CN116466092A - Kit for quantitatively determining uroretinol binding protein - Google Patents
Kit for quantitatively determining uroretinol binding protein Download PDFInfo
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- CN116466092A CN116466092A CN202310276811.3A CN202310276811A CN116466092A CN 116466092 A CN116466092 A CN 116466092A CN 202310276811 A CN202310276811 A CN 202310276811A CN 116466092 A CN116466092 A CN 116466092A
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- binding protein
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- retinol binding
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- 102000014914 Carrier Proteins Human genes 0.000 title description 15
- 108091008324 binding proteins Proteins 0.000 title description 15
- 102000029752 retinol binding Human genes 0.000 claims abstract description 90
- 108091000053 retinol binding Proteins 0.000 claims abstract description 90
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- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 28
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- 238000001514 detection method Methods 0.000 claims abstract description 25
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 22
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- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 36
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- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 description 10
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- 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 8
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- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 2
- 108010071690 Prealbumin Proteins 0.000 description 2
- 102000007584 Prealbumin Human genes 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
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- FXEYZZMDPRBKHU-UHFFFAOYSA-N 5-methylnonan-5-ylbenzene Chemical compound CCCCC(C)(CCCC)C1=CC=CC=C1 FXEYZZMDPRBKHU-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
-
- 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
- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
-
- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- 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
- G01N2333/775—Apolipopeptides
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
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- Hematology (AREA)
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- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
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- General Physics & Mathematics (AREA)
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- Cell Biology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kit for quantitatively determining a urea retinol binding protein, which belongs to the technical field of biological detection, and comprises a reagent 1 and a reagent 2; reagent 1 comprises: 45-55mM of tris (hydroxymethyl) aminomethane, 5-7g/L of sodium chloride, 4.5-5.5g/L of bovine serum albumin, 0.1-0.3ml/L of biological preservative and 8-12g/L of surfactant: reagent 2 comprises: 45-55mM of tris (hydroxymethyl) aminomethane, 45-55mL/L of anti-human retinol binding protein antibody latex particles, 4.5-5.5g/L of bovine serum albumin and 0.1-0.3mL/L of biological preservative. The invention can avoid the aggregation of anti-human retinol binding protein antibody latex particles in the storage process, improves the accuracy and precision of detection results, has longer storage time of the kit, and high detection sensitivity, realizes the simultaneous detection of blood and urine, and has simple operation and lower cost.
Description
Technical Field
The invention belongs to the technical field of biological detection, and relates to a kit for quantitatively determining a urea retinol binding protein.
Background
Retinol Binding Protein (RBP) is a small molecular weight lipocalin synthesized by the liver and having a molecular weight of 21KD, which plays a role in transporting retinol from hepatocytes to tissues and cells of the body in vivo, and serum RBP transports retinol in such a way as to combine with Prealbumin (PA) and retinol to form a macromolecular complex which cannot be filtered through the filter membrane of glomeruli and thus exists in human blood; when the complex reaches the target cell, retinol is absorbed by the target cell, RBP is dissociated into small molecular weight free state and enters serum, glomerular filtration can be carried out, the RBP enters into renal tubule and is absorbed by epithelial cells to be converted into amino acid for organism reuse, and in addition, a very small part of RBP is not absorbed and is directly discharged out of the body along with urine.
Clinically, the levels of serum RBP and urine RBP can be used as effective detection indexes of liver and kidney injury of organisms, diabetes and other syndromes. RBP is synthesized in the liver, and if the liver synthesis function is impaired, the serum RBP concentration is reduced, and the reduction degree is highly correlated with the liver damage, and the more serious the liver damage is, the greater the reduction degree is, and the lower the liver cirrhosis is. The RBP has short half-life and sensitive response change, and is more suitable for being used as an index of early liver function injury.
Retinol binding protein is metabolized in the kidney, so urinary RBP levels are of great importance for early diagnosis of kidney disease. Some diseases (such as diabetes, hypertension, etc.) cause the glomerulus function of the organism to be impaired, glomerular filtration is hindered, and RBP accumulates in blood to cause the serum RBP content to be increased; in addition, certain kidney diseases (such as viral infections) can cause impairment of the reabsorption function of the body's tubular ducts, and RBP in body fluid cannot be reabsorbed after entering the kidneys, and all of the RBP is excreted outside the body with urine, resulting in an increase in urinary RBP content.
