A kit and its application in detecting vascular endothelial growth factor in anterior chamber fluid

Abstract

The present invention relates to the technical field of aqueous humor antigen detection, and specifically to a kit and its application in detecting vascular endothelial growth factor in anterior chamber fluid, wherein the kit contains the following components which are stored independently: an enzyme reactant, a magnetic separation reagent, a stabilizing enhancer, a calibrator, a cleaning concentrate and a substrate solution. The kit provided by the present invention can accurately detect the content of vascular endothelial growth factor in anterior chamber fluid, has a small sample requirement, a short detection time, a wide linear range, and when mutant alkaline phosphatase is used as a magnetic particle blocking agent, the background value can be significantly reduced, thereby reducing the minimum detection limit of the reagent, and has important application value for the diagnosis of VEGF-related ophthalmic diseases.

Description

Technical Field

[0001] The invention relates to the technical field of aqueous humor antigen detection, and in particular to a kit and an application thereof in detecting vascular endothelial growth factor in anterior chamber fluid. Background Art

[0002] In the field of ophthalmic diseases, although the precise pathogenesis of most eye diseases is still not fully understood, key pathogenic molecules of some important blinding diseases have been discovered in recent years, among which vascular endothelial growth factor (VEGF) is the most typical.

[0003] Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is an important pro-angiogenic cytokine in the body. It acts on the glycosylated cell mitogens of endothelial cells to enhance vascular permeability, induce angiogenesis and vasculogenesis, promote endothelial cell growth, promote cell migration, and inhibit cell apoptosis.

[0004] VEGF at physiological concentrations is an essential cytokine for human development, injury repair and maintenance of normal physiological functions, but its overexpression is one of the important mechanisms of malignant tumor growth and ocular neovascular diseases.

[0005] Ocular neovascular diseases closely related to increased VEGF expression include neovascular age-related macular degeneration (nAMD, also called wet AMD), idiopathic polypoidal choroidal vasculopathy (IPCV), proliferative diabetic retinopathy (PDR), diabetic macular edema (DME), retinal vein occlusion (RVO), myopic choroidal neovascularization (myopicCNV), neovascular glaucoma, idiopathic choroidal neovascularization (iCNV) and idiopathic juxtafoveal telangiectasia (IJRT), etc. In addition, macular edema, Coats disease and retinopathy of prematurity (ROP) caused by various retinal diseases are also related to increased VEGF.

[0006] At present, the diagnosis and monitoring technology of intraocular diseases is mainly limited to physical diagnosis, imaging diagnosis and pathological diagnosis. Since the eyeball is a transparent and superficial organ, the physical diagnosis and imaging diagnosis technology of the eye are relatively well developed.

[0007] Although traditional experimental diagnostic techniques have long been widely used in systemic diseases and other large organ diseases, for example, the testing of body fluids such as blood, urine, cerebrospinal fluid, pleural effusion, and peritoneal effusion has long become a clinical routine, but intraocular fluid is limited by sampling difficulties and small sample sizes, and various traditional body fluid diagnostic techniques have not been used in the field of ophthalmology.

[0008] In addition, due to the influence of the blood-aqueous humor barrier, the protein content in the anterior chamber fluid is much lower than that in the blood. The minimum detection limits of the VEGF determination methods reported by Yeo et al. (Clin. Chem. 1992, 38: 71), Houcket et al. (J Biol. Chem. 1992, 267: 260-31) and Hanatani et al. (Biosci. Biotechnol. Biochem. 1995, 59: 1985) were relatively high, at the μg / mL-ng / mL level.

[0009] It can be seen that the development of a new detection technology with lower sample requirements and shorter detection time for diagnosing VEGF-related ophthalmic diseases has important application value. Summary of the invention

[0010] The purpose of the present invention is to overcome the defects of the existing detection technology for diagnosing VEGF-related diseases, such as high sample demand, long detection time and narrow linear range.

