CN117805397A - Method for detecting free VEGF - Google Patents
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Abstract
The present application provides a method of detecting free VEGF in a blood sample of a subject following administration of bevacizumab, ranibizumab or aflibercept, said blood sample comprising VEGF that binds to bevacizumab, ranibizumab or aflibercept and free VEGF. The present application also provides the use of bevacizumab, ranibizumab or aflibercept for detecting free VEGF in a blood sample of a subject following administration of bevacizumab, ranibizumab or aflibercept.
Description
Technical Field
The present application relates to the field of biological sample detection. In particular, the present application relates to detecting free VEGF in biological samples.
Background
Wet age-related macular degeneration (AMD), also known as neovascular AMD, has a great deal of relevance to the formation of new blood vessels, characterized by the formation of Choroidal Neovascularization (CNV), accounts for 20% of AMD incidence, although not as large, but is a major cause of vision impairment in more than 90% of patients. Wet AMD can incorporate fundus hemorrhage and exudation, which in turn can cause scarring, damage to the macular structure, and affect visual function. Wet AMD progresses rapidly, and if untreated, over 40% of patients develop lesions in both eyes within 5 years. About 90% of patients in this category have vision impairment caused by exudates.
The generation of new blood vessels is associated with vascular endothelial growth factor (vascular endothelial growth factor, VEGF). VEGF, also known as vascular permeability factor (vascular permeability factor, VPF), is a highly specific pro-vascular endothelial growth factor with the effects of promoting increased vascular permeability, extracellular matrix degeneration, vascular endothelial cell migration, proliferation and angiogenesis, including VEGF-a, VEGF-b, VEGF-c, VEGF-d, VEGF-e (viral coding) and placental growth factor (PIGF). VEGF-c and VEGF-d are mainly involved in the regulation of lymphangiogenesis. Given the dominant role of VEGF-a in regulating angiogenesis and disease, it is the physiologically most relevant VEGF subtype. The generation of new blood vessels transports nutrients to tissues and organs and eliminates metabolites, thus playing a key role in maintaining homeostasis of the body. However, uncontrolled vascular growth can confer a growth advantage to tumor cells, and can also promote diabetic retinopathy.
Clinical studies have shown that a small trend in the incidence of serious adverse events, which may be associated with systemic VEGF inhibition, was observed in ranibizumab (Lucentis, genentech) dosed groups, reflecting the trend of severe arterial thromboembolic events and, to a lesser extent, the trend of severe non-ocular bleeding. Moreover, a slightly increased overall trend of severe arterial thromboembolic events was observed in subjects treated with 0.5mg ranibizumab per month compared to other subjects. The level of inhibition of VEGF following administration is therefore detected as an indication of safety.
Currently, commercial kits have a sensitivity of 15.6pg/mL and cannot determine whether free VEGF is present. Therefore, there is a need to establish a method for detecting free VEGF, which is used to indicate the relationship between the efficacy of VEGF monoclonal antibodies and VEGF trap drugs and the occurrence rate of adverse events.
Disclosure of Invention
In a first aspect, the present application provides a method of detecting free VEGF in a blood sample of a subject following administration of bevacizumab, ranibizumab or aflibercept, said blood sample comprising VEGF that binds to bevacizumab, ranibizumab or aflibercept and free VEGF, said method comprising:
(1) Fixing bevacizumab, ranibizumab or albesiex on a solid phase;
(2) Adding a blood sample of the subject after administration of bevacizumab, ranibizumab or aflibercept to the solid phase of step (1);
(3) Adding a detectably labeled anti-VEGF antibody to the blood sample of step (2), wherein the binding epitope of the detectably labeled anti-VEGF antibody to VEGF does not overlap with the binding epitope of bevacizumab, ranibizumab or albesieadditional to VEGF;
(4) The intensity of the detectable label is detected to determine the amount of free VEGF in the blood sample.
In some embodiments, the detectable label is biotin.
In some embodiments, detecting the intensity of the detectable label further comprises adding horseradish peroxidase-labeled streptavidin.
In some embodiments, the anti-VEGF antibody in step (3) is human VEGF 165 An antibody.
In some embodiments, the detectably labeled anti-VEGF antibody of step (3) is human VEGF 165 Biotinylated antibody, namely biotinylated anti-VEGF antibody BAF293.
In some embodiments, the bevacizumab is avastin.
In some embodiments, the subject is administered bevacizumab, and bevacizumab is immobilized on a solid phase in step (1).
In some embodiments, the subject is administered bevacizumab and the aflibercept in step (1) is immobilized on a solid phase.
