CN112557669A - Kit and application thereof in detection of vascular endothelial growth factor in anterior chamber fluid - Google Patents
Kit and application thereof in detection of vascular endothelial growth factor in anterior chamber fluid Download PDFInfo
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- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 title claims abstract description 38
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 title claims abstract description 38
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- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 title claims abstract 7
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/475—Assays involving growth factors
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/16—Ophthalmology
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Abstract
The invention relates to the technical field of aqueous humor antigen detection, in particular to a kit and application thereof in detecting vascular endothelial growth factor in anterior chamber liquid, wherein the kit contains the following components which are respectively and independently stored: enzyme reactant, magnetic separation reagent, stability enhancer, calibrator, cleaning concentrated solution and substrate solution. The kit provided by the invention can accurately detect the content of the vascular endothelial growth factor in the anterior chamber liquid, has small sample demand, short detection time and wide linear range, can obviously reduce the background value when the mutant alkaline phosphatase is used as the magnetic particle blocking agent, further reduces the minimum detection limit of the reagent, and has important application value for diagnosing VEGF related ophthalmic diseases.
Description
Technical Field
The invention relates to the technical field of aqueous humor antigen detection, in particular to a kit and application thereof in detecting vascular endothelial growth factor in anterior chamber liquid.
Background
In the field of ophthalmic diseases, although the precise pathogenesis of most ocular diseases is not completely understood, in recent years some key causative molecules of important blinding diseases have been discovered, most typically Vascular Endothelial Growth Factor (VEGF).
Vascular endothelial cell growth factor (VEGF), also known as vascular permeability factor, is an important in vivo angiogenic cytokine, and plays a role in enhancing vascular permeability, inducing angiogenesis and angiogenesis, endothelial cell growth, promoting cell migration, inhibiting apoptosis, etc. by acting on glycosylated cells of endothelial cells, mitogen.
VEGF at physiological concentrations is a cytokine essential for human development, injury repair and maintenance of normal physiological function, but its overexpression is one of the important mechanisms for malignant tumor growth and ocular neovascular diseases.
Ocular neovascular diseases closely associated with increased VEGF expression include neovascular age-related macular degeneration (nAMD, also known as wet AMD), Idiopathic Polypoidal Choroidal Vasculopathy (IPCV), Proliferative Diabetic Retinopathy (PDR), Diabetic Macular Edema (DME), Retinal Vein Occlusion (RVO), myopic choroidal neovessels (myopicCNV), neovascular glaucoma, idiopathic choroidal neovessels (iccnv), and idiopathic parafoveal telangiectasia (IJRT), among others. In addition, the increase of VEGF has been associated with the onset of macular edema, Coats' disease, retinopathy of prematurity (ROP), etc., which are caused by various retinal diseases.
Currently, the diagnosis and monitoring techniques for internal eye diseases are mainly limited to physical diagnosis, imaging diagnosis and pathological diagnosis. As the eyeball is a transparent and superficial organ, the physical diagnosis and the imaging diagnosis of the eye are well developed.
Although the conventional experimental diagnosis techniques have been widely used for the examination of systemic diseases and other large organ diseases, for example, the examination of body fluids such as blood, urine, cerebrospinal fluid, pleural effusion, abdominal effusion and the like has become a clinical routine, the intraocular fluid is limited by the difficulty in sampling and the small sample size, and various conventional body fluid diagnosis techniques have not been applied in the field of ophthalmology.
In addition, the protein content in the anterior chamber fluid is much lower than in blood due to the blood-aqueous humor barrier. The VEGF assay reported by Yeoetal (Clin. chem.1992, 38: 71), Houcketal (JBiol. chem.1992, 267: 26031) and Hanatanieal (biosci. Biotechnol. biochem.1995, 59: 1985), et al, all have high minimum detection limits, on the μ g/mL-ng/mL scale.
Therefore, the development of a new detection technology for diagnosing the VEGF related ophthalmic diseases with lower sample demand and shorter detection time has important application value.
Disclosure of Invention
The invention aims to overcome the defects of high sample demand, long detection time and narrow linear range of the existing detection technology for diagnosing VEGF related diseases.
