Kit and its preparation method and application

Abstract

The present invention discloses a kit, a preparation method and an application thereof, relating to the technical field of immunoassay analysis. The kit is used for detecting the content of soluble fms-like tyrosine kinase-1, and the kit includes: a first reagent, including streptavidin magnetic beads and a first diluent; a second reagent, including an enzyme-labeled soluble fms-like tyrosine kinase-1 monoclonal antibody and a second diluent; a third reagent, including NHS-LC-biotin labeled with a soluble fms-like tyrosine kinase-1 monoclonal antibody and a third diluent; a calibrator and a luminescent substrate. The kit proposed by the present invention has high sensitivity, good stability and repeatability, and strong anti-interference ability when detecting the content of soluble fms-like tyrosine kinase-1.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay analysis, and particularly relates to a kit, a preparation method thereof, and an application thereof. Background Art

[0002] sFlt-1 (soluble fms-like tyrosine kinase-1) is a rare splicing form of vascular endothelial growth receptor-1 (fms-like tyrosine kinase receptor 1, Flt-1), which only retains the ligand-binding region and does not have tyrosine kinase activity. Its molecular weight is 63.6 KD and it is produced by trophoblasts during pregnancy. Medical research at home and abroad shows that sFlt-1 inhibits angiogenesis by affecting the functions of pro-angiogenic factors VEGF (vascular endothelial growth factor) and PIGF (placental growth factor); the expressions of VEGF and PIGF in the placenta of patients with PE (preeclampsia) are decreased, and it is inferred that the reduction of VEGF family protein expression affects the differentiation and invasion of placental trophoblasts, and even leads to placental hypoxia reaction. Therefore, it is inferred that sFlt-1 participates in the pathogenesis of PE by antagonizing the biological functions of VEGF and PIGF. Compared with normal pregnant women, the blood concentration of sFlt-1 significantly increases 5 weeks before the appearance of PE symptoms, while the concentration of PIGF decreases at 13-16 weeks of pregnancy. Therefore, the predictive effect of the concentrations of sFlt-1 and PIGF in pregnant women's serum on PE has become a research hotspot. Current research shows that sFlt-1 / PlGF can be used as an effective parameter for diagnosing and predicting PE. The increase of sFlt-1 / PlGF is closely related to the clinical need for delivery. sFlt-1 / PlGF is suitable for close monitoring of patients with high-risk preeclampsia to help clinical decision-making.

[0003] There are various methods for measuring the content of sFlt-1. Currently, there are mainly immunoturbidimetry, fluorescence immunochromatography, chemiluminescence immunoassay, etc. For immunoturbidimetry, this method has high requirements for samples and poor anti-interference ability. There will be large test deviations for hemolytic samples and lipid samples, and the sensitivity is relatively low; fluorescence immunochromatography is simple, efficient, and easy to operate, and does not require large-scale instruments. However, due to the limitations of the membrane medium, there are defects such as large test deviations, susceptibility to environmental influence, and low sensitivity. Summary of the Invention

[0004] The main object of the present invention is to propose a kit, a preparation method thereof, and an application thereof, aiming to provide a kit for detecting the content of soluble fms-like tyrosine kinase-1 with high sensitivity, good stability and repeatability, and strong anti-interference ability.

[0005] To achieve the above object, the present invention proposes a kit for detecting the content of soluble fms-like tyrosine kinase-1, and the kit includes:

[0006] The first reagent, comprising streptavidin magnetic beads and a first diluent;

[0007] The second reagent, comprising an enzyme-labeled soluble fms-like tyrosine kinase-1 monoclonal antibody and a second diluent;

[0008] The third reagent, comprising NHS-LC-biotin labeled soluble fms-like tyrosine kinase-1 monoclonal antibody and a third diluent;

[0009] Calibrators and luminescent substrates.

[0010] Optionally, the first diluent includes a buffer, a surfactant, a protein, a preservative, salts, amino acids, saccharides, and a high molecular polymer.

[0011] Optionally, the buffer is 2-morpholinoethanesulfonic acid, and the concentration of 2-morpholinoethanesulfonic acid in the first diluent is 20 mM to 100 mM; and / or,

[0012] The surfactant is any one of Tween 20, Tween 80, Triton 100, and Triton X405, and the concentration of the surfactant in the first diluent is 0.01% to 0.1% (v / v); and / or,

[0013] The protein is bovine serum albumin, and the concentration of bovine serum albumin in the first diluent is 1% to 3% (w / v); and / or,

[0014] The preservative is Proclin 300; and / or,

[0015] The salts are NaCl; and / or,

[0016] The amino acid is glycine, and the concentration of glycine in the first diluent is 0.05% to 0.2% (w / v); and / or,

[0017] The saccharide is trehalose or sucrose, and the concentration of the saccharide in the first diluent is 0.1% to 2% (w / v); and / or,

[0018] The high molecular polymer is PEG modified with BSA, and the concentration of the high molecular polymer in the first diluent is 0.01% to 2% (w / v).

[0019] Optionally, the first diluent comprises: 50 mM 2-morpholinoethanesulfonic acid, 0.05% Tween 20 (v / v), 0.1% Proclin 300 (v / v), 0.9% NaCl (w / v), 1% bovine serum albumin (w / v), 0.1% glycine (w / v), 0.5% trehalose (w / v), and 0.05% BSA-modified PEG (w / v), and the pH value of the first diluent is 6.5.

[0020] Optionally, the second diluent comprises: 50 mM to 100 mM 2-morpholinoethanesulfonic acid, 0.05% to 0.2% Tween 20 (v / v), 0.1% Proclin 300 (v / v), 0.9% NaCl (w / v), and 1% to 3% bovine serum albumin (w / v); and / or,

[0021] The pH of the first diluent is 6.5 to 8.0; and / or,

[0022] The pH of the second diluent is 6.5 to 7.2; and / or,

[0023] The third diluent comprises 50 mM 2-morpholinoethanesulfonic acid, 0.05% Tween 20 (v / v), 0.1% Proclin 300 (v / v), 0.9% NaCl (w / v), and 1.5% bovine serum albumin (w / v), and the pH value of the third diluent is 6.8.

