Kit for detecting human VEGF level in biological sample

Abstract

The invention discloses a combined reagent, the combined reagent is obtained through a first mouse monoclonal antibody and a second mouse monoclonal antibody, a heavy chain variable region of the first mouse monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO: 1, and a light chain variable region of the first mouse monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO: 2; a heavy chain variable region of the second mouse monoclonal antibody contains an amino acid sequence as shown in SEQ ID NO: 3, and a light chain variable region of the second mouse monoclonal antibody contains an amino acid sequence as shown in SEQ ID NO: 4. The invention also provides a kit and a method for detecting the human VEGF level in a biological sample.

Description

Technical Field

[0001] The present invention relates to the field of immunology, and in particular, to a combination reagent, a kit based on the combination reagent, and a method for detecting the level of human VEGF in a biological sample. Background Art

[0002] Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a homodimeric glycoprotein composed of two identical polypeptide chains cross-linked by disulfide bonds. It has the functions of promoting the proliferation of vascular endothelial cells, stimulating the formation of new blood vessels in the body, promoting the increase of vascular permeability, and maintaining the normal state and integrity of blood vessels. A large number of studies have shown that VEGF is expressed in almost all tumor tissues. The expression of VEGF is significantly correlated with the malignancy, progression, metastasis and prognosis of the tumor. The activity of tumor angiogenesis is an important factor affecting the proliferation of tumor cells. As one of the most important factors promoting tumor angiogenesis, VEGF can reflect the occurrence, development and transformation of tumors. It is currently recognized as one of the most promising tumor screening and auxiliary diagnostic reagents.

[0003] ELISA kits are usually used to detect the level of human VEGF in biological samples. Specifically, the sample, standard, and HRP-labeled detection antibody are added to the coated microwells pre-coated with VEGF antibodies in sequence, incubated and thoroughly washed. The substrate TMB is used for color development. TMB turns blue under the catalysis of peroxidase and converts to the final yellow under the action of acid. The depth of color is positively correlated with the VEGF in the sample. The absorbance (OD value) is measured at a wavelength of 450nm using an enzyme reader to calculate the sample concentration.

[0004] How to improve detection sensitivity is a technical problem that needs to be solved urgently in this field. Summary of the invention

[0005] The present invention aims to solve one of the technical problems in the related art to a certain extent. To this end, the present invention provides a combination reagent, a kit based on the combination reagent and a method for detecting the level of human VEGF in a biological sample.

[0006] As a first aspect of the present invention, a combination reagent is provided, characterized in that the combination reagent is obtained by a first mouse monoclonal antibody and a second mouse monoclonal antibody, the heavy chain variable region of the first mouse monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region of the first mouse monoclonal antibody contains the amino acid sequence shown in SEQ ID NO: 2; the heavy chain variable region of the second mouse monoclonal antibody contains the amino acid sequence shown in SEQ ID NO: 3, and the light chain variable region of the second mouse monoclonal antibody contains the amino acid sequence shown in SEQ ID NO: 4.

[0007] Optionally, the combination reagent comprises the first mouse monoclonal antibody labeled with biotin and the second mouse monoclonal antibody labeled with a set marker, and the set marker is selected from any one of a radioactive isotope, a fluorescein and an enzyme substrate.

[0008] Optionally, the enzyme substrate comprises streptavidin-horseradish peroxidase, or alkaline phosphatase; The radioactive isotope includes iodine; The fluorescein includes rhodamine-type fluorescein or quantum dots.

[0009] As a second aspect of the present invention, there is provided a kit for detecting the level of human VEGF in a biological sample, the kit comprising a capture antibody and a detection antibody, wherein the capture antibody comprises a first mouse monoclonal antibody, the heavy chain variable region of the first mouse monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region of the first mouse monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 2; The detection antibody comprises a second mouse monoclonal antibody, the heavy chain variable region of the second mouse monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 3, and the light chain variable region of the second mouse monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 4.

[0010] Optionally, the capture antibody is the first mouse monoclonal antibody labeled with biotin; the detection antibody is the second mouse monoclonal antibody labeled with a set marker, and the set marker is selected from any one of a radioactive isotope, a fluorescein and an enzyme substrate.