In order to achieve the above object, a first aspect of the present invention provides a kit comprising the following components, each of which is stored independently:
enzyme reactant, magnetic separation reagent, stability enhancer, calibrator, cleaning concentrated solution and substrate solution;
the enzyme reactant contains vascular endothelial growth factor specific antibody I marked by alkaline phosphatase; wherein, in the labeling process, the activation ratio of the antibody I is 60 times, the activation ratio of the alkaline phosphatase is 20 times, and the coupling ratio of the antibody I to the alkaline phosphatase is 1: 0.75;
the magnetic separation reagent contains 0.05 mass percent of magnetic particles, and the magnetic particles are coated with a vascular endothelial growth factor specific antibody II;
the antibody I is a VEGF165/VEGFA labeled antibody of Sino Biological company with the model number of 11066-R010, and the antibody II is a VEGF165/VEGFA coated antibody of Sino Biological company with the model number of 11066-R012.
In a second aspect, the present invention provides the use of a kit according to the first aspect above for the detection of vascular endothelial growth factor in anterior chamber fluid.
Compared with the existing detection technology for diagnosing VEGF related diseases, the kit for detecting the vascular endothelial growth factor in the anterior chamber liquid provided by the invention has at least the following advantages:
(1) the kit provided by the invention can accurately detect the content of the vascular endothelial growth factor in the anterior chamber fluid, and the sample demand is small, and only 20 mu L of trace intraocular fluid sample is needed;
(2) compared with the traditional ELISA method which needs 2 hours or even overnight incubation time, the kit provided by the invention can complete incubation within 30 minutes when in use, can complete detection within 1 hour, and greatly saves time;
(3) the linear range is wide, and the linear range is better in the measurement range of 0-5000 pg/mL.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
As described above, the first aspect of the present invention provides a kit comprising the following components, each of which is stored independently:
enzyme reactant, magnetic separation reagent, stability enhancer, calibrator, cleaning concentrated solution and substrate solution;
the enzyme reactant contains vascular endothelial growth factor specific antibody I marked by alkaline phosphatase; wherein, in the labeling process, the activation ratio of the antibody I is 60 times, the activation ratio of the alkaline phosphatase is 20 times, and the coupling ratio of the antibody I to the alkaline phosphatase is 1: 0.75;
the magnetic separation reagent contains 0.05 mass percent of magnetic particles, and the magnetic particles are coated with a vascular endothelial growth factor specific antibody II;
the antibody I is a VEGF165/VEGFA labeled antibody of Sino Biological company with the model number of 11066-R010, and the antibody II is a VEGF165/VEGFA coated antibody of Sino Biological company with the model number of 11066-R012.
In the present invention, the vascular endothelial growth factor-specific antibody I labeled with alkaline phosphatase and the vascular endothelial growth factor-specific antibody II coated on the magnetic fine particles in the enzyme reaction mixture cannot be used interchangeably.
In the invention, the magnetic particles are made of magnetic particles produced by JSR Life Sciences company, and the model number of the magnetic particles is MagnosphereTM MS300/Carboxyl。
In the invention, the activation ratio is the molar ratio between the activating agent and the activated substance; the coupling ratio is the molar ratio of the antibody I to the alkaline phosphatase.
Preferably, the activation ratio of the magnetic particles is 50 times, and the antibody coating rate is 50 mug/mg.
Preferably, the blocking agent in the magnetic separation reagent is mutant alkaline phosphatase (AP mutain).
In the invention, the process of coating the antibody on the magnetic particles comprises a sealing process, and the sealing agent is an important material in the sealing process.
Preferably, the mutant alkaline phosphatase of the present invention is a mutant alkaline phosphatase (AP mutain) manufactured by Roche, model 04781007103.
In the invention, the antibody coating rate is calculated according to the mass concentration and the volume of the antibody and the magnetic particles; the sealant is a component in the magnetic particles.
Preferably, the amount of the blocking agent is 100mg of blocking agent per 1g of magnetic particles.