[0011] In order to achieve the above object, the first aspect of the present invention provides a kit, which contains the following components which are stored independently:

[0012] Enzyme reagents, magnetic separation reagents, stability enhancers, calibrators, wash concentrates, and substrate solutions;

[0013] The enzyme reactant contains a vascular endothelial growth factor-specific antibody I labeled with alkaline phosphatase; wherein, during 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;

[0014] The magnetic separation reagent contains 0.05 mass % of magnetic particles, and the magnetic particles are coated with vascular endothelial growth factor-specific antibody II;

[0015] The antibody I is a VEGF165 / VEGFA labeled antibody with model number 11066-R010 from Sino Biological Company, and the antibody II is a VEGF165 / VEGFA coated antibody with model number 11066-R012 from Sino Biological Company.

[0016] The second aspect of the present invention provides use of the kit described in the first aspect in detecting vascular endothelial growth factor in anterior chamber fluid.

[0017] Compared with the existing detection technology for diagnosing VEGF-related diseases, the kit for detecting vascular endothelial growth factor in anterior chamber fluid provided by the present invention has at least the following advantages:

[0018] (1) The kit provided by the present invention can accurately detect the content of vascular endothelial growth factor in the anterior chamber fluid, and at the same time, the sample requirement is small, only 20 μL of intraocular fluid sample is required;

[0019] (2) Compared with the traditional ELISA method which requires an incubation time of 2 hours or even overnight, the kit provided by the present invention only takes 30 minutes to complete the incubation and can complete the detection within 1 hour, which greatly saves time;

[0020] (3) The linear range is wide, with good linearity in the measurement range of 0-5000pg / mL.

[0021] Other features and advantages of the present invention will be described in detail in the following detailed description. DETAILED DESCRIPTION

[0022] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0023] As mentioned above, the first aspect of the present invention provides a kit, which contains the following components which are stored independently:

[0024] Enzyme reagents, magnetic separation reagents, stability enhancers, calibrators, wash concentrates, and substrate solutions;

[0025] The enzyme reactant contains a vascular endothelial growth factor-specific antibody I labeled with alkaline phosphatase; wherein, during 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;

[0026] The magnetic separation reagent contains 0.05 mass % of magnetic particles, and the magnetic particles are coated with vascular endothelial growth factor-specific antibody II;

[0027] The antibody I is a VEGF165 / VEGFA labeled antibody with model number 11066-R010 from Sino Biological Company, and the antibody II is a VEGF165 / VEGFA coated antibody with model number 11066-R012 from Sino Biological Company.

[0028] In the present invention, the vascular endothelial growth factor-specific antibody I labeled with alkaline phosphatase in the enzyme reaction and the vascular endothelial growth factor-specific antibody II coated on the magnetic microparticles cannot be used interchangeably.

[0029] In the present invention, the magnetic particles are produced by JSR Life Sciences, and the model is Magnosphere TM MS300 / Carboxyl.

[0030] In the present invention, the activation ratio is the molar ratio between the activator and the activated substance; the coupling ratio is the molar ratio between the antibody I and the alkaline phosphatase.

[0031] Preferably, the activation ratio of the magnetic particles is 50 times, and the antibody coating rate is 50 μg / mg.

[0032] Preferably, the blocking agent in the magnetic separation reagent is mutant alkaline phosphatase (AP mutain).

[0033] In the present invention, the process of coating the antibody on the magnetic particles includes a blocking process, and the blocking agent is an important material in the blocking process.

[0034] Preferably, the mutant alkaline phosphatase of the present invention is a mutant alkaline phosphatase (APmutain) produced by Roche, model number 04781007103.

[0035] In the present invention, the antibody coating rate is calculated based on the mass concentration and volume of the antibody and the magnetic particles; the blocking agent is a component in the magnetic particles.

[0036] Preferably, the amount of the blocking agent is 100 mg blocking agent / 1 g magnetic particles.