[0011] Optionally, the enzyme substrate comprises streptavidin-horseradish peroxidase, or alkaline phosphatase; The radioactive isotope includes iodine; The fluorescein includes rhodamine-type fluorescein or quantum dots.

[0012] As a third aspect of the present invention, a method for detecting the level of human VEGF in a biological sample is provided, wherein the method comprises: contacting the biological sample with an immobilized capture antibody and incubating them together, wherein the immobilized capture antibody comprises a capture antibody immobilized to a solid support, the capture antibody comprises a first mouse monoclonal antibody, the heavy chain variable region of the first mouse monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region of the first mouse monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 2; separating the biological sample from the immobilized capture antibody; Contacting the complex of the immobilized capture antibody and the target molecule human VEGF with a detection antibody capable of binding to the epitope region of human VEGF, wherein the detection antibody comprises a second mouse monoclonal antibody, wherein the heavy chain variable region of the second mouse monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 3, and the light chain variable region of the second mouse monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 4; The level of human VEGF bound by the immobilized capture antibody is determined.

[0013] Optionally, the capture antibody is the first mouse monoclonal antibody labeled with biotin; the detection antibody is the second mouse monoclonal antibody labeled with a set marker, and the set marker is selected from any one of a radioactive isotope, a fluorescein and an enzyme substrate.

[0014] Optionally, the enzyme substrate comprises streptavidin-horseradish peroxidase, or alkaline phosphatase; The radioactive isotope includes iodine; The fluorescein includes rhodamine-type fluorescein or quantum dots.

[0015] The combined reagent provided in the embodiment of the present invention can be used in a kit. The amino acid sequences of the variable regions (including the heavy chain variable region and the light chain variable region) of the first mouse monoclonal antibody and the variable regions (including the heavy chain variable region and the light chain variable region) of the second mouse monoclonal antibody will specifically bind to the antigenic determinant of the antigen, and the two are mainly bound by non-covalent bonds, thereby achieving the purpose of specific recognition of the antigen.

[0016] In addition, when the combination reagent is used in the test kit, the first mouse monoclonal antibody is labeled with biotin, and the second mouse monoclonal antibody is labeled with a set marker, and the set marker (especially streptavidin) has a very high affinity with biotin, and its dissociation constant is about 10-15M, which is one of the strongest non-covalent interactions known in nature. Therefore, the test kit has good stability. In addition, streptavidin is a tetrameric protein, and each molecule can bind to four biotin molecules. This allows a streptavidin molecule to simultaneously bind to multiple detection molecules labeled with biotin, thereby achieving signal amplification. Therefore, the test kit provided in an embodiment of the present invention has high sensitivity. DETAILED DESCRIPTION

[0018] References to "one embodiment" or "example" or "example" in this specification mean that a particular feature, structure, or characteristic described in conjunction with the embodiment itself may be included in at least one embodiment disclosed herein. The appearance of the phrase "in one embodiment" in various places in the specification does not necessarily refer to the same embodiment.

[0020] As a first aspect of the present invention, a combination reagent is provided, wherein the combination reagent is obtained by a first mouse monoclonal antibody and a second mouse monoclonal antibody, the heavy chain variable region of the first mouse monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region of the first mouse monoclonal antibody contains the amino acid sequence shown in SEQ ID NO: 2; the heavy chain variable region of the second mouse monoclonal antibody contains the amino acid sequence shown in SEQ ID NO: 3, and the light chain variable region of the second mouse monoclonal antibody contains the amino acid sequence shown in SEQ ID NO: 4.

[0021] The combined reagent can be used to prepare a detection antibody in a kit for detecting the level of human VEGF in a biological sample.

[0022] The Complementarity Determining Region (CDR) in the variable region of a monoclonal antibody is a key structure for antigen-antibody specific binding. The amino acid sequence of CDR is highly diverse, and different CDRs can form binding pockets or surfaces of different shapes, sizes, and chemical properties. When a monoclonal antibody binds to an antigen, CDR acts like a "molecular recognition fingerprint" that achieves specific binding through precise complementarity with the antigen epitope in shape and chemical groups.

[0023] Although the framework region (FR) of a monoclonal antibody does not directly participate in antigen binding, it provides a stable structural basis for CDR. The amino acid sequence of FR is relatively conservative, and its correct folding and spatial positioning can ensure that CDR is in the appropriate position and conformation, so that the antibody can specifically bind to the antigen epitope in the correct way. If the structure of FR is abnormal, it may cause the spatial position of CDR to change, thereby affecting the specificity of antibody-antigen binding.

