CN111190003A - Retinol binding protein detection kit and preparation method thereof - Google Patents
Retinol binding protein detection kit and preparation method thereof Download PDFInfo
- Publication number
- CN111190003A CN111190003A CN202010233799.4A CN202010233799A CN111190003A CN 111190003 A CN111190003 A CN 111190003A CN 202010233799 A CN202010233799 A CN 202010233799A CN 111190003 A CN111190003 A CN 111190003A
- Authority
- CN
- China
- Prior art keywords
- reagent
- buffer solution
- binding protein
- retinol binding
- detection kit
- 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
- 102000029752 retinol binding Human genes 0.000 title claims abstract description 63
- 108091000053 retinol binding Proteins 0.000 title claims abstract description 63
- 238000002331 protein detection Methods 0.000 title claims abstract description 22
- 238000002360 preparation method Methods 0.000 title abstract description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 87
- 239000007853 buffer solution Substances 0.000 claims abstract description 39
- 239000003755 preservative agent Substances 0.000 claims abstract description 17
- 239000003381 stabilizer Substances 0.000 claims abstract description 17
- 229910017053 inorganic salt Inorganic materials 0.000 claims abstract description 16
- 150000002500 ions Chemical class 0.000 claims abstract description 16
- 230000002335 preservative effect Effects 0.000 claims abstract description 16
- 239000000375 suspending agent Substances 0.000 claims abstract description 9
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical group [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 38
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 26
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 24
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 18
- 229940098773 bovine serum albumin Drugs 0.000 claims description 18
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 13
- 239000011780 sodium chloride Substances 0.000 claims description 13
- 239000008118 PEG 6000 Substances 0.000 claims description 12
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 claims description 12
- HDSBZMRLPLPFLQ-UHFFFAOYSA-N Propylene glycol alginate Chemical group OC1C(O)C(OC)OC(C(O)=O)C1OC1C(O)C(O)C(C)C(C(=O)OCC(C)O)O1 HDSBZMRLPLPFLQ-UHFFFAOYSA-N 0.000 claims description 12
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical group OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 claims description 12
- 239000001103 potassium chloride Substances 0.000 claims description 12
- 239000000770 propane-1,2-diol alginate Substances 0.000 claims description 12
- 235000010409 propane-1,2-diol alginate Nutrition 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 10
- 239000004471 Glycine Substances 0.000 claims description 8
- 239000001913 cellulose Substances 0.000 claims description 8
- 229920002678 cellulose Polymers 0.000 claims description 8
- 239000012528 membrane Substances 0.000 claims description 8
- 239000007987 MES buffer Substances 0.000 claims description 7
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 239000007993 MOPS buffer Substances 0.000 claims description 4
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 4
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 4
- 229910021538 borax Inorganic materials 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 239000004328 sodium tetraborate Substances 0.000 claims description 4
- 235000010339 sodium tetraborate Nutrition 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 2
- QYYMDNHUJFIDDQ-UHFFFAOYSA-N 5-chloro-2-methyl-1,2-thiazol-3-one;2-methyl-1,2-thiazol-3-one Chemical group CN1SC=CC1=O.CN1SC(Cl)=CC1=O QYYMDNHUJFIDDQ-UHFFFAOYSA-N 0.000 claims description 2
- 229920002307 Dextran Chemical group 0.000 claims description 2
- 239000006173 Good's buffer Substances 0.000 claims description 2
- 239000007832 Na2SO4 Substances 0.000 claims description 2
- 229920002594 Polyethylene Glycol 8000 Chemical group 0.000 claims description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 2
- 239000001110 calcium chloride Substances 0.000 claims description 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 2
- 229910052939 potassium sulfate Inorganic materials 0.000 claims description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 2
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 2
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical group [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 claims description 2
- 229940033663 thimerosal Drugs 0.000 claims description 2
- 238000002731 protein assay Methods 0.000 claims 1
- 238000001514 detection method Methods 0.000 description 16
- 238000002835 absorbance Methods 0.000 description 15
- 239000000523 sample Substances 0.000 description 14
- 239000000872 buffer Substances 0.000 description 8
- 230000008859 change Effects 0.000 description 7
- 238000005259 measurement Methods 0.000 description 7
- 230000035945 sensitivity Effects 0.000 description 7
- 238000003908 quality control method Methods 0.000 description 6
