CN111044734A - Detection kit for detecting vascular endothelial growth factor, and preparation method and use method thereof - Google Patents
Detection kit for detecting vascular endothelial growth factor, and preparation method and use method thereof Download PDFInfo
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Abstract
The invention relates to a detection kit for detecting vascular endothelial growth factor, a preparation method and a use method thereof, the kit comprises a biotinylated VEGF monoclonal antibody, an alkaline phosphatase-labeled VEGF monoclonal antibody and streptavidin magnetic beads, and is suitable for a full-automatic chemiluminescence immunoassay analyzer Lumiart-II-1.
Description
Technical Field
The invention relates to a detection kit, in particular to a detection kit for detecting vascular endothelial growth factor, a preparation method and a use method thereof.
Background
The growth, development and metastasis of tumor cells are multifactorial, multistage, involving intricate relationships between tumor cells and the host, and the mechanism is not clear. In recent years, research shows that the generation of new blood vessels is closely related to the development of tumors and metastasis. The vascular endothelial growth factor (vascular endothelial growth factor) is a functional signal transduction protein with high biological activity, which can act on vascular endothelial cells to cause the proliferation, migration and lumen formation of the vascular endothelial cells, participate in angiogenesis and increase the permeability of capillary vessels, cause abnormal growth of tumor blood vessels, prevent the effective delivery of antitumor drugs into tumor tissues and stimulate the increase of new-born blood vessel growth factors. The expression of VEGF in blood of almost all solid tumor patients is higher than that of healthy people, so that the VEGF-free anti-tumor marker is a brand-new broad-spectrum tumor marker which is researched more in recent years, and for a subject with high expression of VEGF in blood, the VEGF-free anti-tumor marker can be pertinently screened together with other tumor markers according to the physical signs of the subject, so that the detection rate can be greatly improved, and certain tumor tissue sources can be determined.
VEGF has a relative molecular weight of 45kDa and is a highly glycosylated basic protein, two identical subunits are bound by disulfide bonds, and form different isoforms due to different exon splicing, VEGF has at least five different isoforms, and VEGF is named as: VEGF121, VEGF145, VEGF165, VEGF189, VEGF 206. Among them, VEGF121, VEGF145 and VEGF165 are secreted soluble proteins, which directly act on vascular endothelial cells to promote vascular endothelial proliferation and increase vascular permeability, and VEGF165 is the main existing form of VEGF.
In recent years, research and application of various immunoassay techniques have been rapidly developed, and they are widely used for auxiliary diagnosis of clinical diseases and the like. At present, the method for detecting human serum VEGF is mainly an enzyme-linked immunosorbent assay, but the detection sensitivity is low, the detection range is narrow, the accuracy is not high, the operation is more complicated, and the like.
Disclosure of Invention
The invention aims to solve the problems and provides a detection kit for detecting vascular endothelial growth factor, a preparation method and a use method thereof.
The purpose of the invention is realized by the following technical scheme:
a detection kit for detecting vascular endothelial growth factor comprises a biotinylation VEGF monoclonal antibody, an alkaline phosphatase-labeled VEGF monoclonal antibody and streptavidin magnetic beads.
Preferably, in the biotinylated VEGF monoclonal antibody, the molar mass ratio of the VEGF monoclonal antibody to biotin is 1: 45-55.
Preferably, in the alkaline phosphatase-labeled VEGF monoclonal antibody, the mass ratio of the VEGF monoclonal antibody to the alkaline phosphatase marker is 1: 1-2.
Preferably, the particle size of the streptavidin magnetic bead is 0.5-1.5 microns.
A preparation method of a detection kit for detecting vascular endothelial growth factor comprises the following steps:
dissolving biotin and a VEGF monoclonal antibody in a solvent, and desalting by using a desalting column to obtain a biotinylated VEGF monoclonal antibody;
coupling alkaline phosphatase and VEGF monoclonal antibody by a sodium periodate method, and desalting by a desalting column to obtain the VEGF monoclonal antibody marked by the alkaline phosphatase.
