the buffer 5 comprises dipotassium hydrogen phosphate; the buffer solution 6 comprises tris (hydroxymethyl) aminomethane, sodium chloride, bovine serum albumin and tween 20.
In some embodiments, the specific formulations and methods of preparation of buffers 1-9 are as follows:
(ii) buffer solution 1
14.8-15.1g of ethanolamine and 5.8-6.0g of NaCl are weighed and added into a certain amount of purified water to be stirred until the ethanolamine and the NaCl are completely dissolved, the pH value is adjusted to be 7.3-7.6, and the volume is fixed to 1000 ml. Filtration was performed with a 0.22 μm filter.
TABLE 1 buffer 1 formulation
| Name of raw materials | Weighing volume |
| Ethanolamine | 14.8-15.1g |
| Sodium chloride | 5.8-6.0g |
| pH value | 7.3-7.6 |
| Purified water | The volume is up to 1000mL |
② buffer solution 2
75g of glycine is weighed and added into a certain amount of purified water to be stirred until the glycine is completely dissolved, and the volume is up to 1000 ml. Filtration was performed with a 0.22 μm filter.
TABLE 2 buffer 2 formulation
| Name of raw materials | Weighing volume |
| Glycine | 75g |
| Purified water | The volume is up to 1000mL |
203.3g of MgCl were weighed 2 ·6H 2 Adding O into a certain amount of purified water, stirring until the O is completely dissolved, and metering to 1000 ml. Filtration was performed with a 0.22 μm filter.
TABLE 3 buffer 3 formulation
| Name of raw materials | Weighing volume |
| Magnesium chloride hexahydrate | 203.3g |
| Purified water | The volume is up to 1000mL |
Weighing 7.0-10.0g of Na 2 B 4 O 7 ·10H 2 Adding O into a certain amount of purified water, stirring until the O is completely dissolved, adjusting the pH value to be 9.0-11.0, and metering to 1000 ml. Filtration was performed with a 0.22 μm filter.
TABLE 4 buffer 4 formulation
| Name of raw materials | Weighing volume |
| Sodium tetraborate decahydrate | 7.0-10.0g |
| pH value | 9.0-11.0 |
| Purified water | The volume is up to 1000mL |
Buffer solution 5
Weighing 470-530g of K 2 HPO 4 Adding into a certain amount of purified water, stirring to dissolve completelyAdjusting the pH value to 9.0-11.0 and fixing the volume to 1000 ml. Filtration was performed with a 0.22 μm filter.
TABLE 5 buffer 5 formulation
| Name of raw materials | Weighing volume |
| Dipotassium hydrogen phosphate | 470-530g |
| pH value | 9.0-11.0 |
| Purified water | The volume is up to 1000mL |
Weighing 7.5-8.0g of Tris, 9.0g of NaCl and 3.0-10.0g of bovine serum albumin, adding the Tris into a certain amount of purified water, stirring until the Tris is completely dissolved, weighing 5-20mL of Tween 20, adding the Tween 20 into the container, adjusting the pH value to be 7.3-7.8, and fixing the volume to 1000 mL. Filtration was performed with a 0.22 μm filter.
TABLE 6 buffer 6 formulation
| Name of raw materials | Weighing volume |
| Tris (hydroxymethyl) aminomethane | 7.5-8.0g |
| Sodium chloride | 9.0g |
| Bovine serum albumin | 3.0-10.0g |
| Tween 20 | 5-20mL |
| pH value | 7.3-7.8 |
| Purified water | The volume is up to 1000mL |
(iv) buffer solution 7
Weighing 12.0-15.0g of Tris, 5.0-50g of bovine serum albumin and 1.0-30g of glycine, adding into a certain amount of purified water, stirring until the mixture is completely dissolved, adjusting the pH value to 7.6-8.8, and fixing the volume to 1000 ml. Filtration was performed with a 0.22 μm filter.
TABLE 7 buffer 7 formulation
| Name of raw materials | Weighing amount |
| Tris (hydroxymethyl) aminomethane | 12.0-15.0g |
| Bovine serum albumin | 5.0-50g |
| Glycine | 1.0-30g |
| pH value | 7.6-8.8 |
| Purified water | The volume is up to 1000mL |
(iii) buffer solution 8
Weighing 12.0-15.0g of Tris, 9.0g of NaCl, 1.0-50g of bovine serum albumin, 10-35g of sucrose, 30-120mL of enzyme stabilizer, 0.5-5mL of 1M magnesium chloride solution, 0.5-5mL of 0.1M zinc chloride solution, 5.0-20.0g of glycerol and 5.0-40g of glycine, adding into a certain amount of purified water, stirring until complete dissolution is achieved, adjusting the pH value to be 7.5-9.0 and fixing the volume to 1000 mL. Filtration was performed with a 0.22 μm filter.
TABLE 8 buffer 8 formulation
Wherein, the 0.1M zinc chloride solution is the buffer solution 3.
Weighing 5.6-5.9g of Na 2 HPO 4 ·12H 2 O, 0.55-0.60g NaH 2 PO 4 9.0g of NaCl, 1.0-50g of bovine serum albumin, 80-140g of sucrose, 1.0-5.0g of cellulose salt or cellulose derivative and 5-50g of gelatin are added into a certain amount of purified water and stirred until the materials are completely dissolved, the pH value is adjusted to be 6.2-8.0, and the volume is fixed to 1000 ml. Using a 0.22 μm filterAnd (5) line filtering.