- 238000003556 assay Methods 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- FPIPGXGPPPQFEQ-OVSJKPMPSA-N all-trans-retinol Chemical compound OC\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-OVSJKPMPSA-N 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000001124 body fluid Anatomy 0.000 description 2
- 239000010839 body fluid Substances 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000004064 dysfunction Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 210000001525 retina Anatomy 0.000 description 2
- 238000004879 turbidimetry Methods 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- 229940045997 vitamin a Drugs 0.000 description 2
- FPIPGXGPPPQFEQ-UHFFFAOYSA-N 13-cis retinol Natural products OCC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-UHFFFAOYSA-N 0.000 description 1
- 241000220479 Acacia Species 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 235000010643 Leucaena leucocephala Nutrition 0.000 description 1
- 102000019298 Lipocalin Human genes 0.000 description 1
- 108050006654 Lipocalin Proteins 0.000 description 1
- 229940100609 all-trans-retinol Drugs 0.000 description 1
- 239000011717 all-trans-retinol Substances 0.000 description 1
- 235000019169 all-trans-retinol Nutrition 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 230000024924 glomerular filtration Effects 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000000760 immunoelectrophoresis Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000003002 pH adjusting agent Substances 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 229920003169 water-soluble polymer Polymers 0.000 description 1
- 239000000230 xanthan gum Substances 0.000 description 1
- 235000010493 xanthan gum Nutrition 0.000 description 1
- 229920001285 xanthan gum Polymers 0.000 description 1
- 229940082509 xanthan gum Drugs 0.000 description 1
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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (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)
- Peptides Or Proteins (AREA)
Abstract
A retinol binding protein detection kit and a preparation method thereof comprise a reagent R1 and a reagent R2 which are independent of each other, wherein the reagent R1 comprises a buffer solution, inorganic salt ions, an accelerator, a stabilizer and a preservative, and the reagent R2 comprises a buffer solution, inorganic salt ions, a preservative, a stabilizer, a suspending agent and an anti-human retinol binding protein antibody.
Description
Technical Field
The invention belongs to the field of medical immunization in-vitro diagnosis, and particularly relates to a retinol binding protein detection kit and a preparation method thereof.
Background
Retinol binding protein is a small molecule lipocalin (molecular weight 21000 daltons) synthesized in the liver. Retinol binding protein, when synthesized in the liver, transports all-trans retinol (vitamin a) to epithelial tissues, providing vitamin a to the retina. Research shows that RBP exists not only in retina, but also in human blood, urine and other body fluids, and the content of RBP is closely related to the health condition of the kidney and the viscera. Currently, serum RBP detection is an early indicator for the assessment of liver function impairment and glomerular filtration dysfunction, while urine RBP detection is an early marker for the assessment of proximal tubular dysfunction. At present, enzyme-linked immunosorbent assay, immunotransmission turbidimetry, radioimmunoassay, immunoelectrophoresis and the like are used as detection methods of retinol binding protein. The immunoturbidimetry utilizes a full-automatic biochemical analyzer for detection, has no radioactive source, is relatively environment-friendly, has good stability, precision and accuracy, and is widely applied to clinical routine detection. The transmission immunoturbidimetry and the scattering immunoturbidimetry can be used for measuring the RBP content on a full-automatic biochemical analyzer, the detection method is simple and rapid, the accuracy of the detection result is high, the method has certain resistance to common interference factors such as hemolysis, yellow pox and blood fat to a certain degree, can meet the requirement of clinical detection, and is the most widely used method for measuring the retinol binding protein at present.
However, the concentration of RBP in body fluid is low, so that the specificity and sensitivity are poor, and the wide application in clinic is inconvenient. The detection principle of the immunoturbidimetry is that an insoluble antigen-antibody complex is generated through reaction and generates certain turbidity, and the turbidity reflects the content of the retinol binding protein in a sample, so that the defect of low sensitivity inevitably occurs in the conventional retinol binding protein kit.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a retinol binding protein detection kit which can be directly applied to a full-automatic biochemical analyzer, has strong result accuracy, high sensitivity and convenient use and is convenient for large-scale popularization.