Preferably, the stability enhancer contains Tris (hydroxymethyl) aminomethane (Tris), NaCl, tween20, Bovine Serum Albumin (BSA), mutant alkaline phosphatase (AP mutain), and Proclin 300.
In the present invention, the mutant alkaline phosphatase in the stability enhancer and the mutant alkaline phosphatase as the blocking agent are the same substance, and the effect in both components is to reduce non-specific adsorption.
The invention uses the mutant alkaline phosphatase as the sealant in the magnetic particle coating process, can obviously reduce the background value, further reduce the lowest detection limit of the reagent, can detect that the concentration of the vascular endothelial growth factor in the anterior chamber liquid is lower than 5pg/mL, and has important application value for diagnosing VEGF related ophthalmic diseases.
In the present invention, Proclin300 is a preservative.
Preferably, in the stability enhancer, the concentration of tris is 25mmol/L, the concentration of NaCl is 150mmol/L, the concentration of tween20 is 0.05 vol%, the concentration of bovine serum albumin is 1 mass%, the concentration of mutant alkaline phosphatase is 100 μ g/mL, and the concentration of Proclin300 is 0.1 vol%.
Preferably, in the kit, the calibrator is formulated with the international reference substance NIBSC02/286 for VEGF165, and the calibrator comprises VEGF165 in the following concentrations of samples: 0pg/mL, 6.9pg/mL, 20.6pg/mL, 61.7pg/mL, 185.2pg/mL, 555.6pg/mL, 1666.7pg/mL, 5000 pg/mL.
Preferably, in the kit, the cleaning concentrated solution contains tris, NaCl, tween20, triton 100 and Proclin 300;
in the cleaning concentrated solution, the concentration of the tris is 50mmol/L, the concentration of the NaCl is 875mmol/L, the concentration of the Tween20 is 0.15 volume%, the concentration of the Triton 100 is 0.05 volume%, and the concentration of the Proclin300 is 0.1 volume%.
Preferably, in the kit, the substrate solution is purchased from Shenzhen Meykater company under the cargo number of Intlus-A04.
As mentioned above, a second aspect of the present invention provides the use of a kit according to the first aspect as described above for the detection of vascular endothelial growth factor in anterior chamber fluid.
The present invention will be described in detail below by way of examples.
In the following examples, room temperature was 22 ℃ to 25 ℃ unless otherwise specified.
In the following examples, unless otherwise specified, the laboratory instruments and raw materials are commercially available.
Laboratory apparatus
Ultraviolet spectrophotometer: nanodrop One, Thermo corporation.
Blood blending instrument: KJMR IIA, health care medical company.
Raw materials
Vascular endothelial growth factor specific antibody (denoted antibody I): VEGF165/VEGFA labeled antibody, 11066-R010, Sino Biological.
Vascular endothelial growth factor specific antibody (denoted antibody II): VEGF165/VEGFA coated antibody, 11066-R012, Sino Biological.
Vascular endothelial growth factor165 international reference substance: vascular endoscopic Growth Factor165, 02/286, NIBSC.
Traut's Reagent (Traut's Reagent): 2-Iminosulfane hydrochloride, 26101, THERMO PIERCE.
SMCC: succinimidyl 4- [ N-maleimidomethyl ] cyclohexane-1-carboxylate, 22360, THERMO PIERCE.
NEM: n-ethylmaleimide 23030, THERMO PIERCE.
Ea (ethanolamine): ethanolamine, 398136-.
EDC: carbodiimide hydrochloride, 22980, THERMO PIERCE.
DMSO, DMSO: dimethylsulfoxide, D158550, Sigma company.
Alkaline phosphatase (alkaline phosphatase): AP, high activity (highlyactive), recombinant (recombiant), CR, 03535452103, Roche.
AP polypeptide: mutant alkaline phosphatase, 04781007103, Roche.
Tween 20: tween20, 0777-1L, Amresco Inc.
Proclin 300: 48914-U, Sigma.