[0024] Optionally, the second diluent comprises: 50 mM 2-morpholinoethanesulfonic acid, 0.05% Tween 20 (v / v), 0.1% Proclin 300 (v / v), 0.9% NaCl (w / v), and 1.5% bovine serum albumin (w / v), and the pH value of the second diluent is 6.8; and / or,

[0025] In the first reagent, the concentration of streptavidin magnetic beads is 0.05 mg / mL to 0.2 mg / mL; and / or,

[0026] In the first reagent, the particle size of the streptavidin magnetic beads is 1.5 μm.

[0027] Optionally, in the second reagent, the enzyme of the enzyme-labeled soluble fms-like tyrosine kinase-1 monoclonal antibody is alkaline phosphatase; and / or,

[0028] In the third reagent, the concentration of NHS-LC-biotin labeled with soluble fms-like tyrosine kinase-1 monoclonal antibody is 0.1 μg / mL to 0.3 μg / mL.

[0029] Optionally, the calibrator is a buffer added with soluble fms-like tyrosine kinase-1 antigen; and / or,

[0030] The luminescent substrate is AMPPD.

[0031] The present invention further provides a method for preparing the kit as described above, comprising the following steps:

[0032] Add streptavidin magnetic beads to the first diluent, mix evenly, perform magnetic separation and washing, and then resuspend in the first diluent to obtain the first reagent;

[0033] After activating the soluble fms-like tyrosine kinase-1 monoclonal antibody, mixing and reacting it with an enzyme solution and purifying, an enzyme-labeled soluble fms-like tyrosine kinase-1 monoclonal antibody is prepared. Mix the enzyme-labeled soluble fms-like tyrosine kinase-1 monoclonal antibody evenly with the second diluent to obtain the second reagent;

[0034] Dissolve NHS-LC-biotin in dimethyl sulfoxide to form an NHS-LC-biotin solution. Add soluble fms-like tyrosine kinase-1 to PBS buffer, mix evenly, and mix and react with the NHS-LC-biotin solution. After purification, NHS-LC-biotin labeled with soluble fms-like tyrosine kinase-1 monoclonal antibody is obtained. Mix the NHS-LC-biotin labeled with soluble fms-like tyrosine kinase-1 monoclonal antibody evenly with the third diluent to obtain the third reagent.

[0035] The present invention further provides a method for detecting the content of soluble fms-like tyrosine kinase-1. Using the kit as described above, the method for detecting the content of soluble fms-like tyrosine kinase-1 comprises the following steps:

[0036] Mix the test sample evenly with the second reagent, add the first reagent and the third reagent, mix and incubate to obtain a mixture;

[0037] After subjecting the mixture to magnetic separation and washing, add the luminescent substrate and mix evenly to obtain a test solution;

[0038] Detect the luminescence intensity of the test solution;

[0039] Calculate the content of soluble fms-like tyrosine kinase-1 based on the luminescence intensity.

[0040] In the technical solution provided by the present invention, an enzyme-catalyzed chemiluminescence immunoassay method is used, and a one-step reaction mode is adopted. According to the principle of the double-antibody sandwich method, under the action of an external magnetic field, the complex formed by the immune reaction is separated from other unbound substances. After removing the supernatant, the magnetic particle complex is washed, a luminescent substrate is added, and the luminescence intensity of the reaction is detected by a luminometer. The luminescence intensity is proportional to the content of the antigen to be detected, and the concentration of the antigen to be detected in the sample can be calculated using the corresponding calculation method. Through this enzyme-catalyzed chemiluminescence immunoassay method, a detection kit with high detection sensitivity, stable detection results, good repeatability, high sensitivity, and strong anti-interference ability is provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or in the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0042] Figure 1 It is a comparison diagram of one-step blocking and two-step blocking in the embodiment of the present invention;

[0043] Figure 2 It is a detection result diagram of the linear range of the kit in Example 1 of the present invention;

[0044] Figure 3 It is a comparison diagram of clinical sample tests of the kit in Example 1 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0045] The following will clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, rather than all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0046] In addition, the technical solutions between the various embodiments can be combined with each other, but it must be based on the fact that those of ordinary skill in the art can implement them. When the combination of technical solutions results in contradictions or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0047] For those conditions not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0048] There are various methods for measuring the content of sFlt-1. Currently, there are mainly immunoturbidimetry, fluorescence immunochromatography, chemiluminescent immunoassay, etc. Immunoturbidimetry has high requirements for samples and poor anti-interference ability. There will be large test deviations for hemolytic samples and blood lipid samples, and the sensitivity is relatively low. Fluorescence immunochromatography is simple, efficient and easy to operate, and does not require large-scale instruments. However, due to the limitations of the membrane medium, there are defects such as large test deviations, easy to be affected by the environment, and low sensitivity.

[0049] In view of this, the present invention provides a kit, its preparation method and application, aiming to provide a kit for detecting the content of soluble fms-like tyrosine kinase-1 with high sensitivity, good stability and repeatability, and strong anti-interference ability.

[0050] In an embodiment of the kit provided by the present invention, the kit is used to detect the content of soluble fms-like tyrosine kinase-1, and the kit includes: a first reagent, including streptavidin magnetic beads and a first diluent; a second reagent, including an enzyme-labeled soluble fms-like tyrosine kinase-1 monoclonal antibody and a second diluent; a third reagent, including NHS-LC-biotin labeled with a soluble fms-like tyrosine kinase-1 monoclonal antibody and a third diluent; a calibrator and a luminescent substrate.

[0051] In the technical solution provided by the present invention, the enzyme-catalyzed chemiluminescent immunoassay method is used, and a one-step reaction mode is adopted. According to the principle of the sandwich immunoassay method, under the action of an external magnetic field, the complex formed by the immune reaction is separated from other unbound substances. After removing the supernatant, the magnetic particle complex is washed, and a luminescent substrate is added. The luminescence intensity of the reaction is detected by a luminometer. The luminescence intensity is proportional to the content of the antigen to be measured, and the concentration of the antigen to be measured in the sample can be calculated using the corresponding calculation method. The present invention adopts a streptavidin-NHS-LC-biotin system to amplify the signal value and can effectively improve the sensitivity of the reagent. Through this enzyme-catalyzed chemiluminescent immunoassay method, a detection kit with high detection sensitivity, stable detection results, good repeatability, high sensitivity and strong anti-interference ability is provided.