A retinol binding protein detection kit comprising a reagent R1 and a reagent R2 independently from each other;
the reagent R1 comprises the following components in parts by weight:
buffer solution 20-200 mmol/L
10-40 g/L of accelerator
5-15 g/L stabilizer
0.5-1.5 g/L of preservative
The mass percentage content of the inorganic salt ions is 0.5-10% of the total mass;
the reagent R2 comprises the following components in parts by weight:
buffer solution 20-200 mmol/L
0.5-1.5 g/L of preservative
5-15 g/L stabilizer
10-50 mmol/L suspending agent
0.5-3.0 g/L of anti-human retinol binding protein antibody
The mass percentage content of the inorganic salt ions is 0.5-10% of the total mass.
The preservative in the reagent R1 and the preservative in the reagent R2 refer to reagents which can inhibit bacterial and microbial contamination in the reagents, and have the effects of preserving and sterilizing the reagents. The stabilizer in the reagent R1 and the stabilizer in the reagent R2 can maintain the activity of the anti-human retinol binding protein antibody.
Preferably, the buffer solution in the reagent R1 is selected from any one or a combination of more of MES buffer solution, Tris-HCl buffer solution, MOPS buffer solution, PBS buffer solution, glycine buffer solution and borax buffer solution, and the pH value is 7.0-9.0;
the buffer solution in the reagent R2 is selected from one or more of PBS buffer solution, borax buffer solution, glycine buffer solution, Hepes buffer solution, GOODS buffer solution and MOPS buffer solution, and the pH value is 7.0-9.0.
Further, the buffer solution in the reagent R1 is MES buffer solution; the buffer solution in the reagent R2 is PBS buffer solution.
The buffer solution in the reagent R1 and the buffer solution in the reagent R2 may be pH-adjusted by using various pH adjusting agents known in the art.
Preferably, the inorganic salt ion in the reagent R1 is selected from KCl, NaCl, MgCl2、CaCl2Any one or a combination of more of;
the inorganic salt ions in the reagent R2 are selected from NaCl, KCl and Na2CO3、Na2SO4、K2SO4Any one or a combination of more of them.
Furthermore, the inorganic salt ions in the reagent R1 and the inorganic salt ions in the reagent R2 are NaCl, so that the system can be kept stable for a long time, and the method has the advantages of low price and readily available raw materials.
Preferably, the preservative in the reagent R1 and the reagent R2 is sodium azide, thimerosal or ProClin 300.
Furthermore, the preservatives in the reagent R1 and the reagent R2 are sodium azide, so that the preservative has excellent preservative and bactericidal properties.
Preferably, the accelerator in the reagent R1 is PEG6000, PEG8000 or dextran.
Further, the accelerator in the reagent R1 is PEG6000, and since PEG6000 is a nonionic water-soluble polymer and has relatively high solubility in water, the viscosity of the reagent R1 can be adjusted, and the binding of antigen and antibody molecules into a complex is promoted.
Preferably, the stabilizer in the reagent R1 and the stabilizer in the reagent R2 are both bovine serum albumin, and the stabilizers can protect the activity of the anti-human retinol binding protein.
Preferably, the suspending agent in the reagent R2 is acacia, glycerol, xanthan gum or propylene glycol alginate.
Furthermore, the suspending agent in the reagent R2 is propylene glycol alginate, so that the antigen-antibody complex can be suspended after the sample is added, and the reaction accuracy is improved.