Preferably, the specific preparation method of the biotinylated VEGF monoclonal antibody is as follows:
diluting the VEGF monoclonal antibody with PBS buffer solution, weighing biotin, dissolving the biotin in DMSO, mixing the two solutions at room temperature, desalting the mixture with a centrifugal desalting column, and collecting the liquid in a centrifuge tube to a storage tube to obtain the biotinylated VEGF monoclonal antibody.
Preferably, the specific preparation method of the VEGF monoclonal antibody marked by alkaline phosphatase comprises the following steps:
(1) preparing alkaline phosphatase into a solution by using purified water, adding an equal volume of sodium periodate purified water solution, and reacting for a period of time at low temperature in a dark place;
(2) adding an ethylene glycol solution with the same volume as the alkaline phosphatase solution, reacting for a period of time in a low-temperature dark place, adding the VEGF monoclonal antibody, desalting by using a centrifugal desalting column, and collecting liquid in a centrifugal tube;
(3) adding a sodium borohydride solution, reacting for a period of time in a low-temperature dark place, adding a saturated ammonium sulfate solution, reacting for a period of time in a low-temperature dark place, centrifuging, and removing a supernatant to obtain a precipitate;
(4) dissolving the precipitate in PBS, desalting with a centrifugal desalting column, and collecting the liquid in a centrifugal tube to obtain the VEGF monoclonal antibody marked by alkaline phosphatase.
Preferably, a PD SpinTrap G-25 centrifugal desalting column is used for desalting treatment, and the desalting column is pretreated by PBS in the desalting process;
obtaining biotinylation VEGF monoclonal antibody or VEGF monoclonal antibody marked by alkaline phosphatase, adding glycerol, mixing uniformly, and storing at the temperature lower than-20 ℃.
Preferably, the concentration of alkaline phosphatase in the step (1) is 10 mg/mL; the concentration of the sodium periodate is 12.8 mg/mL; the temperature of the low-temperature light-proof reaction is 4 ℃, and the reaction time is 0.5 hour;
the glycol solution in the step (2) is 1% by volume; the temperature of the low-temperature light-proof reaction is 4 ℃, and the reaction time is 45 minutes;
and (3) adding 20 mu L of sodium borohydride solution into each mg of antibody, wherein the concentration of the sodium borohydride solution is 5mg/mL, the temperature of the low-temperature light-resistant reaction is 4 ℃, and the reaction time is 2 hours.
A method for using a detection kit for detecting vascular endothelial growth factors comprises the steps of taking a full-automatic chemiluminescence immunoassay analyzer as a detection tool, sequentially adding a sample, a biotinylated VEGF monoclonal antibody and an alkaline phosphatase-labeled VEGF monoclonal antibody into a reaction cup, reacting for a period of time, adding streptavidin magnetic beads, reacting for a period of time, carrying out magnetic separation, cleaning for a plurality of times by using a cleaning solution, conveying the reaction cup into a dark room, adding a chemiluminescence substrate solution for carrying out a luminescence reaction, and recording a luminescence value. The reaction principle is that VEGF in a sample is combined with an alkaline phosphatase-labeled antibody and a biotinylated antibody to form an antibody-antigen-antibody-enzyme complex, and the complex is combined with magnetic beads. Then, the magnetic beads were separated using a magnetic separator and components not bound to the solid phase magnetic beads were removed. Finally, a chemiluminescent substrate is added, alkaline phosphatase which is combined with the solid phase can promote the substrate to emit photons, the luminous intensity of the substrate is in direct proportion to the content of VEGF in the sample, and the content of VEGF in the sample is calculated through a calibration curve.
Compared with the traditional ELISA kit, the kit has the following remarkable advantages.
The kit adopts streptavidin magnetic microspheres as a solid phase carrier, the streptavidin molecules are composed of 4 same peptide chains, each peptide chain can be combined with one biotin, so that one streptavidin molecule can be combined with 4 biotin molecules with high specificity, when a biotinylated antibody-antigen-antibody-enzyme compound is combined with magnetic beads, photons are emitted by exciting a substrate, signals of trace antigens can be amplified by thousands of times, and the sensitivity is obviously improved compared with an ELISA method.
The preparation of the biotinylated antibody and the preparation process of the alkaline phosphatase labeled antibody are simple, the labeling process is stable, the prepared conjugate is stable in structure and higher in titer, and the cost required by a single test is lower.