TABLE 9 buffer 9 formulation
| Name of raw materials | Weighing volume |
| Disodium hydrogen phosphate dodecahydrate | 5.6-5.9g |
| Sodium dihydrogen phosphate | 0.55-0.60g |
| Sodium chloride | 9.0g |
| Bovine serum albumin | 1.0-50g |
| Sucrose | 80-140g |
| Cellulose salts or cellulose derivatives | 1.0-5.0g |
| Gelatin | 5.0-50g |
| pH value | 6.2-8.0 |
| Purified water | The volume is up to 1000mL |
In some embodiments, the preparation of the enzyme-labeled GFAP antibody conjugate specifically comprises the steps of:
(1) activation of antibodies
The activation of the antibody needs to be carried out in a hundred thousand grade clean room. 4-8 mg of 2-iminothiolane hydrochloride (2IT) was weighed and dissolved in buffer 1 to 13.76 mg/mL. Adding the 2IT solution into the antibody solution for activation according to the molar ratio of the 2IT to the antibody of 15: 1-30: 1 (namely, adding 1mg of the antibody into 10-20 mu l of the 2IT solution). After shaking and mixing, the mixture was reacted at room temperature for 30 minutes. And stopping activation, adding the buffer solution 2 into the antibody solution according to the proportion that 1mg of the antibody is added into 5-20 mu l of the buffer solution 2, and reacting for 10min at room temperature. Excess 2IT was removed using a PD10 desalting column and the activated antibody was collected.
(2) Activation of alkaline phosphatase (ALP)
The activation of ALP is carried out in a hundred thousand grade clean room. 2-4 mg of (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester (SMCC) was weighed out and dissolved in Dimethylformamide (DMF) to a concentration of 6.69 mg/mlL. The SMCC solution is added into the ALP solution according to the molar ratio of the SMCC to the ALP of 15: 1-60: 1 (namely, 8.5-34.5 mu l of the SMCC solution is added into 1mg of the ALP). After shaking and mixing, the mixture was reacted at room temperature for 30 minutes. After the activation was terminated, buffer 2 was added to the ALP solution at a ratio of 1mg of ALP to 10 to 50. mu.l of buffer 2, and the mixture was reacted at room temperature for 10 min. Excess SMCC was removed using a PD10 desalting column and the ALP was collected after activation.
(3) Linking of antibody and ALP
The linking of the antibody and ALP was performed in a hundred thousand grade clean room. The ALP solution is added to the antibody solution in a mass ratio of 1: 2-1: 1 (i.e., 1.0mg of antibody is added to 1.0-2.0 mg of ALP). After shaking and mixing evenly, the mixture reacts for 12 to 18 hours at the temperature of between 2 and 8 ℃.
(4) Termination and purification of antibody conjugates
The termination and purification of the antibody conjugate is carried out in a hundred thousand grade clean room. 1-10mg of maleimide was weighed out and dissolved in DMF to 9.7 mg/mL. At a ratio of 1/10, the solution was diluted with buffer 1 to give a 0.97mg/mL maleimide solution. This solution was added in a ratio of 1mg of the mixture obtained in step (3) to 10. mu.l of 0.97mg/mL maleimide solution, and reacted at room temperature for 15 minutes. mu.L of ethanolamine was measured accurately and dissolved in buffer 1 to 100 mM. That is, 994. mu.L of buffer 1 was added to 6. mu.L of ethanolamine. Adding 10-50 mul of 100mM ethanolamine solution into 1mg of antibody, and shaking and mixing uniformly. And concentrating the antibody conjugate to be purified to 0.5-2 mg/mL by 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.
The preparation method of the GFAP antibody magnetic particle conjugate specifically comprises the following steps:
after washing the magnetic particles with buffer 4, the particles were resuspended to 5 mg/mL. Adding the antibody into the magnetic particle solution according to the mass ratio of the magnetic particles to the antibody of 100: 1-100: 10, adding the buffer solution 4 into the mixture according to the volume mass ratio of the buffer solution 4 to the magnetic particles of 100: 1-100: 10, and reacting for 10min at room temperature. Adding the buffer solution 5 into the mixture according to the volume mass ratio of the buffer solution 5 to the magnetic particles of 100: 1-1000: 10, and reacting for 16-24 hours at 37 ℃.
The magnetic particle conjugates were washed with buffer 6 and resuspended to 5 mg/mL. Reacting for 16-24 hours at 37 ℃. The magnetic particle conjugates were washed with buffer 8 and resuspended to 10 mg/mL. The product is antibody magnetic particle conjugate.
The technical effects obtained by the invention are as follows:
1. compared with an imaging detection means (mainly CT), the method can objectively reflect the real situation of a sample and reduce misjudgment and missed judgment caused by subjective judgment.
2. The magnetic particle chemiluminescence method used in the invention can enable the detection sensitivity to reach the picogram level (10) -12 g/mL), while CT relies on pixels to achieve higher resolution. Because the invention detects the brain injury specific marker, the detection window is much earlier than CT. In the case of CT negativity, that isNormal and mild TBI patients were effectively distinguished.
3. The invention uses a full-automatic instrument for detection, and accurate results can be obtained only by adding a serum sample for 30 minutes. The CT detection time is long, and generally needs to wait for 4 hours before the detection result can be obtained.
4. The invention uses the concentration value to judge, and the obtained result can know whether the patient is sick or not, thus having stronger objectivity. The CT detection needs doctors to read the films, and has strong subjective judgment according to the business level of the doctors, so that the missed judgment and the misjudgment are easily caused.
