Kit for detecting human vegf levels in a biological sample

Abstract

The application discloses a combined reagent, which is obtained by a first mouse monoclonal antibody and a second mouse monoclonal antibody, wherein 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 contains 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 application further provides a kit and a method for detecting a human VEGF level in a biological sample.

Description

TECHNICAL FIELD

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

[0002] Vascular endothelial growth factor (VEGF, Vascular Endothelial Growth Factor) is also known as vascular permeability factor, which is a homodimeric glycoprotein composed of two identical polypeptide chains cross-linked by disulfide bond, has the functions of promoting vascular endothelial cell proliferation, stimulating in vivo neovascularization, promoting vascular permeability increase and maintaining vascular normal state and integrity. A large number of studies have shown that there is expression of VEGF in almost all tumor tissues. The expression of VEGF is significantly correlated with the malignant degree, progression, metastasis and prognosis of tumor. The active degree of tumor angiogenesis is an important factor affecting tumor cell proliferation, as one of the most important promoting factors of tumor angiogenesis, VEGF can reflect the occurrence, development and metastasis of tumor, and is one of the currently recognized tumor screening and auxiliary diagnostic reagents with application prospect.

[0003] Generally, the human VEGF level in a biological sample is detected by using an ELISA kit, specifically, a sample, a standard, an HRP-labeled detection antibody are sequentially added into a coated micro-well coated with VEGF antibody, and then incubated and washed thoroughly. Color development is performed by using a substrate TMB, TMB is converted into blue under the catalysis of peroxidase, and is converted into final yellow under the action of acid. The color depth is positively correlated with the VEGF in the sample. The absorbance (OD value) is measured by using an enzyme-labeled instrument at 450 nm wavelength, and the sample concentration is calculated.

[0004] How to improve the detection sensitivity is a technical problem to be solved in the field. SUMMARY

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

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

[0007] Optionally, the combination reagent comprises the first murine monoclonal antibody labeled by biotin and the second murine monoclonal antibody labeled by a set marker selected from any one of a radioisotope, a fluorescein and an enzyme substrate.

[0008] Optionally, the enzyme substrate comprises streptavidin-horseradish peroxidase or alkaline phosphatase.

[0009] The radioisotope comprises iodine element.

[0010] The fluorescein comprises rhodamine or quantum dots.

[0011] As a second aspect of the present application, a kit for detecting human VEGF level in a biological sample is provided, the kit comprising a capture antibody and a detection antibody, wherein the capture antibody comprises a first murine monoclonal antibody, the heavy chain variable region of the first murine monoclonal antibody comprising an amino acid sequence as shown in SEQ ID NO: 1, the light chain variable region of the first murine monoclonal antibody comprising an amino acid sequence as shown in SEQ ID NO: 2.

[0012] The detection antibody comprises a second murine monoclonal antibody, the heavy chain variable region of the second murine monoclonal antibody comprising an amino acid sequence as shown in SEQ ID NO: 3, the light chain variable region of the second murine monoclonal antibody comprising an amino acid sequence as shown in SEQ ID NO: 4.

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

[0014] Optionally, the enzyme substrate comprises streptavidin-horseradish peroxidase or alkaline phosphatase.

[0015] The radioisotope comprises iodine element.

[0016] The fluorophore includes a rhodamine fluorophore or a quantum dot.

[0017] As a third aspect of the present application, a method for detecting the level of human VEGF in a biological sample is provided, wherein the method comprises:

[0018] contacting the biological sample with a solidified capture antibody and incubating together, wherein the solidified capture antibody comprises a capture antibody immobilized to a solid phase carrier, the capture antibody comprising a first murine monoclonal antibody, a heavy chain variable region of the first murine monoclonal antibody comprising an amino acid sequence as shown in SEQ ID NO: 1, and a light chain variable region of the first murine monoclonal antibody comprising an amino acid sequence as shown in SEQ ID NO: 2;

[0019] separating the biological sample from the solidified capture antibody;

[0020] contacting a complex of the solidified capture antibody and a target molecule human VEGF with a detection antibody, the detection antibody being capable of binding to an epitope region of human VEGF, the detection antibody comprising a second murine monoclonal antibody, a heavy chain variable region of the second murine monoclonal antibody comprising an amino acid sequence as shown in SEQ ID NO: 3, and a light chain variable region of the second murine monoclonal antibody comprising an amino acid sequence as shown in SEQ ID NO: 4;

[0021] determining the level of human VEGF bound by the solidified capture antibody.

