CN113999309A

CN113999309A - VEGF (vascular endothelial growth factor) monoclonal antibody, kit, preparation method and application thereof

Info

Publication number: CN113999309A
Application number: CN202011614751.4A
Authority: CN
Inventors: 张陆明
Original assignee: Jiangsu Proway Biotechnology Co ltd
Current assignee: Jiangsu Proway Biotechnology Co ltd
Priority date: 2020-12-30
Filing date: 2020-12-30
Publication date: 2022-02-01

Abstract

The invention is applicable to the technical field of biology, and provides a VEGF monoclonal antibody, a kit, a preparation method and application thereof. The heavy chain variable region amino acid sequence of the VEGF monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 1; the amino acid sequence of the variable region of the light chain of the VEGF monoclonal antibody is shown as SEQ ID NO. 5. The VEGF monoclonal antibody has high affinity, strong specificity and good biological activity in vitro. The detection kit can be used as a therapeutic effect evaluation tool of VEGF/VEGFR pathway tumor targeting drugs.

Description

VEGF (vascular endothelial growth factor) monoclonal antibody, kit, preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a VEGF monoclonal antibody, a kit, a preparation method and application thereof.

Background

Blood vesselEndothelial Growth Factor (VEGF) is a dimeric cationic glycoprotein with high biological activity. The human VEGF gene maps to 6p21.3, and consists of 8 exons and 7 introns, with a full length of 14 Kb. Through the different splicing modes (Alternative splicing) of mRNA transcribed by genes, 4 main isomers are encoded, namely VEGF121, VEGF165, VEGF189 and VEGF206, which are respectively composed of 121, 165, 189 and 206 amino acids. VEGF, an isoform that primarily mediates Angiogenesis (angiogenisis) and Vasculogenesis (Vasculogenesis), is a powerful specific endothelial mitogen that mediates endothelial cell proliferation, budding, migration, and lumen formation. In 1994, Kondo Nishida et al first demonstrated cancer and VEGF relationships, and according to Folkman's hypothesis that solid tumors would not exceed 1-3mm in size due to oxygen and other nutrient supply limitations if they had no new angiogenesis³. Vascular Endothelial Growth Factor (VEGF) is a key protein factor that promotes tumor angiogenesis.

The target therapeutic drug Bevacizumab (Bevacizumab) is a humanized monoclonal antibody of VEGF, and can be combined with various subtypes and active degradation products of VEGF. Humanized bevacizumab was later commercialized by Genentech under the name Avastin. In view of the remarkable efficacy of bevacizumab in clinical trials for treating metastatic colorectal cancer, bevacizumab was approved by the U.S. Food and Drug Administration (FDA) to be marketed in 2004 for first-line treatment of metastatic colorectal cancer and became the first approved VEGF inhibitor in the world, and bevacizumab has been approved by FDA for first-line treatment of metastatic colorectal cancer, metastatic breast cancer, advanced non-small cell lung cancer, and metastatic renal cell carcinoma. In addition, the application of bevacizumab in other malignant tumors such as colorectal cancer, gastric cancer, esophageal cancer and the like has encouraging results. In 2013, Roche/Genentech, USA, is conducting clinical trials of the VEGF detection kit, and the US FDA clinical trial approval number is NCT 01663727. In recent years, researches show that the concentration of VEGF in serum of a tumor patient has an important relation with the aspects of tumor disease progression, treatment effect evaluation, disease outcome and the like, and has great application value in the aspects of disease diagnosis, disease treatment prediction and the like.

Research and application of a labeling immunoassay technology have been rapidly developed in the last decade, and the labeling immunoassay technology has been widely applied to various fields of biomedical basic theory research and clinical disease diagnosis. Methods for detecting serological indicators mainly include radioisotope immunoassays, enzyme-linked immunosorbent assays, and chemiluminescent immunoassays. The methods can be used as a primary screening test and a confirmation test, wherein the chemiluminescence method has the advantages of wide detection linear range, simple detection instrument, convenient operation and the like.