5. The invention improves the buffer system (buffer solution 9) of the magnetic particle carrier in the magnetic particle luminescence method. The magnetic particles are particles with a certain mass and mainly comprise FeO and Fe 2 O 3 The diameter of the magnetic particle antibody conjugate is 1-4 microns, the magnetic particle antibody conjugate is insoluble in water, and the magnetic particle antibody conjugate has certain hydrophilicity due to the connection of protein. Due to gravity, the magnetic particle antibody conjugate will sink rapidly in an aqueous medium. After a certain time, the mixture can be hardened even, and the difficulty of mixing uniformly again after hardening is high.
firstly, the magnetic particle antibody conjugate does not sink visible to naked eyes within 7 days in a normal temperature environment;
secondly, the reagent B keeps good fluidity in the normal temperature environment. The absorption quantity cannot be influenced when the instrument performs absorption operation, and the solution cannot have wall-hanging residue when being uniformly mixed.
And thirdly, the reagent B can be changed into gel under the environment of 2-8 ℃, and the suspension property of the magnetic particle antibody conjugate can be maintained within 6 months.
In addition, the glycerin in the buffer solution 8, the cellulose salt or cellulose derivative and the gelatin in the buffer solution 9 have a synergistic effect under the condition of a specific proportioning, so that the uniformity and the dispersibility of the magnetic particles in the buffer solution 9 during the reaction can be further improved, and the method is favorable for the immune reaction.
And fourthly, the test is carried out in the 13 th month, and although the magnetic particle antibody conjugate is completely settled, the magnetic particle antibody conjugate is extremely easy to mix evenly. Experimental data showed that the repeatability result CV at month 14 was less than 8% and the deviation from the accuracy result at month 0 was no more than 10%. The comparative group showed a repeatability CV of greater than 30% at month 14 with accuracy results deviating by more than 70% from month 0.
Drawings
FIG. 1 is a schematic view of a GFAP detection reagent strip, in which the portions corresponding to 1 to 15 are shown in detail in Table 11;
FIG. 2 is a reaction scheme of the present invention;
FIG. 3 is a process flow diagram of the present invention.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It should be noted that the enzyme stabilizer used in the present invention is specifically EAS04 of sirolimus, and the other raw materials are all common commercial products, and therefore the source thereof is not particularly limited.
1. The detection principle is as follows: the kit provided by the invention adopts a double-antibody sandwich method to measure the GFAP content. GFAP in the sample binds to the antibody in reagent A and the GFAP antibody in reagent B to form a "sandwich" structure. Upon washing, the luminescent substrate is enzymatically cleaved by the enzymes in the complex to form unstable excited state intermediates which emit photons when they return to the ground state. The number of photons generated is positively correlated with the concentration of GFAP in the sample.
2. Components
2.1 kit Components
The GFAP kit consists of a detection reagent strip, a calibrator, a quality control material and a two-dimensional code. The detection reagent strip is composed of a series of solutions and accessories into a whole and can independently detect a sample. The calibrator is prepared from GFAP antigen with two concentrations and buffer solution and is used for calibrating a standard curve; the quality control product is prepared from GFAP antigen with two concentrations and buffer solution; the standard curve of the current batch is recorded in the two-dimensional code.
TABLE 10 major Components of the kit
| The main components of the kit | Loading capacity |
| |
10 strips |
| Quality control product | 200μL×1 |
| |
200μL×1 |
| |
200μL×1 |
| Box label two- |
1 is provided with |
1.2 reagent strip Components
A schematic diagram of the GFAP detection reagent strip is shown in figure 1, and the detection reagent strip is composed of a reagent A, a reagent B, a cleaning solution, a luminescent substrate, a reading hole, an elution sleeve and a suction head. The reagent A is a GFAP antibody solution containing a certain concentration of alkaline phosphatase label; the reagent B is GFAP antibody solution containing magnetic particles with certain concentration for marking; 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 11 main Components of the test strips
3. Production process
3.1 production of calibrator and quality control Material
The GFAP recombinant protein was used as a starting material for a calibrator. The samples were dissolved in buffer 7 and mixed well to prepare 2 calibrators at concentrations of 20pg/mL and 160 pg/mL.
The GFAP recombinant protein is used as a raw material of a quality control product. Dissolving with buffer solution 7, and mixing to obtain quality control product. The concentration was 40 pg/mL.
3.2 production of reagent A
An enzyme-labeled GFAP antibody conjugate was used as a raw material of the reagent A. The mixture was mixed well with buffer 8 to prepare reagent A.
3.3 production of reagent B
The GFAP antibody magnetic particle conjugate was used as a raw material of reagent B. The mixture was mixed well with buffer 9 to prepare reagent B.
4. Examples and comparative examples
Example 1
A kit, comprising: detecting a reagent strip, a calibrator and a quality control product; the test reagent strip comprises: reagent A and reagent B; see the above components and the contents of the production process.
The preparation method of the enzyme-labeled GFAP antibody conjugate specifically comprises the following steps:
(1) activation of antibodies
The activation of the antibody needs to be carried out in a hundred thousand grade clean room. 5mg of 2-iminothiolane hydrochloride (2IT) was weighed out and dissolved in buffer 1 to 13.76 mg/mL. The 2IT solution is added into the antibody solution for activation according to the molar ratio of 2-IT to the antibody of 20: 1. After shaking and mixing, the mixture was reacted at room temperature for 30 minutes. After the termination of the activation, buffer 2 was added to the antibody solution in a ratio of 1mg of the antibody to 10. mu.l of buffer 2, and the reaction was carried out at room temperature for 10 min. Excess 2IT was removed using a PD10 desalting column and the activated antibody was collected.