[0022] Optionally, the capture antibody is the first murine monoclonal antibody labeled with biotin; and the detection antibody is the second murine monoclonal antibody labeled with a set marker selected from any one of a radioisotope, a fluorophore, and an enzyme substrate.

[0023] Optionally, the enzyme substrate includes streptavidin-horseradish peroxidase, or alkaline phosphatase.

[0024] The radioisotope includes an iodine element.

[0025] The fluorophore includes a rhodamine fluorophore or a quantum dot.

[0026] The combination reagent provided by the embodiment of the present application 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 murine monoclonal antibody and the second murine monoclonal antibody specifically bind to the antigenic determinant of the antigen, and are mainly combined through non-covalent bonds, so as to achieve the purpose of specifically recognizing the antigen.

[0027] In addition, when the combination reagent is used in a kit, the first mouse monoclonal antibody is labeled with biotin, and the second mouse monoclonal antibody is labeled with a setting marker. The setting marker (especially streptavidin) has a very high affinity between biotin, and the dissociation constant is about 10-15M, which is one of the strongest non-covalent interactions in nature. Therefore, the kit has good stability. In addition, streptavidin is a tetrameric protein, and each molecule can bind four biotin molecules. This allows a streptavidin molecule to bind multiple biotin-labeled detection molecules at the same time, thereby amplifying the signal. Therefore, the kit provided by the embodiment of the present application has high sensitivity. DETAILED DESCRIPTION

[0028] In this specification, "one embodiment" or "an embodiment" or "example" or "exemplary" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. The appearances of the phrase "in one embodiment" in various places in the specification do not necessarily refer to the same embodiment.

[0029] As a first aspect of the present application, 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 an amino acid sequence as shown in SEQ ID NO: 1, and the light chain variable region of the first mouse monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO: 2; the heavy chain variable region of the second mouse monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO: 3, and the light chain variable region of the second mouse monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO: 4.

[0030] The combination reagent can be used as a detection antibody in a kit for detecting the level of human VEGF in a biological sample.

[0031] The complementarity determining region (CDR) in the variable region of the monoclonal antibody is the key structure of the specific binding of antigen-antibody. The amino acid sequence of CDR has high diversity, and different CDRs can form binding pockets or surfaces of different shapes, sizes and chemical properties. When the monoclonal antibody binds to the antigen, the CDR acts like a "molecular recognition fingerprint", which realizes specific binding through precise complementarity in shape and chemical groups with the antigen epitope.

[0032] The frame region (FR) of the monoclonal antibody does not directly participate in antigen binding, but it provides a stable structural basis for the CDR. The amino acid sequence of the FR is relatively conservative, and its correct folding and spatial positioning can ensure that the CDR is in the right position and conformation, so that the antibody can specifically bind to the epitope of the antigen in the correct way. If the structure of the FR is abnormal, the spatial position of the CDR may change, thereby affecting the specificity of the antibody and the antigen binding.

[0033] In the embodiment of the present application, 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 specifically bind to the antigenic determinant of the antigen, and the two are mainly combined through non-covalent bonds, so as to achieve the purpose of specifically recognizing the antigen.

[0034] Optionally, the combined reagent comprises the first mouse monoclonal antibody labeled with biotin and the second mouse monoclonal antibody labeled with a set marker. The set marker is selected from any one of a radioisotope, a fluorescein and an enzyme substrate.