[0024] In the embodiment of the present invention, the amino acid sequences of the variable region (including the heavy chain variable region and the light chain variable region) of the first mouse monoclonal antibody and the variable region (including the heavy chain variable region and the light chain variable region) of the second mouse monoclonal antibody will specifically bind to the antigenic determinant of the antigen, and the two are mainly bound by non-covalent bonds, thereby achieving the purpose of specific recognition of the antigen.

[0025] Optionally, the combination reagent comprises the first mouse monoclonal antibody labeled with biotin and the second mouse monoclonal antibody labeled with a set marker, wherein the set marker is selected from any one of a radioactive isotope, a fluorescein and an enzyme substrate.

[0026] Optionally, the set marker is streptavidin-horseradish peroxidase.

[0027] When the combination reagent is used in the test kit, the first mouse monoclonal antibody is labeled with biotin, and the second mouse monoclonal antibody is labeled with a set marker. The set marker can be streptavidin, which has a very high affinity with biotin, and its dissociation constant is about 10-15M, which is one of the strongest non-covalent interactions known in nature. Therefore, the test kit has good stability. In addition, streptavidin is a tetrameric protein, and each molecule can bind to four biotin molecules. This allows a streptavidin molecule to simultaneously bind to multiple detection molecules labeled with biotin, thereby achieving signal amplification. Therefore, the test kit provided in the embodiment of the present invention has high sensitivity.

[0028] As another optional embodiment, the enzyme substrate may also be alkaline phosphatase.

[0029] Of course, the present invention is not limited thereto. When the second mouse monoclonal antibody is labeled with a radioactive isotope, the radioactive isotope may be iodine.

[0030] When fluorescein is used to label the second mouse monoclonal antibody, the fluorescein may be rhodamine-type fluorescein or quantum dots.

[0031] As a second aspect of the present invention, a kit for detecting the level of human VEGF in a biological sample is provided, the kit comprising a capture antibody and a detection antibody.

[0032] The capture antibody comprises a first mouse monoclonal antibody, the heavy chain variable region of the first mouse monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region of the first mouse monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 2.

[0033] The detection antibody comprises a second mouse monoclonal antibody, the heavy chain variable region of the second mouse monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 3, and the light chain variable region of the second mouse monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 4.

[0034] In an embodiment of the present invention, the capture antibody can be immobilized on an enzyme-linked immunosorbent assay (ELISA) microplate.

[0035] Furthermore, the capture antibody is the first mouse monoclonal antibody labeled with biotin; the detection antibody is the second mouse monoclonal antibody labeled with a set marker, and the set marker is selected from any one of a radioactive isotope, a fluorescein, and an enzyme substrate.

[0036] Furthermore, the kit may also include a VEGF calibrator, a sample diluent, a concentrated washing solution, a color developing solution A, a color developing solution B, and a stop solution.

[0037] As a third aspect of the present invention, a method for detecting the level of human VEGF in a biological sample is provided, wherein the method comprises: contacting the biological sample with an immobilized capture antibody and incubating them together, wherein the immobilized capture antibody comprises a capture antibody immobilized to a solid support, the capture antibody comprises a first mouse monoclonal antibody, the heavy chain variable region of the first mouse monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region of the first mouse monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 2; separating the biological sample from the immobilized capture antibody; Contacting the complex of the immobilized capture antibody and the target molecule human VEGF with a detection antibody capable of binding to the epitope region of human VEGF, wherein the detection antibody comprises a second mouse monoclonal antibody, wherein the heavy chain variable region of the second mouse monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 3, and the light chain variable region of the second mouse monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 4; The level of human VEGF bound by the immobilized capture antibody is determined.

[0038] Wherein, the solid phase carrier can be an enzyme-linked immunosorbent assay (ELISA) microplate.

[0039] Optionally, the capture antibody is the first mouse monoclonal antibody labeled with biotin; the detection antibody is the second mouse monoclonal antibody labeled with a set marker, and the set marker is selected from any one of a radioactive isotope, a fluorescein and an enzyme substrate.