The RBP detection methods widely used clinically include enzyme-linked immunosorbent assay (ELISA), unidirectional immunodiffusion RID, immune turbidimetry and the like. The detection limit of the enzyme-linked immunosorbent method is poor and time-consuming. Unidirectional immunodiffusion is mainly manually operated, the test repeatability is poor, the detection accuracy is not high, and the unidirectional immunodiffusion is not used as a quantitative detection method in clinic. The method commonly adopted for detecting a large number of samples in clinic at present is an immunoturbidimetry method, but due to low signals, the sensitivity is low, and the blood and urine co-detection required in the market at present cannot be achieved.
There are latex-enhanced immunoturbidimetry for measuring RBP, latex-enhanced immunoturbidimetry based on double particle size, latex-enhanced immunoturbidimetry based on double monoclonal antibody, and latex-enhanced immunoturbidimetry based on a monoclonal antibody and a polyclonal antibody in the current market, although the sensitivity is high enough to achieve blood and urine co-detection, the reagent production cost is high, which is unfavorable for market popularization.
Disclosure of Invention
In order to solve the technical problems, the invention provides a kit for quantitatively determining the urea retinol binding protein, which has low cost and high sensitivity and can detect RBP in blood at the same time.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a kit for quantitatively determining a uroretinol binding protein, comprising a reagent 1 and a reagent 2;
the reagent 1 comprises: 45-55mM of tris (hydroxymethyl) aminomethane, 5-7g/L of sodium chloride, 4.5-5.5g/L of bovine serum albumin, 0.1-0.3ml/L of biological preservative and 8-12g/L of surfactant:
the reagent 2 comprises: 45-55mM of tris (hydroxymethyl) aminomethane, 45-55mL/L of anti-human retinol binding protein antibody latex particles, 4.5-5.5g/L of bovine serum albumin and 0.1-0.3mL/L of biological preservative.
The biological preservative is at least one of NaN3, sodium benzoate, proclin-200, proclin-300 or Proclin500; preferably, the biological preservative is Proclin-300.
The surfactant is at least one of triton X-100, tween 20, tween 60 and polyoxyethylene lauryl ether-35; preferably, the surfactant is polyoxyethylene lauryl ether-35.
The anti-human retinol binding protein antibody latex particles are polymethyl methacrylate latex particles coated by the anti-human retinol binding protein antibody, wherein the volume ratio of the anti-human retinol binding protein antibody to the polymethyl methacrylate latex particles is 1:4-7; further preferably, the volume ratio of the anti-human retinol binding protein antibody to polymethyl methacrylate latex particles is 1:6.
Preferably, the particle size of the polymethyl methacrylate latex particles is 60-120nm.
Preferably, the method for preparing the anti-human retinol binding protein antibody latex particles of the present invention comprises the following steps:
(1) Dissolving an anti-human retinol binding protein antibody in MES buffer solution with pH of 6.5 to prepare an anti-human retinol binding protein antibody solution with concentration of 1.0 mL/mL;
(2) Dissolving polymethyl methacrylate latex particles in 0.1mol/L MES buffer with the concentration of 1% (w/v), adding 10mg/mL polyaspartic acid and 10mg/mL N, N-methylene bisacrylamide, incubating, centrifuging, discarding the supernatant, washing latex microspheres 3-4 times with the MES buffer, adding the MES buffer, and shaking and re-suspending to obtain latex microsphere suspension with the concentration of 1% (w/v);
(3) Mixing the anti-human retinol binding protein antibody solution obtained in the step (1) and the latex microsphere suspension obtained in the step (2), incubating, adding 4 mu l/mL of 2-hydroxy ethylamine, mixing uniformly, sealing, centrifuging, discarding the supernatant, washing 3-4 times with MES buffer solution, adding MES buffer solution, shaking and re-suspending to obtain the anti-human retinol binding protein antibody latex particles.