PD10 column: disposable PD-10Desalting Column, dextran G-25 chromatography medium, 1.0-2.5mL sample (Disposable PD-10Desalting Column, with Sephadex G-25resin,1.0-2.5mL samples), 17085101, GE company.
Zeba column 7K 0.5 mL: zebaTMCentrifugal Desalting Columns (Spin Desalting Columns), 7K MWCO, 0.5mL, 89882, THERMO PIERCE.
Zeba column 7K 2 mL: zebaTMCentrifugal Desalting Columns (Spin Desalting Columns), 7K MWCO, 2mL, 89890, THERMO PIERCE.
Magnetic fine particles: magnosphereTMMS300/Carboxyl, MS300 CA, JSR Life Sciences, Inc.
Example 1
(1) Preparation of enzyme reactant
(a) Antibody treatment
PD10 column equilibrium: equilibrating PD10 column with TSE85 buffer, 5mL each, and repeating 5 times;
antibody loading: taking a specific antibody (the trade mark is 11066-R010, purchased from Sino Biological company) of the vascular endothelial growth factor for marking, wherein the mass is 1mg, the concentration of the antibody is 1mg/mL, and the volume of the antibody is 1000 mu L; adding the antibody into the balanced PD10 column, and supplementing 1500 mu L of TSE85 buffer solution;
and (3) antibody recovery: add 3mL of TSE85 buffer to PD10 column and recover the antibody by gravity;
and (3) antibody concentration detection: detecting A280 by using an ultraviolet spectrophotometer, wherein the concentration (mg/mL) of the detected antibody is A280/1.36;
antibody recovery calculation: the recovery rate of the antibody is detection mass (mg)/feeding mass (mg) × 100%;
wherein the detection mass (mg) is detection antibody concentration (mg/mL) × antibody volume (mL), and the dosing mass (mg) is vial antibody concentration (mg/mL) × antibody volume (mL);
concentration: concentrating the treated antibody to 5mg/mL by using a protein concentration tube, wherein the volume of the antibody is 200uL, and the antibody is reserved at 2-8 ℃;
(b) antibody activation
Weighing a certain mass of Traut's Reagent, and preparing to 13.76mg/mL (for activation within 2 min) by using TSE85 buffer solution;
adding the prepared Traut's reagent into the antibody, wherein the volume of the added Traut's reagent is 4 mu L; the molar ratio of Traut's reagent to antibody was 60: 1;
reacting at room temperature for 30 min;
PD10 column equilibrium: equilibrating PD10 column with TSE73 buffer, 5mL each, and repeating 5 times;
and (3) terminating the reaction: adding 4 mu L of 1mol/L glycine into the reactant to obtain an activated antibody;
and (3) purification: adding the activated antibody into a PD10 column, supplementing TSE73 buffer solution to 2500 mu L, adding 3mL of TSE73 buffer solution, and recovering the antibody by gravity;
and (3) antibody concentration detection: detecting A280 by using an ultraviolet spectrophotometer, wherein the concentration (mg/mL) of the detected antibody is A280/1.36;
(c) alkaline phosphatase treatment and activation
Taking 1mg of alkaline phosphatase, adding alkaline phosphatase dialysate to adjust the concentration to 10 mg/mL;
weighing a certain mass of SMCC, and preparing the SMCC into 27.56mg/mL (used for activation within 2 min) by using DMSO;
the prepared SMCC reagent (27.56mg/mL) is added into alkaline phosphatase, and the adding volume is 4.5 uL; the molar ratio of SMCC reagent to alkaline phosphatase is 20: 1;
reacting at room temperature for 30 min;
PD10 column equilibrium: equilibrating the PD10 column with TSMZ73 buffer, 5mL each, repeated 5 times;
and (3) terminating the reaction: adding 4.5 mu L of 1mol/L glycine into the reaction mixture;
and (3) purification: alkaline phosphatase was recovered by gravity by adding alkaline phosphatase to a PD10 column, adding TSMZ73 buffer to 2500. mu.L, and adding 3mL of TSMZ73 buffer;
and (3) detecting the concentration of alkaline phosphatase: detecting A280 by using an ultraviolet spectrophotometer, wherein the concentration of alkaline phosphatase (mg/mL) is A280/1;
(d) reaction and termination of post-activation antibody with post-activation alkaline phosphatase