(2) Activation of alkaline phosphatase (ALP)
The activation of ALP is carried out in a hundred thousand grade clean room. 2-4 mg of (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester (SMCC) was weighed out and dissolved in Dimethylformamide (DMF) to a concentration of 6.69 mg/mlL. The SMCC solution was added to the ALP solution at a molar ratio of SMCC to ALP of 40: 1. After shaking and mixing, the mixture was reacted at room temperature for 30 minutes. After termination of the activation, buffer 2 was added to the ALP solution in a ratio of 1mg of ALP to 30. mu.l of buffer 2, and the reaction was carried out at room temperature for 10 min. Excess SMCC was removed using a PD10 desalting column and the ALP was collected after activation.
(3) Linking of antibody and ALP
The linking of the antibody and ALP was carried out in a hundred thousand grade clean room. ALP solution was added to the antibody solution at a mass ratio of 1:2 of antibody to ALP (i.e., 1.0mg of antibody to 2.0mg of ALP). After shaking and mixing, the mixture was reacted at 48 ℃ for 15 hours.
(4) Termination and purification of antibody conjugates
The termination and purification of the antibody conjugate is carried out in a hundred thousand grade clean room. 5mg of maleimide was weighed out and dissolved in DMF to 9.7 mg/mL. At a ratio of 1/10, the solution was diluted with buffer 1 to give a 0.97mg/mL maleimide solution. This solution was added in a ratio of 1mg of the mixture obtained in step (3) to 10. mu.l of 0.97mg/mL maleimide solution, and reacted at room temperature for 15 minutes. mu.L of ethanolamine was measured accurately and dissolved in buffer 1 to 100 mM. That is, 994. mu.L of buffer 1 was added to 6. mu.L of ethanolamine. The solution was added in a ratio of 1mg of the antibody to 30. mu.l of 100mM ethanolamine solution, and mixed by shaking. The antibody conjugate to be purified was concentrated to 1mg/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.
The preparation method of the GFAP antibody magnetic particle conjugate specifically comprises the following steps:
after washing the magnetic particles with buffer 4, the particles were resuspended to 5 mg/mL. Adding the antibody into the magnetic particle solution according to the mass ratio of the magnetic particles to the antibody of 20:1, adding the buffer solution 4 into the mixture according to the volume mass ratio of the buffer solution 4 to the magnetic particles of 20:1, and reacting for 10min at room temperature. Buffer 5 is added into the mixture according to the volume mass ratio of the buffer 5 to the magnetic particles being 20:1, and the reaction is carried out for 20 hours in an environment at 37 ℃.
The magnetic particle conjugates were washed with buffer 6 and resuspended to 5 mg/mL. The reaction was carried out at 37 ℃ for 20 hours. The magnetic particle conjugates were washed with buffer 8 and resuspended to 10 mg/mL. The product is antibody magnetic particle conjugate.
The specific formulation and preparation method of the related buffer solution 1-9 are shown as follows:
(ii) buffer solution 1
15.0g of ethanolamine and 5.9g of NaCl are weighed and added into a certain amount of purified water to be stirred until the ethanolamine and the NaCl are completely dissolved, the pH value is adjusted to be 7.3-7.6, and the volume is fixed to 1000 ml. Filtration was performed with a 0.22 μm filter.
TABLE 12 buffer 1 formulation
| Name of raw materials | Weighing volume |
| Ethanolamine | 15.0g |
| Sodium chloride | 5.9g |
| pH value | 7.3-7.6 |
| Purified water | The volume is up to 1000mL |
② buffer solution 2
75g of glycine is weighed and added into a certain amount of purified water to be stirred until the glycine is completely dissolved, and the volume is up to 1000 ml. Filtration was performed with a 0.22 μm filter.
TABLE 13 buffer 2 formulation
| Name of raw materials | Weighing amount |
| Glycine | 75g |
| Purified water | The volume is up to 1000mL |
③ buffer solution 3
203.3g of MgCl were weighed 2 ·6H 2 O additionAdding into a certain amount of purified water, stirring to dissolve completely, and metering to 1000 ml. Filtration was performed with a 0.22 μm filter.
TABLE 14 buffer 3 formulation
Fourthly buffer solution 4
8.0g of Na are weighed 2 B 4 O 7 ·10H 2 Adding O into a certain amount of purified water, stirring until the O is completely dissolved, adjusting the pH value to be 9.0-11.0, and metering to 1000 ml. Filtration was performed with a 0.22 μm filter.
TABLE 15 buffer 4 formulation
| Name of raw materials | Weighing volume |
| Sodium tetraborate decahydrate | 8.0g |
| pH value | 9.0-11.0 |
| Purified water | The volume is up to 1000mL |
Buffer solution 5
500g of K are weighed 2 HPO 4 Adding into a certain amount of pureDissolving the materials in water, stirring until the materials are completely dissolved, adjusting the pH value to be 9.0-11.0, and fixing the volume to 1000 ml. Filtration was performed with a 0.22 μm filter.
TABLE 16 buffer 5 formulation
| Name of raw materials | Weighing volume |
| Dipotassium hydrogen phosphate | 500g |
| pH value | 9.0-11.0 |
| Purified water | The volume is up to 1000mL |
Weighing 7.8g of Tris, 9.0g of NaCl and 5.0g of bovine serum albumin, adding the Tris into a certain amount of purified water, stirring until the Tris is completely dissolved, weighing 12mL of Tween 20, adding the Tween into the container, adjusting the pH value to be 7.3-7.8, and fixing the volume to 1000 mL. Filtration was performed with a 0.22 μm filter.
