Application of BMP-2 detection kit in preparation of product for predicting diabetic retinopathy treatment effect

Abstract

The invention discloses application of a kit for detecting BMP-2 (bone morphogenetic protein-2) in preparation of a product for predicting the therapeutic effect of diabetic retinopathy. The method has good accuracy, sensitivity and specificity, and compared with existing clinical indexes, the method can better reflect the curative effect essence. The quantitative prediction of the curative effect can be realized by quantitatively detecting the BMP-2 concentration. In addition, a doctor can be helped to screen out a patient possibly having good response to treatment before treatment and make an individualized scheme, medical resource waste caused by invalid treatment is reduced, repeated injection of anti-VEGF drugs to a patient having no response is avoided, the adverse reaction risk is reduced, and the clinical value is remarkable.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of a kit for detecting BMP-2 in the preparation of products for predicting the treatment effect of diabetic retinopathy. Background Technology

[0002] Diabetic retinopathy (DR) is one of the most common microvascular complications of diabetes. Its pathological mechanisms involve retinal angiogenesis, inflammatory response, and disruption of the blood-retinal barrier, which can lead to vision loss or even blindness. Current clinical treatments for DR include anti-vascular endothelial growth factor (anti-VEGF) injections, laser photocoagulation, and vitrectomy. However, the efficacy varies significantly among individuals: some patients experience significant improvement in lesions and vision after treatment, while others show no response or a weak response, or even disease progression.

[0003] Current methods for predicting the efficacy of diabetic retinopathy (DR) treatment mainly rely on clinical indicators (such as the duration of diabetes, blood glucose control level, and baseline visual acuity) and imaging examinations (such as fundus fluorescein angiography and optical coherence tomography). For example, clinicians often use the number of retinal neovascularizations at baseline to assess the response rate to anti-VEGF therapy, or the degree of macular edema to predict the effectiveness of laser treatment.

[0004] However, existing methods for predicting the efficacy of DR have the following drawbacks: (1) Low specificity and sensitivity: Clinical indicators (such as blood glucose) are greatly affected by short-term fluctuations and have a weak direct correlation with the efficacy of DR; Imaging examinations can only reflect the morphology of lesions and cannot predict the treatment response in the early stages. For example, pre-existing neovascularization may not respond to drugs, but imaging cannot distinguish this. (2) Failure to reflect pathological mechanisms: The core of the difference in the efficacy of DR lies in the individual's molecular response to treatment, such as changes in the expression of angiogenesis-related factors. However, existing technologies do not involve key molecular markers and it is difficult to explain the difference in efficacy from a mechanistic perspective. (3) Leading to blind treatment decisions: Due to the lack of accurate prediction methods, some patients may receive ineffective treatment, increasing medical costs and the risk of adverse reactions, such as the risk of intraocular inflammation caused by anti-VEGF drugs. Summary of the Invention

[0005] The purpose of this invention is to address the above-mentioned technical problems by providing a technical solution that can accurately predict the treatment effect of diabetic retinopathy.

[0006] To achieve the above-mentioned objectives, this invention provides a kit for detecting BMP-2 and its application in the preparation of products for predicting the treatment efficacy of diabetic retinopathy.

[0007] This invention enables the prediction of the therapeutic effect of diabetic retinopathy by detecting the expression level of bone morphogenetic protein-2 (BMP-2).

[0008] Preferably, the kit includes: a capture antibody that specifically binds to BMP-2, a detection antibody, an enzyme-labeled secondary antibody, a substrate, a BMP-2 standard, a sample diluent, and a washing solution.

[0009] Preferably, the capture antibody that specifically binds to BMP-2 is an anti-human BMP-2 polyclonal antibody.

[0010] Preferably, the detection antibody is a biotin-labeled anti-human BMP-2 polyclonal antibody.

[0011] Preferably, the enzyme-labeled secondary antibody is streptavidin-horseradish peroxidase (HRP)-labeled anti-mouse / rabbit IgG.