[0040] As described above, the enzyme substrate includes streptavidin-horseradish peroxidase or alkaline phosphatase; the radioactive isotope includes iodine; and the fluorescein includes rhodamine fluorescein or quantum dots.

[0041] In the embodiment of the present invention, the human vascular endothelial growth factor expression vector can be constructed by the following method: Search for VEGF on the NCBI website 165 The amino acid sequence of the gene was expressed by adding a histidine tag to the N-terminus or C-terminus of the amino acid. The codons were optimized in the CHO expression system using codon optimization software. After gene synthesis, the gene was cloned into the pcDNA3.1 expression vector using the BamH I and Xho I restriction endonuclease sites to construct pcDNA3.1-VEGF. 165 Expression plasmids.

[0042] Expression of human vascular endothelial growth factor can be produced by the following method: pcDNA3.1-VEGF 165 The expression plasmid was transformed into CHO cells, and positive cell lines were screened by G418 pressure to establish a stable expression of VEGF 165 The positive cell lines of VEGF were identified and expanded. SDS-PAGE and Western Blot showed that the transfected CHO cells could stably secrete and express VEGF. 165 The relative molecular mass of the obtained recombinant human vascular endothelial growth factor was about 23 kDa, which was consistent with the prediction.

[0043] Preparation Example Preparation Example 1 This preparation example provides a method for preparing a monoclonal antibody, comprising: VEGF 165 The protein and Freund's adjuvant were mixed in equal volumes and emulsified using a homogenizer. The immunization dose was 50 μg / mouse, and multiple subcutaneous injections were performed. After 2 weeks, a booster immunization was performed, and the immunization dose was 50 μg / mouse, and multiple subcutaneous injections were performed. Blood was collected from the tail of mice every other week and centrifuged to separate serum. The titer of antibodies in the tail serum was tested by indirect ELISA, and mice with higher titers of antibodies in vivo were selected for hybridoma cell fusion experiments. After about 7 days of HAT screening culture, the supernatant of positive hybridoma cells was taken for indirect ELISA detection. 165 Protein-coated microplates were used to screen out positive hybridoma cell lines with high specificity and good affinity, and two rounds of cell subcloning were performed to clone monoclonal cell lines; Monoclonal antibodies were prepared by inducing ascites in mice and purified using Protein A affinity chromatography.

[0044] The above method was used to prepare the first mouse monoclonal antibody and the second mouse monoclonal antibody.

[0045] Preparation Example 2 This preparation example provides a method for preparing a biotinylated mouse anti-human VEGF monoclonal antibody (i.e., a step of labeling a first mouse monoclonal antibody with biotin), comprising: Take sulfo-NHS-biotin out of the refrigerator and equilibrate it to room temperature. Take 1-3 mg of sulfo-NHS-biotin and add it to 200-400 µL of pure water and shake it gently to activate it. Dilute the VEGF monoclonal antibody (first mouse monoclonal antibody) obtained in Preparation Example 1 with 10 mmol / L PBS buffer to a concentration of 1-3 mg / mL; Immediately add 50-100 µL of activated biotin solution slowly to 1-3 mL of antibody solution, react at room temperature for 20-40 min, and dialyze the reaction solution with 10 mmol / L PBS buffer at 4°C for 3 days, changing the dialyzate solution every day; The biotinylated monoclonal antibody conjugate (ie, the biotinylated mouse anti-human VEGF monoclonal antibody) was collected, an appropriate amount of glycerol was added, and the mixture was stored at -20°C for later use.

[0046] Preparation Example 3 This preparation example provides a method for preparing an ELISA microplate, the method comprising: Dilute the biotinylated mouse anti-human VEGF monoclonal antibody obtained in Preparation Example 1 with carbonate buffer (formula 0.1-0.2 g / L NaCO3, 3.5-4.0 g / L NaHCO3, pH 9.0-10.0); The optimal coating concentration was determined to be 2.5 mg / mL using the square array method; Take a detachable 96-well ELISA plate, add 100 μL to each well, and coat at 4°C for 24 h; Wash 5 times with PBST (formula 0.2-0.3 g / L KH2PO4, 3.5-4 g / L Na2HPO4, 7-10 g / L NaCl, 0.2-0.4 g / L KCl, 0.05% Tween-20, pH 7.0-8.0); Add 200 μL of blocking solution to each well and block at 37°C for 30-60 min; The plate was washed thoroughly with PBST and dried thoroughly to prepare an ELISA microplate.