In some embodiments, the subject is administered bevacizumab, and ranibizumab in step (1) is immobilized on a solid phase.
In some embodiments, the subject is administered albesipine and bevacizumab is immobilized on a solid phase in step (1).
In some embodiments, the subject is administered aflibercept and the aflibercept in step (1) is immobilized on a solid phase.
In some embodiments, the subject is administered bevacizumab, and ranibizumab in step (1) is immobilized on a solid phase.
In some embodiments, the immobilization of bevacizumab, ranibizumab or albespride in step (1) on the solid phase does not affect the binding of free VEGF in the blood sample in step (2) to bevacizumab, ranibizumab or al Bai Xi pride immobilized on the solid phase.
In some embodiments, the binding site for the anti-VEGF antibody with a detectable label to bind to VEGF is different from the binding site for bevacizumab, ranibizumab or albespride to bind to VEGF, and binding of the anti-VEGF antibody with a detectable label to VEGF does not affect binding of bevacizumab, ranibizumab or albespride to VEGF on a solid phase.
In some embodiments, binding of any of bevacizumab, ranibizumab, and aflibercept to VEGF prevents binding of bevacizumab, ranibizumab, or aflibercept to the bound VEGF on the solid phase.
In a second aspect, the present application provides the use of bevacizumab, ranibizumab or aflibercept for detecting free VEGF in a blood sample of a subject following administration of bevacizumab, ranibizumab or aflibercept.
Detailed Description
The capture drug immobilized on the solid phase can determine that the detected VEGF is VEGF that is not bound to the drug, i.e., free VEGF. The captured medicine is easy to obtain, has low cost and can accelerate the detection process. The method is simple to operate, good in repeatability and suitable for detecting free VEGF in biological samples on a large scale.
Current anti-VEGF drugs include ranibizumab, albesipine, and bevacizumab for super indications.
Abelmosipu is a VEGFR receptor domain-Fc fusion protein. Abelmoschus contains 2 domains. Bevacizumab may be Avastin.
In a first aspect, the present application provides a method of detecting free VEGF in a blood sample of a subject following administration of bevacizumab, ranibizumab or aflibercept, said blood sample comprising VEGF that binds to bevacizumab, ranibizumab or aflibercept and free VEGF, said method comprising:
(1) Fixing bevacizumab, ranibizumab or albesiex on a solid phase;
(2) Adding a blood sample of the subject after administration of bevacizumab, ranibizumab or aflibercept to the solid phase of step (1);
(3) Adding a detectably labeled anti-VEGF antibody to the blood sample of step (2), wherein the binding epitope of the detectably labeled anti-VEGF antibody to VEGF does not overlap with the binding epitope of bevacizumab, ranibizumab or albesieadditional to VEGF;
(4) The intensity of the detectable label is detected to determine the amount of free VEGF in the blood sample.
In some embodiments, the subject blood sample after administration of bevacizumab, ranibizumab, or aflibercept is added to the solid phase of step (1) such that free VEGF in the blood sample binds to bevacizumab, ranibizumab, or aflibercept immobilized on the solid phase and VEGF in the blood sample that binds to bevacizumab, ranibizumab, or aflibercept does not bind to bevacizumab, ranibizumab, or aflibercept immobilized on the solid phase.
In some embodiments, the anti-VEGF antibody with a detectable label is added to the blood sample of step (2) and washed with a PBST wash; the anti-VEGF antibody with a detectable label is combined with the free VEGF combined with the bevacizumab, the ranibizumab or the Abelmoschus immobilized on a solid phase in the step (2).
The PBST wash was pH7.4 phosphate buffer containing 0.05% Tween-20, abbreviated as PBST.
In some embodiments, the detectable label is biotin.
In some embodiments, detecting the intensity of the detectable label further comprises adding horseradish peroxidase-labeled streptavidin.
In some embodiments, the anti-VEGF antibody in step (3) is human VEGF 165 An antibody.
In some embodiments, the detectably labeled anti-VEGF antibody of step (3) is human VEGF 165 Biotinylated antibodies.
Human VEGF 165 The biotinylated antibody is preferably purchased from R&D Human/Primate VEGF 165 Biotinylated Antibody, which is an antigen affinity purified polyclonal sheep IgG (accession number: BAF 293).
In some embodiments, the bevacizumab is avastin.
In some embodiments, the subject is administered bevacizumab, and bevacizumab is immobilized on a solid phase in step (1).
In some embodiments, the subject is administered bevacizumab and the aflibercept in step (1) is immobilized on a solid phase.
In some embodiments, the subject is administered bevacizumab, and ranibizumab in step (1) is immobilized on a solid phase.