TABLE 17 buffer 6 formulation
| Name of raw materials | Weighing amount |
| Tris (hydroxymethyl) aminomethane | 7.8g |
| Sodium chloride | 9.0g |
| Bovine serum albumin | 5.0g |
| Tween 20 | 12mL |
| pH value | 7.3-7.8 |
| Purified water | The volume is up to 1000mL |
(iv) buffer solution 7
Weighing 13.0g of Tris, 20.0g of bovine serum albumin and 8.0g of glycine, adding into a certain amount of purified water, stirring until the Tris, the bovine serum albumin and the glycine are completely dissolved, adjusting the pH value to 7.6-8.8 and fixing the volume to 1000 ml. Filtration was performed with a 0.22 μm filter.
TABLE 18 buffer 7 formulation
| Name of raw materials | Weighing amount |
| Tris (hydroxymethyl) aminomethane | 13.0g |
| Bovine serum albumin | 20.0g |
| Glycine | 8.0g |
| pH value | 7.6-8.8 |
| Purified water | The volume is up to 1000mL |
(iii) buffer solution 8
Weighing 13.0g of Tris, 9.0g of NaCl, 15.0g of bovine serum albumin, 20.0g of sucrose, 50mL of enzyme stabilizer, 3.5mL of 1M magnesium chloride solution, 3.5mL of 0.1M zinc chloride solution, 15.0g of glycerol and 26.8g of glycine, adding into a certain amount of purified water, stirring until complete dissolution, adjusting the pH value to be 7.5-9.0 and fixing the volume to 1000 mL. Filtration was performed with a 0.22 μm filter.
TABLE 19 buffer 8 formulation
| Name of raw materials | Weighing amount |
| Tris (hydroxymethyl) aminomethane | 13.0g |
| Sodium chloride | 9.0g |
| Bovine serum albumin | 15.0g |
| Sucrose | 20.0g |
| Enzyme stabilizer | 50mL |
| 1M magnesium chloride solution | 3.5mL |
| 0.1M Zinc chloride solution | 3.5mL |
| Glycerol | 15.0g |
| Glycine | 26.8g |
| pH value | 7.5-9.0 |
| Purified water | The volume is up to 1000mL |
Wherein, the 0.1M zinc chloride solution is the buffer solution 3.
5.8g of Na were weighed 2 HPO 4 ·12H 2 O, 0.58g of NaH 2 PO 4 9.0g of NaCl, 15.0g of bovine serum albumin, 100g of sucrose, 3.0g of cellulose salt or cellulose derivative and 15g of gelatin are added into a certain amount of purified water and stirred until the materials are completely dissolved, the pH value is adjusted to be 6.2-8.0, and the volume is fixed to 1000 ml. Filtration was performed with a 0.22 μm filter.
TABLE 20 buffer 9 formulation
| Name of raw materials | Weighing amount |
| Disodium hydrogen phosphate dodecahydrate | 5.8g |
| Sodium dihydrogen phosphate | 0.58g |
| Sodium chloride | 9.0g |
| Bovine serum albumin | 15.0g |
| Sucrose | 100g |
| Sodium carboxymethylcellulose | 3.0g |
| Gelatin | 15g |
| pH value | 6.2-8.0 |
| Purified water | The volume is up to 1000mL |
Example 2
The only difference from example 1 is that buffer 8 comprises: 12.0g/L of tris (hydroxymethyl) aminomethane, 9.0g/L of sodium chloride, 48.8g/L of bovine serum albumin, 10.0g/L of sucrose, 120mL/L of an enzyme stabilizer, 0.5mL/L of a 1M magnesium chloride solution, 5mL/L of a 0.1M zinc chloride solution, 5.0g/L of glycerol and 38g/L of glycine. The buffer 9 includes: 5.6g/L disodium hydrogen phosphate dodecahydrate, 0.6g/L sodium dihydrogen phosphate, 9g/L sodium chloride, 1.2g/L bovine serum albumin, 140g/L sucrose, 1g/L sodium carboxymethylcellulose and 50g/L gelatin.
Example 3
The only difference from example 1 is that buffer 8 comprises: 15.0g/L of tris (hydroxymethyl) aminomethane, 9.0g/L of sodium chloride, 1.0g/L of bovine serum albumin, 35g/L of sucrose, 30.5mL/L of an enzyme stabilizer, 5mL/L of a 1M magnesium chloride solution, 0.5mL/L of a 0.1M zinc chloride solution, 18g/L of glycerol and 5.5g/L of glycine. The buffer 9 includes: 5.9g/L disodium hydrogen phosphate dodecahydrate, 0.55g/L sodium dihydrogen phosphate, 9g/L sodium chloride, 4.8g/L bovine serum albumin, 84g/L sucrose, 4.8g/L sodium carboxymethylcellulose and 5.6g/L gelatin.
Comparative example 1
The only difference from example 1 is that glycerol in buffer 8 was replaced by equal amounts of mannitol and cellulose or cellulose derivative and gelatin in buffer 9 were replaced by equal amounts of bovine bone meal and xanthan gum.
Comparative example 2
The only difference from example 1 was that buffer 8 was replaced with tris buffer (pH 7.4).
5. Detection method
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:
s1 immune response: and adding the 30uL sample, the 50uL reagent B and the 50uL reagent A into the 11 th pore site in sequence, and reacting for 20min at 37 ℃.