[0047] Preparation Example 4 This preparation example provides a method for preparing a coating buffer: Weigh 0.1-0.2g NaCO3 and 3.5-4.0g NaHCO3, fully dissolve them in double distilled water, adjust the pH to 9.0-10.0, and then make up to 1000mL with double distilled water to obtain coating buffer.

[0048] Preparation Example 5 This preparation example provides a method for preparing an enzyme conjugate (i.e., an enzyme-labeled antibody working solution, i.e., a labeled second mouse monoclonal antibody), comprising: Add SA-HRP to sodium acetate buffer, fully dissolve, add 0.1-0.3M NaIO4 solution, mix well, and oxidize at 4°C for 60-90 minutes; Add 150-180 mM ethylene glycol solution and shake in the dark for 60-90 min; The above solution was placed in a dialysis bag and dialyzed with 0.1 M carbonate buffer at 4°C, changing the solution 2-3 times, each time for 2-3 hours; The second mouse monoclonal antibody solution obtained in Preparation Example 1 was placed in a dialysis bag and dialyzed with 0.1 M carbonate buffer at 4°C, with the solution changed 3 times, each time for 2-3 hours; Place IgG antibody in a reaction tube, add activated SA-HRP (mass ratio 1:1-2:1), and react in a dark and oscillating tube for 60-90 minutes; The reaction mixture was placed in a dialysis bag and dialyzed with 1× PBS buffer at 4°C for 1-2 h; Take out the dialyzed reaction mixture into a reaction tube, add sodium cyanoborohydride solution, and react in a dark environment with shaking for 30-60 minutes; Add ethanolamine solution and shake to react for 30-60 minutes in the dark; Ultrafiltration was performed using a 15 mL 100 kDa ultrafiltration tube. Repeat the ultrafiltration 5 times, and 1× PBS solution was added to 0.3-0.5 mL each time. After the ultrafiltration is completed, all volumes of the antibody solution are collected in an EP tube, and the total volume of the solution is adjusted to about 5 mL to obtain the enzyme-labeled antibody working solution (labeled second mouse monoclonal antibody).

[0049] Preparation Example 6 This preparation example provides a method for preparing a concentrated washing solution 20×, comprising: Weigh 160-170 g NaCl, 4-6 g KCl, 70-80 g Na2HPO4·12H2O, and 4-6 g KH2PO4, fully dissolve them with deionized water, add 8-10 mL Tween-20 and mix well, and make up to 1000 mL with deionized water to obtain the concentrated washing solution 20×.

[0050] Preparation Example 7 This preparation example provides a method for preparing a sample diluent, comprising: Weigh 5-8g BSA, 8-10g NaCl, 0.2-0.4g KH2PO4, 3-5g Na2HPO4·12H2O, and 0.2-0.4g KCl, dissolve them in double distilled water in sequence and mix them, add 0.05-0.1% preservative, and dilute to 1000mL with double distilled water to obtain a sample diluent.

[0051] Preparation Example 8 This preparation example provides a method for preparing a color developing solution A, comprising: Weigh 26-27 g of sodium acetate and 3-5 g of citric acid, dissolve them in deionized water in turn and mix them, add 0.6-1 mL of H2O2 solution, and make up to 1000 mL with deionized water to obtain the color developing solution A.

[0052] Preparation Example 9 This preparation example provides a method for preparing a color developing solution B, comprising: Weigh 0.4-0.6 g of disodium ethylenediaminetetraacetate, 2-3 g of citric acid, measure 100 mL of glycerol, take 0.3-0.5 g of TMB and dissolve it in 3-5 mL of DMSO, dissolve them in turn with double distilled water and make up to 1000 mL to obtain color developing solution B.

[0053] Preparation Example 10 This preparation example provides a method for preparing a stop solution, comprising: 400 mL of 5 M sulfuric acid solution was measured and diluted to 1000 mL with double distilled water to obtain the stop solution.