[0037] Preferably, the stabilization enhancer contains tris(hydroxymethyl)aminomethane (Tris), NaCl, Tween 20, bovine serum albumin (BSA), mutant alkaline phosphatase (AP mutain) and Proclin300.

[0038] In the present invention, the mutant alkaline phosphatase in the stabilizing enhancer and the mutant alkaline phosphatase used as the blocking agent are the same substance, and the function of the two components is to reduce non-specific adsorption.

[0039] The present invention uses mutant alkaline phosphatase as a blocking agent in the magnetic particle coating process, which can significantly reduce the background value and thus reduce the minimum detection limit of the reagent. The concentration of vascular endothelial growth factor in the detectable anterior chamber fluid is lower than 5pg / mL, which has important application value for the diagnosis of VEGF-related ophthalmic diseases.

[0040] In the present invention, Proclin 300 is a preservative.

[0041] Preferably, in the stabilizing enhancer, the concentration of tris(hydroxymethyl)aminomethane is 25 mmol / L, the concentration of NaCl is 150 mmol / L, the concentration of Tween 20 is 0.05% by volume, the concentration of bovine serum albumin is 1% by mass, the concentration of mutant alkaline phosphatase is 100 μg / mL, and the concentration of Proclin300 is 0.1% by volume.

[0042] Preferably, in the kit, the calibrator is prepared using the international reference material NIBSC 02 / 286 of VEGF165, and the calibrator contains samples with the following concentrations of VEGF165: 0 pg / mL, 6.9 pg / mL, 20.6 pg / mL, 61.7 pg / mL, 185.2 pg / mL, 555.6 pg / mL, 1666.7 pg / mL, 5000 pg / mL.

[0043] Preferably, in the kit, the cleaning concentrate contains tris(hydroxymethyl)aminomethane, NaCl, Tween 20, Triton 100 and Proclin 300;

[0044] In the cleaning concentrate, the concentration of tris(hydroxymethyl)aminomethane is 50 mmol / L, the concentration of NaCl is 875 mmol / L, the concentration of Tween 20 is 0.15% by volume, the concentration of Triton 100 is 0.05% by volume, and the concentration of Proclin 300 is 0.1% by volume.

[0045] Preferably, in the kit, the substrate solution is a substrate solution with a product number of Intlus-A04 purchased from Shenzhen Meikaite Company.

[0046] As mentioned above, the second aspect of the present invention provides the use of the kit described in the first aspect in detecting vascular endothelial growth factor in anterior chamber fluid.

[0047] The present invention will be described in detail below by way of examples.

[0048] In the following examples, unless otherwise specified, the room temperature is 22°C-25°C.

[0049] In the following examples, unless otherwise specified, the experimental instruments and raw materials involved are commercially available.

[0050] Experimental Instruments

[0051] UV spectrophotometer: Nanodrop One, Thermo Corporation.

[0052] Blood mixer: KJMR IIA, Kangjian Medical Corporation.

[0053] raw material

[0054] Vascular endothelial growth factor-specific antibody (denoted as antibody I): VEGF165 / VEGFA labeling antibody, 11066-R010, Sino Biological Company.

[0055] Vascular endothelial growth factor-specific antibody (denoted as antibody II): VEGF165 / VEGFA coated antibody, 11066-R012, Sino Biological Company.

[0056] Vascular endothelial growth factor 165 international reference material: Vascular Endothelial Growth Factor 165, 02 / 286, NIBSC.

[0057] Traut's Reagent: 2-Iminothiolane hydrochloride, 26101, THERMO PIERCE.

[0058] SMCC: succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate, 22360, THERMOPIERCE.

[0059] NEM: N-ethylmaleimide, 23030, THERMO PIERCE.

[0060] EA (Ethanolamine): Ethanolamine, 398136-500ML, Sigma.

[0061] EDC: carbodiimide hydrochloride, 22980, THERMO PIERCE.

[0062] DMSO: dimethyl sulfoxide, D158550, Sigma.