[0052] Compared with other types of luminescence, the enzyme-catalyzed chemiluminescent method has a fast reaction speed, provides correct and reliable results in a very short time, is beneficial to signal detection, and provides the sensitivity and specificity performance of the kit. The kit of the present invention has a simple structure and low cost, greatly reducing the production cost. Moreover, its operation is simple and can improve the detection efficiency. In addition, the affinity constant of avidin binding to biotin can be one million times that of the antigen-antibody reaction. The dissociation constant of the complex formed by the two is very small, showing irreversible reactivity; and acids, bases, denaturing agents, proteolytic enzymes, and organic solvents do not affect their binding. Therefore, in this application, the stability of the product is high, which can reduce operation errors and improve the accuracy of determination.

[0053] The present invention places no restrictions on the components of the first diluent, the second diluent, and the third diluent. Preferably, the first diluent includes a buffer, a surfactant, a protein, a preservative, salts, amino acids, sugars, and a high molecular polymer. The inclusion of the above substances in the first diluent can effectively improve the sensitivity, stability, repeatability, and anti-interference ability of the kit.

[0054] Specifically, the buffer is 2-(N-morpholino)ethanesulfonic acid (MES), and the concentration of 2-(N-morpholino)ethanesulfonic acid in the first diluent is 20 mM to 100 mM; the surfactant is any one of Tween 20, Tween 80, Triton X-100, and Triton X-405, and the concentration of the surfactant in the first diluent is 0.01% to 0.1% (v / v); the protein is bovine serum albumin (BSA), and the concentration of bovine serum albumin in the first diluent is 1% to 3% (w / v); the preservative is Proclin 300; the salts are NaCl; the amino acid is glycine, and the concentration of glycine in the first diluent is 0.05% to 0.2% (w / v); the sugar is trehalose or sucrose, and the concentration of the sugar in the first diluent is 0.1% to 2% (w / v); the high molecular polymer is PEG modified with BSA, with a molecular weight of 5000 to 20000, and the concentration of the high molecular polymer in the first diluent is 0.01% to 2% (w / v). The selection of the above substances can satisfy only one of them or can satisfy them simultaneously. As a preferred embodiment of the present invention, the above substances satisfy simultaneously, and the obtained kit has the highest sensitivity.

[0055] It should be noted that v / v represents the volume ratio. For example, when the concentration of the surfactant is 0.01%, it means that 0.01 mL of the surfactant is included in 100 mL of the first diluent; w / v represents the mass-volume ratio, with the unit of g / mL. For example, when the concentration of bovine serum albumin is 1%, it means that 1 g of bovine serum albumin is included in 100 mL of the first diluent. The same applies hereinafter and will not be elaborated further.

[0056] Preferably, the first diluent includes: 50 mM 2-(N-morpholino)ethanesulfonic acid, 0.05% Tween 20 (v / v), 0.1% Proclin 300 (v / v), 0.9% NaCl (w / v), 1% bovine serum albumin (w / v), 0.1% glycine (w / v), 0.5% trehalose (w / v), and 0.05% PEG modified with BSA (w / v).

[0057] Under the above substances and ratios, the test effect of the obtained kit is better. Among them, 0.05% PEG (w / v) has an amino group, with a molecular weight of 5000 - 20000. Since PEG is polyethylene glycol, a high molecular polymer, and also a dispersant, it has a certain promoting aggregation property and can promote the binding of antigens and antibodies in the reaction. Because it has an amino group, after being modified by BSA, in this kit, this substance can significantly increase its signal value and play a relatively obvious role in improving its sensitivity. Design experiments to verify that adding 0.05% BSA-modified PEG and not adding it to its diluent, and test the reactivity of the kit. The data is shown in Table 1 below. Adding 0.05% BSA-modified PEG nearly doubles the increase in its signal value, and the sensitivity of the kit is significantly improved.

[0058] Table 1 Influence of the addition of BSA-modified PEG on the sensitivity of the kit

[0059] Diluent Without addition of BSA-modified PEG Addition of 0.05% BSA-modified PEG Concentration (pg / mL) Luminescence value (RLU value) Luminescence value (RLU value) 0 1413 1695 100 52870 103389 1000 624726 1176959

[0060] For the second diluent, preferably, the second diluent includes: 50 mM - 100 mM 2-morpholinoethanesulfonic acid, 0.05% - 0.2% Tween 20 (v / v), 0.1% Proclin 300 (v / v), 0.9% NaCl (w / v), and 1% - 3% bovine serum albumin (w / v). Under the above substances and ratios, the test effect of the obtained kit is better.

[0061] Furthermore, the second diluent includes: 50 mM 2-morpholinoethanesulfonic acid, 0.05% Tween 20 (v / v), 0.1% Proclin 300 (v / v), 0.9% NaCl (w / v), and 1.5% bovine serum albumin (w / v). Under the above substances and ratios, the test effect of the obtained kit is the best.

[0062] For the third diluent, preferably, the third diluent includes 50 mM 2-morpholinoethanesulfonic acid, 0.05% Tween 20 (v / v), 0.1% Proclin 300 (v / v), 0.9% NaCl (w / v), and 1.5% bovine serum albumin (w / v). Under the above substances and ratios, the test effect of the obtained kit is better.

[0063] The present invention also does not limit the pH of the first diluent and the second diluent. Preferably, the pH of the first diluent is 6.5 - 8.0; the pH of the second diluent is 6.5 - 7.2. More preferably, the pH of the first diluent is 6.5; the pH of the second diluent is 6.8, and the pH of the third diluent is 6.8. The above pH values are beneficial to improving the accuracy of the detection of soluble fms-like tyrosine kinase-1 content.

[0064] Regarding the concentration and particle size of streptavidin magnetic beads in the first reagent, the present invention places no restrictions. Preferably, in the first reagent, the concentration of streptavidin magnetic beads is 0.05 mg / mL to 0.2 mg / mL. At the above concentrations, the test results are accurate. Preferably, in the first reagent, the particle size of the streptavidin magnetic beads is 1.5 μm. At the above particle size, it is easier to form a double antibody sandwich structure.