[0054] Example Example 1 A kit comprising: (1) ELISA microplate obtained in Preparation Example 3: Each kit contains 1 ELISA microplate strip containing a detachable coated mouse anti-human VEGF monoclonal antibody, with a specification of 96 wells x 1 plate; (2) VEGF calibrator: lyophilized recombinant human VEGF, 800 pg per bottle; (3) Sample dilution obtained in Preparation Example 7: 20 mL x 1 bottle; (4) 20× of the concentrated washing solution obtained in Preparation Example 6: 50 mL × 1 bottle; (5) Enzyme-labeled antibody working solution obtained in Preparation Example 5, 20 mL × 1 bottle; (6) Color development solution A obtained in Preparation Example 8, 10 mL x 1 bottle; (7) Color development solution B obtained in Preparation Example 9, 10 mL x 1 bottle; (8) Prepare the stop solution obtained in Example 10, 5 mL x 1 bottle.

[0055] Example 2 A method for detecting human serum VEGF, comprising: (1) Solution preparation: Determine the number of strips required based on the number of experimental wells (serum samples and VEGF calibrators) and dilute the concentrated wash solution 20-fold with distilled or deionized water to prepare 1× wash solution.

[0056] (2) Preparation of standard curve: Thoroughly mix the VEGF calibrator stock solution, taking the final volume of 250μL for each concentration as an example. Take 250μL of VEGF calibrator stock solution, add 250μL of sample diluent and mix well (S7), and use it as the highest concentration of the standard curve, 400pg / mL, and mark the tube wall. Take 6 centrifuge tubes (S6-S1) and arrange them in sequence. First, add 250μL of sample diluent to each tube, then take 250μL from centrifuge tube S7 and add it to S6, and gently blow to mix; take 250μL from centrifuge tube S6 and add it to S5, and gently blow to mix, and so on to perform multiple dilutions of the calibrator. S0 is the sample diluent.

[0057] (3) Sample addition: Take out the required number of microwells and dilute the serum sample with the sample diluent in a 1:1 ratio, 200 μL / well, ensuring that the final volume of each sample after dilution is ≥ 250 μL. Mix all samples thoroughly to avoid bubbles. Add 200 μL of VEGF calibrator and sample to be tested to the corresponding wells, cover with sealing film, and incubate at 37°C for 30-60 min.

[0058] (4) Washing: Remove the liquid from the wells and add 300 μL of 1× washing solution to each well. Discard the liquid in the wells and dry on filter paper. Repeat 5 times.

[0059] (5) Add enzyme: Add 200 μL of enzyme-labeled antibody working solution to each well, cover with sealing film, and incubate at 37°C for 30-60 min.

[0060] (6) Washing: Remove the liquid from the wells and add 300 μL of 1× washing solution to each well. Discard the liquid in the wells and dry on filter paper. Repeat 5 times.

[0061] (7) Color development: Add 100 μL of color development solution A and 100 μL of color development solution B to each well, shake gently to mix, seal the plate and incubate at 37°C in the dark for 10-15 min.

[0062] (8) Determination: Add 50 μL of stop solution to each well, gently shake to mix, and place the plate on an ELISA reader at a wavelength of 450 nm to measure the absorbance of each well.

[0063] (9) Calculation: Draw a standard curve based on the measured values ​​and concentrations of the VEGF calibrator, and then find the corresponding concentration based on the absorbance value of the sample. Since the serum sample was diluted 1:1, the result was multiplied by 2 as the final test result.

[0064] (10) Data processing: Fitting type: four-parameter logistic fitting; Coordinate selection: XY.

[0065] Test Case The VEGF corporate reference product was tested three times, and the relative deviation was calculated. The accuracy index test results of the double antibody sandwich ELISA kit prepared in this example are shown in Table 1.

[0066] Table 1 Accuracy test results

[0067] Conclusion: The relative deviation of the measurement results is within the range of ±10%, and all three results meet the requirements. The accuracy of the above three batches of test kits is qualified.

[0068] (3) Precision 10 serum samples were mixed and tested 20 times continuously using the double antibody sandwich ELISA kit prepared in Example 1, and tested once a day for 20 consecutive days. The results showed that the intra-batch and inter-batch CV% were 8.25% and 7.33%, respectively, both within the allowable error range (CV% < 15%), with high precision and good repeatability.

[0069] (4) Stability The double antibody sandwich ELISA kit prepared in this example was stored at 37°C for 0d, 3d and 8d, respectively, and then the high-level samples and the low-level samples were tested respectively. The CV% of the high-level samples was 10.62%, and the CV% of the low-level samples was 8.57%, both less than 15%. The shelf life can reach 10-12 months when stored at 4°C, indicating that the kit has good stability.