[0035] Optionally, the set marker is streptavidin-horseradish peroxidase.

[0036] When the combined reagent is used in a 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 extremely high affinity with biotin, and the dissociation constant is about 10-15M, which is one of the strongest non-covalent interactions in nature. Therefore, the kit has good stability. In addition, streptavidin is a tetramer protein, and each molecule can bind four biotin molecules. This allows a streptavidin molecule to bind multiple biotin-labeled detection molecules at the same time, thereby amplifying the signal. Therefore, the kit provided in the embodiment of the present application has high sensitivity.

[0037] As another optional embodiment, the enzyme substrate can also be alkaline phosphatase.

[0038] Of course, the present application is not limited thereto, and when the second mouse monoclonal antibody is labeled with a radioisotope, the radioisotope can be selected from iodine elements.

[0039] When the fluorescein is used to label the second mouse monoclonal antibody, the fluorescein can be selected from rhodamine fluorescein or quantum dots.

[0040] As a second aspect of the present application, a kit for detecting human VEGF level in a biological sample is provided, the kit comprising a capture antibody and a detection antibody.

[0041] The capture antibody comprises a first murine monoclonal antibody, a heavy chain variable region of the first murine monoclonal antibody comprising an amino acid sequence as shown in SEQ ID NO: 1, and a light chain variable region of the first murine monoclonal antibody comprising an amino acid sequence as shown in SEQ ID NO: 2.

[0042] The detection antibody comprises a second murine monoclonal antibody, a heavy chain variable region of the second murine monoclonal antibody comprising an amino acid sequence as shown in SEQ ID NO: 3, and a light chain variable region of the second murine monoclonal antibody comprising an amino acid sequence as shown in SEQ ID NO: 4.

[0043] In an embodiment of the present application, the capture antibody can be immobilized to an Enzyme-Linked Immunosorbent Assays (ELISA) microplate.

[0044] Further, the capture antibody is the first murine monoclonal antibody labeled with biotin; and the detection antibody is the second murine monoclonal antibody labeled with a set of labels selected from any one of a radioisotope, a fluorophore, and an enzyme substrate.

[0045] Further, the kit can further comprise a VEGF calibrator, a sample diluent, a concentrated washing solution, a color developing solution A, a color developing solution B, and a stop solution.

[0046] As a third aspect of the present application, a method for detecting human VEGF level in a biological sample is provided, wherein the method comprises:

[0047] contacting the biological sample with a solidified capture antibody and incubating together, wherein the solidified capture antibody comprises a capture antibody immobilized to a solid phase carrier, the capture antibody comprising a first murine monoclonal antibody, a heavy chain variable region of the first murine monoclonal antibody comprising an amino acid sequence as shown in SEQ ID NO: 1, and a light chain variable region of the first murine monoclonal antibody comprising an amino acid sequence as shown in SEQ ID NO: 2;

[0048] separating the biological sample from the solidified capture antibody;

[0049] contacting the complex of the solidified capture antibody and the target molecule human VEGF with a detection antibody, the detection antibody being capable of binding to an epitope region of human VEGF, the detection antibody comprising a second murine monoclonal antibody, a heavy chain variable region of the second murine monoclonal antibody comprising an amino acid sequence as shown in SEQ ID NO: 3, and a light chain variable region of the second murine monoclonal antibody comprising an amino acid sequence as shown in SEQ ID NO: 4;

[0050] determining the level of human VEGF bound by the solidified capture antibody.

[0051] The solid phase carrier can be an enzyme-linked immunosorbent assay (ELISA) microplate.

[0052] Optionally, the capture antibody is the first murine monoclonal antibody labeled with biotin; and the detection antibody is the second murine monoclonal antibody labeled with a set marker selected from any one of a radioisotope, a fluorescein and an enzyme substrate.

[0053] The enzyme substrate includes streptavidin-horseradish peroxidase or alkaline phosphatase, the radioisotope includes iodine, and the fluorescein includes rhodamine or quantum dots.