Preferably, the reagent R1 consists of MES buffer, NaCl, PEG6000, bovine serum albumin and sodium azide;
the reagent R2 consists of a PBS buffer solution, NaCl, sodium azide, bovine serum albumin, propylene glycol alginate and an anti-human retinol binding protein antibody;
the reagent R1 comprises the following components in parts by weight:
reagent R1:
MES buffer 50mmol/L
NaCl 0.5％
PEG6000 25g/L
Bovine serum albumin 10g/L
1g/L of sodium azide;
the reagent R2 comprises the following components in parts by weight:
PBS buffer 50mmol/L
NaCl 0.5％
Sodium azide 1g/L
Bovine serum albumin 10g/L
Propylene glycol alginate 30mmol/L
Anti-human retinol binding protein antibody 3.0g/L
A preparation method of a retinol binding protein detection kit comprises the following steps:
the method comprises the following steps: sequentially adding 0.5-10% of inorganic salt ions, 10-40 g/L of accelerator, 5-15 g/L of stabilizer and 0.5-1.5 g/L of preservative into 20-200 mmol/L of buffer solution, stirring and mixing uniformly, and filtering through a 0.22 micron mixed cellulose membrane to obtain a reagent R1;
step two: 0.5-10% of inorganic salt ions, 10-50 mmol/L of suspending agent, 5-15 g/L of stabilizing agent, 0.5-1.5 g/L of preservative and 0.5-3.0 g/L of anti-human retinol binding protein antibody are sequentially added into 20-200 mmol/L of buffer solution, uniformly stirred and mixed, and filtered through a 0.22 micron mixed cellulose membrane to obtain a reagent R2.
Compared with the prior art, the invention has the beneficial effects that:
1. the kit has the advantages of simple and rapid detection, high sensitivity, good accuracy and precision and low production cost.
2. The retinol binding protein detection method adopted by the invention is an immune transmission turbidimetry method, so that the retinol binding protein detection is more economical, convenient and rapid, and the method is suitable for automatic biochemical analyzers in most hospitals, and particularly can realize rapid quantitative detection in emergency treatment.
3. According to the invention, propylene glycol alginate is innovatively added into the reagent R2 as a suspending agent, so that an antigen-antibody complex generated during reaction after a sample is added is suspended and uniformly dispersed, and can be detected even at a low concentration, and the detection sensitivity of the reagent is greatly improved.
Detailed Description
Example 1
A retinol binding protein detection kit comprising a reagent R1 and a reagent R2 independently from each other;
the reagent R1 comprises the following components in parts by weight:
MES buffer 50mmol/L
NaCl 0.5％
PEG6000 25g/L
Bovine serum albumin 10g/L
1g/L of sodium azide;
the reagent R2 comprises the following components in parts by weight:
PBS buffer 50mmol/L
NaCl 0.5％
Sodium azide 1g/L
Bovine serum albumin 10g/L
Propylene glycol alginate 30mmol/L
Anti-human retinol binding protein antibody 3.0g/L
Example 2
A retinol binding protein detection kit comprising a reagent R1 and a reagent R2 independently from each other;
the reagent R1 comprises the following components in parts by weight:
Tris-HCl buffer 75mmol/L
KCl 0.5％
PEG6000 25g/L
Bovine serum albumin 10g/L
1g/L of sodium azide;
the reagent R2 comprises the following components in parts by weight:
glycine buffer 50mmol/L
KCl 0.5％
Sodium azide 1g/L
Bovine serum albumin 10g/L
Propylene glycol alginate 20mmol/L
Anti-human retinol binding protein antibody 2.0g/L
Example 3
A preparation method and a using method of a retinol binding protein detection kit comprise the following steps:
the method comprises the following steps: sequentially adding 0.5% of NaCl, 25g/L of PEG6000, 10g/L of bovine serum albumin and 1g/L of sodium azide into 50mmol/L of MES buffer solution, stirring and mixing uniformly, and filtering through a 0.22-micron mixed cellulose membrane to obtain a reagent R1;
step two: 0.5% NaCl, 1g/L sodium azide, 10g/L bovine serum albumin, 30mmol/L propylene glycol alginate and 3.0g/L anti-human retinol binding protein antibody are sequentially added into 50mmol/L PBS buffer solution, stirred and mixed uniformly, and filtered by a 0.22 micron mixed cellulose membrane to obtain a reagent R2.
