CN111808191A

CN111808191A - Antibody pair for detecting VEGF content in serum and application thereof

Info

Publication number: CN111808191A
Application number: CN202010389678.9A
Authority: CN
Inventors: 刘继来; 刘静; 程勇; 王征
Original assignee: Langfang Tian Guang Biological Technology Co ltd
Current assignee: Langfang Tian Guang Biological Technology Co ltd
Priority date: 2020-05-11
Filing date: 2020-05-11
Publication date: 2020-10-23
Also published as: CN113150157A

Abstract

The application provides an antibody pair for detecting VEGF content in serum and application thereof, comprising a monoclonal antibody AntiVEGF _ N combined with an N-terminal epitope of VEGF, namely an amino acid sequence shown in SEQ ID NO. 2, and a monoclonal antibody AntiVEGF _ C combined with a C-terminal epitope of VEGF, namely an amino acid sequence shown in SEQ ID NO. 3, wherein the monoclonal antibody AntiVEGF _ N comprises a light chain variable region of the amino acid sequence shown in SEQ ID NO. 7 and a heavy chain variable region of the amino acid sequence shown in SEQ ID NO. 11, and the AntiVEGF _ C comprises a light chain variable region of the amino acid sequence shown in SEQ ID NO. 17 and a heavy chain variable region of the amino acid sequence shown in SEQ ID NO. 21. The VEGF antigen is divided into two sequences of an N end (SEQ ID NO:2) and a C end (SEQ ID NO:3), mice are immunized by the two sequences respectively, antibody pairs with high specificity and high sensitivity, namely AntiVEGF _ N and AntiVEGF _ C are screened, and the content of VEGF in serum can be effectively determined by the antibody pairs, so that the broad-spectrum early-stage screening of tumors is facilitated, and the accuracy of early-stage screening of tumors is greatly improved.

Description

Antibody pair for detecting VEGF content in serum and application thereof

Technical Field

The application relates to the field of medicines, in particular to an antibody pair for detecting the content of VEGF in serum and application thereof.

Background

Vascular Endothelial Growth Factor (VEGF) is a functional glycoprotein with high biological activity. It is a specific mitogen of endothelial cells with strong physiological action, can act on vascular endothelial cells to proliferate, migrate and form lumens, participate in angiogenesis and increase capillary permeability; it can cause abnormal growth of tumor blood vessel, prevent the effective delivery of antitumor drug into tumor tissue, and stimulate the increase of new blood vessel growth factor.

VEGF was first purified in 1983 by Senger et al [ Senger DR, PerruzziCA, FederJ.A. high purity retained vascular factor secreted by a variety of human and animal vascular cells [ J ]. CancerRes, 1986, 46 (11): 5629-5632. it was found that a 23kD single-chain protein is composed of two 8 exons and 7 introns. 1994, Kondo [ Kondo S, A sano M, Matsuo K, et a1. vacuum endothiolia l growth fac-tor/vacuum l ar ease factored in the series of pot or bearing M and cancer substrates [ J ]. Biochim Biophys Acta, 1994, 1221 (2): 211-214, et al, have first reported that serum levels of VEGF in cancer patients are higher than those of normal controls, and thus have initiated experimental clinical studies of VEGF blood (including serum and plasma) levels and changes in VEGF during tumor development, progression, and treatment and have obtained a large amount of research data. In recent years, VEGF has been widely studied as a substance that can respond to the development and change of tumors. In searching the VEGF-related literature in more than 280 English and 230 Chinese, more than 95% of the literature supports the fact that serum VEGF levels are increased in tumor patients. Tumor types cover almost all of the majority of solid tumors and some non-solid tumors in the clinic today. VEGF blood levels are increased not only throughout the tumor stage but also from stage 0 to 1 in its early stages, and conventional tumor markers differ significantly only between stage 4 cancer and normal controls. VEGF is currently considered to be the most meaningful marker for broad-spectrum and early screening of tumors. Because the antigen of VEGF is large, the difficulty of preparing the antigen is large, and at present, no paired antibody aiming at VEGF clinical detection is reported.