Products aiming at VEGF/VEGFR pathway targeted drug monitoring and curative effect evaluation in the prior art are blank at home at present, and the specificity, affinity and other aspects of similar products are possible to be further improved, so that the invention provides a VEGF monoclonal antibody with high affinity and strong specificity, a kit, a preparation method and application thereof.

Disclosure of Invention

The embodiment of the invention aims to provide a VEGF monoclonal antibody, a kit, a preparation method and application thereof, and aims to solve the problems in the prior art pointed out in the background art.

The embodiment of the invention is realized by that, a VEGF monoclonal antibody,

the heavy chain variable region amino acid sequence of the VEGF monoclonal antibody is the amino acid sequence shown in SEQ ID NO.1, or the amino acid sequence with the same function formed by replacing, deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 1.

The light chain variable region amino acid sequence of the VEGF monoclonal antibody is the amino acid sequence shown in SEQ ID NO.5, or the amino acid sequence with the same function formed by replacing, deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 5.

As another preferred scheme of the embodiment of the invention, the amino acid sequences of the heavy chain hypervariable region CDR-H1, the heavy chain hypervariable region CDR-H2 and the heavy chain hypervariable region CDR-H3 of the VEGF monoclonal antibody are respectively the amino acid sequences shown in SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4, or the amino acid sequences shown in SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 are formed by replacing, deleting and/or adding one or more amino acid residues to form the amino acid sequences with the same functions.

As another preferred scheme of the embodiment of the invention, the light chain hypervariable region CDR-L1, the light chain hypervariable region CDR-L2 and the light chain hypervariable region CDR-L3 amino acid sequences of the VEGF monoclonal antibody are respectively the amino acid sequences shown in SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8, or the amino acid sequences shown in SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8 are formed by replacing, deleting and/or adding one or more amino acid residues to form amino acid sequences with the same functions.

Another objective of the embodiments of the present invention is to provide a method for preparing the VEGF monoclonal antibody, comprising the following steps:

firstly, preparing a VEGF protein antigen;

secondly, preparing a VEGF polyclonal antibody;

thirdly, preparing a VEGF monoclonal antibody;

fourthly, screening monoclonal antibodies: establishing a dose response curve, and screening an optimal monoclonal antibody;

fifthly, the monoclonal antibody gene in the hybridoma is regulated:

extracting total RNA of the hybridoma cells;

carrying out reverse transcription synthesis by taking OligodT as a primer to obtain cDNA;

carrying out PCR amplification by taking the synthesized cDNA as a template;

obtaining a gene sequence for coding a heavy chain variable region and a gene sequence for coding a light chain variable region;

carrying out codon optimization and synthesis on the heavy chain variable region gene sequence, the encoding light chain variable region gene sequence and the constant region of the antibody to respectively obtain a heavy chain and a light chain of the complete antibody gene;

cloning the obtained heavy chain and light chain genes into pMD18-T expression vectors respectively;

transfecting the obtained plasmid into a competent cell;

collecting cell supernatant, purifying and concentrating to obtain the VEGF monoclonal antibody.

As another preferred embodiment of the present invention, the PCR amplification conditions are: denaturation at 95 deg.C for 5min, 55 deg.C for 1min, and 72 deg.C for 2min for 35 cycles; finally, the extension is carried out for 10min at 72 ℃.

Another purpose of the embodiment of the invention is to provide a VEGF detection kit, which contains the VEGF monoclonal antibody.