Preferably, the reagent 1 comprises: 48-52mM of tris (hydroxymethyl) aminomethane, 5.5-6.5g/L of sodium chloride, 4.6-5.4g/L of bovine serum albumin, 0.15-0.25ml/L of biological preservative and 9-11g/L of surfactant: further preferably, the reagent 1 comprises: 50mM tris (hydroxymethyl) aminomethane, 6.0g/L sodium chloride, 4.8g/L bovine serum albumin, 0.18ml/L biological preservative and 9.5g/L surfactant.
In order to prevent aggregation of anti-human retinol binding protein antibody latex particles, said reagent 2 further comprises gelatin, glycine and raffinose, specifically, gelatin is 10-15g/L, glycine is 8-12g/L and raffinose is 20-25g/L.
Further preferably, the reagent 2 further comprises: 48-52mM of tris (hydroxymethyl) aminomethane, 46-54mL/L of anti-human retinol binding protein latex particles, 4.6-5.4g/L of bovine serum albumin, 11-14g/L of gelatin, 8.5-11g/L of glycine, 21-24g/L of raffinose and 0.15-0.25mL/L of biological preservative; still further preferably, the reagent 2 further comprises: 50mM tris, 50mL/L anti-human retinol binding protein latex particles antibody latex particles, 4.8g/L bovine serum albumin, 12g/L gelatin, 10g/L glycine, 22g/L raffinose and 0.18mL/L biological preservative.
In another aspect, the present invention provides a method for detecting retinol binding protein, which is to detect the content of retinol binding protein in urine and/or serum by using the above-mentioned kit.
Specifically, the detection method comprises the following steps:
(1) Drawing a standard curve: absorbance change value Δa=a of the calibrator Calibration of -A Blank space The corresponding concentration C is calibrated to be the abscissa, and a calibration curve is drawn;
(2) Measuring a sample: mixing the sample and the reagent R1 uniformly, incubating for 5min at 37 ℃, adding the reagent R2, mixing uniformly, standing for 30s, detecting the absorbance A1 at the wavelength of 600nm, reacting at the constant temperature of 37 ℃ for 5min, ending the reaction, detecting the absorbance A2 at the wavelength of 600nm, and calculating the concentration of the retinol binding protein in the sample by using the absorbance change value delta A=A2-A1 according to the standard curve drawn in the step (1).
The beneficial effects of the invention are as follows:
the kit for quantitatively determining the urea retinol binding protein provided by the invention has the advantages that gelatin, glycine and raffinose are added in the reaction system, so that the aggregation of anti-human retinol binding protein antibody latex particles in the storage process can be avoided, the mixing is easy, the accuracy and precision of a detection result are improved, and the storage time of the kit is longer.
The ascorbic acid is less than or equal to 300mg/L, the combined bilirubin is less than or equal to 300mg/L, and the triglyceride is less than or equal to 11.3mmol/L in the sample, so that the test result is not obvious.
In addition, the invention has high detection sensitivity, realizes the simultaneous detection of blood and urine, and has simple operation and lower economic cost.
Detailed Description
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.
The following examples, unless otherwise specified, do not contain other components not explicitly indicated except for unavoidable impurities. The raw materials used in each example were commercially available products.
(1) Dissolving an anti-human retinol binding protein antibody in MES buffer solution with pH of 6.5 to prepare an anti-human retinol binding protein antibody solution with concentration of 1.0 mL/mL;
(2) Dissolving polymethyl methacrylate latex particles with the particle size of 60nm in 0.1mol/L MES buffer solution with the concentration of 1% (w/v), adding 10mg/mL polyaspartic acid and 10mg/mL N, N-methylene bisacrylamide, incubating at 20 ℃ for 30min, centrifuging at 10000rpm for 20min, discarding the supernatant, washing latex microspheres with the same volume of MES buffer solution for 3-4 times, removing the redundant cross-linking agent, dissolving the latex particles in the MES buffer solution after washing, and oscillating for 15min at 25 ℃ for resuspension to prepare a latex microsphere suspension with the concentration of 1% (w/v);
(3) Mixing the anti-human retinol binding protein antibody solution obtained in the step (1) and the latex microsphere suspension obtained in the step (2) according to the mass ratio of 1:4, incubating for 2 hours at 37 ℃, adding 4 μl/mL of 2-hydroxyethylamine, mixing uniformly, sealing for 30 minutes at 37 ℃, centrifuging for 20 minutes at 10000rpm, discarding the supernatant, washing for 3-4 times with the same volume of MES buffer, removing unbound antibody, adding the MES buffer, and shaking for 15 minutes at 25 ℃ for resuspension to obtain the anti-human retinol binding protein antibody latex particles.