[0012] Preferably, the substrate is TMB (3,3′,5,5′-tetramethylbenzidine).

[0013] Preferably, the BMP-2 standard is recombinant human BMP-2.

[0014] Preferably, the sample diluent consists of: 0.01M PBS at pH 7.4, 1% w / v BSA, and 0.05% w / v Tween-20.

[0015] Preferably, the washing solution consists of: 0.01M PBS at pH 7.4 and 0.1% w / v Tween-20.

[0016] Preferably, the terminating solution is 2M H2SO4.

[0017] Preferably, the kit is used to detect the concentration of BMP-2 in biological samples of DR patients before treatment.

[0018] Preferably, the biological sample is serum or tears.

[0019] Preferably, the patient's response rate to treatment is predicted based on the BMP-2 detection results: when the BMP-2 concentration is <200 pg / mL, it indicates a good treatment response; when the BMP-2 concentration is ≥200 pg / mL, it indicates a poor treatment response.

[0020] Preferably, the treatment is drug therapy or surgical treatment. The drug is preferably an anti-VEGF drug, but is not limited thereto. The surgery is preferably laser photocoagulation, but is not limited thereto.

[0021] The beneficial effects of this invention are as follows:

[0022] (1) High specificity: BMP-2 directly participates in the proliferation of vascular endothelial cells and the repair of the blood-retinal barrier during the pathological process of DR. Its expression level is directly related to the effect of angiogenesis inhibition and the degree of inflammation relief after treatment. Compared with existing clinical indicators, it can better reflect the essence of efficacy.

[0023] (2) Precise prediction: By quantitatively detecting the concentration of BMP-2, the efficacy can be quantitatively predicted.

[0024] (3) Significant clinical value: It helps doctors screen out patients who may respond well to treatment before treatment and develop individualized plans. For example, anti-VEGF treatment is preferred for patients with low BMP-2, and combination therapy is adjusted for patients with high BMP-2. This reduces the waste of medical resources caused by ineffective treatment, avoids repeated injection of anti-VEGF drugs for unresponsive patients, and reduces the risk of adverse reactions. Detailed Implementation

[0025] The technical solution of the present invention will be further described below with reference to specific embodiments, but the scope of protection is not limited thereto.

[0026] It is worth noting that, unless otherwise specified, the instruments or reagents used in the embodiments are all conventional instruments or reagents in the art and can be obtained through commercial purchase. The specific experimental operations involved in the text are all understandable or known by those skilled in the art based on their common knowledge or conventional technical means, and will not be described in detail here.

[0027] 1. Sample collection:

[0028] Serum samples were collected from 50 patients with DR before treatment: fasting venous blood, the supernatant was collected after centrifugation and stored at -80℃.

[0029] 2. Kit preparation

[0030] The reagent kit components of this invention are as follows:

[0031] (1) Capture antibody that specifically binds to BMP-2

[0032] Anti-human BMP-2 polyclonal antibody (clone number ab14933, Abcam) is used to specifically bind BMP-2 in samples. 50 ng is coated per well of a 96-well plate (concentration 1 μg / mL, 50 μL of sample added per well).

[0033] (2) Antibody detection

[0034] Biotin-labeled anti-human BMP-2 polyclonal antibody (catalog number ULAA013Ca71, Wuhan Yunclone Technology Co., Ltd.) can bind to another epitope of BMP-2 for signal amplification. The working concentration is 0.5 μg / mL. The kit contains a 100 μg / mL stock solution, which should be diluted 1:200 by volume with sample diluent before use.

[0035] (3) Enzyme-labeled secondary antibody

[0036] Streptavidin-horseradish peroxidase (HRP)-labeled anti-mouse / rabbit IgG binds to biotin in the detection antibody, catalyzing a colorimetric reaction of the substrate. The working concentration is 1:5000. The kit contains a 5 mg / mL stock solution; dilute with sample diluent before use.