Another objective of the embodiments of the present invention is to provide a method for preparing the VEGF detection kit, comprising the following steps:

diluting the VEGF monoclonal antibody with a buffer solution, and adding streptavidin magnetic beads for reaction;

washing a buffer solution;

adding PBS confining liquid containing fetal calf serum, performing magnetic separation after reaction, and removing supernatant;

after being washed by buffer solution, the magnetic particles are suspended in the preservation buffer solution of PBS containing bovine serum albumin to prepare VEGF-coated magnetic beads;

diluting a plurality of gradients by VEGF protein antigen according to a proportion, and subpackaging to prepare a VEGF calibrator;

adding the VEGF polyclonal antibody into an acridine ester solution, and carrying out a light-resistant reaction;

taking out after the reaction is finished, adding a lysine salt solution, and continuing the reaction in a dark place;

purifying to obtain a VEGF acridinium ester standard antibody;

adding a serum sample and a calibrator into the VEGF-coated magnetic beads, adding a VEGF acridinium ester standard antibody, and incubating to form an antibody-antigen-labeled antibody compound;

adding HNO respectively₃、H₂O₂Immediately putting the luminescence excitation liquid A, NaOH and Triton-100 luminescence excitation liquid B into a chemiluminescence immunoassay instrument, and detecting the luminescence intensity of each hole;

and calculating the content of VEGF in the sample according to the response curve.

As another preferred embodiment of the present invention, PBS, tween-20 washing buffer was used.

As another preferred embodiment of the present invention, VEGF acridinium ester standard antibodies are obtained by purification on a G-25 desalting column.

The embodiment of the invention also aims to provide application of the VEGF detection kit in preparation of a VEGF targeting drug curative effect detection kit.

The VEGF monoclonal antibody has higher affinity, strong specificity and good biological activity in vitro; the detection kit containing the VEGF monoclonal antibody has the advantages of simple detection method, strong accuracy and low cost. The VEGF protein detection kit established by the VEGF monoclonal antibody and the VEGF polyclonal antibody can be used as a monitoring tool of VEGF/VEGFR pathway tumor targeted drugs.

Drawings

FIG. 1 is a schematic diagram of a VEGF detection dose-response system.

FIG. 2 is a scatter diagram of VEGF monoclonal antibody constitution control serum detection.

FIG. 3 is a schematic diagram of the dynamic change of serum VEGF before and after two groups of TACE are detected by using the kit of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Specific implementations of the present invention are described in detail below with reference to specific embodiments.

Example 1

The embodiment provides a VEGF monoclonal antibody, wherein the heavy chain variable region amino acid sequence of the VEGF monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 1; or an amino acid sequence with the same function formed by replacing, deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 1;

the amino acid sequences of a heavy chain hypervariable region CDR-H1, a heavy chain hypervariable region CDR-H2 and a heavy chain hypervariable region CDR-H3 of the VEGF monoclonal antibody are respectively the amino acid sequences shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4; or the amino acid sequence shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 is formed into an amino acid sequence with the same function by replacing, deleting and/or adding one or more amino acid residues;

the amino acid sequence of the variable region of the light chain of the VEGF monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 5; or an amino acid sequence with the same function formed by replacing, deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 5;

the light chain hypervariable region CDR-L1, the light chain hypervariable region CDR-L2 and the light chain hypervariable region CDR-L3 of the VEGF monoclonal antibody have amino acid sequences shown in SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8 respectively; or the amino acid sequence shown in SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8 is formed into an amino acid sequence with the same function by replacing, deleting and/or adding one or more amino acid residues.

SEQ ID NO.1：

SEQ ID NO.2：

SEQ ID NO.3：

SEQ ID NO.4：

SEQ ID NO.5：

SEQ ID NO.6：

SEQ ID NO.7：

Tyr Gln Gln

1

SEQ ID NO.8：

Example 2

The embodiment provides a preparation method of a VEGF monoclonal antibody, which comprises the following steps:

firstly, preparing VEGF protein antigen:

(1) designing a pair of primers according to the sequence of human VEGF (KT768148.1) in GenBank database:

the upstream primer is vF: 5'-ATGAACTTTCTGCTCTCTTGGGTACA-3', respectively;

the downstream primer is vR: GCAGATGTGACAAGCCAAGGCGGTG-3';

the sites of the upstream primer and the downstream primer are anastomosed with the corresponding multiple cloning sites on the mammalian cell high-efficiency expression plasmid vector pSec/WG;

the cloning plasmid containing human VEGF gene fragment is synthesized by whole gene, VEGF gene specific amplification is carried out by Pyrobest DNA polymerase with the plasmid as a template, and the PCR amplification conditions are as follows: denaturation at 95 deg.C for 5 min; 94 ℃ for 1 min; 2min at 52 ℃; 72 ℃, 1min, 35 cycles; extending for 10min at 72 ℃;

carrying out gel recovery, chloroform extraction, ethanol precipitation and TE dissolution on the amplified PCR product to obtain pSec/WG plasmid for later use;

carrying out Pci I and Xho I double enzyme digestion on the recovered VEGF gene and pSec/WG plasmid respectively, and recovering by a gel electrophoresis method;

respectively purifying the PCR enzyme digestion product and the recovery product of the vector again, mixing VEGF and pSec/WG according to the molar ratio of 1:1, dissolving the mixture by using TE, reacting the mixture for 12 hours at the temperature of 16 ℃, and placing the mixture at the temperature of 70 ℃ for 10min to terminate the reaction;

connecting the reaction product with DH-5 alpha competent cells, screening out positive clones by an ampicillin (1mg/mL) antibody, amplifying, extracting recombinant VEGF/pSec/WG plasmid, and carrying out enzyme digestion and sequencing identification for later use;

(2) transfecting recombinant VEGF/pSec/WG plasmid to a eukaryotic expression cell line Flp-In CHO by using a liposome reagent Lipofectamine 2000, and placing the transfected cells In a hygromycin-containing culture medium for culture and screening;

untransfected Flp-In CHO cell lines were cultured In complete Hams F12 medium (containing 10% FBS, 2mM L-glutamine) supplemented with 1% qing/streptomycin and 100. mu.g/mL Zeocin-transfected recombinant plasmid VEGF/pSec/WG Flp-In CHO cells because VEGF gene insertion inactivated the Zeocin resistance gene In the host cell genome but at the same time brought In the hygromycin resistance gene (Hyg +), so that host cells transfected with recombinant plasmid could be cultured In hygromycin-containing medium, whereas untransfected host cells could not survive under these conditions; culturing in complete Hams F12 culture medium containing 800 μ g/mL hygromycin, and screening out corresponding recombinant gene expression clone in 6-7 days;

culturing Flp-In CHO cells transfected with VEGF/pSec/WG recombinant plasmid In UltraCHO serum-free medium, and collecting cell culture supernatant 1 time every 3-4 days;

centrifuging the collected culture supernatant at 10000r/min for 5 minutes, and removing the precipitate; adding 0.02% NaN into the supernatant₃And all possible cell debris was removed by filtration through a 0.22 μm filter;

the column was packed with l.0mL of resin and equilibrated with approximately 10 volumes of PBS (pH 7.2); passing the supernatant containing the target Protein through a Protein G-Sepharose 4B column for 2 times; 20-30 volumes of PBS (pH 7.2) column wash; 28 μ L of 1.25M Tris-HCl (pH 8.0) was added to each collection vial beforehand; eluting with 100mM Glycine-HCl (pH 3.0) eluent, and collecting in the collection tubules at a rate of 1.0rnl per tube to neutralize the eluent in the collection tubules immediately; usually the protein is eluted in the 3 rd to 10 th tube, with peaks around 3, 4, 5; collecting all the eluate with A280 > 0.01, and concentrating with centrifugal filter column of Ultrafree 15(MWCO 10000, Millipore); the expression level is 50mg/L, SDS-PAGE electrophoresis identification is carried out, and the purity of the purified VEGF protein is more than 98 percent; after the protein is quantified, filtering and sterilizing through a 0.22 mu m filter membrane, and storing at-20 ℃ for later use;