Example 2 preparation of anti-human retinol binding protein antibody latex particles
This embodiment differs from embodiment 1 only in that: the particle size of the polymethyl methacrylate latex particles in the step (2) is 100nm; and the mass ratio in the step (3) is 1:6.
Example 3 preparation of anti-human retinol binding protein antibody latex particles
This embodiment differs from embodiment 1 only in that: the particle size of the polymethyl methacrylate latex particles in the step (2) is 120nm; and the mass ratio in the step (3) is 1:7.
Example 4 kit for quantitative determination of uroretinol binding protein
The kit for quantitatively determining the urea retinol binding protein comprises a reagent 1 and a reagent 2, wherein the specific preparation components of the reagent 1 and the reagent 2 are as follows:
the reagent 1: 45mM of tris (hydroxymethyl) aminomethane, 5g/L of sodium chloride, 4.5g/L of bovine serum albumin, 0.1ml/L of biological preservative and 8g/L of surfactant:
the reagent 2 comprises: 45mM tris (hydroxymethyl) aminomethane, 45mL/L anti-human retinol binding protein antibody latex particles, 4.5g/L bovine serum albumin and 0.1mL/L biological preservative;
the biological preservative is Proclin-300; the surfactant is polyoxyethylene lauryl ether-35; the anti-human retinol binding protein antibody latex particles were prepared as described in example 2.
Example 5 kit for quantitative determination of uroretinol binding protein
The kit for quantitatively determining the urea retinol binding protein comprises a reagent 1 and a reagent 2, wherein the specific preparation components of the reagent 1 and the reagent 2 are as follows:
the reagent 1: tris 55mM, sodium chloride 7g/L, bovine serum albumin 5.5g/L, biological preservative 0.3ml/L and surfactant 12g/L:
the reagent 2 comprises: tris 55mM, anti-human retinol binding protein antibody latex particles 55mL/L, bovine serum albumin 5.5g/L and biological preservative 0.3mL/L.
The biological preservative is Proclin-300; the surfactant is polyoxyethylene lauryl ether-35; the anti-human retinol binding protein antibody latex particles were prepared as described in example 2.
Example 6 kit for quantitative determination of uroretinol binding protein
The kit for quantitatively determining the urea retinol binding protein comprises a reagent 1 and a reagent 2, wherein the specific preparation components of the reagent 1 and the reagent 2 are as follows:
the reagent 1: 48mM tris (hydroxymethyl) aminomethane, 5.5g/L sodium chloride, 4.6g/L bovine serum albumin, 0.15ml/L biological preservative and 9g/L surfactant:
the reagent 2: 48mM tris, 46mL/L anti-human retinol binding protein latex particles antibody latex particles, 4.6g/L bovine serum albumin, 11g/L gelatin, 8.5g/L glycine, 21g/L raffinose and 0.15mL/L biological preservative.
The biological preservative is Proclin-300; the surfactant is polyoxyethylene lauryl ether-35; the anti-human retinol binding protein antibody latex particles were prepared as described in example 2.
Example 7 kit for quantitative determination of uroretinol binding protein
The kit for quantitatively determining the urea retinol binding protein comprises a reagent 1 and a reagent 2, wherein the specific preparation components of the reagent 1 and the reagent 2 are as follows:
the reagent 1: 52mM tris (hydroxymethyl) aminomethane, 6.5g/L sodium chloride, 5.4g/L bovine serum albumin, 0.25ml/L biological preservative and 11g/L surfactant:
the reagent 2: 52mM tris, 54mL/L anti-human retinol binding protein latex particles antibody latex particles, 5.4g/L bovine serum albumin, 14g/L gelatin, 11g/L glycine, 24g/L raffinose and 0.25mL/L biological preservative.