[0054] In the embodiment of the present application, the human vascular endothelial growth factor expression vector can be constructed by the following method:

[0055] The amino acid sequence of VEGF is found on the NCBI website, and a histidine tag is added at the N- or C-terminus of the amino acid. 165 The codon is optimized in the CHO expression system by using a codon optimization software, and after gene synthesis, the gene is cloned into a pcDNA3.1 expression vector by using BamH I and Xho I restriction endonuclease enzyme cleavage sites to construct a pcDNA3.1-VEGF 165 expression plasmid.

[0056] The human vascular endothelial growth factor expression can be produced by the following method:

[0057] The pcDNA3.1-VEGF 165 expression plasmid is transformed into CHO cells, and positive cell strains are selected by G418 pressure screening to establish a positive cell strain stably expressing VEGF 165 protein, and the positive cell strain is expanded and cultured. After transfection, the CHO cells can stably secrete and express VEGF 165The relative molecular weight of the obtained recombinant human vascular endothelial growth factor is about 23 kDa, which is consistent with the prediction.

[0058] Preparation Example

[0059] Preparation Example 1

[0060] The present preparation example provides a preparation method of a monoclonal antibody, comprising:

[0061] The VEGF 165 The protein is mixed with an equal volume of Freund's adjuvant and emulsified by a homogenizer. The immunization amount is 50 μg per mouse, and the mice are subcutaneously injected at multiple points. Two weeks later, the mice are boosted by subcutaneous injection at multiple points, and the immunization amount is 50 μg per mouse.

[0062] Every other week, the mouse tail blood is collected and centrifuged to separate the serum. The antibody titer in the tail serum is detected by indirect ELISA, and the mouse with a high antibody level titer is selected for hybridoma cell fusion experiment.

[0063] After about 7 days of HAT screening medium screening culture, the supernatant of the positive hybridoma cells is detected by indirect ELISA, and the VEGF 165 The protein is coated on a microplate, and the positive hybridoma cell strain with high specificity and good affinity is screened out. Two rounds of cell subcloning are performed to clone the monoclonal cell strain.

[0064] The preparation of the monoclonal antibody is performed by the mouse in vivo ascites induction method, and the Protein A affinity chromatography column is used for the purification of the monoclonal antibody.

[0065] Using the above method, a first mouse monoclonal antibody and a second mouse monoclonal antibody are respectively prepared.

[0066] Preparation Example 2

[0067] The present preparation example provides a preparation method of a biotinylated mouse anti-human VEGF monoclonal antibody (i.e., the step of labeling the first mouse monoclonal antibody with biotin), comprising:

[0068] Sulfo-NHS-biotin is taken out of the refrigerator and equilibrated to room temperature. 1-3 mg of sulfo-NHS-biotin is added to 200-400 μL of pure water and shaken gently to activate.

[0069] The VEGF monoclonal antibody (first mouse monoclonal antibody) obtained in Preparation Example 1 is diluted with 10 mmol / L PBS buffer to a concentration of 1-3 mg / mL.

[0070] Immediately add 50-100 μL of the activated biotin solution to 1-3 mL of the antibody solution, and react at room temperature for 20-40 min. The reaction solution is dialyzed against 10 mmol / L PBS buffer at 4°C for 3 days, and the dialysis solution is changed every day.

[0071] The biotinylated monoclonal antibody conjugate (i.e., the biotinylated mouse anti-human VEGF monoclonal antibody) is collected, an appropriate amount of glycerol is added, and the mixture is stored at -20°C for later use.