preparation of VEGF polyclonal antibody

(1) Using male big ear rabbit as immune animal, firstly injecting 10mg BCG vaccine to stimulate animal, using VEGF protein as immune antigen, and starting immunization after one week; 4-6 points of subcutaneous injection are injected below feet, and Freund's complete adjuvant is used as an immunologic adjuvant to immunize animals for four times; 1mg of VEGF protein is taken each time, equivalent Freund's complete adjuvant and antigen solution are respectively sucked into two injectors for full emulsification for 1 hour, and foot subcutaneous injection is carried out, and the interval is two weeks each time;

taking auricular venous blood to detect titer by an ELISA method, performing carotid bleeding when the ratio reaches 1:40000, centrifuging at 5000rpm to take serum, and purifying by DEAE ion exchange to obtain crude serum polyclonal antibody for later use;

(2) diluting the serum crude polyclonal antibody to 1mg/mL by using 0.5mol/L PBS (pH 7.5), preparing 3mL of CNBr-Sepharose 4B agarose gel, coupling 10mg VEGF protein antigen by using a coupling agent, reacting at room temperature for 4 hours to prepare a VEGF antibody affinity chromatographic column, purifying the serum crude polyclonal antibody on the column, eluting and collecting, determining an antibody OD value at the wavelength of 280nm by using an ultraviolet-visible spectrophotometer, dividing the obtained OD value by 1.35 to obtain the concentration of the determined antibody, adding 40-50% of glycerol, and placing at-20 ℃ for long-term storage;

preparation of VEGF monoclonal antibody

(1) Culturing the above-mentioned VEGF/pSec/WG recombinant plasmid Flp-In CHO cells stably expressed, immunizing 5 female BALA/c mice, injecting 1 × 10 subcutaneously into each BALB/c mouse⁷Cells, immunised 4 times consecutively, each time with 2 weeks intervals; immunizationCollecting blood 7 days later, detecting serum titer by CLIA method, selecting mice with highest titer, and injecting into spleen for 1 × 10⁶Boosting the immunity of each cell, taking the spleen of the mouse 3 days later, grinding the spleen, and counting the spleen cells for later use;

(2) fusing splenocytes and bone marrow cells Sp2/0 according to the ratio of cell count 5:1, inducing by PEG1200, adding the fused cells into a 96-well plate containing a feeder layer for culturing, changing the culture solution by half amount by using HAT culture medium after one week, and observing the cell state after fusion;

screening positive hybridoma cell strains by an indirect CLIA method, selecting 1 strain of hybridoma cell (4G6) with VEGFAb continuous secretion positive rate of more than 98% for amplification culture, and preparing a mouse for intraperitoneal injection, wherein the mouse is intraperitoneally injected with 500 mu L of liquid paraffin 1 week before;

collecting hybridoma cells by centrifugation, suspending with incomplete culture medium, mixing, and adjusting cell number to 1 × 10⁹Performing inoculation on mice at the beginning, performing intraperitoneal injection on each mouse at 500 mu L, extracting ascites from mice with obviously swollen abdomens after 1 week, centrifuging the obtained ascites at 3000r/min for 3 minutes, and collecting supernatant;

(3) purifying ascites with protein G purifying column; balancing the purification column with 0.02mol/L PB buffer solution, adding ascites for sampling, eluting with 0.1mol/L glycine hydrochloric acid buffer solution (pH 2.7), collecting with an EP tube, dialyzing with 0.05mol/LPB, concentrating to obtain VEGF monoclonal antibody, and freezing at-20 deg.C;

fourth, monoclonal antibody screening

(1) Coating the prepared VEGF monoclonal antibody, and diluting the antibody to 2-5 mu g/mL by 0.5mol/L PBS; adding 100 mu L/hole into an enzyme label plate for coating, after overnight at 2-8 ℃, washing with 0.9% NaCl for 3 times, adding a blocking solution containing 1% BSA, blocking at 150 mu L/hole, and airing for later use after overnight at 2-8 ℃;