[0065] In addition, preferably, the enzyme of the enzyme-labeled soluble fms-like tyrosine kinase-1 monoclonal antibody in the second reagent is alkaline phosphatase. Alkaline phosphatase is an enzyme that can dephosphorylate the corresponding substrate, that is, remove the phosphate group on the substrate molecule by hydrolyzing phosphomonoester, and generate phosphate ions and free hydroxyl groups. Such substrates include nucleic acids, proteins, alkaloids, etc. And this process of removing the phosphate group is called dephosphorylation or phosphorylation. Alkaline phosphatase is a type of phosphatase, and the function of phosphatase is the opposite of that of kinase. Kinase is a phosphorylating enzyme that can use energy molecules such as ATP to add a phosphate group to the corresponding substrate molecule. Alkaline phosphatase has the maximum activity in an alkaline environment. For ALP derived from bacteria, its optimal pH is 8.0, while for ALP derived from cattle, it is 8.5.

[0066] Regarding the NHS-LC-biotin in the third reagent, which is succinimidyl 6-(biotinamido)hexanoate, and the concentration of NHS-LC-biotin for labeling the soluble fms-like tyrosine kinase-1 monoclonal antibody, the present invention also places no restrictions. In the third reagent, the concentration of NHS-LC-biotin for labeling the soluble fms-like tyrosine kinase-1 monoclonal antibody is 0.1 μg / mL to 0.3 μg / mL. At the above concentrations, the test results are accurate.

[0067] In addition, preferably, when preparing NHS-LC-biotin for labeling the soluble fms-like tyrosine kinase-1 monoclonal antibody, the molar ratio of the soluble fms-like tyrosine kinase-1 monoclonal antibody to NHS-LC-biotin is 1:10. At the above ratio, the test results are accurate.

[0068] Furthermore, in the embodiments of the present invention, the calibrator is a buffer solution added with soluble fms-like tyrosine kinase-1 antigen. Using the above calibrator, the test results are accurate.

[0069] In addition, preferably, the luminescent substrate includes AMPPD. AMPPD is a 1,2-dioxocyclohexane derivative, which is a latest ultrasensitive alkaline phosphatase substrate in the field of biochemistry, characterized by a fast reaction rate and providing correct and reliable results in a very short time. There are two important parts in its molecular structure. One is the dioxatetracyclic ring connecting the benzene ring and the adamantane, which can break and emit photons; the other is the phosphate group, which maintains the stability of the whole molecular structure. Under normal circumstances, this compound is very stable.

[0070] The present invention further provides a method for preparing the kit as described above, comprising the following steps:

[0071] S10: Add streptavidin magnetic beads to the first diluent, mix evenly, perform magnetic separation and washing, and then resuspend in the first diluent to obtain the first reagent.

[0072] This step is used to prepare the first reagent. The following gives a specific implementation manner of this step:

[0073] Take 10 mg of streptavidin magnetic beads, add 1 mL of the first diluent (50 mM 2-morpholinoethanesulfonic acid, 0.05% Tween 20 (v / v), 0.1% Proclin 300 (v / v), 0.9% NaCl (w / v), 1% bovine serum albumin (w / v), 0.1% glycine (w / v), 0.5% trehalose (w / v) and 0.05% BSA-modified PEG (w / v), pH 6.5), perform magnetic separation and washing, repeat the washing 3 times, and then resuspend in the first diluent. Dilute with the first diluent to a streptavidin magnetic bead concentration of 0.05 mg / mL to 0.2 mg / mL, which is the first reagent.

[0074] S20: After activating the soluble fms-like tyrosine kinase-1 monoclonal antibody, mix it with the enzyme solution for reaction and purification to obtain an enzyme-labeled soluble fms-like tyrosine kinase-1 monoclonal antibody. Mix the enzyme-labeled soluble fms-like tyrosine kinase-1 monoclonal antibody evenly with the second diluent to obtain the second reagent.

[0075] Specifically, in the embodiment of the present invention, the operation of step S20 is as follows:

[0076] Take 100 μL of soluble fms-like tyrosine kinase-1 monoclonal antibody, add 250 μL of PBS buffer with a concentration of 0.1 M and a pH value of 7.0, mix well, add 20 μL of freshly prepared 10.0 mg / mL EDC aqueous solution, activate for 30 minutes, then add 200 μL of alkaline phosphatase solution with a concentration of 5 mg / mL and mix well. Take it out after storing in the dark at room temperature for 2 hours, block the reaction at room temperature for 30 min with 0.1 M Tris buffer containing 1% BSA and a pH value of 7.4, then add 0.5% diethyl phosphate and continue the reaction at room temperature for 30 min. Desalt and purify with a 30 KD ultrafiltration column. Add half glycerol to the collected remaining volume solution and store it at -20 °C for later use to prepare the enzyme-labeled soluble fms-like tyrosine kinase-1 monoclonal antibody. Take the enzyme-labeled soluble fms-like tyrosine kinase-1 monoclonal antibody and dilute it 1:2000 times with the second diluent (50 mM 2-morpholinoethanesulfonic acid, 0.05% Tween 20 (v / v), 0.1% Proclin 300 (v / v), 0.9% NaCl (w / v) and 1.5% bovine serum albumin (w / v), pH value of 6.8) to obtain the second reagent.

[0077] Among them, diethyl phosphate is used as a secondary modification blocking agent, and its function is to modify and block the reaction. Because its phosphate ions are significantly helpful for the stability of the enzyme-labeled reagent of the second reagent, but the phosphate ions in its solution will affect the reaction between alkaline phosphatase and the substrate. The diluent of the second reagent generally selects MES buffer as the buffer instead of PBS buffer. However, because its phosphate ions are beneficial to its stability, diethyl phosphate is selected to be added during the labeling of alkaline phosphatase antibody for phosphorylation blocking treatment to achieve the effect of enhanced stability without affecting its reaction with the substrate.