When the ELISA is developed, due to the fact that the time difference between the addition of the substrate and the addition of the stop solution is generated, data difference caused by inconsistent developing time can be generated inevitably, the chemiluminescence substrate solution used by the kit is low in non-enzymatic hydrolysis degree under an alkaline condition, namely, the background is low, the thermal stability is good, the intensity reaches a peak in 15min, the optical signal intensity is kept consistent within 15-60min, the change is small, and a correct result can be obtained even after 12h of determination.
The time for detecting one sample by the kit is 25 minutes, and compared with 2.5 hours of an ELISA method, the detection efficiency is greatly improved.
Compared with the prior art, the kit is suitable for a full-automatic chemiluminescence immunoassay analyzer Lumiart-II-1, compared with the traditional ELISA kit, the full-automatic operation reduces manual errors, the reaction time is shorter, the sensitivity is higher, high-throughput rapid determination is realized, the accuracy is high, the specificity is strong, the accuracy and the detection efficiency are greatly improved, the kit can be used for early cancer risk screening, the treatment curative effect evaluation and monitoring of tumor patients to guide medication, relapse monitoring, prognosis and the like, and has good application prospect.
Drawings
FIG. 1 is a flowchart of a method for preparing a detection kit according to an embodiment of the present invention;
FIG. 2 is a calibration curve of the kit of example 3 of the present invention (calibrator concentration is in abscissa, RLU is in ordinate);
FIG. 3 is a comparison of the results of the ELISA kit of the embodiment 4 and Beijing Jianping Jinxing in the invention for measuring 40 clinical sera.
Detailed Description
The invention is described in detail below with reference to the figures and specific embodiments.
Example 1
The specific process of preparing the quantitative detection kit for the vascular endothelial growth factor is shown in figure 1.
(1) Preparation of biotinylated VEGF monoclonal antibodies
0.1mg of VEGF monoclonal antibody is taken, diluted to 1mg/mL by PBS buffer solution, 1mg of biotin is weighed and dissolved by DMSO at the concentration of 10mmol/L, and the ratio of the antibody to the biotin 1: 50, mixing at room temperature for 30 minutes, desalting by using a PD SpinTrap G-25 centrifugal desalting column, pretreating the desalting column by using PBS (phosphate buffer solution), adding the obtained biotinylated VEGF monoclonal antibody solution, collecting the liquid in a centrifuge tube to a storage tube to obtain the biotinylated VEGF monoclonal antibody, adding glycerol with the same volume, mixing uniformly, and storing at-20 ℃.
(2) VEGF monoclonal antibody marked by alkaline phosphatase
0.2mg of alkaline phosphatase (ALP) was added to a concentration of 10mg/mL in purified water, and sodium periodate (12.8mg/mL, dissolved in purified water and used as it is) was added in an equal volume to the ALP solution and reacted at 4 ℃ for 30 minutes in the dark. A1% ethylene glycol solution (ready prepared) having the same volume as the ALP solution was added thereto, and the mixture was reacted at 4 ℃ for 45 minutes in the absence of light. Adding 0.1mg of antibody, mixing, desalting with a PDSpinTrap G-25 centrifugal desalting column, pretreating the desalting column with PBS during desalting, collecting the mixed solution in a centrifuge tube, adding 20 μ l of sodium borohydride solution (5mg/mL, purified water is dissolved and is ready for use) per mg of antibody, and reacting at 4 ℃ for 2 hours in a dark place. An equal volume of saturated ammonium sulfate solution was slowly added and the reaction was carried out at 4 ℃ for 2 hours. The mixture was centrifuged (4 ℃ C., 8000rpm, 30 minutes), and the supernatant was discarded. Dissolving the precipitate with appropriate amount of PBS, desalting with PD SpinTrap G-25 centrifugal desalting column, pretreating the desalting column with PBS, collecting the liquid in the centrifuge tube, adding glycerol of the same volume, mixing, and storing at-20 deg.C.
(3) Preparation of vascular endothelial growth factor calibrator and quality control product
Using calibrator and quality control buffer (50mM Tris, 1% BSA, 0.9% NaCl, pH7.4), vascular endothelial growth factor antigen was formulated into calibrator at concentrations of 0pg/mL, 5pg/mL, 25pg/mL, 125pg/mL, 250pg/mL, 1000pg/mL, and quality control at concentrations of 150pg/mL and 500 pg/mL.