Mixing the activated antibody with the activated alkaline phosphatase, and adding 1mol/L MgCl2Reacting at 2-8 ℃ for 22h (MgCl)2Volume (μ L) ═ antibody volume (μ L)/500, where the antibody is the activated antibody obtained in step (b);
the molar ratio of activated antibody/activated alkaline phosphatase was 0.75/1;
weighing a small amount of NEM, and dissolving the NEM in DMF to 9.7 mg/mL;
diluting the NEM with the concentration of 9.7mg/mL by 10 times by using TSMZ73 buffer;
adding the 10-fold diluted NEM obtained above to a marker, NEM volume (μ L) ═ marker volume (μ L)/100;
wherein the marker is a product obtained after the activated antibody and the activated alkaline phosphatase are marked, and the volume of the marker is the sum of the volumes of the activated antibody and the activated alkaline phosphatase;
mixing and standing for 10 min;
preparing 100mmol/L of EA (ammonium iodide) by using purified water, and adding the EA into the marker, wherein the volume (mu L) of the EA is equal to the volume (mu L) of the marker/100;
mixing and standing for 10 min;
(e) marker purification and preservation
PD10 column equilibrium: equilibrating the PD10 column with TSMZ73 buffer, 5mL each, repeated 5 times;
and (3) purification: adding the marker to a PD10 column, adding TSMZA73 buffer to 2500. mu.L, adding 3mL of TSMZA73 buffer, and recovering the marker by gravity;
and (3) antibody concentration detection: detecting A280 by using an ultraviolet spectrophotometer, wherein the concentration (mg/mL) of the detected antibody is A280/1.36;
adding glycerol with the same volume, and preserving at-20 ℃;
(f) reagent preparation
The purified marker was prepared to 2. mu.g/mL using Assaybuffer.
(2) Magnetic separation reagent preparation
(a) Antibody treatment
A0.5 mL Zeba centrifugal desalting column was equilibrated with 100mmol/L MES buffer (hereinafter, the "MES buffer" is referred to as "the buffer" and is referred to as "pH 5.0") at 0.3 mL/time × 3 times, and centrifuged at 1500 × g for 1 min;
1mg of vascular endothelial growth factor specific antibody (10 mg/mL. times.0.1 mL) (trade mark 11066-R012, available from Sino Biological Co.) was passed through a Zeba centrifugal desalting column, and centrifuged at 1500 Xg for 2min, and then the buffer in the antibody was replaced with the MES buffer (10 mg/mL. times.0.1 mL);
(b) magnetic particle treatment and activation
Collecting 200 μ L magnetic microparticles (10% solid content by mass), discarding supernatant, and washing with 400 μ L MES buffer solution for 2 times; adding 200 μ L MES buffer solution, and mixing;
weighing a certain amount of EDC, dissolving in MES buffer solution (ice bath) with the concentration of 19.1mg/mL, and adding 100 μ L; at this time, the molar ratio of EDC to the magnetic fine particles in terms of carboxyl groups contained therein was 50: 1;
supplementing the cold MES buffer solution to 0.4 mL;
reacting for 30min at room temperature on a blood mixing instrument;
adsorbing magnetic particles, discarding supernatant, washing with 0.4mL of the above cold MES buffer for 1 time;
resuspending in 0.2mL of cold MES buffer as described above;
(c) reaction of activated magnetic particles with antibodies
Adding 100 μ L of the antibody obtained in step (a) to 0.2mL of the activated magnetic particles obtained in step (b), and adding the MES buffer to 0.4 mL;
reacting at room temperature for 12h on a blood mixing instrument;
(d) blocking of magnetic particles after antibody coating
Adsorbing magnetic particles, collecting the supernatant in a centrifuge tube (protein concentration to be measured), and washing with MES buffer solution for 2 times (0.4 mL/time);
adding 0.4mL of blocking solution (AP Mutain), and reacting for 4h at room temperature on a blood mixing instrument;
adsorbing the magnetic particles, discarding the supernatant, washing 3 times (0.4 mL/time) with 25mmol/L Tris-HCl pH 7.4;
suspending the mixture in 0.4mL of magnetic particle preservation solution again, wherein the final concentration of the magnetic particles is 50 mg/mL;
(e) reagent preparation
The product was made up to 0.05mg/mL using a Beads buffer.