[0072] Preparation Example 3

[0073] The present preparation example provides a method for preparing an ELISA microplate, which comprises:

[0074] The biotinylated mouse anti-human VEGF monoclonal antibody obtained in Preparation Example 1 is diluted with a carbonate buffer (formula: 0.1-0.2 g / L NaCO3, 3.5-4.0 g / L NaHCO3, pH 9.0-10.0);

[0075] The optimal coating concentration is determined to be 2.5 mg / mL by a square array method;

[0076] A detachable 96-well enzyme-labeled plate is taken, 100 μL is added to each well, and the plate is coated at 4°C for 24 h;

[0077] The plate is washed 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);

[0078] 200 μL of blocking solution is added to each well, and the plate is blocked at 37°C for 30-60 min;

[0079] The plate is washed thoroughly with PBST, and an ELISA microplate is prepared after thorough drying.

[0080] Preparation Example 4

[0081] The present preparation example provides a method for preparing a coating buffer, which comprises:

[0082] 0.1-0.2 g of NaCO3 and 3.5-4.0 g of NaHCO3 are weighed, dissolved in double-distilled water, and adjusted to pH 9.0-10.0. The solution is then diluted with double-distilled water to 1000 mL to obtain the coating buffer.

[0083] Preparation Example 5

[0084] The present preparation example provides a method for preparing an enzyme conjugate (i.e., an enzyme-labeled antibody working solution, which is a second mouse monoclonal antibody labeled with an enzyme), which comprises:

[0085] SA-HRP was added to sodium acetate buffer, dissolved thoroughly, 0.1-0.3M NaIO4 solution was added, mixed, and oxidized at 4°C for 60-90min;

[0086] 150-180mM ethylene glycol solution was added, and the reaction was carried out in the dark for 60-90min;

[0087] The above solution was placed in a dialysis bag, dialyzed at 4°C with 0.1M carbonate buffer, and the solution was changed 2-3 times, each time for 2-3h;

[0088] The second mouse monoclonal antibody solution obtained in Preparation Example 1 was placed in a dialysis bag, dialyzed at 4°C with 0.1M carbonate buffer, and the solution was changed 3 times, each time for 2-3h;

[0089] The IgG antibody was taken into a reaction tube, and activated SA-HRP (mass ratio 1:1-2:1) was added, and the reaction was carried out in the dark for 60-90min;

[0090] The reaction mixture was placed in a dialysis bag, dialyzed at 4°C with 1×PBS buffer for 1-2h;

[0091] The dialyzed reaction mixture was taken into a reaction tube, and sodium cyanoborohydride solution was added, and the reaction was carried out in the dark for 30-60min;

[0092] Ethanolamine solution was added, and the reaction was carried out in the dark for 30-60min;

[0093] Ultrafiltration was performed with 15mL 100kDa ultrafiltration tube, and the ultrafiltration was repeated 5 times, each time adding 1×PBS solution to 0.3-0.5mL;

[0094] After ultrafiltration, all the volumes of antibody solution were collected in an EP tube, and the total volume of the solution was adjusted to about 5mL to obtain the enzyme-labeled antibody working solution (labeled second mouse monoclonal antibody).

[0095] Preparation Example 6

[0096] The present preparation example provides a preparation method of concentrated washing solution 20×, comprising:

[0097] 160-170g of NaCl, 4-6g of KCl, 70-80g of Na2HPO4·12H2O, and 4-6g of KH2PO4 were weighed, dissolved thoroughly with double distilled water, mixed with 8-10mL of Tween-20, and made up to 1000mL with double distilled water to obtain the concentrated washing solution 20×.

[0098] Preparation Example 7

[0099] The present preparation example provides a preparation method of sample diluent, comprising:

[0100] Take 5-8 g of BSA, 8-10 g of NaCl, 0.2-0.4 g of KH2PO4, 3-5 g of Na2HPO4·12H2O, and 0.2-0.4 g of KCl, dissolve them in double distilled water in sequence, mix, add 0.05-0.1 % preservative, and dilute to 1000 mL with double distilled water to obtain a sample diluent.