(2) screening the optimal monoclonal antibody: diluting the prepared VEGF protein antigen, diluting 8 gradients (0, 10, 50, 100, 200, 400, 1000 and 2000pg/mL) according to a proportion, sequentially adding the diluted VEGF protein antigen into the prepared enzyme label, adding a biotinylated polyclonal antibody and streptavidin labeled with horseradish peroxidase into each hole with the volume of 50 mu L, incubating for 1 hour at 37 ℃, washing for 5 times by PBST, adding a chromogenic substrate solution, and detecting an OD value by using an enzyme label instrument;

dose response curves were established with VEGF concentration as X-value on the abscissa and OD as Y-value on the ordinate (see FIG. 1). It can be seen that the dose-response curve established by the VEGF detection kit has a linear coefficient R of more than 0.99, a detection range of 0-800pg/ml and excellent analysis performance. Finally, selecting the VEGF monoclonal antibody with the best evaluation index; the monoclonal antibody as an evaluation standard of a sandwich ELISA detection method simultaneously meets the requirements that an OD value of S0 is less than 0.1, an OD value of S7/S1(P/N) is the largest, a dose-response curve correlation coefficient is more than 0.99, and the detection rate of 30 cases of quality control serum is more than 90%;

fifth, the retrieval of monoclonal antibody genes in hybridomas

(1) Extraction with Trizol reagent 5X 10⁶Total RNA of selected hybridoma cells of (4G 6);

then using OligodT as a primer, carrying out reverse transcription by AMV reverse transcriptase at the temperature of 37 ℃ for 15 minutes, and synthesizing to obtain cDNA;

taking the synthesized cDNA as a template, and carrying out nested PCR amplification aiming at a primer of an RNA sequence and a gene specific primer GSP, wherein the PCR conditions are as follows: denaturation at 95 deg.C for 5min, 55 deg.C for 1min, and 72 deg.C for 2min for 35 cycles; finally, extending for 10min at 72 ℃;

(2) identifying the PCR product by 1% agarose gel electrophoresis, cutting and recovering a target gel strip, calling a target gene, connecting the target gene to a pGEM-T vector, carrying out EcoRI enzyme digestion to identify a positive recombinant plasmid, and determining a DNA sequence to respectively obtain a coding heavy chain variable region gene sequence and a coding light chain variable region gene sequence;

carrying out codon optimization and synthesis on the obtained heavy chain and light chain variable region gene sequences and the constant region of the antibody to respectively obtain a heavy chain and a light chain of a complete antibody gene, and respectively cloning the obtained heavy chain and light chain genes into a pMD18-T expression vector; respectively extracting 150 mu g of plasmids, removing endotoxin (less than 1EU/mg), co-transfecting the obtained 2 plasmids 1:1 into suspension competent TG1 cells with the volume of 50mL, wherein PEI is adopted as a transfection reagent, and the ratio of the plasmids to the PEI is 1: 2;

after 3-7 days of transfection, collecting cell supernatant, detecting whether the antibody expression is correct by SDS-PAGE, purifying by a ProteinA column, and concentrating to obtain qualified VEGF monoclonal antibody with the purity of more than 98%.

Example 3

This example provides a VEGF assay kit containing the VEGF monoclonal antibody of example 1.

Example 4

The embodiment provides a preparation method of a VEGF detection kit, which comprises the following steps:

(1) using streptavidin magnetic beads as an immunoreaction carrier, diluting a VEGF monoclonal antibody to 2.0 mu g/mL by using 0.02mol/L Tris-HCl coupling buffer solution, adding the streptavidin magnetic beads, and reacting in a shaker at 37 ℃ for 30 min;

washing with 0.01mol/L PBS and 0.05% tween-20 washing buffer solution for about 3 times;

adding 1ml of 0.01mol/L PBS blocking solution containing 2% fetal calf serum, reacting in a shaking table for 2h at 37 ℃ and 180rpm, performing magnetic separation, and removing supernatant;

washing with 1ml of 0.01mol/L PBS buffer solution containing 0.1% tween-20 and pH7.4 for 3 times, suspending the magnetic particles in 1ml of 0.01mol/L PBS storage buffer solution containing 1% bovine serum albumin and pH7.4 to obtain VEGF-coated magnetic beads, and keeping at 4 deg.C;