(3) Preparation of a stabilization enhancer
The stability enhancer contains 25mmol/L Tris, 150mmol/L NaCl, 1mmol/L MgCl2、1mmol/L ZnCl20.05 vol% tween20, 1 mass% BSA, 100 μ g/ml amputin and 0.1 vol% Proclin 300.
(4) Preparation of calibrator
The WHO international reference substance NIBSC02/286 was directly reconstituted with purified water to 13. mu.g/mL as described, and then prepared into samples at concentrations (pg/mL) of 0, 6.9, 20.6, 61.7, 185.2, 555.6, 1666.7, 5000, respectively, using STD Buffer.
(5) Experimental procedure
And (3) sequentially adding 20 mu L of the calibrator prepared in the step (4), 50 mu L of the magnetic separation reagent, 50 mu L of the enzyme reactant and 50 mu L of the stability enhancer into the reaction hole, fully and uniformly mixing, reacting at 35 ℃ for 30min, magnetically separating after the reaction is finished, discarding the supernatant, adding 375 mu L of cleaning solution, repeatedly cleaning for 3 times, adding the substrate, uniformly mixing in a dark place, and measuring light.
(6) Making a standard curve
And (3) making a standard curve by using the data measured in the step (5) through a four-parameter or linear fitting method, wherein the standard curve is obtained and is y-3.87311E +06+ ((10715.3-3.87311E +06)/(1+ (x/5082.16) ^ 1.35744)).
In the measurement range of 0-5000pg/mL, the four-parameter fitting correlation coefficient R is more than or equal to 0.9900.
(7) Detection method
And (5) when the sample to be detected is detected, replacing the calibrator with the sample to be detected by adopting the same experimental process as the step (5).
And (4) calculating the measured data by adopting the standard curve in the step (6) to obtain the concentration value of the VEGF in the sample.
The results of the above-described examples are shown below.
TABLE 1 sealing Effect
| Luminous value | BSA blocking | AP Mutain blocking | Is not closed |
| S0(0pg/mL) | 8679 | 3570 | 21010 |
| S1(6.9pg/mL) | 16650 | 9200 | 30770 |
| S1/S0 | 1.92 | 2.58 | 1.46 |
The occlusion effect was evaluated by two parameters, the light emission value at S0 and S1/S0 (signal-to-noise ratio).
As can be seen from the results in Table 1, when AP Mutain is used as a blocking agent, the emission value of S0 is the lowest, and S1/S0 (signal-to-noise ratio) is the highest, indicating that the blocking effect is the best.
TABLE 2 minimum detection limits
The test was carried out using a zero concentration calibrator or a sample diluent as a sample, and the measurement was repeated 20 times to obtain the RLU value (relative luminescence value) of the 20 measurements, as shown in table 2.
Calculating the average value (M) and the Standard Deviation (SD) to obtain M +2SD, and performing two-point regression fitting according to the concentration-RLU value result between the zero-concentration calibrator and the adjacent calibrator to obtain a linear equation: y 602.32x + 4290.
Substituting the RLU value of M +2SD into the equation to obtain a corresponding concentration value, namely the lowest detection limit which is 2.38pg/mL, which indicates that the lowest detection limit of the kit provided by the invention is lower.