[0037] (4) Substrate solution

[0038] TMB (3,3′,5,5′-tetramethylbenzidine) turns blue upon HRP treatment, then yellow upon termination. Its absorbance is positively correlated with the BMP-2 concentration. The working concentration is 0.4 mg / mL, and the solvent is citrate buffer containing 0.003% w / v H2O2.

[0039] (5) BMP-2 Standard

[0040] Recombinant human BMP-2 (purity > 95%), in concentration gradients of 0, 10, 50, 200, 500, and 1000 pg / mL, is used to plot standard curves and quantify the BMP-2 concentration in samples. Each kit contains 1 mL of each concentration.

[0041] (6) Sample diluent

[0042] The kit contains: 0.01M PBS (pH 7.4), 1% w / v BSA, and 0.05% w / v Tween-20. The kit contains 10 mL of each.

[0043] (7) Washing liquid

[0044] The kit contains: 0.01M PBS (pH 7.4) and 0.1% w / v Tween-20. The kit contains 50 mL of each.

[0045] (8) Termination solution

[0046] The component is 2M H2SO4. The kit contains 10 mL of this component.

[0047] 3. Sample preprocessing

[0048] Serum sample: Take 5 mL of fasting venous blood from DR patients, centrifuge at 3000×g for 10 minutes (4℃), collect the supernatant, and store at -80℃; before testing, take it out and warm it again, centrifuge at 12000×g for 5 minutes (4℃), and collect the supernatant for testing.

[0049] Tear sample: Collect 50 μL of the patient's tears using a sterile glass capillary tube, immediately add 150 μL of sample diluent (1:4 volume ratio), mix well, centrifuge at 12000×g for 5 minutes (4℃), and use the supernatant for testing.

[0050] The following describes the testing steps using serum samples as an example. The testing steps for tear samples are the same.

[0051] 4. Detection steps (1) Reagent kit equilibration: Take the reagent kit out of 4℃ and equilibrate at room temperature (25℃) for 30 minutes.

[0052] (2) Sample loading: Add 100 μL of each concentration of standard to the standard wells of the antibody-coated plate, add 100 μL of pretreated sample to the sample wells, and add 100 μL of sample diluent to the blank wells; after sealing the plate, incubate at 37°C for 60 minutes with humidity >80% in the incubator.

[0053] (3) Washing: Discard the liquid in the well, add 300 μL of washing solution to each well, soak for 30 seconds and then discard, repeat 5 times, and finally pat dry the residual liquid in the well.

[0054] (4) Add detection antibody: Add 100 μL of diluted detection antibody to each well, seal the plate and incubate at 37°C for 30 minutes.

[0055] (5) Repeat the washing steps (same as step (3)).

[0056] (6) Add enzyme-labeled secondary antibody: Add 100 μL of diluted enzyme-labeled secondary antibody to each well, seal the plate and incubate at 37°C for 30 minutes.

[0057] (7) Repeat the washing steps (same as step (3)).

[0058] (8) Color development: Add 100 μL of substrate solution to each well and incubate at 37°C for 15 minutes in the dark. At this time, the positive wells will turn blue.

[0059] (9) Termination: Add 50 μL of termination solution to each well and gently shake to mix. At this point, the color will turn yellow.

[0060] (10) Reading: Within 30 minutes, use an ELISA reader to measure the absorbance of each well at a wavelength of 450 nm (zero the blank well).

[0061] (11) Calculation of results: Plot a standard curve based on the absorbance of the standard and calculate the concentration of BMP-2 in the sample.

[0062] (12) Therapeutic efficacy prediction: With 200 pg / mL as the threshold, patients with BMP-2 < 200 pg / mL were predicted to have “good treatment response”, and patients with BMP-2 ≥ 200 pg / mL were predicted to have “poor treatment response”.

[0063] 5. Verification Results:

[0064] Sample: Serum samples from 50 patients with DR before treatment.