The biological preservative is Proclin-300; the surfactant is polyoxyethylene lauryl ether-35; the anti-human retinol binding protein antibody latex particles were prepared as described in example 2.
Example 8 kit for quantitative determination of uroretinol binding protein
The kit for quantitatively determining the urea retinol binding protein comprises a reagent 1 and a reagent 2, wherein the specific preparation components of the reagent 1 and the reagent 2 are as follows:
the reagent 1: 50mM tris (hydroxymethyl) aminomethane, 6.0g/L sodium chloride, 4.8g/L bovine serum albumin, 0.18ml/L biological preservative and 9.5g/L surfactant:
the reagent 2: 50mM tris, 50mL/L anti-human retinol binding protein latex particles antibody latex particles, 4.8g/L bovine serum albumin, 12g/L gelatin, 10g/L glycine, 22g/L raffinose and 0.18mL/L biological preservative.
The biological preservative is Proclin-300; the surfactant is polyoxyethylene lauryl ether-35; the anti-human retinol binding protein antibody latex particles were prepared as described in example 2.
Example 9 kit for quantitative determination of uroretinol binding protein
This embodiment differs from embodiment 8 in that: the biological preservative is Proclin500; the surfactant is triton X-100; the anti-human retinol binding protein antibody latex particles were prepared as described in example 1.
Example 10 kit for quantitative determination of uroretinol binding protein
This embodiment differs from embodiment 8 in that: the biological preservative is Proclin-200; the surfactant is Tween 60; the anti-human retinol binding protein antibody latex particles were prepared as described in example 3.
Comparative example 1 kit for quantitative determination of uroretinol binding protein
This embodiment differs from embodiment 8 in that: reagent 2 is different (the dosage of gelatin, glycine and raffinose in reagent 2 is different); the method comprises the following steps:
the reagent 2: 50mM tris, 50mL/L anti-human retinol binding protein latex particles antibody latex particles, 4.8g/L bovine serum albumin, 20g/L gelatin, 14g/L glycine, 10g/L raffinose and 0.18mL/L biological preservative.
Comparative example 2 kit for quantitative determination of uroretinol binding protein
This embodiment differs from embodiment 8 in that: reagent 2 is different (the dosage of gelatin, glycine and raffinose in reagent 2 is different); the method comprises the following steps:
the reagent 2: 50mM tris, 50mL/L anti-human retinol binding protein latex particles antibody latex particles, 4.8g/L bovine serum albumin, 16g/L gelatin, 2g/L glycine, 27g/L raffinose and 0.18mL/L biological preservative.
Comparative example 3 kit for quantitative determination of uroretinol binding protein
This embodiment differs from embodiment 8 in that: reagent 2 is different (with dibutyl methyl toluene, glycine and dextran sulfate instead of gelatin, glycine and raffinose); the method comprises the following steps:
the reagent 2: 50mM tris, 50mL/L anti-human retinol binding protein latex particles antibody latex particles, 4.8g/L bovine serum albumin, 12g/L dibutylmethyl toluene, 10g/L glycine, 22g/L dextran sulfate and 0.18mL/L biological preservative.
Comparative example 4 kit for quantitative determination of uroretinol binding protein
This embodiment differs from embodiment 8 in that: reagent 2 is different (using cellulose salts and sucrose instead of gelatin and raffinose); the method comprises the following steps:
the reagent 2: 50mM tris, 50mL/L anti-human retinol binding protein latex particles antibody latex particles, 4.8g/L bovine serum albumin, 12g/L cellulose salt, 10g/L glycine, 22g/L sucrose and 0.18mL/L biological preservative.
EXAMPLE 11 detection of uroretinol binding protein
The kit can utilize a full-automatic biochemical analyzer for detection, and the applicable instrument comprises: hitachi 7170/7600/7180/008AS/3500, michael BS2000, dirui CS600, yaban C16000, toshiba TBA120FR/TBA2000FR/TBA-FX8, beckmann AU5800/AU680/AU 2700/DXC 800, siemens ADVIA2400/CH930/Dimension RXL, roche C701/C702.