(2) diluting the prepared VEGF protein antigen with 1% BSA/0.5mol/L PBS, diluting according to a proportion by 6 gradients (10, 50, 100, 200, 400, 800pg/mL), and subpackaging to prepare a VEGF calibrator;

adding 50 μ l of a 0.5mM acridinium ester (4- (2-succinimidylcarboxyl) phenyl-10-methylacridine-9-carboxylate fluorosulfonate) solution to the biotinylated VEGF polyclonal antibody, and reacting in a shaker at 25 ℃ and 180rpm in the absence of light for 20 min;

taking out after the reaction is finished, adding 100 mu l of 10% lysine salt solution, keeping out of the sun at 25 ℃, reacting for 30min at 180rpm in a shaking table;

purifying by using a G-25 desalting column to obtain a VEGF acridinium ester standard antibody;

(3) adding 50 mu L/hole of serum sample and calibrator into VEGF-coated magnetic beads, adding 50 mu L/hole of VEGF acridinium ester standard antibody, incubating at 37 ℃ for 1h to form a solid phase antibody-antigen-labeled antibody complex, washing for 5 times at 0.01mol/LPBS (low pressure polystyrene), and removing unbound VEGF polyclonal antibody and acridinium ester;

respectively adding 0.1mol/L HNO₃，0.1％H₂O₂Immediately putting 50 mul of luminescence excitation liquid A, 0.25mol/L NaOH and 50 mul of 2% Triton-100 luminescence excitation liquid B into a chemiluminescence immunoassay instrument, and detecting the luminescence intensity (RLU) of each hole within the accumulated time of 1.5 s;

the RLU value of the sample increases along with the increase of the concentration of the VEGF, and the content of the VEGF in the sample can be calculated according to the response curve.

Experimental example detection of VEGF Performance of the detection kit

(1) Performance analysis of VEGF detection kit

Repeatability: the intra-batch Coefficient of Variation (CV) of the VEGF detection kit is not more than 10 percent; the inter-batch Coefficient of Variation (CV) is not more than 15%;

analysis sensitivity: the lowest detection limit of the kit is not more than 30 pg/mL;

analysis of specificity: the detection result of specific substances (FGF2 and EGF) at a certain concentration is not more than 50 pg/mL;

linear range: in the concentration range of 0-800pg/mL, the linear correlation coefficient r is not less than 0.9900.

(2) Clinical Performance of VEGF detection kit

20 patients with clinical diagnosis of primary colorectal cancer were randomly divided into an experimental group (group A) and a control group (group B) at a ratio of 1:1, and 200mg of bevacizumab (avastin) was administered to group A1 day before conventional TACE addition; the control group was given only conventional TACE surgery. All patients had blood taken 1 day before, 1 day, 3 days, 5 days, 7 days and 1 month after TACE surgery, plasma VEGF levels were quantitatively determined by chemiluminescence, large intestine-enhanced CT/MRI was reviewed 1 month after TACE surgery, and recent clinical efficacy was assessed according to mRICIST standards. The results of the experiment are shown in figure 3 and the following table.

TABLE 1 serum VEGF values before and after surgery for two groups of TACE (pg/l)

Note: group A: experimental group, bevacizumab in combination with TACE procedure; group B: control group, TACE procedure alone; p1 for cohort a and B comparisons; p2 is a comparison of group a post-operative VEGF values with pre-operative values; p3 is a comparison of group B post-operative VEGF values with pre-operative values. P is less than 0.05; p is less than 0.01.