[0078] The present invention uses one-step blocking (Tris buffer) and two-step blocking (Tris buffer + diethyl phosphate) to conduct comparative experiments for labeling antibodies, and prepares the second reagent after labeling. Divide the second reagent into 5 equal parts, store one part at 2 - 8 °C in the refrigerator, and place the other 4 parts in a 37 °C incubator for acceleration for 1 day, 3 days, 5 days, and 7 days respectively. After the acceleration is completed, take it out and equilibrate to room temperature, use the same first reagent and third reagent to test samples at 3 concentration levels, and evaluate the difference in their stability.

[0079] The test results are shown in Figure 1 , and the acceleration decay rate of its two-step blocking (Tris buffer + diethyl phosphate) is significantly lower than that of one-step blocking (Tris buffer), its stability is significantly better than that of one-step blocking, the signal value of its two-step blocking is slightly lower than that of one-step blocking, but the influence is not great and can meet the performance requirements.

[0080] S30. Dissolve NHS-LC-biotin in dimethyl sulfoxide to form an NHS-LC-biotin solution. Add soluble fms-like tyrosine kinase-1 to PBS buffer and mix evenly. After mixing and reacting with the NHS-LC-biotin solution, purify to obtain NHS-LC-biotin labeled with soluble fms-like tyrosine kinase-1 monoclonal antibody. Mix the NHS-LC-biotin labeled with soluble fms-like tyrosine kinase-1 monoclonal antibody evenly with the third diluent to obtain the third reagent.

[0081] The following gives a specific implementation of this step:

[0082] Weigh 1 mg of NHS-LC-biotin and dissolve it in dimethyl sulfoxide (DMSO) to 10 mM. Take 100 μg of soluble fms-like tyrosine kinase-1 and dilute it to 1 mg / mL with 20 mM PBS buffer (0.05% Triton X-100). Add 1.4 μL of 10 mM NHS-LC-biotin solution (the molar ratio of antibody to NHS-LC-biotin is 1:20). After mixing, place it at room temperature for reaction for 30 min. After the reaction, add 1% volume of 0.1 M Tris-HCl to terminate the reaction for 30 min. After the reaction, dialyze to remove the excess NHS-LC-biotin. Recover the dialysis solution, add an equal volume of glycerol for preservation to obtain NHS-LC-biotin labeled with soluble fms-like tyrosine kinase-1 monoclonal antibody. Dilute the NHS-LC-biotin labeled with soluble fms-like tyrosine kinase-1 monoclonal antibody with the third diluent to a concentration of 0.1 μg / mL - 0.3 μg / mL, which is the third reagent.

[0083] The molecular ratio between the used NHS-LC-biotin and the protein to be biotinylated will vary according to the density of ε-amino groups on the protein surface. Improper selection will affect the labeling efficiency. Several different molecular ratios should be used first to screen the optimal conditions, as shown in Table 2. Excessive NHS-LC-biotin is also unfavorable. The binding sites of the antigen may be blocked, resulting in antibody inactivation. The preferred ratio of antibody to NHS-LC-biotin in this example is 1:20. As the ratio increases, the signal value also increases. It reaches a plateau at a molar ratio of 1:20. When the molar ratio is increased further, due to steric hindrance, the signal value shows a decreasing trend. Therefore, the preferred ratio of antibody to NHS-LC-biotin in this example is 1:20.

[0084] Table 2 Effects of different molar ratios of antibody to NHS-LC-biotin on luminescence value

[0085]

[0086]

[0087] Due to the inaccessibility of the amino group of the antibody, insufficient NHS-LC-biotinylation may occur. In this case, the surfactant Triton X-100 can be added. Triton X-100 can accelerate the collision between antibody molecules and NHS-LC-biotin molecules in the solution, which significantly improves the coupling efficiency of the NHS-LC-biotinylation reaction. In this implementation scheme, several pairs of surfactants were selected for comparative screening, and Table 3 was obtained. The results show that adding 0.05% Triton X-100 to the coupling buffer significantly improves its coupling efficiency and significantly enhances the signal value.

[0088] Table 3 Effects of different surfactants on the coupling efficiency

[0089] Coupling buffer PBS buffer PBS buffer + 0.05% Triton X-100 Concentration (pg / mL) Luminescence value (RLU value) Luminescence value (RLU value) 0 1389 1524 100 52763 82246 1000 652257 928135

[0090] It can be understood that the preparation steps of the above first reagent, second reagent and third reagent are not in a specific order, that is, the present invention does not limit the order of steps S10, S20 and S30.

[0091] The preparation method of the kit proposed by the present invention is simple and convenient, and has all the beneficial effects of the above kit, which will not be elaborated here one by one.

[0092] The present invention further provides a method for detecting the content of soluble fms-like tyrosine kinase-1. Using the kit as described above, the method for detecting the content of soluble fms-like tyrosine kinase-1 includes the following steps:

[0093] S100: Mix the sample to be tested evenly with the second reagent, and add the first reagent and the third reagent, and incubate the mixture to obtain a mixture;

[0094] S200: After magnetically separating and washing the mixture, add a luminescent substrate and mix evenly to obtain a test solution;

[0095] S300: Detect the luminescence intensity of the test solution;

[0096] S400: Calculate the content of soluble fms-like tyrosine kinase-1 according to the luminescence intensity.

[0097] The following gives an example of the method for detecting the content of soluble fms-like tyrosine kinase-1 in the embodiment of the present invention:

[0098] Take the serum as the sample to be tested, use a fully automatic chemiluminescence analyzer (CL-2000) as the detection tool, add 20 μL of the sample to be tested, 100 μL of the second reagent, then add 50 μL of the first reagent and 50 μL of the third reagent, incubate for 5 min, perform magnetic separation and washing. After the washing is completed, the instrument automatically adds 300 μL of the luminescent substrate, then moves the reaction cup to the optoelectronic module to collect the optoelectronic value, and the instrument automatically converts it into concentration and displays the test result.