[0063] Alkaline phosphatase: AP, highly active, recombinant, CR, 03535452103, Roche.

[0064] AP Mutain: mutant alkaline phosphatase, 04781007103, Roche.

[0065] Tween 20: Tween20, 0777-1L, Amresco.

[0066] Proclin300:48914-U, Sigma.

[0067] PD10 column: Disposable PD-10 Desalting Column, with Sephadex G-25 resin, 1.0-2.5 mL samples, 17085101, GE.

[0068] Zeba column 7K 0.5mL: Zeba TM Spin Desalting Columns, 7K MWCO, 0.5 mL, 89882, THERMO PIERCE.

[0069] Zeba column 7K 2mL: Zeba TM Spin Desalting Columns, 7K MWCO, 2 mL, 89890, THERMO PIERCE.

[0070] Magnetic particles: Magnosphere TM MS300 / Carboxyl, MS300 CA, JSR Life Sciences.

[0071] Example 1

[0072] (1) Preparation of enzyme reactants

[0073] (a) Antibody treatment

[0074] PD10 column equilibration: Equilibrate the PD10 column with TSE85 buffer, 5 mL each time, repeat 5 times;

[0075] Antibody loading: Take the vascular endothelial growth factor-specific antibody for labeling (trade name 11066-R010, purchased from Sino Biological Company), with a mass of 1 mg and an antibody concentration of 1 mg / mL. The antibody volume required is 1000 μL; add the antibody to the equilibrated PD10 column and add 1500 μL of TSE85 buffer;

[0076] Antibody recovery: Add 3 mL of TSE85 buffer to the PD10 column and recover the antibody by gravity;

[0077] Antibody concentration detection: A280 was detected using a UV spectrophotometer, and the antibody concentration (mg / mL) = A280 / 1.36;

[0078] Calculation of antibody recovery rate: Antibody recovery rate = test mass (mg) / feed mass (mg) × 100%;

[0079] Among them, detection mass (mg) = detection antibody concentration (mg / mL) × antibody volume (mL), feed mass (mg) = bottle label antibody concentration (mg / mL) × antibody volume (mL);

[0080] Concentration: Use a protein concentrator to concentrate the treated antibody to 5 mg / mL, volume 200uL, and store at 2-8℃ for later use;

[0081] (b) Antibody Activation

[0082] Weigh a certain amount of Traut's Reagent and prepare it to 13.76 mg / mL with TSE85 buffer (for activation within 2 min);

[0083] Add the prepared Traut's reagent to the antibody in a volume of 4 μL; the molar ratio of Traut's reagent to antibody is 60:1;

[0084] React at room temperature for 30 minutes;

[0085] PD10 column equilibration: Equilibrate the PD10 column with TSE73 buffer, 5 mL each time, repeat 5 times;

[0086] Termination reaction: Add 4 μL of 1 mol / L glycine to the reaction to obtain activated antibody;

[0087] Purification: Add the activated antibody to the PD10 column, fill with TSE73 buffer to 2500 μL, add 3 mL of TSE73 buffer, and recover the antibody by gravity;

[0088] Antibody concentration detection: A280 was detected using a UV spectrophotometer, and the antibody concentration (mg / mL) = A280 / 1.36;

[0089] (c) Alkaline phosphatase treatment and activation

[0090] Take 1 mg of alkaline phosphatase and add alkaline phosphatase dialysate to adjust the concentration to 10 mg / mL;

[0091] Weigh a certain amount of SMCC and prepare it to 27.56 mg / mL with DMSO (for activation within 2 min);

[0092] Add the prepared SMCC reagent (27.56 mg / mL) to alkaline phosphatase in a volume of 4.5 uL; the molar ratio of SMCC reagent to alkaline phosphatase is 20:1;

[0093] React at room temperature for 30 minutes;

[0094] PD10 column equilibration: Equilibrate the PD10 column with TSMZ73 buffer, 5 mL each time, repeat 5 times;