In some embodiments, the subject is administered albesipine and bevacizumab is immobilized on a solid phase in step (1).
In some embodiments, the subject is administered aflibercept and the aflibercept in step (1) is immobilized on a solid phase.
In some embodiments, the subject is administered bevacizumab, and ranibizumab in step (1) is immobilized on a solid phase.
In some embodiments, the immobilization of bevacizumab, ranibizumab or albespride in step (1) on the solid phase does not affect the binding of free VEGF in the blood sample in step (2) to bevacizumab, ranibizumab or al Bai Xi pride immobilized on the solid phase.
In some embodiments, the immobilization of bevacizumab on the solid phase in step (1) does not affect the binding of free VEGF in the blood sample in step (2) to the immobilized al Bai Xi prions on the solid phase.
In some embodiments, the immobilization of the aflibercept in step (1) on the solid phase does not affect the binding of free VEGF in the blood sample in step (2) to bevacizumab immobilized on the solid phase.
In some embodiments, the binding site for the anti-VEGF antibody with a detectable label to bind to VEGF is different from the binding site for bevacizumab, ranibizumab or albespride to bind to VEGF, and binding of the anti-VEGF antibody with a detectable label to VEGF does not affect binding of bevacizumab, ranibizumab or albespride to VEGF on a solid phase.
In some embodiments, the binding site for the anti-VEGF antibody with a detectable label to bind VEGF is different from the binding site for the aflibercept to bind VEGF, and binding of the anti-VEGF antibody with a detectable label to VEGF does not affect binding of aflibercept to VEGF on the solid phase.
In some embodiments, the binding site for the anti-VEGF antibody with the detectable label to bind to VEGF is different from the binding site for bevacizumab to bind to VEGF, and binding of the anti-VEGF antibody with the detectable label to VEGF does not affect binding of bevacizumab to VEGF on a solid phase.
In some embodiments, binding of bevacizumab, ranibizumab or aflibercept to VEGF prevents binding of bevacizumab, ranibizumab or aflibercept to the bound VEGF on a solid phase.
In some embodiments, binding of bevacizumab to VEGF prevents binding of aflibercept to the bound VEGF on the solid phase.
In some embodiments, binding of ranibizumab to VEGF prevents binding of bevacizumab to bound VEGF on a solid phase.
In some embodiments, binding of ranibizumab to VEGF prevents binding of aflibercept to the bound VEGF on the solid phase.
In some embodiments, binding of aflibercept to VEGF prevents binding of bevacizumab to the bound VEGF on the solid phase.
In a second aspect, the present application provides the use of bevacizumab, ranibizumab or aflibercept for detecting free VEGF in a blood sample of a subject following administration of bevacizumab, ranibizumab or aflibercept.
Examples
The present application will be described in more detail by way of specific examples. The following examples are provided for illustrative purposes only and are not intended to limit the present application in any way. Those skilled in the art will readily recognize various non-critical parameters that may be altered or modified to produce substantially the same result. The examples are available commercially without labeling the specific source of the reagent.
Example 1: standard curve preparation
Reagent information
Sample: VEGF, (0.2 mg/mL);
coating reagent 1 on solid phase: avastin (Avastin) 25mg/mL;
coating reagent 2 on solid phase: 25mg/mL of Abelmoschus (Zaltrap);
detection reagent (anti-VEGF antibody with detectable label): human VEGF165 biotinylated antibody (R & D, cat. BAF 293) 0.2mg/mL;
enzyme-labeled reagent: SA-HRP;
blocking/dilution: I-Block;
a substrate: TMB solution (Thermo);
stop solution: 1M sulfuric acid solution (UP-pharma);
carbonate buffer: CBS (UP-pharma);
plate wash (PBST wash): PBST pH 7.2-7.4 (UP-Pharma);
TABLE 1 preparation of reagents
Experimental procedure
1. Preparing a coating reagent working solution: coating reagent working solutions were prepared according to the preparation method in table 1 according to the experimental amounts.
2. Coating: and adding 100 mu L/hole of the prepared coating reagent working solution 1 or coating reagent working solution 2 into the ELISA plate, and coating for 16-18h at 2-8 ℃ by using a sealing plate membrane sealing plate.
3. Washing the plate: the ELISA plate was removed, plate washed 3 times with 350. Mu.L/Kong Xi plate and the plate wells were blotted dry on paper towels.
4. Closing: the ELISA plate is added with a sealing solution at a concentration of 300 mu L/hole, a sealing plate is covered by a sealing plate membrane, and the biochemical incubator at 25+/-3 ℃ is incubated for 2 hours+/-10 minutes.