2. Parameter setting of full-automatic biochemical analyzer
a. Detecting the temperature: 37 ℃;
b. detection wavelength: the main wavelength is 340nm, and the sub-wavelength is 700 nm;
c. reaction time: 10min, wherein the incubation time is 5min, the absorbance A1 is read immediately after the reagent R2 is added, the absorbance A2 is read after 5min, and the absorbance change Delta A is calculated to be A2-A1;
d. the reaction direction is as follows: carrying out positive reaction;
3. detection step
a. Uniformly mixing 200 mul of reagent R1 with 20 mul of sample to be detected;
b. incubating the mixed solution at 37 deg.C for 5 min;
c. adding 50 μ l of reagent R2, immediately measuring the read absorbance a1, 5min later, reading the absorbance a2, and calculating the change in absorbance Δ a ═ a2-a 1;
d. based on retinol binding protein (mg/L) = CS × Δ A in the sampleW/ΔAS(mg/L) the concentration of retinol binding protein in the sample was calculated.
Example 4
A preparation and use method of a retinol binding protein detection kit comprises the following steps:
sequentially adding 0.5% of KCl, 25g/L of PEG6000, 10g/L of bovine serum albumin and 1g/L of sodium azide into 75mmol/L of Tris-HCl buffer solution, stirring and mixing uniformly, and filtering through a 0.22 micron mixed cellulose membrane to obtain a reagent R1;
step two: 0.5 percent of KCl, 1g/L of sodium azide, 10g/L of bovine serum albumin, 20mmol/L of propylene glycol alginate and 2.0g/L of anti-human retinol binding protein antibody are sequentially added into 50mmol/L glycine buffer solution, stirred and mixed uniformly and filtered by a 0.22 micron mixed cellulose membrane to obtain a reagent R2.
2. Parameter setting of full-automatic biochemical analyzer
a. Detecting the temperature: 37 ℃;
b. detection wavelength: the main wavelength is 340nm, and the sub-wavelength is 700 nm;
c. reaction time: 10min, wherein the incubation time is 5min, the absorbance A1 is read immediately after the reagent R2 is added, the absorbance A2 is read after 5min, and the absorbance change Delta A is calculated to be A2-A1;
d. the reaction direction is as follows: carrying out positive reaction;
3. detection step
a. Uniformly mixing 200 mul of reagent R1 with 20 mul of sample to be detected;
b. incubating the mixed solution at 37 deg.C for 5 min;
c. adding 50 μ l of reagent R2, immediately measuring the read absorbance a1, 5min later, reading the absorbance a2, and calculating the change in absorbance Δ a ═ a2-a 1;
d. based on retinol binding protein (mg/L) = CS × Δ A in the sampleW/ΔAS(mg/L) the concentration of retinol binding protein in the sample was calculated.
The following tests were performed on the test kits of the examples
1. Sensitivity of the probe
Samples at known concentrations (20. + -.2) mg/L were tested using the kit and the change in absorbance at the parameters specified for the kit was recorded. The change in absorbance converted to a retinol binding protein content of 20 mg/L. Table 1 shows the results of the measurement of the sample using the kit for measuring retinol binding protein prepared in example 1 and the kit for measuring retinol binding protein prepared in example 2, and the results are shown in table 1:
TABLE 1
| 1 st time (Δ A) | 2 nd (Δ A) | 3 rd time (Δ A) | Mean value (Delta A) | |
| Example 1 | 0.1396 | 0.1409 | 0.1407 | 0.1404 |
| Example 2 | 0.1315 | 0.1332 | 0.1347 | 0.1331 |
As is clear from Table 1, the kit for measuring retinol binding protein prepared in the present invention has a large measurement result Δ A of the change in absorbance of a sample, and thus the measurement sensitivity is high, and example 1 is the most preferable choice.
2. Accuracy of
Table 2 shows the results of the measurement of the quality control product 1 by using the kit for measuring retinol binding protein prepared in example 1 and the kit for measuring retinol binding protein prepared in example 2, wherein the concentration of retinol binding protein in the quality control product 1 is 35mg/L, and the measurement results are shown in table 2:
TABLE 2
| 1 st time (mg/L) | 2 nd (mg/L) | 3 rd time (mg/L) | Mean value (mg/L) | Deviation (%) | |
| Example 1 | 34.03 | 35.58 | 34.15 | 34.59 | -1.18% |
| Example 2 | 32.37 | 33.14 | 34.86 | 33.46 | -4.41% |
As is clear from Table 2, the kit for assaying retinol binding protein prepared in the present invention has a high assay accuracy because the assay result of quality control 1 is less deviated, and example 1 is the most preferable choice.