S2, magnetic separation and cleaning: adding 300 mu L of cleaning solution into the No. 12 hole, sucking the mixture containing the magnetic particles out of the No. 11 hole by using magnetic force, and demagnetizing the No. 12 hole. After 2min of cleaning. Magnetic separation and washing were performed 1 time at positions 13 and 14, respectively.
S3 reading value: adding 150uL of luminescent substrate into the hole site No. 15, sucking the mixture containing magnetic particles out of the hole site No. 14 by magnetic force, and demagnetizing the hole site No. 15. The relative luminescence intensity (RLU) was measured using a self-developed instrument after the luminescence of the alkaline phosphatase-catalyzed luminescent substrate.
And S4, obtaining a GFAP concentration-luminous value standard curve according to the detected value of the calibrator. The curve was fitted using a four parameter Logistic equation.
And 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.
The reaction flow chart and the process flow chart of the invention are shown in detail in figures 2 and 3.
6. Detecting the index
5.1 accuracy
A Glial Fibrillary Acidic Protein (GFAP) solution (A) at a concentration of about 800pg/mL (tolerance. + -. 10%) was added to a sample B at a concentration ranging from 0pg/mL to 10pg/mL, at a volume ratio of 1:9 between the added GFAP antigen and the sample B, and the recovery R was calculated according to equation (1) and should be in the range of 85% to 115%.
In the formula:
r is the recovery rate;
v is the volume of the sample A liquid;
v0 is the volume of the serum sample B liquid;
c is the average value of 3 measurements after the serum sample B liquid is added into the A liquid;
c0 is the average value of 3 measurements of the serum sample B liquid;
CS is the concentration of sample A solution.
5.2 blank limit
The sample without any analyte was tested 20 times repeatedly to obtain concentration values of 20 test results,calculating the average value thereof
And Standard Deviation (SD). Mean value of
The blank limit is obtained, and the result is less than or equal to 5 pg/mL.
5.3 Linear region
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 calculated i ). In diluted concentration (x) i ) As independent variable, the mean value (y) of the results is determined i ) 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 more than or equal to 0.990 within a linear interval of 10-320 pg/mL.
In the formula:
r is a correlation coefficient;
x i is a dilution ratio;
y i determining a mean value for each sample;
5.4 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 was CV ≦ 10%.
In the formula: s is the standard deviation of the sample test values;
5.5 run-to-run 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
And standard deviation SD, and obtaining Coefficient of Variation (CV) according to formula (3), wherein the result CV is less than or equal to 15%.
5.6 difference between calibrator and quality control bottle
Detecting 10 bottles of calibrator (or quality control material) of the same batch for 1 time respectively, calculating according to formula (5), and determining the mean value of the results
And standard deviation (S1). Continuously measuring for 10 times with 1 bottle of the above 10 bottles of calibrators (or quality control products), and calculating the mean value of the results
And standard deviation (S2), calculating the CV% of repeatability between bottles according to the formulas (6) and (7), and the CV of the measurement result should be less than 10%.
(Note: when S1< S2, let CV bottle be 0)
In the formula:
s is the standard deviation.
7. The result of the detection
(1) Accuracy of
TABLE 21
| Examples of the invention | Percent recovery |
| Example 1 | 98 |
| Example 2 | 103 |
| Example 3 | 92 |
| Comparative example 1 | 67 |
| Comparative example 2 | 45 |
(2) Margin limit
TABLE 22
(3) Linear interval
TABLE 23
| Examples of the invention | Coefficient of correlation r |
| Example 1 | 0.9994 |
| Example 2 | 0.9916 |
| Example 3 | 0.9952 |
| Comparative example 1 | 0.8713 |
| Comparative example 2 | 0.8521 |
(4) Repeatability of
Watch 24
(5) Difference between batches
TABLE 25
(6) Difference between bottles
Watch 26
Finally, it should be noted that the above-mentioned contents are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, and that the simple modifications or equivalent substitutions of the technical solutions of the present invention by those of ordinary skill in the art can be made without departing from the spirit and scope of the technical solutions of the present invention.
Claims (9)
1. A buffer, characterized in that: the method comprises the following steps: buffer 8 and buffer 9; the buffer solution 8 includes: tris (hydroxymethyl) aminomethane, sodium chloride, bovine serum albumin, sucrose, an enzyme stabilizer, a 1M magnesium chloride solution, a 0.1M zinc chloride solution, glycerol, and glycine; the buffer 9 includes: disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate, sodium chloride, bovine serum albumin, sucrose, cellulose salt or cellulose derivative, and gelatin; the cellulose derivative comprises one or more of methyl cellulose, croscarmellose sodium, carboxymethyl cellulose and hydroxyethyl cellulose.
2. The buffer of claim 1, wherein: the buffer solution 8 includes: 12.0-15.0g/L of trihydroxymethyl aminomethane, 9.0g/L of sodium chloride, 1.0-50g/L of bovine serum albumin, 10.0-35g/L of cane sugar, 30-120mL/L of enzyme stabilizer, 0.5-5mL/L of 1M magnesium chloride solution, 0.5-5mL/L of 0.1M zinc chloride solution, 5.0-20.0g/L of glycerol and 5.0-40g/L of glycine.
3. The buffer of claim 1, wherein: the buffer solution 9 comprises: 5.6-5.9g/L disodium hydrogen phosphate dodecahydrate, 0.55-0.6g/L sodium dihydrogen phosphate, 9g/L sodium chloride, 1-5g/L bovine serum albumin, 80-140g/L sucrose, 1-5g/L cellulose salt or cellulose derivative, and 5-50g/L gelatin.