[0065] Detection indicators: Simultaneous detection of BMP-2 (this kit), VEGF (human VEGF ELISA kit, R&D Systems, catalog number DY293B), and IL-6 (human IL-6 ELISA kit, Abcam, catalog number ab222503).

[0066] Joint detection model: A multi-indicator model is established using Logistic regression, as shown in the following formula:

[0067] Logit(P)=0.02×BMP-2+0.01×VEGF-0.03×IL-6-5.2

[0068] Where P represents the probability of a good treatment response.

[0069] Evaluation metric: Prediction accuracy (number of correctly predicted cases / total number of cases × 100%).

[0070] The efficacy of anti-VEGF drug treatment was tracked in 50 patients for 3 months to verify the predictive accuracy.

[0071] When BMP-2 was tested alone, there were 32 cases in the BMP-2 < 200 pg / mL group, of which 28 cases had lesion shrinkage ≥ 50%, with a response rate of 87.5%. In the BMP-2 ≥ 200 pg / mL group, there were 18 cases, of which only 5 cases responded, with a response rate of 27.8% and a predictive accuracy of 82%.

[0072] The combined detection of BMP-2, VEGF, and IL-6, and the calculation of the probability of a good treatment response using the above model formula, achieved an accuracy rate of 90%.

[0073] 6. Sensitivity and Specificity

[0074] (1) Research subjects:

[0075] DR group: 50 patients diagnosed with DR (meeting international clinical grading standards);

[0076] Control group: 50 healthy individuals (without diabetes or eye diseases) and 30 patients with non-DR eye diseases (cataracts, glaucoma).

[0077] (2) Detection indicators: BMP-2 (this kit), fasting blood glucose (biochemical method), number of neovascularization in the fundus (fluorescence angiography).

[0078] (3) Evaluation indicators:

[0079] Sensitivity = the proportion of patients in the DR group with BMP-2 ≥ 200 pg / mL;

[0080] Specificity = the proportion of BMP-2 < 200 pg / mL in the control group.

[0081] The results are shown in Table 1 below.

[0082] Table 1. Sensitivity and Specificity Results

[0083] index Sensitivity Specific (healthy individuals + non-DR eye disease) BMP-2 detection 86％(43 / 50) 90％(72 / 80) fasting blood glucose 50％(25 / 50) 60％(48 / 80) Number of new blood vessels in the fundus 70％(35 / 50) 75％(60 / 80)

[0084] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Therefore, any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. Application of a kit for detecting BMP-2 in the preparation of products for predicting the treatment efficacy of diabetic retinopathy.

2. The application according to claim 1, characterized in that, The kit includes: a capture antibody that specifically binds to BMP-2, a detection antibody, an enzyme-labeled secondary antibody, a substrate, BMP-2 standards, a sample diluent, a washing buffer, and a stop solution.

3. The application according to claim 1, characterized in that, The capture antibody that specifically binds to BMP-2 is an anti-human BMP-2 polyclonal antibody.

4. The application according to claim 1, characterized in that, The detection antibody is a biotin-labeled anti-human BMP-2 polyclonal antibody.

5. The application according to claim 1, characterized in that, The enzyme-labeled secondary antibody is streptavidin-horseradish peroxidase-labeled anti-mouse / rabbit IgG.

6. The application according to claim 1, characterized in that, The substrate is 3,3′,5,5′-tetramethylbenzidine.

7. The application according to claim 1, characterized in that, The concentration of BMP-2 in the patient's biological sample before treatment was detected using the kit.

8. The application according to claim 7, characterized in that, The biological sample is serum or tears.

9. The application according to claim 1, characterized in that, The patient's response rate to treatment can be predicted based on the BMP-2 test results: a BMP-2 concentration <200 pg / mL indicates a good treatment response; a BMP-2 concentration ≥200 pg / mL indicates a poor treatment response.

10. The application according to claim 1, characterized in that, The treatment described is anti-VEGF drug therapy.