Table 3 shows the results of the measurement of the quality control 2 using the kit for measuring retinol binding protein prepared in example 1 and the kit for measuring retinol binding protein prepared in example 2, wherein the concentration of retinol binding protein in the quality control 2 is 86mg/L, and the measurement results are shown in table 2:
TABLE 3
| 1 st time (mg/L) | 2 nd (mg/L) | 3 rd time (mg/L) | Mean value (mg/L) | Deviation (%) | |
| Example 1 | 86.04 | 85.78 | 85.89 | 85.90 | -0.11% |
| Example 2 | 83.53 | 82.76 | 84.68 | 83.66 | -2.72% |
As is clear from Table 3, the kit for assaying retinol binding protein prepared in the present invention has a high assay accuracy because the assay result of quality control 2 is less deviated, and example 1 is the most preferable choice.
3. Precision degree
Table 4 shows the results of the SD and CV calculations performed on the same test sample by the kit for measuring retinol binding protein prepared in example 3 and the same test sample by the kit for measuring retinol binding protein prepared in example 4, as follows:
TABLE 4
| 1 st time | 2 nd time | 3 rd time | 4 th time | 5 th time | 6 th time | 7 th time | 8 th time | 9 th time | 10 th time | Mean value | Standard deviation SD | Coefficient of variation CV (%) | |
| Example 3 | 50.10 | 50.32 | 50.17 | 50.75 | 50.10 | 50.25 | 49.94 | 49.96 | 50.45 | 50.60 | 50.26 | 0.268 | 0.53% |
| Example 4 | 49.99 | 50.34 | 50.10 | 51.39 | 49.72 | 51.12 | 49.14 | 49.86 | 50.04 | 49.98 | 50.17 | 0.656 | 1.31% |
As is apparent from Table 4, the kit for assaying retinol binding protein prepared in the present invention has a high precision, and as is apparent from tables 1, 2 and 3, example 3 is the most preferred choice.
Example 5
A retinol binding protein detection kit comprising a reagent R1 and a reagent R2 independently from each other;
the reagent R1 comprises the following components in parts by weight:
Tris-HCl buffer 20mmol/L
KCl 10％
PEG6000 10g/L
Bovine serum albumin 5g/L
Sodium azide 0.5 g/L;
the reagent R2 comprises the following components in parts by weight:
glycine buffer 20mmol/L
KCl 10％
Sodium azide 0.5g/L
Bovine serum albumin 5g/L
Propylene glycol alginate 10mmol/L
Anti-human retinol binding protein antibody 0.5g/L
Example 6
A retinol binding protein detection kit comprising a reagent R1 and a reagent R2 independently from each other;
the reagent R1 comprises the following components in parts by weight:
Tris-HCl buffer 200mmol/L
KCl 5％
PEG6000 40g/L
Bovine serum albumin 15g/L
Sodium azide is 1.5 g/L;
the reagent R2 comprises the following components in parts by weight:
glycine buffer 200mmol/L
KCl 5％
Sodium azide 1.5g/L
Bovine serum albumin 15g/L
Propylene glycol alginate 50mmol/L
Anti-human retinol binding protein antibody 0.5 g/L.
Claims (8)
1. A retinol binding protein assay kit, which is characterized in that: comprises a reagent R1 and a reagent R2 which are independent of each other;
the reagent R1 comprises the following components in parts by weight:
buffer solution 20-200 mmol/L
10-40 g/L of accelerator
5-15 g/L stabilizer
0.5-1.5 g/L of preservative
The mass percentage content of the inorganic salt ions is 0.5-10% of the total mass;
the reagent R2 comprises the following components in parts by weight:
buffer solution 20-200 mmol/L
0.5-1.5 g/L of preservative
5-15 g/L stabilizer
10-50 mmol/L suspending agent
0.5-3.0 g/L of anti-human retinol binding protein antibody
The mass percentage content of the inorganic salt ions is 0.5-10% of the total mass.