[0099] The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments and the accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0100] Example 1 Kit

[0101] First reagent: Streptavidin magnetic beads and the first diluent [50 mM 2-morpholinoethanesulfonic acid, 0.05% Tween 20 (v / v), 0.1% Proclin 300 (v / v), 0.9% NaCl (w / v), 1% bovine serum albumin (w / v), 0.1% glycine (w / v), 0.5% trehalose (w / v) and 0.05% PEG modified with BSA (w / v), pH value is 6.5], the concentration of streptavidin magnetic beads is 0.05 mg / mL, and the particle size of streptavidin magnetic beads is 1.5 μm;

[0102] Second reagent: Enzyme-labeled soluble fms-like tyrosine kinase-1 monoclonal antibody and the second diluent [50 mM 2-morpholinoethanesulfonic acid, 0.05% Tween 20 (v / v), 0.1% Proclin 300 (v / v), 0.9% NaCl (w / v) and 1.5% bovine serum albumin (w / v), pH value is 6.8], the enzyme is alkaline phosphatase;

[0103] Third reagent: NHS-LC-biotin labeled soluble fms-like tyrosine kinase-1 monoclonal antibody and the third diluent [50 mM 2-morpholinoethanesulfonic acid, 0.05% Tween 20 (v / v), 0.1% Proclin 300 (v / v), 0.9% NaCl (w / v) and 1.5% bovine serum albumin (w / v), pH value is 6.8], the concentration of NHS-LC-biotin labeled soluble fms-like tyrosine kinase-1 monoclonal antibody is 0.1 μg / mL, and the molar ratio of soluble fms-like tyrosine kinase-1 monoclonal antibody to NHS-LC-biotin is 1:20;

[0104] Luminescent substrate: AMPPD;

[0105] Calibrator: Buffer added with soluble fms-like tyrosine kinase-1 antigen.

[0106] Example 2 Kit

[0107] First Reagent: Streptavidin magnetic beads and a first diluent [100 mM 2-morpholinoethanesulfonic acid, 0.01% Tween 80 (v / v), 0.1% Proclin 300 (v / v), 0.9% NaCl (w / v), 3% bovine serum albumin (w / v), 0.05% glycine (w / v), 0.1% sucrose (w / v), and 0.01% BSA-modified PEG (w / v), pH 8.0]. The concentration of streptavidin magnetic beads is 0.2 mg / mL, and the particle size of streptavidin magnetic beads is 1.5 μm;

[0108] Second Reagent: Enzyme-labeled soluble fms-like tyrosine kinase-1 monoclonal antibody and a second diluent [100 mM 2-morpholinoethanesulfonic acid, 0.2% Tween 20 (v / v), 0.1% Proclin 300 (v / v), 0.9% NaCl (w / v), and 1% bovine serum albumin (w / v), pH 6.5]. The enzyme is alkaline phosphatase;

[0109] Third Reagent: NHS-LC-biotin labeled soluble fms-like tyrosine kinase-1 monoclonal antibody and a third diluent [50 mM 2-morpholinoethanesulfonic acid, 0.05% Tween 20 (v / v), 0.1% Proclin 300 (v / v), 0.9% NaCl (w / v), and 1.5% bovine serum albumin (w / v), pH 6.8]. The concentration of NHS-LC-biotin labeled soluble fms-like tyrosine kinase-1 monoclonal antibody is 0.3 μg / mL, and the molar ratio of soluble fms-like tyrosine kinase-1 monoclonal antibody to NHS-LC-biotin is 1:20;

[0110] Luminescent Substrate: AMPPD;

[0111] Calibrator: Buffer added with soluble fms-like tyrosine kinase-1 antigen.

[0112] Kit of Example 3

[0113] First Reagent: Streptavidin magnetic beads and a first diluent [20 mM 2-morpholinoethanesulfonic acid, 0.1% Triton X-100 (v / v), 0.1% Proclin 300 (v / v), 0.9% NaCl (w / v), 2% bovine serum albumin (w / v), 0.2% glycine (w / v), 2% trehalose (w / v), and 2% BSA-modified PEG (w / v), pH 8.0]. The concentration of streptavidin magnetic beads is 0.1 mg / mL, and the particle size of streptavidin magnetic beads is 1.5 μm;

[0114] Second Reagent: Enzyme-labeled soluble fms-like tyrosine kinase-1 monoclonal antibody and second diluent [75 mM 2-morpholinoethanesulfonic acid, 0.1% Tween 20 (v / v), 0.1% Proclin 300 (v / v), 0.9% NaCl (w / v), and 3% bovine serum albumin (w / v), pH 7.2]; the enzyme is alkaline phosphatase.

[0115] Third Reagent: NHS-LC-biotin labeled soluble fms-like tyrosine kinase-1 monoclonal antibody and third diluent [50 mM 2-morpholinoethanesulfonic acid, 0.05% Tween 20 (v / v), 0.1% Proclin 300 (v / v), 0.9% NaCl (w / v), and 1.5% bovine serum albumin (w / v), pH 6.8]; the concentration of NHS-LC-biotin labeled soluble fms-like tyrosine kinase-1 monoclonal antibody is 0.2 μg / mL, and the molar ratio of soluble fms-like tyrosine kinase-1 monoclonal antibody to NHS-LC-biotin is 1:20.

[0116] Luminescent Substrate: AMPPD

[0117] Calibrator: Buffer added with soluble fms-like tyrosine kinase-1 antigen.

[0118] Preparation of the Kit in Example 4

[0119] Prepare the kit with the substances in the ratio of Example 1, including the following steps:

[0120] (1) Preparation of the First Reagent: Take 10 mg of streptavidin magnetic beads, add 1 mL of the first diluent (50 mM 2-morpholinoethanesulfonic acid, 0.05% Tween 20 (v / v), 0.1% Proclin 300 (v / v), 0.9% NaCl (w / v), 1% bovine serum albumin (w / v), 0.1% glycine (w / v), 0.5% trehalose (w / v), and 0.05% BSA-modified PEG (w / v), pH 6.5), perform magnetic separation and washing, repeat the washing 3 times, then resuspend in the first diluent, and dilute with the first diluent to a streptavidin magnetic bead concentration of 0.05 mg / mL, which is the first reagent.