[0095] Termination of the reaction: Add 4.5 μL of 1 mol / L glycine to the reaction;

[0096] Purification: Add alkaline phosphatase to PD10 column, fill with TSMZ73 buffer to 2500 μL, add 3 mL of TSMZ73 buffer, and recover alkaline phosphatase by gravity;

[0097] Alkaline phosphatase concentration detection: Use UV spectrophotometer to detect A280, and the detected alkaline phosphatase concentration (mg / mL) = A280 / 1;

[0098] (d) Reaction and termination of activated antibody and activated alkaline phosphatase

[0099] The activated antibody and activated alkaline phosphatase were mixed, 1 mol / L MgCl2 was added, and the mixture was reacted at 2-8°C for 22 h (MgCl2 volume (μL) = antibody volume (μL) / 500, wherein the antibody is the activated antibody obtained in step (b));

[0100] The molar ratio of activated antibody / activated alkaline phosphatase = 0.75 / 1;

[0101] Weigh a small amount of NEM and dissolve it in DMF to 9.7 mg / mL;

[0102] The above NEM with a concentration of 9.7 mg / mL was diluted 10 times with TSMZ73 buffer;

[0103] Add the 10-fold diluted NEM obtained above to the marker, NEM volume (μL) = marker volume (μL) / 100;

[0104] Wherein, the marker is a product obtained by labeling activated antibodies and activated alkaline phosphatase, and the volume of the marker is the sum of the volumes of the activated antibodies and activated alkaline phosphatase;

[0105] Mix well and let stand for 10 minutes;

[0106] Prepare 100mmol / L EA with purified water and add it to the marker, EA volume (μL) = marker volume (μL) / 100;

[0107] Mix well and let stand for 10 minutes;

[0108] (e) Marker purification and storage

[0109] PD10 column equilibration: Equilibrate the PD10 column with TSMZ73 buffer, 5 mL each time, repeat 5 times;

[0110] Purification: Add the marker to the PD10 column, fill up to 2500 μL with TSMZA73 buffer, add 3 mL of TSMZA73 buffer, and recover the marker by gravity;

[0111] Antibody concentration detection: A280 was detected using a UV spectrophotometer, and the antibody concentration (mg / mL) = A280 / 1.36;

[0112] Add an equal volume of glycerol and store at -20°C;

[0113] (f) Reagent preparation

[0114] The purified marker was prepared at 2 μg / mL using Assay buffer.

[0115] (2) Preparation of magnetic separation reagent

[0116] (a) Antibody treatment

[0117] Take a 0.5 mL Zeba centrifugal desalting column, equilibrate it with 100 mmol / L MES buffer at pH 5.0 (hereinafter referred to as "the above MES buffer"), 0.3 mL / time × 3 times, centrifuge at 1500 × g for 1 min each time;

[0118] 1 mg of vascular endothelial growth factor-specific antibody (10 mg / mL × 0.1 mL) (trade number 11066-R012, purchased from Sino Biological) was passed through a Zeba centrifugal desalting column, centrifuged at 1500 × g for 2 min, and then the buffer in the antibody was replaced with the above-mentioned MES buffer, 10 mg / mL × 0.1 mL;

[0119] (b) Magnetic particle treatment and activation

[0120] Take 200 μL of magnetic microparticles (10% by mass solid content), discard the supernatant, wash twice with 400 μL of the above-mentioned MES buffer; add 200 μL of the above-mentioned MES buffer and mix thoroughly;

[0121] Weigh a certain amount of EDC, dissolve it in the above MES buffer (ice bath) to a concentration of 19.1 mg / mL, and add 100 μL; at this time, the molar ratio of EDC to the amount of magnetic particles calculated based on the carboxyl groups contained therein is 50:1;

[0122] Make up to 0.4 mL of the cold MES buffer;

[0123] Incubate the mixture on a blood mixer at room temperature for 30 min;