5. Preparing a sample: see table 2.
6. Washing the plate: the ELISA plate was removed, plate washed 3 times with 350. Mu.L/Kong Xi plate and the plate wells were blotted dry on paper towels.
7. Sample adding: the prepared sample is added into 100 mu L/hole, a membrane sealing plate is sealed, and the biochemical incubator is incubated for 2 hours plus or minus 5 minutes at 25 plus or minus 3 ℃.
8. Preparing a detection reagent working solution: according to the experimental dosage, the working solution of the detection reagent is prepared according to the preparation method in the table 1.
9. Washing the plate: the ELISA plate was removed, incubated with plate wash at 350. Mu.L/Kong Xi plate 3 times for 30s each, and the plate wells were dried on paper towels.
10. Adding a detection reagent working solution: the detection reagent working solution is added into the reaction kettle at 100 mu L/hole, the sealing plate is covered by a sealing plate, and the biochemical incubator at 25+/-3 ℃ is incubated for 1 h+/-5 min.
11. Preparing an enzyme-labeled reagent working solution: the enzyme-labeled reagent working solution was prepared according to the preparation method in table 1 according to the experimental amount.
12. Washing the plate: the ELISA plate was removed, incubated with plate wash at 350. Mu.L/Kong Xi plate 3 times for 30s each, and the plate wells were dried on paper towels.
13. And (3) adding an enzyme-labeled reagent working solution: and adding enzyme-labeled reagent working solution into 100 mu L/hole, sealing a plate membrane sealing plate, and incubating in a biochemical incubator at 25+/-3 ℃ for 1 h+/-5 min.
14. Washing the plate: the ELISA plate was removed, incubated with plate wash at 350. Mu.L/Kong Xi plate 3 times for 30s each, and the plate wells were dried on paper towels.
15. Adding a substrate: and adding the prepared substrate working solution into 100 mu L/hole, and carrying out light-proof reaction at room temperature for 15-20 min (adjusting the color development time according to the color shade).
16. Adding a stop solution: adding a stop solution into 50 mu L of each well; slight shaking ensures that the edges of each well are free of blue-green phenomena.
17. And (3) detection: reading absorbance within 10min by using an enzyme-labeled instrument, detecting the wavelength to be 450nm and the reference wavelength to be 630nm; the original data is printed and saved.
TABLE 2-1 preparation of standard curve samples
Remarks: VEGF stock solutions at 0.2mg/mL were prepared using I-Block to give standard curve samples at concentrations of 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500 and 1000pg/mL according to the above table.
TABLE 2-2 validation of sample formulation
Remarks: VEGF stock solutions at 0.2mg/mL were prepared using I-Block to verify samples at concentrations of 7.81, 20.0, 65.0, 375 and 500 pg/mL according to the above table.
Table 3: board picture
Remarks: STD: standard curve samples; VS: the samples were validated.
Table 4: raw data of plate map
Table 5: standard curve fitting result
Remarks: STD: standard curve samples; VS: the samples were validated.
Example 2: free VEGF pattern test
Bevacizumab (i.e., avastin) was added at various concentrations to samples containing VEGF, and free VEGF was detected using the experimental procedure in example 1, wherein the coating reagent was Avastin at a concentration of 0.25 μg/mL.
Table 6-1: avastin drug concentration formulation table
Note that: the formulated Avastin samples were mixed in equal volumes with the VEGF samples formulated according to Table 2-1 and tested by adding to the microwell plates according to the lower panel diagram.
TABLE 6-2 sample plate
Note that: the tail-A1 sample is VEGF sample and Avastin with the equal volume mixing final concentration of 500 mug/mL of Avastin of 1000 mug/mL; -A2 is 100 μg/mL Avastin, -A3 is 10 μg/mL Avastin, -A4 is 1 μg/mL Avastin, -A5 is 0.1 μg/mL Avastin.
Table 7: free VEGF mode test result analysis
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Remarks: STD: standard curve samples; VS: the sample was validated and BQL indicated that no value was detected.
As can be seen from the results in table 7, free VEGF decreased with increasing concentration of the added drug. Thus, the concentration detected was indicated to be the free VEGF concentration.
The sensitivity of the method is 3.9pg/mL, which is higher than that of a commercial kit of 15.6 pg/mL. The coating reagent fixed on the solid phase is a medicament on the market, is easy to obtain, and can solve the problems of long antibody preparation period and high cost of the short plate. The method has low cost, the cost of each plate is within 100 yuan, and the cost of the commercial kit is about 5000 yuan. The method is suitable for screening of a large number of samples, and can accelerate the marketing process of new drugs while saving cost and having high sensitivity.