[0070] (5) Anti-interference capability Different concentrations of hematin, ascorbic acid, bilirubin and cholesterol were added to two samples with VEGF concentrations of 50 pg / mL and 200 pg / mL, respectively. The group without addition was used as the control. The relative deviation between the added group and the control group was less than 5%, which was the acceptable interference standard. The maximum concentration levels of different interfering substances were tested. The results are shown in Table 2.

[0071] Table 2 Analysis of the results of the maximum concentration level of interferences

[0072] Conclusion: When the maximum concentration level of the interfering substance is as shown in Table 2, the interference with the detection result is not obvious, which proves that the double antibody sandwich ELISA kit prepared in this example has good anti-interference ability.

[0073] The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that the present invention includes but is not limited to the contents described in the above specific embodiments. Any modification that does not deviate from the functional and structural principles of the present invention will be included in the scope of the claims.

Claims

1. A combination reagent, characterized in that: The combination reagent is obtained by a first mouse monoclonal antibody and a second mouse monoclonal antibody, wherein the heavy chain variable region of the first mouse monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region of the first mouse monoclonal antibody contains the amino acid sequence shown in SEQ ID NO: 2; the heavy chain variable region of the second mouse monoclonal antibody contains the amino acid sequence shown in SEQ ID NO: 3, and the light chain variable region of the second mouse monoclonal antibody contains the amino acid sequence shown in SEQ ID NO:

4.

2. The combined reagent according to claim 1, characterized in that: The combination reagent comprises the first mouse monoclonal antibody labeled with biotin and the second mouse monoclonal antibody labeled with a set marker, wherein the set marker is selected from any one of a radioactive isotope, a fluorescein and an enzyme substrate.

3. The combined reagent according to claim 2, characterized in that: The enzyme substrate includes streptavidin-horseradish peroxidase, or alkaline phosphatase; The radioactive isotope includes iodine; The fluorescein includes rhodamine-type fluorescein or quantum dots.

4. A kit for detecting the level of human VEGF in a biological sample, the kit comprising a capture antibody and a detection antibody, characterized in that: The capture antibody comprises a first murine monoclonal antibody, the heavy chain variable region of the first murine monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region of the first murine monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 2; The detection antibody comprises a second mouse monoclonal antibody, the heavy chain variable region of the second mouse monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 3, and the light chain variable region of the second mouse monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:

4.

5. The kit according to claim 4, characterized in that The capture antibody is the first mouse monoclonal antibody labeled with biotin; the detection antibody is the second mouse monoclonal antibody labeled with a set marker, and the set marker is selected from any one of radioactive isotopes, fluorescein and enzyme substrates.

6. The kit according to claim 5, characterized in that The enzyme substrate includes streptavidin-horseradish peroxidase, or alkaline phosphatase; The radioactive isotope includes iodine; The fluorescein includes rhodamine-type fluorescein or quantum dots.

7. A method for detecting the level of human VEGF in a biological sample, characterized in that: The method comprises: contacting the biological sample with an immobilized capture antibody and incubating them together, wherein the immobilized capture antibody comprises a capture antibody immobilized to a solid support, the capture antibody comprises a first mouse monoclonal antibody, the heavy chain variable region of the first mouse monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region of the first mouse monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 2; separating the biological sample from the immobilized capture antibody; Contacting the complex of the immobilized capture antibody and the target molecule human VEGF with a detection antibody capable of binding to the epitope region of human VEGF, wherein the detection antibody comprises a second mouse monoclonal antibody, wherein the heavy chain variable region of the second mouse monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 3, and the light chain variable region of the second mouse monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 4; The level of human VEGF bound by the immobilized capture antibody is determined.

8. The method according to claim 7, characterized in that The capture antibody is the first mouse monoclonal antibody labeled with biotin; the detection antibody is the second mouse monoclonal antibody labeled with a set marker, and the set marker is selected from any one of radioactive isotopes, fluorescein and enzyme substrates.

9. The method according to claim 8, characterized in that The enzyme substrate includes streptavidin-horseradish peroxidase, or alkaline phosphatase; The radioactive isotope includes iodine; The fluorescein includes rhodamine-type fluorescein or quantum dots.

Images