[0101] Preparation Example 8

[0102] The present preparation example provides a preparation method of color developing solution A, comprising:

[0103] Take 26-27 g of sodium acetate and 3-5 g of citric acid, dissolve them in double distilled water in sequence, mix, add 0.6-1 mL of H2O2 solution, dilute to 1000 mL with double distilled water to obtain the color developing solution A.

[0104] Preparation Example 9

[0105] The present preparation example provides a preparation method of color developing solution B, comprising:

[0106] Take 0.4-0.6 g of disodium ethylenediaminetetraacetate and 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 double distilled water in sequence, dilute to 1000 mL to obtain the color developing solution B.

[0107] Preparation Example 10

[0108] The present preparation example provides a preparation method of a termination solution, comprising:

[0109] Measure 400 mL of 5M sulfuric acid solution, dilute to 1000 mL with double distilled water to obtain the termination solution.

[0110] Example

[0111] Example 1

[0112] A kit, comprising:

[0113] (1) the ELISA microplate obtained in Preparation Example 3: each kit contains 1 piece of ELISA microplate strip containing detachable coated mouse anti-human VEGF monoclonal antibody, with a specification of 96 wells x 1 piece;

[0114] (2) VEGF calibrator: lyophilized recombinant human VEGF, 800 pg x 1 bottle;

[0115] (3) sample diluent obtained in Preparation Example 7: 20 mL x 1 bottle;

[0116] (4) concentrated washing solution 20x obtained in Preparation Example 6: 50 mL x 1 bottle;

[0117] (5) Enzyme-labeled antibody working solution obtained in Preparation Example 5, 20 mL x 1 bottle;

[0118] (6) Color developing solution A obtained in Preparation Example 8, 10 mL x 1 bottle;

[0119] (7) Color developing solution B obtained in Preparation Example 9, 10 mL x 1 bottle;

[0120] (8) Termination solution obtained in Preparation Example 10, 5 mL x 1 bottle.

[0121] Example 2

[0122] A method for detecting VEGF in human serum, comprising:

[0123] (1) Preparation of solution: according to the number of experimental holes (serum samples and VEGF calibrators), determine the required number of strips, dilute the concentrated washing solution with distilled water or deionized water 20 times to prepare 1x washing solution.

[0124] (2) Preparation of standard curve: mix the VEGF calibrator stock solution well, for example, prepare a final volume of 250 μL at each concentration. Take 250 μL of the VEGF calibrator stock solution, mix with 250 μL of sample diluent (S7), and use it as the highest concentration 400 pg / mL of the standard curve, and mark the tube wall. Take 6 centrifuge tubes (S6-S1) in turn, first add 250 μL of sample diluent to each, then take 250 μL from centrifuge tube S7 and add it to S6, mix gently; take 250 μL from centrifuge tube S6 and add it to S5, mix gently, and so on for the dilution of the calibrators, and S0 is the sample diluent.

[0125] (3) Sample addition: take out the required number of microwells, dilute the serum samples with sample diluent at a ratio of 1:1, 200 μL / well, and ensure that the final volume after dilution of each sample is ≥250 μL. Mix all samples well to avoid air bubbles, and add 200 μL of VEGF calibrator and test sample to the corresponding wells, cover with a sealing film, and incubate at 37°C for 30-60 min.

[0126] (4) Washing: remove the liquid in the wells, add 300 μL of 1x washing solution to each well without stopping, discard the liquid in the wells and dry on filter paper, and repeat 5 times.

[0127] (5) Enzyme addition: add 200 μL of enzyme-labeled antibody working solution to each well, cover with a sealing film, and incubate at 37°C for 30-60 min.

[0128] (6) Washing: remove the liquid in the wells, add 300 μL of 1x washing solution to each well without stopping, discard the liquid in the wells and dry on filter paper, and repeat 5 times.

[0129] (7) Color development: 100 μL of color developing solution A and color developing solution B were added into each well in turn, mixed gently, and incubated at 37°C for 10-15 min.

[0130] (8) Measurement: 50 μL of termination solution was added into each well, mixed gently, and the absorbance value of each well was measured at 450 nm.