Example 3: testing of free VEGF in blood samples based on different combinations of added drug and coating reagent
The concentration of free VEGF was measured following the experimental procedure of example 1, except that the standard curve sample and the validation sample in example 1 were exchanged for the specific sample of this example. The specific sample used in this example was a blood sample taken from a subject, and 10. Mu.g/mL, 1. Mu.g/mL, and 0.1. Mu.g/mL of bevacizumab (Avastin), ranibizumab (Lucentis), or Abelmoschus (Zaltrap) were added to the blood sample.
The combination of coating agent and drug added is as follows:
(1) The drug added was 10 μg/mL, 1 μg/mL or 0.1 μg/mL bevacizumab and the coating agent was 0.25 μg/mL bevacizumab;
(2) The drug added was 10 μg/mL, 1 μg/mL or 0.1 μg/mL bevacizumab and the coating agent was 0.25 μg/mL Abelmoschus;
(3) The drug added was 10 μg/mL, 1 μg/mL or 0.1 μg/mL bevacizumab and the coating agent was 0.25 μg/mL ranibizumab;
(4) The drug added was 10 μg/mL, 1 μg/mL and 0.1 μg/mL of Abelmoschus and the coating agent was 0.25 μg/mL of bevacizumab;
(5) The drug added was 10 μg/mL, 1 μg/mL and 0.1 μg/mL of aflibercept and the coating agent was 0.25 μg/mL of aflibercept; and
(6) The drug added was 10 μg/mL, 1 μg/mL and 0.1 μg/mL bevacizumab and the coating agent was 0.25 μg/mL ranibizumab.
The amounts of free VEGF detected in the blood samples are shown in Table 8, which shows that all six combinations are effective in detecting free VEGF in the blood samples.
Table 8: free VEGF assay results
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Claims (10)
1. A method of detecting free VEGF in a blood sample of a subject following administration of bevacizumab, ranibizumab or aflibercept, said blood sample comprising VEGF that binds to bevacizumab, ranibizumab or aflibercept and free VEGF, said method comprising:
(1) Fixing bevacizumab, ranibizumab or albesiex on a solid phase;
(2) Adding a blood sample of the subject after administration of bevacizumab, ranibizumab or aflibercept to the solid phase of step (1);
(3) Adding a detectably labeled anti-VEGF antibody to the blood sample of step (2), wherein the binding epitope of the detectably labeled anti-VEGF antibody to VEGF does not overlap with the binding epitope of bevacizumab, ranibizumab or albesieadditional to VEGF;
(4) The intensity of the detectable label is detected to determine the amount of free VEGF in the blood sample.
2. The method of claim 1, wherein the detectable label is biotin.
3. The method of claim 2, wherein detecting the intensity of the detectable label further comprises adding horseradish peroxidase-labeled streptavidin.
4. The method of claim 1, wherein the anti-VEGF antibody in step (3) is human VEGF 165 An antibody.
5. The method of claim 1, wherein the anti-VEGF antibody with a detectable label in step (3) is the biotinylated anti-VEGF antibody BAF293.
6. The method of claim 1, wherein,
the subject is administered bevacizumab, and bevacizumab is immobilized on a solid phase in step (1);
the subject is administered bevacizumab and the aflibercept in step (1) is immobilized on a solid phase;
the subject is administered bevacizumab, and in step (1) ranibizumab is immobilized on a solid phase;
the subject is administered with aflibercept and bevacizumab in step (1) is immobilized on a solid phase;
the subject is administered with aflibercept and the aflibercept in step (1) is immobilized on a solid phase; or (b)
The subject is administered bevacizumab, and in step (1) ranibizumab is immobilized on a solid phase.
7. The method of claim 1, wherein the immobilization of bevacizumab, ranibizumab or albespride in step (1) on the solid phase does not affect the binding of free VEGF in the blood sample in step (2) to bevacizumab, ranibizumab or al Bai Xi pride immobilized on the solid phase.
8. The method of claim 1, wherein binding of the detectably labeled anti-VEGF antibody to VEGF does not affect binding of bevacizumab, ranibizumab or albesiex to VEGF on a solid phase.
9. The method of claim 1, wherein binding of any one of bevacizumab, ranibizumab and aflibercept to VEGF prevents binding of bevacizumab, ranibizumab or aflibercept to the bound VEGF on a solid phase.
10. Use of bevacizumab, ranibizumab or aflibercept for detecting free VEGF in a blood sample of a subject following administration of bevacizumab, ranibizumab or aflibercept.
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