The detection is specifically carried out according to the following steps:
(1) Drawing a standard curve: absorbance change value Δa=a of the calibrator Calibration of -A Blank space The corresponding concentration C is calibrated to be the abscissa, and a calibration curve is drawn;
(2) Measuring a sample: mixing 2 mu L of the sample and 180 mu L of the reagent R1 uniformly, incubating for 5min at 37 ℃, adding 60 mu L of the reagent R2, mixing uniformly, standing for 30s, detecting absorbance A1 at a wavelength of 600nm, reacting at a constant temperature of 37 ℃ for 5min, ending the reaction, detecting absorbance A2 at a wavelength of 600nm, and calculating the concentration of retinol binding protein in the sample by using absorbance change value delta A=A2-A1 according to the standard curve drawn in the step (1).
The sample is a fresh urine sample, the urine sample is collected and centrifuged, and the supernatant is taken for detection.
The standard substance comprises the following components: six concentration gradients of 0mg/L, 0.5mg/L, 1mg/L, 5mg/L, 30mg/L and 60mg/L of retinol binding protein, 4.8g/L of bovine serum albumin and 0.18ml/L of liquid biological preservative; the liquid preservative is a preservative corresponding to the kit.
The quality control product comprises: retinol binding protein 50mL/L, bovine serum albumin 4.8g/L, liquid biological preservative; the liquid preservative is a preservative corresponding to the kit.
Example 12 Performance analysis of the kit
The kits of examples 1 to 10 and comparative examples 1 to 4 were left at 2 to 8℃for 24 months, and the precision and accuracy of the kits were examined, and the results are shown in Table 1 and Table 2.
A. And (3) precision detection: the same sample (0.5-1 mg/L) was continuously extracted 10 times, and the measurement was performed according to the above procedure, and the average value, standard Deviation (SD) and Coefficient of Variation (CV) of the measurement were calculated, CV= (standard deviation/average value) ×100%, and the results are shown in Table 1, CV is usually used to measure the precision of one measurement method, and the smaller CV value means the better precision of the result of the measurement method. For clinical chemistry test projects, a method precision of less than 5% CV is recognized as acceptable.
B. And (3) accuracy detection: the measurement was performed with a quality control product having a retinol binding protein content of 50mg/L, repeated 5 times, and the average value was taken, and the measurement results are shown in Table 2. For clinical chemistry test projects, a relative deviation (CB) of no more than ±15% is considered to have excellent accuracy.
TABLE 1 precision measurement results
TABLE 2 accuracy test results
The reagent 2 of the invention examples 4-10 still has good suspension property under the environment of 2-8 ℃ for 24 months, and is very easy to mix, while the reagent 2 of the invention comparative examples 1-4 has sedimentation at 18 months. As can be seen from tables 1 and 2 above, the precision CV at 24 months of the kits of examples 4-10 was less than 5% and the accuracy CB was less than.+ -. 15%; however, the kit of comparative examples 1-4 has a precision CV of greater than 5% and an accuracy CB of greater than.+ -. 15% at 24 months, indicating that the reagent 2 of the present invention improves the fluidity and stability of the anti-human retinol binding protein latex particle antibody latex particles, providing precision and accuracy of the assay.
Example 13 interference experiment
Three potential interferents, namely ascorbic acid, combined bilirubin, free bilirubin and triglyceride, are selected, and 250mg/L, 300mg/L and 350mg/L of ascorbic acid are respectively added into a quality control product; 250mg/L, 300mg/L, 350mg/L of conjugated bilirubin; 10.3mmol/L, 11.3mmol/L, 12.3mmol/L triglycerides. The control group was a quality control without any interfering substances, and was determined 5 times by using the kit described in example 8 and the detection method described in example 11, and the average value was taken, and it was found that when the ascorbic acid concentration reached 300mg/L, the combined bilirubin concentration reached 300mg/L, and the triglyceride concentration reached 11.3mmol/L, there was no significant interference to the test results, and the relative deviation CB was less than ±15%.
The invention has been further described above in connection with specific embodiments, which are exemplary only and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.