4. Use of the buffer of any of claims 1-3 in a glial fibrillary acidic protein assay kit.
5. Use according to claim 4, characterized in that: the kit comprises: detecting a reagent strip, a calibrator and a quality control product; the test reagent strip comprises: reagent A and reagent B;
the production of the reagent A comprises the following steps: uniformly mixing the enzyme-labeled GFAP antibody conjugate with the buffer solution 8 to obtain the enzyme-labeled GFAP antibody conjugate; the buffer solution 8 includes: tris (hydroxymethyl) aminomethane, sodium chloride, bovine serum albumin, sucrose, an enzyme stabilizer, a magnesium chloride solution, a zinc chloride solution, glycerol, glycine and water;
the production of the reagent B comprises: mixing the buffer solution 9 with the GFAP antibody magnetic particle conjugate to obtain the GFAP antibody magnetic particle conjugate; the buffer 9 comprises disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate, sodium chloride, bovine serum albumin, sucrose, cellulose salt or cellulose derivative, and gelatin.
6. Use according to claim 5, characterized in that: the production of the calibrator and the quality control product is as follows: and mixing and dissolving the buffer solution 7 and the GFAP recombinant protein, and diluting to prepare the recombinant protein.
7. Use according to claim 5, characterized in that: the preparation of the enzyme-labeled GFAP antibody conjugate specifically comprises the following steps:
(1) activation of the antibody: dissolving 2-iminosulfane hydrochloride by using a buffer solution, adding a GFAP antibody solution for activation, uniformly mixing, and reacting; after the activation is stopped, adding a buffer solution 2, reacting, and collecting the activated GFAP antibody;
(2) activation of alkaline phosphatase: dissolving (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester in dimethylformamide, adding into alkaline phosphatase solution, and reacting after mixing; after stopping activation, adding a buffer solution 2, reacting, and collecting the activated alkaline phosphatase;
(3) linking of antibody and ALP: uniformly mixing the GFAP antibody obtained in the step I and the alkaline phosphatase obtained in the step II, and reacting to obtain a mixture;
(4) termination and purification of antibody conjugates: dissolving maleimide with dimethylformamide, diluting with buffer solution 1 to obtain maleimide solution, mixing the maleimide solution with the mixture obtained in the step (3), and reacting to obtain a mixed solution; dissolving ethanolamine by using buffer solution 1, adding the dissolved ethanolamine into the mixed solution, uniformly mixing to obtain a GFAP antibody conjugate to be purified, and concentrating and purifying the GFAP antibody conjugate to obtain an enzyme-labeled GFAP antibody conjugate;
the preparation method of the GFAP antibody magnetic particle conjugate specifically comprises the following steps:
and cleaning the magnetic particles by using a buffer solution 4, then carrying out heavy suspension, adding a GFAP antibody, then adding the buffer solution 4, adding a buffer solution 5 after reaction, carrying out reaction, adding a buffer solution 6, carrying out cleaning, carrying out heavy suspension, carrying out reaction, cleaning by using a buffer solution 8, and carrying out heavy suspension to obtain the GFAP antibody magnetic particle conjugate.
8. Use according to claim 6, characterized in that: the buffer solution 7 comprises tris (hydroxymethyl) aminomethane, bovine serum albumin and glycine.
9. Use according to claim 7, characterized in that: the buffer solution 1 comprises ethanolamine and sodium chloride; the buffer solution 2 comprises glycine; the buffer 3 comprises magnesium chloride hexahydrate; the buffer 4 comprises sodium tetraborate decahydrate; the buffer 5 comprises dipotassium hydrogen phosphate; the buffer solution 6 comprises tris (hydroxymethyl) aminomethane, sodium chloride, bovine serum albumin and tween 20.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210554502.3A CN114994332A (en) | 2022-05-20 | 2022-05-20 | Application of buffer solution in glial fibrillary acidic protein detection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210554502.3A CN114994332A (en) | 2022-05-20 | 2022-05-20 | Application of buffer solution in glial fibrillary acidic protein detection kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114994332A true CN114994332A (en) | 2022-09-02 |
Family
ID=83026555
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210554502.3A Pending CN114994332A (en) | 2022-05-20 | 2022-05-20 | Application of buffer solution in glial fibrillary acidic protein detection kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114994332A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116990513A (en) * | 2023-09-26 | 2023-11-03 | 北京美联泰科生物技术有限公司 | Chemiluminescent detection method of pepsinogen 1 |
| CN117330765A (en) * | 2023-09-26 | 2024-01-02 | 北京美联泰科生物技术有限公司 | Chemiluminescent detection method for gastrin 17 |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005040207A1 (en) * | 2003-10-20 | 2005-05-06 | F. Hoffmann-La Roche Ag | Tyrosine phosphorylation of cdk inhibitor proteins of the cip/kip family |
| CN108373494A (en) * | 2018-02-27 | 2018-08-07 | 武汉伊艾博科技有限公司 | A kind of protection technique preventing Proteolysis of recombinant proteins |