[0131] (9) Calculation: a standard curve was plotted according to the measured values and concentrations of the VEGF calibration sample, and the corresponding concentration was found according to the absorbance value of the sample. Since the serum sample was diluted by 1:1, the result was multiplied by 2 as the final detection result.

[0132] (10) Data processing: fitting type: four-parameter logistic fitting; coordinate selection: X-Y.

[0133] Test Example

[0134] The VEGF enterprise reference sample was repeatedly detected for 3 times, and the relative deviation was calculated. The detection result of the accuracy index of the double-antibody sandwich ELISA kit prepared in the embodiment is shown in Table 1.

[0135] Table 1: Accuracy detection result

[0136]

[0137] Conclusion: the relative deviation of the measurement result is within ±10%, and the results of 3 times meet the requirements. The accuracy of the above 3 batches of kits is qualified.

[0138] (3) Precision

[0139] Ten serum samples were mixed, and the double-antibody sandwich ELISA kit prepared in Example 1 was used for continuous detection for 20 times. In addition, it was detected once a day for 20 days. The results showed that the CV% of intra-batch and inter-batch was 8.25% and 7.33% respectively, which were within the allowable error range (CV% < 15%), and the precision was high and the repeatability was good.

[0140] (4) Stability

[0141] The double-antibody sandwich ELISA kit prepared in the embodiment was stored at 37°C for 0 d, 3 d and 8 d respectively, and then high-level samples and low-level samples were detected. The CV% of the high-level sample was 10.62%, and the CV% of the low-level sample was 8.57%, both of which were less than 15%. The storage period at 4°C could reach 10-12 months, indicating that the kit had good stability.

[0142] (5) Anti-interference ability

[0143] In two samples with VEGF concentration of 50 pg / mL and 200 pg / mL, different concentrations of hematin, ascorbic acid, bilirubin and cholesterol were added respectively, and the unadded group was used as a control. The relative deviation of both the added group and the control group was less than 5%, which was the interference acceptable standard. The maximum concentration level of different interferents was tested, and the results are shown in Table 2.

[0144] Table 2 Analysis of the detection results of the maximum concentration level of the interferents

[0145]

[0146] Conclusion: When the maximum concentration level of the interferents is as shown in Table 2, the detection results are not obviously interfered, which proves that the double-antibody sandwich ELISA kit prepared in the embodiment has good anti-interference ability.

[0147] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited to this. Those skilled in the art should understand that the present application includes but is not limited to the content described in the above specific embodiment. Any modification that does not deviate from the functional and structural principles of the present application 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 murine monoclonal antibody and a second murine monoclonal antibody, wherein 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 contains the amino acid sequence shown in SEQ ID NO: 2; the heavy chain variable region of the second murine monoclonal antibody contains the amino acid sequence shown in SEQ ID NO: 3, and the light chain variable region of the second murine monoclonal antibody contains the amino acid sequence shown in SEQ ID NO:

4.

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

3. The combination 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 human VEGF levels in a biological sample, 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 comprising the amino acid sequence set forth in SEQ ID NO: 1, and the light chain variable region of the first murine monoclonal antibody comprising the amino acid sequence set forth 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 radioisotopes, 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 murine monoclonal antibody, the heavy chain variable region of the first murine monoclonal antibody comprises the amino acid sequence set forth in SEQ ID NO: 1, and the light chain variable region of the first murine monoclonal antibody comprises the amino acid sequence set forth 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 an epitope region of human VEGF, wherein the detection antibody comprises a second murine monoclonal antibody, wherein the heavy chain variable region of the second murine monoclonal antibody comprises the amino acid sequence set forth in SEQ ID NO: 3, and the light chain variable region of the second murine monoclonal antibody comprises the amino acid sequence set forth in SEQ ID NO: 4; The level of human VEGF bound by the immobilized capture antibody was 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 radioisotopes, 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.

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