2. The retinol binding protein detection kit according to claim 1, wherein: the buffer solution in the reagent R1 is selected from any one or combination of MES buffer solution, Tris-HCl buffer solution, MOPS buffer solution, PBS buffer solution, glycine buffer solution and borax buffer solution, and the pH value is 7.0-9.0;
the buffer solution in the reagent R2 is selected from one or more of PBS buffer solution, borax buffer solution, glycine buffer solution, Hepes buffer solution, GOODS buffer solution and MOPS buffer solution, and the pH value is 7.0-9.0.
3. The retinol binding protein detection kit according to claim 1, wherein: the inorganic salt ions in the reagent R1 are selected from KCl, NaCl, MgCl2、CaCl2Any one or a combination of more of;
the inorganic salt ions in the reagent R2 are selected from NaCl, KCl and Na2CO3、Na2SO4、K2SO4Any one or a combination of more of them.
4. The retinol binding protein detection kit according to claim 1, wherein: the preservative in the reagent R1 and the reagent R2 is sodium azide, thimerosal or ProClin 300.
5. The retinol binding protein detection kit according to claim 1, wherein: the accelerator in the reagent R1 is PEG6000, PEG8000 or dextran.
6. The retinol binding protein detection kit according to claim 1, wherein: the stabilizer in the reagent R1 and the stabilizer in the reagent R2 are both bovine serum albumin.
7. The retinol binding protein detection kit according to claim 1, wherein: the suspending agent in the reagent R2 is propylene glycol alginate.
8. A method for preparing the retinol binding protein detection kit as defined in claim 1, which comprises the steps of: the method comprises the following steps:
the method comprises the following steps: sequentially adding 0.5-10% of inorganic salt ions, 10-40 g/L of accelerator, 5-15 g/L of stabilizer and 0.5-1.5 g/L of preservative into 20-200 mmol/L of buffer solution, stirring and mixing uniformly, and filtering through a 0.22 micron mixed cellulose membrane to obtain a reagent R1;
step two: 0.5-10% of inorganic salt ions, 10-50 mmol/L of suspending agent, 5-15 g/L of stabilizing agent, 0.5-1.5 g/L of preservative and 0.5-3.0 g/L of anti-human retinol binding protein antibody are sequentially added into 20-200 mmol/L of buffer solution, uniformly stirred and mixed, and filtered through a 0.22 micron mixed cellulose membrane to obtain a reagent R2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010233799.4A CN111190003A (en) | 2020-03-30 | 2020-03-30 | Retinol binding protein detection kit and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010233799.4A CN111190003A (en) | 2020-03-30 | 2020-03-30 | Retinol binding protein detection kit and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111190003A true CN111190003A (en) | 2020-05-22 |
Family
ID=70705490
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010233799.4A Pending CN111190003A (en) | 2020-03-30 | 2020-03-30 | Retinol binding protein detection kit and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111190003A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112129949A (en) * | 2020-08-18 | 2020-12-25 | 兰州百源基因技术有限公司 | Retinol binding protein detection kit, preparation method and use method thereof |
| CN113777329A (en) * | 2021-09-16 | 2021-12-10 | 何秋富 | Retinol binding protein assay kit and preparation method thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2879624A1 (en) * | 2012-07-24 | 2014-01-30 | Nissan Chemical Industries, Ltd. | Culture medium composition, and method for culturing cell or tissue using said composition |
| CN105486875A (en) * | 2016-01-26 | 2016-04-13 | 宁波天康生物科技有限公司 | Retinol conjugated protein detection kit |
| CN106093423A (en) * | 2016-05-31 | 2016-11-09 | 安徽伊普诺康生物技术股份有限公司 | A kind of test kit measuring retinol binding protein and preparation method thereof |
| CN106872718A (en) * | 2017-04-26 | 2017-06-20 | 吉林省富生医疗器械有限公司 | A kind of microdose urine protein detection kit and preparation method thereof |
| CN107490696A (en) * | 2017-08-10 | 2017-12-19 | 迈克生物股份有限公司 | A kind of Retinal-binding protein detection kit and detection method |