| CN109521004A (en) * | 2018-11-21 | 2019-03-26 | 北京利德曼生化股份有限公司 | For detecting the magnetic microparticle separating chemiluminescence immunoassay of glial fibrillary acid protein (GFAP) |
| CN112485445A (en) * | 2020-11-16 | 2021-03-12 | 北京美联泰科生物技术有限公司 | Kit for quantitatively detecting GFAP and application thereof |
| CN112630430A (en) * | 2020-11-16 | 2021-04-09 | 北京美联泰科生物技术有限公司 | Kit for quantitatively detecting UCHL-1 and application thereof |
| CN112710825A (en) * | 2020-12-15 | 2021-04-27 | 深圳天辰医疗科技有限公司 | Alkaline phosphatase enzyme labeling buffer solution and alkaline phosphatase enzyme labeling reagent |
| CN113203867A (en) * | 2021-04-28 | 2021-08-03 | 北京美联泰科生物技术有限公司 | Buffer solution suitable for insulin detection kit |
| US20210363562A1 (en) * | 2020-05-19 | 2021-11-25 | Kura Biotech, Inc. | One-step formulation for all-in-one hydrolysis solution |
-
2022
- 2022-05-20 CN CN202210554502.3A patent/CN114994332A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005040207A1 (en) * | 2003-10-20 | 2005-05-06 | F. Hoffmann-La Roche Ag | Tyrosine phosphorylation of cdk inhibitor proteins of the cip/kip family |
| CN108373494A (en) * | 2018-02-27 | 2018-08-07 | 武汉伊艾博科技有限公司 | A kind of protection technique preventing Proteolysis of recombinant proteins |
| CN109521004A (en) * | 2018-11-21 | 2019-03-26 | 北京利德曼生化股份有限公司 | For detecting the magnetic microparticle separating chemiluminescence immunoassay of glial fibrillary acid protein (GFAP) |
| US20210363562A1 (en) * | 2020-05-19 | 2021-11-25 | Kura Biotech, Inc. | One-step formulation for all-in-one hydrolysis solution |
| CN112485445A (en) * | 2020-11-16 | 2021-03-12 | 北京美联泰科生物技术有限公司 | Kit for quantitatively detecting GFAP and application thereof |
| CN112630430A (en) * | 2020-11-16 | 2021-04-09 | 北京美联泰科生物技术有限公司 | Kit for quantitatively detecting UCHL-1 and application thereof |
| CN112710825A (en) * | 2020-12-15 | 2021-04-27 | 深圳天辰医疗科技有限公司 | Alkaline phosphatase enzyme labeling buffer solution and alkaline phosphatase enzyme labeling reagent |
| CN113203867A (en) * | 2021-04-28 | 2021-08-03 | 北京美联泰科生物技术有限公司 | Buffer solution suitable for insulin detection kit |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116990513A (en) * | 2023-09-26 | 2023-11-03 | 北京美联泰科生物技术有限公司 | Chemiluminescent detection method of pepsinogen 1 |
| CN116990513B (en) * | 2023-09-26 | 2023-12-26 | 北京美联泰科生物技术有限公司 | Chemiluminescent detection method of pepsinogen 1 |
| CN117330765A (en) * | 2023-09-26 | 2024-01-02 | 北京美联泰科生物技术有限公司 | Chemiluminescent detection method for gastrin 17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112630430B (en) | Kit for quantitatively detecting UCHL-1 and application thereof | |
| CN112485445B (en) | Kit for quantitatively detecting GFAP and application thereof | |
| CN114994332A (en) | Application of buffer solution in glial fibrillary acidic protein detection kit | |
| CN111735965B (en) | Myocardial troponin I detection reagent, preparation method and myocardial troponin I detection kit | |
| CN109187973B (en) | Chemiluminescence immunoassay kit for detecting neuron specific enolase | |
| CN109580955A (en) | For detecting the magnetic microparticle separating chemiluminescence immunoassay of Tau albumen (TAU) | |
| CN113092786B (en) | Buffer solution and application thereof in central nervous system specific protein detection kit | |
| CN104634980A (en) | Cardiac troponin I (cTn I) hypersensitive detection kit and hypersensitive detection method | |
| ES2761427T3 (en) | Procedures to reduce interference | |
| CN109580956A (en) | For detecting the magnetic microparticle separating chemiluminescence immunoassay of amyloid beta (A β) | |
| CN108398563A (en) | A kind of double labelling magnetic bead time-resolved fluoroimmunoassay quantitatively detects cTnI and H-FABP kits | |
| CN109270272A (en) | A kind of kit and preparation method thereof detecting the citrullinated vimentin antibodies of anti-saltant type | |
| CN116990513B (en) | Chemiluminescent detection method of pepsinogen 1 | |
| CN202916286U (en) | Latex enhanced turbidimetric immunoassay kit for quantitatively detecting procalcitonin (PCT) | |
| CN102253215A (en) | Theophylline homogeneous enzyme immunoassay kit and preparation method thereof | |
| CN113933521A (en) | Magnetic particle chemiluminescence detection kit and preparation method and application thereof | |
| CN108717117A (en) | A kind of vancomycin immunologic function test reagent and its preparation and detection method | |
| CN114966055B (en) | Application of buffer solution in colloidal fiber acidic protein detection kit | |
| CN114624448B (en) | Kit for detecting acidic protein in glial fibers | |
| CN104215778B (en) | Crosslinked magnetic particle of a kind of C peptide monoclonal antibody and preparation method thereof and the detection kit that comprises it | |
| CN113702647B (en) | Human growth differentiation factor 15 instant detection kit, preparation method and application thereof | |
| CN115166229A (en) | Direct chemiluminescence method kit for determining content of neurofilament light chain protein and preparation method thereof | |
| CN117405890B (en) | Chemiluminescent detection kit and method for pepsinogen II | |
| CN115166244A (en) | Application of buffer solution in PGP9.5 detection kit | |
| CN111381046A (en) | Calprotectin chemiluminescence immunoassay kit and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |