Indirect ELISA (enzyme-linked immuno sorbent assay) method for detecting concinercept in human serum, anterior aqueous humor and vitreous humor

Abstract

The invention provides an indirect ELISA (Enzyme-Linked Immuno Sorbent Assay) method for detecting canbercept in human serum, anterior aqueous humor and vitreous humor, which comprises the following specific steps: diluting recombinant human VEGF165 to 0.5 mg / mL by using a coating solution, and adding the diluted recombinant human VEGF165 into an ELISA plate according to 90-110mL / hole for coating; after washing the plate, incubating for 0.9-1.1 hours at room temperature by using 290-310mL of confining liquid per hole to finish the confining; after washing the plate again, adding a sample and washing the plate; a detection antibody is added, a Super Block solution is selected as a diluent to dilute the detection antibody, and the dilution ratio is 1: 4000; washing the plate, developing and terminating the reaction; and the OD value is read at the dual wavelengths of 450-630 nm by using a microplate reader. The method can be applied to three different clinical samples of human serum, anterior aqueous humor and vitreous humor, has the advantages of high sensitivity, good repeatability, high accuracy and the like, and is wide in application range.

Description

Technical Field

[0001] This invention belongs to the field of biology and medicine, and relates to an indirect ELISA method for detecting conbercept in human serum, aqueous humor and vitreous humor. Background Technology

[0002] Conbercept is a novel recombinant fusion protein composed of the second domain of human VEGFR1, the third and fourth domains of VEGFR2, and the Fc fragment of human IgG1. It binds with high affinity to multiple members of the vascular endothelial growth factor (VEGF) family, thereby blocking their binding to receptors. Conbercept is approved for the treatment of various neovascularization-related diseases of the fundus, including neovascular age-related macular degeneration (nAMD), choroidal neovascularization in pathological myopia (pmCNV), diabetic macular edema (DME), and retinal vein occlusion-related macular edema (RVO-ME). In clinical applications, conbercept is administered via intravitreal injection, and its efficacy and safety are closely dependent on the drug concentrations in the ocular environment and systemic circulation. Monitoring the drug concentrations of conbercept in serum, aqueous humor, and vitreous humor is crucial for revealing its pharmacokinetic characteristics, guiding individualized medication, and optimizing clinical treatment regimens.

[0003] Enzyme-linked immunosorbent assay (ELISA) is a commonly used method for detecting biological macromolecules, widely applied in clinical and research fields due to its high sensitivity, strong specificity, and ease of operation. Indirect ELISA is a common form, its basic principle being the immobilization and capture of antigens or antibodies on a solid-phase carrier, followed by a colorimetric reaction between the antibody and enzyme label to ultimately achieve quantitative detection of the target molecule. Currently, there is a lack of a highly sensitive indirect ELISA method applicable to both serum samples and common ophthalmic clinical specimens (such as aqueous humor and vitreous humor) for the detection of conbercept. Existing literature and methods largely focus on the detection of conbercept in serum, and are not specifically designed for clinical samples. Research on quantitative methods for intraocular fluid samples is limited, and there is still room for improvement in terms of detection sensitivity, repeatability, and ease of operation.

[0004] Therefore, developing an indirect ELISA method for conbercept that can be applied simultaneously to serum, aqueous humor, and vitreous fluid samples would have advantages such as high sensitivity, good repeatability, and simple operation. It could not only meet the needs of clinical drug concentration monitoring, but also be used to detect the concentration of conbercept in ophthalmic surgical specimens, providing experimental evidence for clinical efficacy and safety assessment, and thus has important application value. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide an indirect ELISA method for detecting conbercept in human serum, aqueous humor, and vitreous fluid. This method solves the problems of insufficient sensitivity, limited applicability, and poor reproducibility and ease of operation of conbercept in ophthalmic samples in existing technologies. This method is suitable for three different clinical samples, exhibits high sensitivity, good reproducibility, high accuracy, and wide applicability.

[0006] To achieve the above objectives, the present invention provides an indirect ELISA method, the steps of which include: An indirect ELISA method for detecting conbercept in human serum, aqueous humor, and vitreous humor, comprising the following steps: 1) Coating protein: Recombinant human VEGF was coated with a coating solution. 165 Dilute to 0.4-0.6 mg / mL and add 90-110 mL / well to an ELISA plate for coating. 2) Washing: Place the ELISA plate in a plate washer, add 290~310mL / well of washing buffer, shake for 1~2 minutes, then remove the washing buffer. Repeat the above steps 2~4 times. 3) Blocking: Blocking was completed by incubating at room temperature for 0.9-1.1 h with 290-310 mL of blocking solution per well; the blocking solution was SuperBlock solution. 4) Washing: Place the ELISA plate in a plate washer, add 290~310mL / well of washing buffer, shake for 1~2 minutes, then remove the washing buffer. Repeat the above steps 2~4 times. 5) Sample addition: Dilute the analyte 10-100 times with Super Block solution and add it to the wells in the same arrangement as the plate, 100-120 mL / well. Incubate in a microplate shaker at 400-450 rpm at room temperature for 1-1.2 h. The analyte may include clinical serum samples, clinical pre-clinical aqueous humor samples, or clinical vitreous fluid. 6) Washing: Place the ELISA plate in a plate washer, add 290~310mL / well of washing buffer, shake for 1~2 minutes, then remove the washing buffer. Repeat the above steps 2~4 times. 7) Add detection antibody: Dilute the horseradish peroxidase (HRP)-labeled anti-human IgG-Fc antibody with diluent, add 100-120 mL / well according to the well plate arrangement, and incubate in a microplate shaker at 400-450 rpm at room temperature for 1-1.2 h; use Super Block solution as diluent to dilute the detection antibody at a dilution ratio of 1:3000-4000. 8) Washing the plate: Place the ELISA plate in a plate washer, add 290~310mL / well of washing buffer, shake for 1~2 minutes, then remove the washing buffer. Repeat the above steps 2~4 times. 9) Color development: Add TMB single-component color development solution according to the well plate layout, 100~120mL / well, and incubate at room temperature in the dark for 9~11min; 10) Termination: Add the stop solution according to the well plate layout, adding 50~55mL to each well to terminate the colorimetric reaction; 11) Microplate reader readings: Use a microplate reader to detect OD values ​​at dual wavelengths from 450nm to 630nm. Statistically analyze the measured values, plot curves, and process the data.

[0007] Preferably, the coating solution in step 1) is a phosphate buffer solution, and the coating process is: incubating in a refrigerator at 2~8ºC for 20~26 hours.

[0008] As a preferred embodiment of the present invention, in step 1), recombinant human VEGF is treated with 1×PBS. 165 Dilute to 0.5 mg / mL.

[0009] As a preferred embodiment of the present invention, in step 1), 100 mL / well is added to the ELISA plate and incubated in a 4ºC refrigerator for 24 hours.

[0010] Preferably, the room temperature mentioned in steps 3), 5), 7) and 9) above is 10ºC to 30ºC, and more preferably 25ºC to 26ºC.

[0011] As a preferred embodiment of the present invention, in step 3), 300 mL of blocking solution is used per well, and the mixture is incubated at room temperature for 1 hour.

[0012] In a preferred embodiment of the present invention, in steps 2), 4), 6), and 8), the ELISA plate is placed in a plate washer, the program is set, 300 mL / well is added, and the plate is shaken for 1 min.

[0013] In a preferred embodiment of the present invention, the washing solution in step 4) is 0.05% PBST; As a preferred embodiment of the present invention, step 5) sample loading: the analyte is diluted 10-fold with Super Block solution and loaded into a 96-well plate at 100 mL / well. The plate is incubated in a microplate constant temperature shaker at a vibration rate of 400 rpm at room temperature for 1 hour. The analyte includes clinical serum samples, clinical pre-clinical aqueous humor samples, or clinical vitreous fluid.

[0014] As a preferred embodiment of the present invention, the terminating solution in step 7) is 2 mol / L sulfuric acid.

[0015] Preferably, in step (7), a commercially available Super Block solution is selected as the diluent to dilute the detection antibody. The commercially available Super Block solution is a multi-component composite blocking buffer. Compared with traditional single-component blocking solutions, it can more effectively reduce non-specific binding, thereby improving the sensitivity and reliability of the experiment.

[0016] Preferably, the perforated plate is a 96-hole plate.

[0017] An indirect ELISA method for detecting conbercept in human serum, aqueous humor, or vitreous humor was developed. Sensitivity and repeatability tests were performed, and the method was validated. The results met domestic and international regulatory guidelines, including but not limited to the "Guideline for Validation of Quantitative Analysis Methods for Biological Samples" and "ICH M10 BIOANALYTICAL METHODVALIDATION AND STUDY SAMPLE ANALYSIS Guidance for Industry."

[0018] This invention provides an indirect ELISA method for detecting conbercept in human serum, aqueous humor, and vitreous humor. The standard curve concentration range is 0.25–16 ng / mL. This reduced concentration range significantly improves the sensitivity of the method, allowing for the detection of more low-concentration samples in actual testing. The method changes the incubation conditions from 37°C to room temperature (generally 10°C–30°C, preferably 25°C–26°C), making the experiment more convenient and eliminating the need for special heating equipment. The results exhibit higher stability and reproducibility. Simultaneously, non-specific binding is reduced, lowering the background value. Both the blocking buffer and sample diluent are adjusted to commercially available Super Block solutions, which offer excellent blocking efficiency, minimize non-specific adsorption between antibodies, and significantly improve the signal-to-noise ratio. Super Block solutions, as commercially available ready-to-use reagents, exhibit minimal batch-to-batch variation and excellent stability and consistency, thus improving the success rate of the method, the reliability of the data, and saving time and labor costs.

[0019] This invention optimizes and establishes methods, conducts sensitivity and repeatability tests, and validates the methodology, ensuring accuracy and repeatability while improving sensitivity. The method of this invention offers advantages such as ease of operation, high sensitivity, good repeatability, and simultaneous quantitative analysis of multiple samples. Clinically, it can be used for monitoring blood drug concentrations during medication in patients with neovascular age-related macular degeneration (nAMD), choroidal neovascularization in pathological myopia (pmCNV), diabetic macular edema (DME), and retinal vein occlusion (RVO), as well as for detecting drug concentrations in specimens excised during ophthalmic surgery, guiding individualized clinical medication.

[0020] Compared with the prior art, the present invention has the following beneficial effects: 1. High detection sensitivity: It can accurately quantify low concentrations of conbercept in samples from different sources such as serum, aqueous humor and vitreous fluid, with concentrations as low as 0.3 ng / mL.

[0021] 2. Good method repeatability: By optimizing coating, sealing and sample loading, the stability and repeatability of the results are guaranteed. The difference between replicates (CV%) can be controlled within 2%, and the recovery rate (RE%) can be controlled within 1% to 10%. The acceptance criteria are CV% ≤ 20% and RE% ≤ 20%.

[0022] 3. The established indirect ELISA method for detecting conbercept in human serum, aqueous humor, and vitreous humor was validated. Results showed that the method exhibited good linearity within the range of 0.25–16 ng / mL, with a linearity coefficient R0. 2 All values ​​were greater than 0.99, and the lower limit of quantitation (LLOQ) was 0.3 ng / mL. The intra- and inter-batch precision and accuracy, stability under different conditions, and dilution reliability were all within ±15%. The established indirect ELISA method can meet the needs of quantitative analysis of clinical samples.

[0023] 4. Simple operation: The testing steps are standardized and the conditions are clear, making it easy to carry out routinely in clinical laboratories.

[0024] 5. Wide range of applications: It can be used not only for detecting the drug concentration of conbercept in ophthalmic surgical specimens, but also for monitoring blood drug concentration during patient treatment, assisting doctors in evaluating efficacy and safety, and providing a basis for individualized dosing regimens.

[0025] The detailed structure of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. Attached Figure Description

[0026] Figure 1 Fitting curves for standard curves at different dilution factors; Figure 2Fitting curves for standard curves of different sealing solutions; Figure 3 Standard curve fitting curves for different antibody dilution ratios; Figure 4 The standard curve fitting curves for different standard concentrations of Conbercept are shown. Detailed Implementation

[0027] Specific embodiments of the present invention are given below. These specific embodiments are only used to further illustrate the present invention in detail and do not limit the scope of protection of the claims of the present invention.

[0028] The sources of the reagents and raw materials used in this invention are described below: Table 1 Raw Material Source Table

[0029] Example 1 The indirect ELISA method for detecting conbercept in human serum, aqueous humor, and vitreous humor of this invention was obtained through creative effort. In the early stages of establishing this method, the inventors conducted numerous experiments and repeated explorations, ultimately obtaining the detection method of this invention, which specifically includes the following steps: 1) Coating protein: Recombinant human VEGF was coated with 1×PBS. 165 Dilute to 0.5 mg / mL, add 100 mL / well to an ELISA plate, and incubate overnight at 2–8°C for 24 hours. 2) Washing: Place the ELISA plate in the plate washer, set the program to 300 mL / well, add washing buffer, shake for 1 min, remove the washing buffer, and repeat the above steps 3 times. 3) Blocking: 300 mL of blocking solution per well, incubated at room temperature for 1 hour; the room temperature is approximately 25ºC. 4) Washing: Place the ELISA plate in the plate washer, set the program to 300 mL / well, add washing buffer, shake for 1 min, remove the washing buffer, and repeat the above steps 3 times. 5) Add the sample: Dilute Conbercept to each concentration and add blank diluent (negative control) to the wells of a 96-well plate in a constant temperature shaker at 400 rpm for 1 hour at room temperature. The diluent is commercially available Super Block. 6) Washing the plate: Place the ELISA plate in the plate washer, set the program to 300 mL / well, add the washing buffer, shake for 1 min, and then remove the washing buffer. Repeat the above steps 3 times. 7) Add detection antibody: Dilute the HRP-labeled anti-human IgG-Fc antibody with diluent, add 100 mL / well in a 96-well plate, and incubate in a microplate shaker at 400 rpm at room temperature for 1 hour. 8) Washing: Place the ELISA plate in the plate washer, set the program to 300 mL / well, add washing buffer, shake for 1 min, remove the washing buffer, and repeat the above steps 3 times. 9) Color development: Add TMB single-component color development solution, 100 mL / well, according to the 96-well plate layout, and incubate at room temperature in the dark for 10 ± 1 min; 10) Termination: Add ELISA stop solution to each well according to the 96-well plate layout, 50 mL; 11) Microplate reader reading: Use a microplate reader to read OD values ​​at a detection wavelength of 450nm and a reference wavelength of 630nm, and statistically analyze the measured values, plot curves, and process the data.

[0030] Meaning of standard curves and their coefficients: A standard curve is composed of standard samples of known concentrations of conbercept diluted in a diluent, representing a specific concentration range. It reflects the relationship between conbercept concentration and the response value of the analytical platform. In actual analysis, a four-parameter logistic curve is used for fitting. The formula is as follows:

[0031] Note: A: Minimum value; B: Slope; C: Half-maximum effective concentration; D: Maximum value; Y: OD value of microplate reader reading; X: Conbercept concentration.

[0032] Sensitivity test: The limit of detection (LOD) is the lowest detectable concentration of an analyte and is an indicator of the sensitivity of the method of this invention; the lower the LOD, the better the sensitivity. In the sensitivity test of this method, the analyte concentrations were set at 0.900 ng / mL, 0.600 ng / mL, 0.400 ng / mL, and 0.300 ng / mL, with three parallel samples for each concentration. A blank dilution was used as a negative control, and the cut-off value was set to negative control group A. 450 Three times the value, when the detected value is ≥ Cut-off, through the standard curve (according to Figure 4 ) Calculate the concentration of the analyte back.

[0033] Effect experiment: The CV% and RE% at each concentration point were less than 10% (CV% acceptance standard ≤ 20%; RE% acceptance standard ≤ 20%), as shown in Table 2.

[0034] Table 2. Results of sensitivity tests at various concentrations

[0035] Repeatability testing: Intra-batch repeatability and inter-batch repeatability experiments were performed separately. For intra-batch repeatability experiments, five concentrations were set up: 12 ng / mL, 9 ng / mL, 2.5 ng / mL, 0.90 ng / mL, 0.30 ng / mL, and a negative control. Three parallel samples were used for each concentration. Blank dilution was used as a negative control. The intra-batch coefficient of variation was calculated for different concentrations. For inter-batch repeatability experiments, three batches were performed using different batches of antibodies and different ELISA plates. The inter-batch coefficient of variation was calculated for each concentration.

[0036] Experimental results: The intra-assay and inter-assay coefficients of variation of the indirect ELISA method for detecting conbercept in human serum, aqueous humor and vitreous humor established in this invention are all within 15%, with 70% of the coefficients of variation being within 10% (as shown in Tables 3-6). The calculated limit of detection (LOD) is 0.136 ng / mL, which indicates that this method has high sensitivity and good repeatability in intra-assay and inter-assay detection.

[0037] Table 3 Precision and accuracy within the batch

[0038] Table 4. Precision and accuracy between batches

[0039] Table 5 Calculation of Standard Curve

[0040] Table 6 Summary of Standard Curve Parameters

[0041] In the embodiments of this invention, the sensitivity test, repeatability experiment, and methodological validation analysis are as follows: This indirect ELISA method can detect conbercept at a minimum sample concentration of 0.3 ng / mL. The repeatability experiment results show that the coefficients of variation within and between batches are both less than 15%, indicating that the method has good repeatability in detection. Methodological validation results show that conbercept exhibits good linearity in the range of 0.25–16 ng / mL, with a linear correlation coefficient R0. 2 All values ​​were greater than 0.99; the precision and accuracy within batches and batches, the parallelism of real samples and spiked samples were all within ±20%, and the established indirect ELISA method can meet the needs of quantitative analysis of samples.

[0042] The quantitation range consists of the lowest concentration control sample (LLOQ) and the highest concentration control sample (ULOQ). The analytical range (the range of the standard curve) consists of at least 6 concentration points, including LLOQ and ULOQ. In the scheme of this invention, the analytical range is between 0.25 ng and 16 ng / mL. We selected the lower limit of quantitation (LLOQ) as 0.3 ng / mL and the upper limit of quantitation (ULOQ) as 12 ng / mL. An additional concentration point is added at each end of the standard curve to improve the curve fitting.

[0043] Comparative Example 1 The specific detection method is the same as in Example 1, except that in step 5), a different diluent is used for detection, and the results are as follows. Figure 1 As shown, Figure 1 Fitting curves to standard curves at different dilution factors, from Figure 1 It can be seen that: 1) By performing four-parameter fitting on two curves comparing different dilution methods, it can be seen that all concentration points of the standard curve fall on the curve and both curves exhibit an S-shape. 2) By comparing the upper ends of the two curves, when the minimum dilution factor is 100, the curve shows an upward trend at the high concentration point, which is beneficial to the pass rate of the first concentration of the standard curve; when the minimum dilution factor is 10, the response value OD range of the curve is still within the range of 0.3 to 3.2, which meets the method validation requirements. Therefore, the minimum dilution factor is set to 10. In actual testing, the dilution factor is usually between 10 and 100, depending on the situation.

[0044] Comparative Example 2 The specific detection method is the same as in Example 1, except that a different blocking solution is used in step 3) of the sample addition step. The results are as follows: Figure 2 As shown, Figure 2 For the standard curve fitting curves of different sealing liquids, from Figure 2 It can be seen that: 1) Perform four-parameter fitting on the three curves, and the results are as follows: Figure 2 As shown, all three curves can be fitted. Compared with the other two blocking solutions, the curve using the commercial Super Block (STD3) for blocking is lower at low concentrations and higher at high concentrations. 2) By comparing the Blank values ​​of the three, it can be seen that the background value of using commercial Super Block as the sealing liquid is the smallest and the sealing effect is the best, as shown in Table 7.

[0045] Table 7 Blank values ​​for different sealing solutions

[0046] As shown in Table 7, using commercially available Super Block as the sealing fluid results in good sealing efficiency and significantly improves the signal-to-noise ratio.

[0047] Comparative Example 3 The specific detection method is the same as in Example 1, except that step 7) uses different antibody dilution ratios for detection, and the results are as follows. Figure 3 As shown, Figure 3 For the standard curve fitting curves of different antibody dilution ratios, from Figure 3 It can be seen that: 1) Fit the signal values ​​of the antibody at different dilution ratios, such as... Figure 3 As shown, when the antibody dilution ratio is 1:4000, the signal value shows a clear gradient and the curve exhibits a standard S-shape. Therefore, the fitting effect is significantly better than that when the antibody dilution ratio is 1:2000. 2) As shown in Table 8, by comparing the zero concentration signals of the two, it can be clearly observed that the detection antibody decreased by 3 times at 1:4000. The higher the dilution ratio of the detection antibody, the weaker the non-specific binding phenomenon and the higher the sensitivity. The sensitivity was significantly improved. Therefore, a dilution ratio of 1:4000 was selected for subsequent experiments.

[0048] Table 8 compares the signal values ​​of antibodies at dilution ratios of 1:4000 and 1:2000.

[0049] Example 2 This invention provides an indirect ELISA method for detecting conbercept in human serum, aqueous humor, and vitreous humor. The specific steps are the same as in Example 1, except that the sample loading step includes: diluting conbercept to various concentrations with blank human serum, loading 100 mL / well into a 96-well plate, and incubating at 400 rpm in a microplate shaker at room temperature for 1 hour. The remaining steps are the same as in Example 1.

[0050] Table 9. Detection results of human serum samples for Conbercept.

[0051] Table 10. Detection results of human serum samples containing Conbercept.

[0052] As can be seen from the data in Tables 9-10, the method of the present invention has high sensitivity, good repeatability and high accuracy when detecting human serum samples.

[0053] Example 3 This invention provides an indirect ELISA method for detecting conbercept in human serum, aqueous humor, and vitreous humor. The specific steps are the same as in Example 1, except that the sample loading step includes: diluting conbercept with blank human aqueous humor to various concentrations, loading the sample into a 96-well plate at 100 mL / well, and incubating it in a microplate shaker at 400 rpm at room temperature for 1 hour; the remaining steps are the same as in Example 1.

[0054] Table 11 Detection results of human anterior atrial fluid samples from Compercept.

[0055] Table 12 Detection results of human anterior atrial fluid samples from Compercept.

[0056] As can be seen from the data in Tables 11-12, the method of the present invention has high sensitivity, good repeatability and high accuracy when detecting human anterior aqueous humor samples.

[0057] Example 4 This invention provides an indirect ELISA method for detecting conbercept in human serum, aqueous humor, and vitreous humor. The specific steps are the same as in Example 1, except that the sample loading step includes: diluting conbercept to various concentrations with blank human vitreous humor, loading 100 mL / well into a 96-well plate, and incubating at 400 rpm in a microplate shaker at room temperature for 1 hour. The remaining steps are the same as in Example 1.

[0058] Table 13 Detection results of vitreous fluid samples from patients treated with Compercept.

[0059] Table 14 Detection results of vitreous fluid samples from patients treated with Compercept.

[0060] As can be seen from the data in Tables 13-14, the method of the present invention has high sensitivity, good repeatability and high accuracy when detecting human vitreous fluid samples.

[0061] Example 5 Parallelism testing of spiked samples for the indirect ELISA method for detecting conbercept in human serum, aqueous humor, and vitreous fluid: The specific detection method is the same as in Example 1, except that parallelism experiments were performed on blank serum, blank aqueous humor and blank vitreous fluid. 500 ng / mL of Conbercept standard was added to blank serum, blank aqueous humor and blank vitreous fluid respectively, and gradient dilutions of 40, 80, 120, 160 and 240 times were set. The coefficient of variation of the overall back-calculated concentration was calculated.

[0062] Experimental results: The coefficients of variation of the indirect ELISA method for detecting conbercept in human serum, aqueous humor and vitreous humor established in this invention are all within 20% (as shown in Table 15). This indicates that the parallelism of the three matrices can be achieved when the method established in this invention is used for sample detection.

[0063] Table 15 Parallelism of spiked serum, aqueous humor, and vitreous fluid

[0064] Example 6 The above method was used to detect conbercept in clinical serum samples: This invention provides an indirect ELISA method for detecting conbercept in human serum. The specific steps are the same as in Example 1, except that the sample loading step includes: diluting both serum 1 and serum 2 from patients receiving conbercept treatment 10-fold, loading them into a 96-well plate at 100 mL / well, and incubating in a microplate shaker at 400 rpm at room temperature for 1 hour. The remaining steps are the same as in Example 1. The OD value is read from the microplate reader, and the concentration of conbercept in the clinical patient's serum is calculated based on the standard curve.

[0065] Efficacy Experiment: In real sample testing, this method uses a four-parameter logistic curve to fit the standard curve of the day. The concentration of conbercept in the patient's serum was calculated as follows: Table 16. Conbercept concentrations in two clinical serum samples

[0066] Example 7 The above method was used to detect conbercept in preclinical aqueous humor samples: This invention provides an indirect ELISA method for detecting conbercept in human anterior aqueous humor. The specific steps are the same as in Example 1, except that the sample loading step includes: diluting anterior aqueous humor 1 (from a patient receiving conbercept treatment) 10-fold and anterior aqueous humor 2 (from a patient receiving conbercept treatment) 50-fold, loading 100 mL / well into a 96-well plate, and incubating at 400 rpm in a microplate shaker at room temperature for 1 hour. The remaining steps are the same as in Example 1. The OD value is read from the microplate reader, and the concentration of conbercept in the anterior aqueous humor of the clinical patient is calculated based on the standard curve.

[0067] Efficacy Experiment: In real sample testing, this method uses a four-parameter logistic curve to fit the standard curve of the day. The concentration of conbercept in the patient's anterior aqueous humor is calculated as follows: Table 17. Conbercept concentrations in two preclinical atrial fluid samples

[0068] Example 8 The above method was used to detect Conbercept in clinical vitreous fluid samples: This invention provides an indirect ELISA method for detecting conbercept in human anterior aqueous humor. The specific steps are the same as in Example 1, except that the sample addition step includes: diluting vitreous fluid 1 (from a patient receiving conbercept treatment) 10-fold and vitreous fluid 2 (from a patient receiving conbercept treatment) 20-fold, adding 100 mL / well of each well in a 96-well plate, and incubating at 400 rpm in a microplate shaker at room temperature for 1 hour. The remaining steps are the same as in Example 1. The OD value is read from the microplate reader, and the concentration of conbercept in the anterior aqueous humor of the clinical patient is calculated based on the standard curve.

[0069] Efficacy Experiment: In real sample testing, this method uses a four-parameter logistic curve to fit the standard curve of the day. The concentration of conbercept in the patient's anterior aqueous humor is calculated as follows: Table 18. Conbercept concentrations in two clinical vitreous fluid samples

[0070] The above description is a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and concept of the present invention, should be covered within the scope of protection of the claims of the present invention.