[0121] (2) Preparation of the second reagent: Take 100 μL of soluble fms-like tyrosine kinase-1 monoclonal antibody, add 250 μL of PBS buffer with a concentration of 0.1 M and a pH value of 7.0, mix well, add 20 μL of freshly prepared 10.0 mg / mL EDC aqueous solution, activate for 30 minutes, then add 200 μL of alkaline phosphatase solution with a concentration of 5 mg / mL and mix well. Take it out after storing in the dark at room temperature for 2 hours, block the reaction at room temperature for 30 min with 0.1 M Tris buffer containing 1% BSA and a pH value of 7.4, then add 0.5% diethyl phosphate and continue the reaction at room temperature for 30 min. Desalt and purify with a 30 KD ultrafiltration column. Add half glycerol to the collected remaining volume solution and store it at -20 °C for later use to obtain the enzyme-labeled soluble fms-like tyrosine kinase-1 monoclonal antibody. Take the enzyme-labeled soluble fms-like tyrosine kinase-1 monoclonal antibody and dilute it 2000-fold with the second diluent (50 mM 2-morpholinoethanesulfonic acid, 0.05% Tween 20 (v / v), 0.1% Proclin 300 (v / v), 0.9% NaCl (w / v), and 1.5% bovine serum albumin (w / v), pH value of 6.8), which is the second reagent.

[0122] (3) Preparation of the third reagent: Weigh 1 mg of NHS-LC-biotin, dissolve it in dimethyl sulfoxide (DMSO) to 10 mM. Take 100 μg of soluble fms-like tyrosine kinase-1 and dilute it to 1 mg / mL with 20 mM PBS (0.05% Triton X-100) buffer. Add 1.4 μL of 10 mM NHS-LC-biotin solution, mix well and place it at room temperature for reaction for 30 min. After the reaction, add 1% volume of 0.1 M Tris-HCl to terminate the reaction for 30 min. After the reaction, dialyze to remove the excess NHS-LC-biotin. Recover the dialysate, add an equal volume of glycerol for storage to obtain the NHS-LC-biotin-labeled soluble fms-like tyrosine kinase-1 monoclonal antibody. Dilute the NHS-LC-biotin-labeled soluble fms-like tyrosine kinase-1 monoclonal antibody with the third diluent to a concentration of 0.1 μg / mL, which is the third reagent.

[0123] The preparation methods of the kits in Examples 2 and 3 are similar and will not be elaborated here.

[0124] Detection of the content of soluble fms-like tyrosine kinase-1 in Example 5

[0125] Take the serum as the test sample, use a fully automatic chemiluminescence analyzer (CL-2000) as the detection tool, add 20 μL of the test sample, 100 μL of the second reagent, then add 50 μL of the first reagent and 50 μL of the third reagent, incubate for 5 min, perform magnetic separation and washing. After the washing is completed, the instrument automatically adds 300 μL of the luminescent substrate, then moves the reaction cup to the optoelectronic module to collect the optoelectronic value, and the instrument automatically converts it into a concentration and displays the test result.

[0126] 1. Detection of sensitivity

[0127] Refer to the experimental protocol recommended by the CLSI EP17-A document, calculate the sensitivity of the soluble fms-like tyrosine kinase-1 (sFlt-1) enzymatic assay kit in Example 1. Take the soluble fms-like tyrosine kinase-1 (sFlt-1) calibrator with a concentration of zero as the test sample for detection, repeat the determination 20 times, calculate its mean value M and standard deviation SD value, take an adjacent calibrator and repeat the test 3 times, calculate its mean value. According to the concentration-RLU value results between the zero-concentration test sample and the adjacent calibrator, perform two-point regression fitting to obtain a linear equation. Substitute the RLU value of M + 2SD into the above equation, and the sensitivity of the soluble fms-like tyrosine kinase-1 (sFlt-1) assay kit is obtained as 10 pg / mL.

[0128] 2. Linear range

[0129] Dilute the high-value sample close to the upper limit of the linear range to 6 concentration gradients of 50000 pg / mL (±20%), 40000 pg / mL (±20%), 30000 pg / mL (±20%), 20000 pg / mL (±20%), 10000 pg / mL (±20%), 10 pg / mL (±40%) according to a certain ratio. Among them, the low-value concentration sample must be close to the lower limit of the linear range. Repeat the detection of each concentration sample 3 times with the kit in Example 1, calculate its average value, perform linear fitting on the average value of the results and the dilution ratio by the least squares method, and calculate the linear correlation coefficient r. The results are shown in Table 4 and Figure 2 as shown.

[0130] Table 4 Detection results of the linear range of the kit in Example 1

[0131]

[0132] The calculated linear correlation coefficient r = 0.9995. The linear range of this kit is 10 - 50000 pg / mL. Specifically, Figure 2 where the abscissa is the theoretical concentration value (pg / mL), the ordinate is the test concentration value (pg / mL), and the linear equation is y = 1.014x - 224.09, indicating good linearity.

[0133] 3. Clinical sample test comparison

[0134] The kit of Example 1 and the Roche kit were used to detect 66 clinical sera simultaneously. The test results are shown in Figure 3 . Taking the results measured by the Roche kit as the abscissa X, with the concentration in pg / mL, and taking the results measured by the kit of Example 1 of the present invention as the ordinate y, with the concentration unit in pg / mL, a regression equation was made. The correlation equation is: y = 0.9801x + 15.169, the correlation coefficient is 0.9986, the K value is 0.9801, and its correlation is very good.

[0135] 4. Repeatability determination

[0136] The kit of Example 1 was used to detect the repeatability samples with concentrations of 2500 pg / ml (±20%) and 10000 pg / ml (±20%) on the test instrument that had been successfully calibrated with the calibrator according to the operating procedure. Each was detected 10 times repeatedly. The average value M and standard deviation SD of the 10 measurement results were calculated. According to the formula CV = SD / M × 100%, the coefficient of variation CV was obtained. The test results are shown in Table 5 below.

[0137] Table 5 Repeatability determination of the kit of Example 1

[0138]

[0139] As can be seen from Table 5, the standard deviation SD and coefficient of variation CV of the kit of Example 1 are relatively small, indicating that the repeatability of the kit test is good.

[0140] 5. Anti-interference ability determination

[0141] The kit of Example 1 was taken, and referring to the relevant standards of CLSI, the effects of endogenous interfering substances such as triglyceride, bilirubin, hemoglobin, total protein, RF, HAMA, etc. and different anticoagulants on the detection were evaluated. According to the evaluation, the results are shown in Table 6 below.