[0124] Discard the supernatant after adsorbing the magnetic particles, and wash once with 0.4 mL of the cold MES buffer mentioned above;

[0125] Resuspend in 0.2 mL of cold MES buffer;

[0126] (c) Reaction of activated magnetic particles with antibodies

[0127] Add 100 μL of the antibody obtained in step (a) to 0.2 mL of the activated magnetic particles obtained in step (b), and make up the amount of the above MES buffer to 0.4 mL;

[0128] The reaction was carried out overnight at room temperature on a blood mixer for 12 h;

[0129] (d) Blocking of magnetic particles after antibody coating

[0130] Adsorb the magnetic particles, collect the supernatant in a centrifuge tube (protein concentration to be measured), and wash twice with the above-mentioned MES buffer (0.4 mL / time);

[0131] Add 0.4 mL of blocking solution (blocking buffer AP Mutain) and react on a blood mixer at room temperature for 4 h;

[0132] The supernatant was discarded after adsorption of magnetic particles, and the particles were washed three times with 25 mmol / L Tris-HCl, pH 7.4 (0.4 mL / time);

[0133] Resuspend in 0.4 mL magnetic particle storage solution, the final concentration of magnetic particles is 50 mg / mL;

[0134] (e) Reagent preparation

[0135] The above product was prepared with Beads buffer to 0.05 mg / mL.

[0136] (3) Preparation of stabilizing enhancer

[0137] The stabilization enhancer contains 25mmol / L Tris, 150mmol / L NaCl, 1mmol / L MgCl2, 1mmol / L ZnCl2, 0.05vol% tween20, 1wt% BSA, 100μg / mL APMutain and 0.1vol% Proclin300.

[0138] (4) Preparation of Calibrators

[0139] The WHO international reference material NIBSC 02 / 286 was directly reconstituted to 13 μg / mL with purified water according to the instructions, and then prepared with STD Buffer into samples with concentrations (pg / mL) of 0, 6.9, 20.6, 61.7, 185.2, 555.6, 1666.7, and 5000.

[0140] (5) Experimental process

[0141] Add 20 μL of the calibrant prepared in step (4), 50 μL of magnetic separation reagent, 50 μL of enzyme reactant, and 50 μL of stabilization enhancer to the reaction wells in sequence, mix thoroughly, and react at 35°C for 30 min. After the reaction is completed, magnetic separation is performed and the supernatant is discarded. Add 375 μL of cleaning solution, repeat the washing process 3 times, add the substrate, mix thoroughly in the dark, and measure the light.

[0142] (6) Preparation of standard curve

[0143] The data measured in step (5) are used to prepare a standard curve using a four-parameter or linear fitting method, and the obtained standard curve is y=3.87311E+06+((10715.3-3.87311E+06) / (1+(x / 5082.16)^1.35744)).

[0144] In the measurement range of 0-5000pg / mL, the four-parameter fitting correlation coefficient R≥0.9900.

[0145] (7) Detection method

[0146] When testing the sample to be tested, the same experimental process as step (5) is adopted, and the calibrator is replaced by the sample to be tested.

[0147] The measured data is calculated using the standard curve described in step (6) to obtain the concentration value of VEGF in the sample.

[0148] The test results of the above examples are shown below.

[0149] Table 1 Sealing effect

[0150] Luminous value BSA Blocking AP Mutain Closed Not closed S0(0pg / mL) 8679 3570 21010 S1 (6.9 pg / mL) 16650 9200 30770 S1 / S0 1.92 2.58 1.46

[0151] The blocking effect was evaluated by the luminescence value of S0 and S1 / S0 (signal-to-noise ratio).

[0152] From the results in Table 1, it can be seen that when AP Mutain is used as a blocking agent, the luminescence value of S0 is the lowest and S1 / S0 (signal-to-noise ratio) is the highest, indicating that its blocking effect is the best.