Claims

1. An indirect ELISA method for detecting conbercept in human serum, aqueous humor, and vitreous humor, characterized in that, The specific steps include: 1) Coating protein: Recombinant human VEGF was coated with a coating solution. 165 Dilute to 0.4-0.6 mg / mL and add 90-110 mL / well to an ELISA plate for coating. 2) Washing: Place the ELISA plate in a plate washer, add 290~310mL / well of washing buffer, shake for 1~2 minutes, then remove the washing buffer. Repeat the above steps 2~4 times. 3) Blocking: Blocking was completed by incubating at room temperature for 0.9-1.1 h with 290-310 mL of blocking solution per well; the blocking solution was SuperBlock solution. 4) Washing: Place the ELISA plate in a plate washer, add 290~310mL / well of washing buffer, shake for 1~2 minutes, then remove the washing buffer. Repeat the above steps 2~4 times. 5) Sample addition: Dilute the analyte 10-100 times with Super Block solution and add it to the wells in the same arrangement as the plate, 100-120 mL / well. Incubate in a microplate shaker at 400-450 rpm at room temperature for 1-1.2 h. The analyte may include clinical serum samples, clinical pre-clinical aqueous humor samples, or clinical vitreous fluid. 6) Washing: Place the ELISA plate in a plate washer, add 290~310mL / well of washing buffer, shake for 1~2 minutes, then remove the washing buffer. Repeat the above steps 2~4 times. 7) Add detection antibody: Dilute the horseradish peroxidase (HRP)-labeled anti-human IgG-Fc antibody with diluent, add 100-120 mL / well according to the well plate arrangement, and incubate in a microplate shaker at 400-450 rpm at room temperature for 1-1.2 h; use Super Block solution as diluent to dilute the detection antibody at a dilution ratio of 1:3000-4000. 8) Washing the plate: Place the ELISA plate in a plate washer, add 290~310mL / well of washing buffer, shake for 1~2 minutes, then remove the washing buffer. Repeat the above steps 2~4 times. 9) Color development: Add TMB single-component color development solution according to the well plate layout, 100~120mL / well, and incubate at room temperature in the dark for 9~11 min; 10) Termination: Add the stop solution according to the well plate layout, adding 50~55mL to each well to terminate the colorimetric reaction; 11) Microplate reader reading: Use a microplate reader to detect OD values ​​at dual wavelengths from 450nm to 630nm.

2. The indirect ELISA method for detecting conbercept in human serum, aqueous humor, and vitreous humor according to claim 1, characterized in that: The coating solution mentioned in step 1) is a phosphate buffer solution, and the coating process is as follows: incubate in a refrigerator at 2~8ºC for 20~26 hours.

3. The indirect ELISA method for detecting conbercept in human serum, aqueous humor, and vitreous humor according to claim 2, characterized in that: In step 1), recombinant human VEGF was used in 1' PBS. 165 Dilute to 0.5 mg / mL, add 100 mL / well to an ELISA plate, and incubate at 4ºC for 24 hours.

4. An indirect ELISA method for detecting conbercept in human serum, aqueous humor, and vitreous humor according to any one of claims 1-3, characterized in that: The room temperature mentioned in steps 3), 5), 7) and 9) above is 10ºC to 30ºC.

5. An indirect ELISA method for detecting conbercept in human serum, aqueous humor, and vitreous humor according to any one of claims 1-3, characterized in that: In step 3), use 300 mL of blocking solution per well and incubate at room temperature for 1 hour.

6. An indirect ELISA method for detecting conbercept in human serum, aqueous humor, and vitreous humor according to any one of claims 1-3, characterized in that, In steps 2), 4), 6), and 8), place the ELISA plate into the plate washer, set the program to 300 mL / well, add the washing buffer, and shake for 1 min.

7. An indirect ELISA method for detecting conbercept in human serum, aqueous humor, and vitreous humor according to any one of claims 1-3, characterized in that: The washing solution mentioned in step 4) is 0.05% PBST.

8. An indirect ELISA method for detecting conbercept in human serum, aqueous humor, and vitreous humor according to any one of claims 1-3, characterized in that: Step 5) Sample addition: Dilute the analyte 10-fold with Super Block solution and add 100 mL / well in a 96-well plate arrangement. Incubate in a microplate shaker at 400 rpm at room temperature for 1 hour. The analyte may include clinical serum samples, preclinical aqueous humor samples, or clinical vitreous fluid.

9. An indirect ELISA method for detecting conbercept in human serum, aqueous humor, and vitreous humor according to any one of claims 1-3, characterized in that: The terminating solution mentioned in step 7) is 2 mol / L sulfuric acid.

10. An indirect ELISA method for detecting conbercept in human serum, aqueous humor, and vitreous humor according to any one of claims 1-3, characterized in that: The perforated plate is a 96-hole plate.

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