[0142] Table 6 Anti-interference ability determination of the kit of Example 1

[0143] Interferent Added concentration Relative deviation of test results Bilirubin 10 mg / dL Within ±10% Hemoglobin 500 mg / dL Within ±10% Triglyceride 1000 mg / dL Within ±10% Total protein 10 g / dL Within ±10% Rheumatoid factor 1000 IU / mL Within ±10% HAMA 40 ng / mL Within ±10%

[0144] As can be seen from Table 6, the kit of Example 1 of the present invention has strong anti-interference ability against endogenous interfering substances such as triglyceride, bilirubin, hemoglobin, total protein, RF, HAMA, etc.

[0145] In summary, the kit proposed by the present invention has high sensitivity and accuracy, good stability and repeatability, and strong anti-interference ability when detecting the content of soluble fms-like tyrosine kinase-1, and can be widely used in the detection of the content of soluble fms-like tyrosine kinase-1.

[0146] The above are only the preferred embodiments of the present invention, and do not thereby limit the patent scope of the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the patent protection scope of the present invention.

Claims

1. A kit, characterized in that, For detecting the content of soluble fms-like tyrosine kinase-1, the kit includes: The first reagent, including streptavidin magnetic beads and a first diluent; The second reagent, including an enzyme-labeled monoclonal antibody against soluble fms-like tyrosine kinase-1 and a second diluent; The third reagent, including NHS-LC-biotin labeled monoclonal antibody against soluble fms-like tyrosine kinase-1 and a third diluent; Calibrators and a luminescent substrate; Wherein, the first diluent includes a buffer, a surfactant, a protein, a preservative, salts, amino acids, saccharides, and a high molecular polymer; The buffer is 2-morpholinoethanesulfonic acid, and the concentration of 2-morpholinoethanesulfonic acid in the first diluent is 20 mM to 100 mM; and / or, The surfactant is any one of Tween 20, Tween 80, Triton X-100, and Triton X-405, and the concentration of the surfactant in the first diluent is 0.01% to 0.1% (v / v); and / or, The protein is bovine serum albumin, and the concentration of bovine serum protein in the first diluent is 1% to 3% (w / v); and / or, The preservative is Proclin 300; and / or, The salts are NaCl; and / or, The amino acid is glycine, and the concentration of glycine in the first diluent is 0.05% to 0.2% (w / v); and / or, The saccharide is trehalose or sucrose, and the concentration of the saccharide in the first diluent is 0.1% to 2% (w / v); and / or, The high molecular polymer is PEG modified with BSA, and the concentration of the high molecular polymer in the first diluent is 0.01% to 2% (w / v); The third diluent includes PBS buffer and Triton X-100.

2. The kit according to claim 1, wherein The first diluent includes: 50 mM 2-morpholinoethanesulfonic acid, 0.05% Tween 20 (v / v), 0.1% Proclin 300 (v / v), 0.9% NaCl (w / v), 1% bovine serum albumin (w / v), 0.1% glycine (w / v), 0.5% trehalose (w / v), and 0.05% PEG modified with BSA (w / v), and the pH value of the first diluent is 6.

5.

3. The kit according to claim 1, characterized in that, The second diluent includes: 50 mM to 100 mM 2-morpholinoethanesulfonic acid, 0.05% to 0.2% Tween 20 (v / v), 0.1% Proclin 300 (v / v), 0.9% NaCl (w / v), and 1% to 3% bovine serum albumin (w / v); and / or, The pH of the first diluent is 6.5 to 8.0; and / or, The pH of the second diluent is 6.5 to 7.2; and / or, The third diluent includes 50 mM 2-morpholinoethanesulfonic acid, 0.05% Tween 20 (v / v), 0.1% Proclin 300 (v / v), 0.9% NaCl (w / v), and 1.5% bovine serum albumin (w / v), and the pH value of the third diluent is 6.

8.

4. The kit according to claim 1, wherein The second diluent comprises: 50 mM 2-morpholinoethanesulfonic acid, 0.05% Tween 20 (v / v), 0.1% Proclin 300 (v / v), 0.9% NaCl (w / v), and 1.5% bovine serum albumin (w / v), and the pH value of the second diluent is 6.8; and / or, In the first reagent, the concentration of streptavidin magnetic beads is 0.05 mg / mL to 0.2 mg / mL; and / or, In the first reagent, the particle size of the streptavidin magnetic beads is 1.5 μm.

5. The kit according to claim 1, wherein In the second reagent, the enzyme of the enzyme-labeled soluble fms-like tyrosine kinase-1 monoclonal antibody is alkaline phosphatase; and / or, In the third reagent, the concentration of NHS-LC-biotin labeled with soluble fms-like tyrosine kinase-1 monoclonal antibody is 0.1 μg / mL to 0.3 μg / mL.

6. The kit according to claim 1, wherein The calibrator is a buffer solution added with soluble fms-like tyrosine kinase-1 antigen; and / or, The luminescent substrate is AMPPD.

7. A method for preparing a kit according to any one of claims 1-6, characterized in that, Comprising the following steps: Adding streptavidin magnetic beads into the first diluent, mixing evenly, performing magnetic separation and washing, and then resuspending in the first diluent to obtain the first reagent; Activating the soluble fms-like tyrosine kinase-1 monoclonal antibody, mixing and reacting with the enzyme solution, and purifying to obtain the enzyme-labeled soluble fms-like tyrosine kinase-1 monoclonal antibody. Mixing the enzyme-labeled soluble fms-like tyrosine kinase-1 monoclonal antibody evenly with the second diluent to obtain the second reagent; Dissolving NHS-LC-biotin in dimethyl sulfoxide to form an NHS-LC-biotin solution, adding soluble fms-like tyrosine kinase-1 into PBS buffer solution, mixing evenly, and mixing and reacting with the NHS-LC-biotin solution. After purification, the NHS-LC-biotin labeled with soluble fms-like tyrosine kinase-1 monoclonal antibody is obtained. Mixing the NHS-LC-biotin labeled with soluble fms-like tyrosine kinase-1 monoclonal antibody evenly with the third diluent to obtain the third reagent.

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