Example 2
Quantitative detection method of vascular endothelial growth factor
A full-automatic chemiluminescence immunoassay analyzer (Lumiart-II-1) is used as a detection tool, the detection principle is a double-antibody sandwich method, 50 mu L of a sample, 50 mu L of biotinylated VEGF monoclonal antibody and 50 mu L of alkaline phosphatase-labeled VEGF monoclonal antibody are sequentially added into a disposable reaction cup, the reaction is carried out for 15 minutes, 25 mu L of streptavidin magnetic beads are added, the reaction is carried out for 5 minutes, magnetic separation is carried out, cleaning solution is washed for 5 times, a reaction cup is sent into a dark room by a mechanical arm, 200 mu L of chemiluminescence substrate liquid (APS-5) is added for luminescence reaction, and finally, the luminescence value is recorded.
Example 3
Performance evaluation of blood vessel endothelium growth factor magnetic particle chemiluminescence immunoassay kit
The VEGF calibrator was assayed as described in example 2 and a standard curve was plotted as shown in FIG. 2.
Detection of sensitivity: the experimental scheme is recommended by referring to CLSI EP17-A document, and the sensitivity of the blood vessel endothelial growth factor magnetic particle chemiluminescence immunoassay kit is calculated and is obtained to be 2 pg/mL.
And (3) linear detection: and performing linear analysis on the vascular endothelial growth factor with the concentration of 3.91pg/mL, 7.81pg/mL, 15.63pg/mL, 31.25pg/mL, 62.5pg/mL, 125pg/mL, 250pg/mL, 500pg/mL and 1000pg/mL, calculating a linear correlation coefficient, wherein r is 0.9996, and the linear detection range of the kit is 3.91-1000 pg/mL.
And (3) detecting the precision: and (3) taking VEGF quality control products with the concentrations, respectively carrying out 10 parallels on each concentration, detecting by using three batches of kit, and calculating the precision of the kit between batches, wherein the result shows that the precision of the kit between batches is less than 5%.
The anti-interference performance is as follows: taking mixed serum, respectively adding interference substances including 10mg/dL bilirubin, 125mg/dL hemoglobin and 2000mg/dL triglyceride, wherein the adding proportion is as follows: 20, respectively measuring the measured values of the mixed blood and the mixed serum added with various interference substances, and calculating the deviation between the measured values, wherein the deviation is within an acceptable range of +/-10 percent, and the result shows that the anti-interference performance meets the requirements.
Tracing the calibrator: after a VEGF WHO standard product (VEGF165, NIBSC code:02/286) is redissolved, PBS is used for diluting 5 concentration points of a corresponding calibrator by times, namely 5pg/mL, 25pg/mL, 125pg/mL, 250pg/mL and 1000pg/mL, the series of standards are used for detecting actual concentration values of each point by a chemiluminescence method based on a working calibrator, a linear correlation coefficient r is calculated to be 0.9991, and the ratio of the actual concentration value to a labeled value is 0.9-1.1.
Example 4
Contrast experiment of vascular endothelial growth factor detection kit
The kit and the traditional ELISA kit are respectively used for detecting 40 cases of clinical serum, the results show that the measured values of the two cases of clinical serum have no obvious difference through linear correlation analysis, the correlation r is 0.97, the clinical compliance rate has good consistency, and the comparative results in the table 1 show that the sensitivity and the detection range of the kit are superior to those of the ELISA kit, the operation is simpler and more convenient, and the detection time is shorter.
TABLE 1 comparison of the kit with the same products already on the market
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Claims (10)
1. A detection kit for detecting vascular endothelial growth factor is characterized by comprising a biotinylation VEGF monoclonal antibody, an alkaline phosphatase-labeled VEGF monoclonal antibody and streptavidin magnetic beads.
2. The detection kit for detecting vascular endothelial growth factor according to claim 1, wherein the molar mass ratio of the VEGF monoclonal antibody to the biotin in the biotinylated VEGF monoclonal antibody is 1: 45-55.