FIG. 3 is a schematic diagram of the dynamic change of serum VEGF before and after two groups of TACE are detected by using the kit of the present invention. Serum VEGF levels were increased gradually at 1, 3, and 5 days post-surgery in both groups, compared to pre-surgery, and were statistically significant (P values of 0.048, 0.011, and 0.036, respectively), and were not statistically different at 30 days post-surgery compared to pre-surgery (P ═ 0.298).

TACE post-operative 1 month trial group was evaluated according to mRICIST criteria: CR: 0 case, PR 3 case, SD: example 6, PD: 1 time; total effective rate (PR + CR + SD): 90 percent; control group: CR: case 0, PR: example 1, SD: 7 cases, PD: 2, example; the total effective rate is as follows: 80 percent.

After the operation, transient intestinal dysfunction appears in both groups of patients, but the difference has no statistical significance (P is more than 0.05), and no obvious bevacizumab-related side reaction appears in the experimental group after bevacizumab is applied, which indicates that the tolerance of the bevacizumab and TACE combination treatment patients is good.

The serum VEGF level of a patient with primary colorectal cancer after TACE operation is increased, and the serum VEGF level of the patient after TACE operation can be reduced by jointly applying bevacizumab.

FIG. 2 is a scatter diagram of VEGF monoclonal antibody constitution control serum detection. As can be seen, the specificity of the VEGF monoclonal antibody to the colorectal cancer quality control serum is 93.3%, and the specificity is good.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

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<213> Artificial Sequence (Artificial Sequence)

<400> 10

gcagatgtga caagccaagg cggtg 25

Claims

1. A VEGF monoclonal antibody, characterized in that,

the heavy chain variable region amino acid sequence of the VEGF monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 1;

the amino acid sequence of the variable region of the light chain of the VEGF monoclonal antibody is shown as SEQ ID NO. 5.

2. The VEGF monoclonal antibody of claim 1,

the amino acid sequences of the heavy chain hypervariable region CDR-H1, the heavy chain hypervariable region CDR-H2 and the heavy chain hypervariable region CDR-H3 of the VEGF monoclonal antibody are respectively the amino acid sequences shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4.

3. The VEGF monoclonal antibody of claim 1,

the light chain hypervariable region CDR-L1, the light chain hypervariable region CDR-L2 and the light chain hypervariable region CDR-L3 of the VEGF monoclonal antibody have amino acid sequences shown in SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8 respectively.

4. A method for preparing a VEGF monoclonal antibody according to any one of claims 1 to 3, comprising the steps of:

firstly, preparing a VEGF protein antigen;

secondly, preparing a VEGF polyclonal antibody;

thirdly, preparing a VEGF monoclonal antibody;

fifthly, the monoclonal antibody gene in the hybridoma is regulated:

extracting total RNA of the hybridoma cells;

carrying out PCR amplification by taking the synthesized cDNA as a template;

transfecting the obtained plasmid into a competent cell;

5. The method for preparing VEGF monoclonal antibody according to claim 4, characterized in that the PCR amplification conditions are as follows: denaturation at 95 deg.C for 5min, 55 deg.C for 1min, and 72 deg.C for 2min for 35 cycles; finally, the extension is carried out for 10min at 72 ℃.

6. A VEGF detection kit comprising the VEGF monoclonal antibody of any one of claims 1 to 3.

7. A method of preparing a VEGF detection kit according to claim 6, comprising the steps of:

washing a buffer solution;

purifying to obtain a VEGF acridinium ester standard antibody;

8. The method of preparing a VEGF detection kit of claim 7, wherein the buffer is washed with PBS, tween-20.

9. The method for preparing a VEGF detection kit according to claim 7, wherein the VEGF acridinium ester standard antibody is obtained by purification with a G-25 desalting column.

10. Use of the VEGF assay kit of claim 6 in the preparation of a test kit for assessing the efficacy of a VEGF-targeted drug.