The results show that the kit provided by the invention can accurately detect the content of the vascular endothelial growth factor in the anterior chamber fluid, has the advantages of small sample demand, short detection time and wide linear range, can obviously reduce the background value when the mutant alkaline phosphatase is used as the magnetic particle sealant, further reduces the minimum detection limit of the reagent, and has important application value for diagnosing VEGF related ophthalmic diseases.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (10)
1. A kit, which is characterized by comprising the following components which are independently preserved:
enzyme reactant, magnetic separation reagent, stability enhancer, calibrator, cleaning concentrated solution and substrate solution;
the enzyme reactant contains vascular endothelial growth factor specific antibody I marked by alkaline phosphatase; wherein, in the labeling process, the activation ratio of the antibody I is 60 times, the activation ratio of the alkaline phosphatase is 20 times, and the coupling ratio of the antibody I to the alkaline phosphatase is 1: 0.75;
the magnetic separation reagent contains 0.05 mass percent of magnetic particles, and the magnetic particles are coated with a vascular endothelial growth factor specific antibody II;
the antibody I is a VEGF165/VEGFA labeled antibody of Sino Biological company with the model number of 11066-R010, and the antibody II is a VEGF165/VEGFA coated antibody of Sino Biological company with the model number of 11066-R012.
2. The kit according to claim 1, wherein the magnetic microparticles have an activation ratio of 50 times and an antibody coating rate of 50 μ g/mg.
3. The kit according to claim 1 or 2, wherein the blocking agent in the magnetic separation reagent is a mutant alkaline phosphatase.
4. The kit according to claim 3, wherein the amount of the blocking agent is 100mg of blocking agent per 1g of magnetic microparticles.
5. The kit according to any one of claims 1 to 4, wherein the stability enhancer comprises tris, NaCl, Tween20, bovine serum albumin, mutant alkaline phosphatase, and Proclin 300.
6. The kit according to claim 5, wherein in the stability enhancer, the concentration of tris is 25mmol/L, the concentration of NaCl is 150mmol/L, the concentration of Tween20 is 0.05 vol%, the concentration of bovine serum albumin is 1 mass%, the concentration of mutant alkaline phosphatase is 100 μ g/mL, and the concentration of Proclin300 is 0.1 vol%.
7. The kit of any one of claims 1 to 6, wherein the calibrator is formulated with the international reference substance NIBSC02/286 for VEGF165 and comprises samples of VEGF165 at the following concentrations: 0pg/mL, 6.9pg/mL, 20.6pg/mL, 61.7pg/mL, 185.2pg/mL, 555.6pg/mL, 1666.7pg/mL, 5000 pg/mL.
8. The kit according to any one of claims 1 to 7, wherein the washing concentrate contains tris, NaCl, tween20, triton 100 and Proclin 300;
in the cleaning concentrated solution, the concentration of the tris is 50mmol/L, the concentration of the NaCl is 875mmol/L, the concentration of the Tween20 is 0.15 volume%, the concentration of the Triton 100 is 0.05 volume%, and the concentration of the Proclin300 is 0.1 volume%.
9. The kit according to any one of claims 1 to 8, wherein the substrate solution is a substrate solution purchased from Shenzhen Meykate company under the cargo number Intlus-A04.
10. Use of a kit according to any one of claims 1 to 9 for the detection of vascular endothelial growth factor in anterior chamber fluid.
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| CN113655226A (en) * | 2021-10-20 | 2021-11-16 | 首都医科大学附属北京朝阳医院 | Calibrator matrix for aqueous humor detection and preparation method and application thereof |
| WO2023206134A1 (en) * | 2022-04-27 | 2023-11-02 | Beijing Sightnovo Medical Technology Co., Ltd | Compositions and methods for eye diseases |
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| EP1767942A1 (en) * | 2004-06-14 | 2007-03-28 | Kyowa Medex Co., Ltd. | Immunoassay in which non-specific reaction is suppressed and reagent therefore |
| CN107561287A (en) * | 2017-08-30 | 2018-01-09 | 潍坊市康华生物技术有限公司 | A kind of vascular endothelial growth factor detection kit and its preparation and application |
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| WO2023206134A1 (en) * | 2022-04-27 | 2023-11-02 | Beijing Sightnovo Medical Technology Co., Ltd | Compositions and methods for eye diseases |
| WO2023208086A1 (en) * | 2022-04-27 | 2023-11-02 | Beijing Sightnovo Medical Technology Co., Ltd | Compositions and methods for eye diseases |
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