[0153] Table 2 Minimum detection limit

[0154]

[0155]

[0156] The zero-concentration calibrator or sample diluent was used as the sample for detection, and the measurement was repeated 20 times to obtain the RLU values ​​(relative luminescence values) of the 20 measurement results, as shown in Table 2.

[0157] The mean (M) and standard deviation (SD) were calculated to obtain M+2SD, and a linear equation was obtained by performing a two-point regression fit based on the concentration-RLU value results between the zero-concentration calibrator and the adjacent calibrators: y=602.32x+4290.

[0158] Substituting the RLU value of M+2SD into the above equation, the corresponding concentration value was obtained, which was the minimum detection limit, which was 2.38 pg / mL, indicating that the minimum detection limit of the kit provided by the present invention was low.

[0159] It can be seen from the above results that the kit provided by the present invention can accurately detect the content of vascular endothelial growth factor in the anterior chamber fluid, has a small sample requirement, a short detection time, a wide linear range, and when mutant alkaline phosphatase is used as a magnetic particle blocking agent, it can significantly reduce the background value, thereby reducing the minimum detection limit of the reagent, and has important application value for the diagnosis of VEGF-related ophthalmic diseases.

[0160] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A kit, It is characterized in that The kit contains the following components, each stored separately: Enzyme reagents, magnetic separation reagents, stability enhancers, calibrators, wash concentrates, and substrate solutions; The enzyme reactant contains a vascular endothelial growth factor-specific antibody I labeled with alkaline phosphatase; wherein, during 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 % of magnetic particles, and the magnetic particles are coated with vascular endothelial growth factor-specific antibody II; The blocking agent in the magnetic separation reagent is a mutant alkaline phosphatase; The antibody I is a VEGF165 / VEGFA labeled antibody with model number 11066-R010 from Sino Biological Company, and the antibody II is a VEGF165 / VEGFA coated antibody with model number 11066-R012 from Sino Biological Company; The stabilizing enhancer contains tris(hydroxymethyl)aminomethane, NaCl, Tween 20, bovine serum albumin, mutant alkaline phosphatase and Proclin300; In the stabilizing enhancer, the concentration of tris(hydroxymethyl)aminomethane is 25 mmol / L, the concentration of NaCl is 150 mmol / L, the concentration of Tween 20 is 0.05% by volume, the concentration of bovine serum albumin is 1% by mass, the concentration of mutant alkaline phosphatase is 100 μg / mL, and the concentration of Proclin300 is 0.1% by volume.

2. The kit according to claim 1, in, The activation ratio of the magnetic particles is 50 times, and the antibody coating rate is 50 μg / mg.

3. The kit according to claim 2, in, The amount of the sealing agent used is 100 mg sealing agent / 1 g magnetic particles.

4. The kit according to claim 1 or 2, in, The calibrator is prepared using the international reference material NIBSC02 / 286 of VEGF165, and the calibrator contains samples with the following concentrations of VEGF165: 0 pg / mL, 6.9 pg / mL, 20.6 pg / mL, 61.7 pg / mL, 185.2 pg / mL, 555.6 pg / mL, 1666.7 pg / mL, and 5000 pg / mL.

5. The kit according to claim 1 or 2, in, The cleaning concentrate contains tris(hydroxymethyl)aminomethane, NaCl, Tween 20, Triton 100 and Proclin 300; In the cleaning concentrate, the concentration of tris(hydroxymethyl)aminomethane is 50 mmol / L, the concentration of NaCl is 875 mmol / L, the concentration of Tween 20 is 0.15% by volume, the concentration of Triton 100 is 0.05% by volume, and the concentration of Proclin 300 is 0.1% by volume.

6. The kit according to claim 1 or 2, in, The substrate solution is purchased from Shenzhen Meikaite Company with the product number Intlus-A04.

7. Use of the kit according to any one of claims 1 to 6 for detecting vascular endothelial growth factor in anterior chamber fluid for non-disease diagnosis purposes.

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