Claims (10)
1. A kit for quantitative determination of urinary retinol binding protein, comprising reagent 1 and reagent 2, characterized in that said reagent 1 comprises: 45-55mM of tris (hydroxymethyl) aminomethane, 5-7g/L of sodium chloride, 4.5-5.5g/L of bovine serum albumin, 0.1-0.3ml/L of biological preservative and 8-12g/L of surfactant:
the reagent 2 comprises: 45-55mM of tris (hydroxymethyl) aminomethane, 45-55mL/L of anti-human retinol binding protein antibody latex particles, 4.5-5.5g/L of bovine serum albumin, 10-15g/L of gelatin, 8-12g/L of glycine, 20-25g/L of raffinose and 0.1-0.3mL/L of biological preservative.
2. The kit of claim 1, wherein the biological preservative is NaN 3 At least one of sodium benzoate, proclin-200, proclin-300, or Proclin 500.
3. The kit according to claim 2, wherein the surfactant is at least one of triton X-100, tween 20, tween 60, polyoxyethylene lauryl ether-35.
4. The kit according to claim 1, wherein the anti-human retinol binding protein antibody latex particles are polymethyl methacrylate latex particles coated with the anti-human retinol binding protein antibody, the volume ratio of the anti-human retinol binding protein antibody to the polymethyl methacrylate latex particles is 1:4-7, and the particle size of the polymethyl methacrylate latex particles is 60-120nm.
5. The kit of claim 1, wherein the method of preparing anti-human retinol binding protein antibody latex particles comprises the steps of:
(1) Dissolving an anti-human retinol binding protein antibody in MES buffer solution to prepare an anti-human retinol binding protein antibody solution;
(2) Dissolving polymethyl methacrylate latex particles in an MES buffer solution, adding polyaspartic acid and N, N-methylene bisacrylamide, incubating, centrifuging, discarding supernatant, washing latex microspheres for 3-4 times by using the MES buffer solution, adding the MES buffer solution, and shaking and re-suspending to obtain a latex microsphere suspension;
(3) Mixing the anti-human retinol binding protein antibody solution obtained in the step (1) and the latex microsphere suspension obtained in the step (2), incubating, adding 2-hydroxy ethylamine, mixing, sealing, centrifuging, discarding the supernatant, washing 3-4 times with MES buffer, adding MES buffer, shaking and re-suspending to obtain the anti-human retinol binding protein antibody latex particles.
6. The kit according to claim 1, wherein the concentration of polyaspartic acid in step (2) is 10mg/mL, the concentration of N, N-methylenebisacrylamide is 10mg/mL, and the concentration of 2-hydroxyethylamine is 4 μl/mL.
7. The kit of claim 1, wherein the reagent 1 comprises: 48-52mM of tris (hydroxymethyl) aminomethane, 5.5-6.5g/L of sodium chloride, 4.6-5.4g/L of bovine serum albumin, 0.15-0.25ml/L of biological preservative and 9-11g/L of surfactant.
8. The kit of claim 1, wherein the reagent 2 further comprises: 48-52mM of tris, 46-54mL/L of anti-human retinol binding protein latex particles, 4.6-5.4g/L of bovine serum albumin, 11-14g/L of gelatin, 8.5-11g/L of glycine, 21-24g/L of raffinose and 0.15-0.25mL/L of biological preservative.
9. A method for detecting retinol binding protein using the kit as defined in any one of claims 1-8, wherein said detection method is to detect the content of retinol binding protein in urine and/or serum using the kit.
10. The method of detection according to claim 9, comprising the steps of:
(1) Drawing a standard curve: absorbance change value Δa=a of the calibrator Calibration of -A Blank space The corresponding concentration C is calibrated to be the abscissa, and a calibration curve is drawn;
(2) Measuring a sample: mixing the sample and the reagent R1 uniformly, incubating for 5min at 37 ℃, adding the reagent R2, mixing uniformly, standing for 30s, detecting the absorbance A1 at the wavelength of 600nm, reacting at the constant temperature of 37 ℃ for 5min, ending the reaction, detecting the absorbance A2 at the wavelength of 600nm, and calculating the concentration of the retinol binding protein in the sample by using the absorbance change value delta A=A2-A1 according to the standard curve drawn in the step (1).
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