3. The detection kit for detecting vascular endothelial growth factor according to claim 1, wherein the mass ratio of the VEGF monoclonal antibody to the alkaline phosphatase marker in the alkaline phosphatase labeled VEGF monoclonal antibody is 1: 1-2.
4. The detection kit for detecting VEGF according to claim 1, wherein the streptavidin magnetic beads have a particle size of 0.5-1.5 μm.
5. A method for preparing a test kit for detecting vegf according to claim 1, comprising:
dissolving biotin and a VEGF monoclonal antibody in a solvent, and desalting by using a desalting column to obtain a biotinylated VEGF monoclonal antibody;
coupling alkaline phosphatase and VEGF monoclonal antibody by a sodium periodate method, and desalting by a desalting column to obtain the VEGF monoclonal antibody marked by the alkaline phosphatase.
6. The preparation method of the detection kit for detecting the vascular endothelial growth factor according to claim 5, wherein the specific preparation method of the biotinylated VEGF monoclonal antibody is as follows:
diluting the VEGF monoclonal antibody with PBS buffer solution, weighing biotin, dissolving the biotin in DMSO, mixing the two solutions at room temperature, desalting the mixture with a centrifugal desalting column, and collecting the liquid in a centrifuge tube to a storage tube to obtain the biotinylated VEGF monoclonal antibody.
7. The preparation method of the detection kit for detecting the vascular endothelial growth factor according to claim 5, wherein the specific preparation method of the VEGF monoclonal antibody marked by the alkaline phosphatase comprises the following steps:
(1) preparing alkaline phosphatase into a solution by using purified water, adding an equal volume of sodium periodate purified water solution, and reacting for a period of time at low temperature in a dark place;
(2) adding an ethylene glycol solution with the same volume as the alkaline phosphatase solution, reacting for a period of time in a low-temperature dark place, adding the VEGF monoclonal antibody, desalting by using a centrifugal desalting column, and collecting liquid in a centrifugal tube;
(3) adding a sodium borohydride solution, reacting for a period of time in a low-temperature dark place, adding a saturated ammonium sulfate solution, reacting for a period of time in a low-temperature dark place, centrifuging, and removing a supernatant to obtain a precipitate;
(4) dissolving the precipitate in PBS, desalting with a centrifugal desalting column, and collecting the liquid in a centrifugal tube to obtain the VEGF monoclonal antibody marked by alkaline phosphatase.
8. The method for preparing a detection kit for detecting vascular endothelial growth factor according to claim 6 or 7, wherein the desalting treatment is performed by using a PD SpinTrap G-25 centrifugal desalting column, and the desalting treatment is performed by pretreating the desalting column with PBS;
obtaining biotinylation VEGF monoclonal antibody or VEGF monoclonal antibody marked by alkaline phosphatase, adding glycerol, mixing uniformly, and storing at the temperature lower than-20 ℃.
9. The method for preparing a detection kit for detecting VEGF according to claim 7,
the concentration of alkaline phosphatase in the step (1) is 10 mg/mL; the concentration of the sodium periodate is 12.8 mg/mL; the temperature of the low-temperature light-proof reaction is 4 ℃, and the reaction time is 0.5 hour;
the glycol solution in the step (2) is 1% by volume; the temperature of the low-temperature light-proof reaction is 4 ℃, and the reaction time is 45 minutes;
and (3) adding 20 mu L of sodium borohydride solution into each mg of antibody, wherein the concentration of the sodium borohydride solution is 5mg/mL, the temperature of the low-temperature light-resistant reaction is 4 ℃, and the reaction time is 2 hours.
10. The use method of the detection kit for detecting the vascular endothelial growth factor according to claim 1, characterized in that a full-automatic chemiluminescence immunoassay analyzer is used as a detection tool, a sample, a biotinylated VEGF monoclonal antibody and an alkaline phosphatase-labeled VEGF monoclonal antibody are sequentially added into a reaction cup, the reaction is carried out for a period of time, streptavidin magnetic beads are added, the reaction is carried out for a period of time, magnetic separation is carried out, a cleaning solution is washed for several times, the reaction cup is sent into a dark room, a chemiluminescent substrate solution is added for a luminescent reaction, and a luminescent value is recorded.
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