| CN107533050A (en) * | 2015-03-31 | 2018-01-02 | 美迪恩斯生命科技株式会社 | The measure reagent and assay method of thrombin-antithrombin complex |
-
2020
- 2020-03-30 CN CN202010233799.4A patent/CN111190003A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2879624A1 (en) * | 2012-07-24 | 2014-01-30 | Nissan Chemical Industries, Ltd. | Culture medium composition, and method for culturing cell or tissue using said composition |
| CN107533050A (en) * | 2015-03-31 | 2018-01-02 | 美迪恩斯生命科技株式会社 | The measure reagent and assay method of thrombin-antithrombin complex |
| CN105486875A (en) * | 2016-01-26 | 2016-04-13 | 宁波天康生物科技有限公司 | Retinol conjugated protein detection kit |
| CN106093423A (en) * | 2016-05-31 | 2016-11-09 | 安徽伊普诺康生物技术股份有限公司 | A kind of test kit measuring retinol binding protein and preparation method thereof |
| CN106872718A (en) * | 2017-04-26 | 2017-06-20 | 吉林省富生医疗器械有限公司 | A kind of microdose urine protein detection kit and preparation method thereof |
| CN107490696A (en) * | 2017-08-10 | 2017-12-19 | 迈克生物股份有限公司 | A kind of Retinal-binding protein detection kit and detection method |
Non-Patent Citations (1)
| Title |
|---|
| 王宁;: "免疫比浊法测定血清视黄醇结合蛋白方法学评价" * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112129949A (en) * | 2020-08-18 | 2020-12-25 | 兰州百源基因技术有限公司 | Retinol binding protein detection kit, preparation method and use method thereof |
| CN113777329A (en) * | 2021-09-16 | 2021-12-10 | 何秋富 | Retinol binding protein assay kit and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Doumas et al. | Candidate reference method for determination of total bilirubin in serum: development and validation. | |
| CN108362688B (en) | Detection kit for chemiluminescence of 25-hydroxy vitamin D magnetic particles | |
| JP5362564B2 (en) | Method for measuring cellular hemoglobin | |
| AU601086B2 (en) | Denaturant reagents for convenient determination of hemoglobin derivatives in blood | |
| CN111337691B (en) | Sensitive and stable serum procalcitonin determination kit and preparation method and application thereof | |
| CN106198415A (en) | A kind of test kit measuring glycolated hemoglobin and preparation method thereof | |
| CN112526134A (en) | Chitinase 3-like protein 1 determination kit | |
| CN111057150B (en) | Latex microsphere, application thereof and glycosylated hemoglobin detection kit | |
| CN106872718A (en) | A kind of microdose urine protein detection kit and preparation method thereof | |
| CN111190003A (en) | Retinol binding protein detection kit and preparation method thereof | |
| CN112029817B (en) | Creatinine detection kit and application method thereof | |
| NL8200587A (en) | METHOD FOR AN IMMUNOCHEMICAL ENZYME DETERMINATION. | |
| CN117607459B (en) | Neutrophil gelatinase-associated lipocalin assay kit, and preparation method and application thereof | |
| NIshi et al. | Three turbidimetric methods for determining total protein compared. | |
| CN108627652B (en) | It is a kind of to detect based on simple grain diameter and simultaneously the kit of RBP ELISA in serum and urine specimen | |
| CN112710854B (en) | Anti-interference and stable serum total bilirubin (enzyme method) determination kit and preparation method and application thereof | |
| CN112710853B (en) | Anti-interference and stable serum direct bilirubin (enzyme method) determination kit and preparation method and application thereof | |
| CN107942068B (en) | β2Microglobulin assay kit | |
| CN108717117A (en) | A kind of vancomycin immunologic function test reagent and its preparation and detection method | |
| CN107991500A (en) | Glycosylated hemoglobin detection kit | |
| CN104673878B (en) | Kit for measuring concentration ratio of glycated albumin and albumin by virtue of single system | |
| CN115128282B (en) | Fibronectin detection kit and detection method thereof | |
| CN108508194A (en) | A kind of tobramycin immunologic function test reagent and its preparation and detection method | |
| US4529708A (en) | Assay for the determination of creatinine | |
| CN113125749B (en) | Kit for detecting serum glycosylated albumin |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200522 |
|
| RJ01 | Rejection of invention patent application after publication |