Disclosure of Invention

The present application provides an antibody pair for detecting VEGF content in serum and the use thereof to solve the above technical problems.

The technical scheme adopted by the application is as follows: in a first aspect, the present application provides an antibody pair for detecting VEGF content in serum, characterized in that: the monoclonal antibody AntiVEGF _ N is combined with an N-terminal epitope of VEGF, namely an amino acid sequence shown in SEQ ID NO. 2, and the monoclonal antibody AntiVEGF _ C is combined with a C-terminal epitope of VEGF, namely an amino acid sequence shown in SEQ ID NO. 3, wherein the monoclonal antibody AntiVEGF _ N comprises a light chain variable region of the amino acid sequence shown in SEQ ID NO. 7 and a heavy chain variable region of the amino acid sequence shown in SEQ ID NO. 11, and the AntiVEGF _ C comprises a light chain variable region of the amino acid sequence shown in SEQ ID NO. 17 and a heavy chain variable region of the amino acid sequence shown in SEQ ID NO. 21.

Further, the light chain amino acid sequence of the monoclonal antibody AntiVEGF _ N comprises an LCDR1 shown in SEQ ID NO. 4 or an amino acid sequence at least 90% homologous with the LCDR1, an LCDR2 shown in SEQ ID NO. 5 or an amino acid sequence at least 90% homologous with the LCDR2, and an LCDR3 shown in SEQ ID NO. 6 or an amino acid sequence at least 90% homologous with the LCDR 3; the heavy chain amino acid sequence of the monoclonal antibody AntiVEGF _ N comprises an HCDR1 shown in SEQ ID NO. 8 or an amino acid sequence at least 90% homologous with the HCDR1, an HCDR2 shown in SEQ ID NO. 9 or an amino acid sequence at least 90% homologous with the HCDR2, and an HCDR3 shown in SEQ ID NO. 10 or an amino acid sequence at least 90% homologous with the HCDR 3.

Further, the light chain amino acid sequence of the monoclonal antibody AntiVEGF _ C comprises an LCDR1 shown in SEQ ID NO. 14 or an amino acid sequence at least 90% homologous with the LCDR1, an LCDR2 shown in SEQ ID NO. 15 or an amino acid sequence at least 90% homologous with the LCDR2, and an LCDR3 shown in SEQ ID NO. 16 or an amino acid sequence at least 90% homologous with the LCDR 3; the heavy chain amino acid sequence of the monoclonal antibody AntiVEGF _ C comprises an HCDR1 shown in SEQ ID NO. 18 or an amino acid sequence at least 90% homologous with the HCDR1, an HCDR2 shown in SEQ ID NO. 19 or an amino acid sequence at least 90% homologous with the HCDR2, and an HCDR3 shown in SEQ ID NO. 20 or an amino acid sequence at least 90% homologous with the HCDR 3.

Further, the monoclonal antibodies AntiVEGF _ N and AntiVEGF _ C are IgG type monoclonal antibodies, one is a capture antibody, and the other is a detection antibody.

Further, the AntiVEGF _ N is a capture antibody, and the AntiVEGF _ C is a detection antibody.

Further, the detection marker of the detection antibody is one of detection marker protein, a fluorescent group and a radioactive isotope.

Further, the detection marker protein of the detection antibody comprises horseradish peroxidase, alkaline phosphatase, luciferase, beta-galactosidase, glucose oxidase, lysozyme and malate dehydrogenase, and the detection antibody and the detection marker protein are expressed in a recombinant mode.

Further, the detection method for detecting the antibody includes enzyme-linked immunosorbent assay (ELISA), chemiluminescence detection assay, western blot detection assay, Immunohistochemistry (IHC) assay and immunochromatography detection assay.

Further, the antibody pair is an antibody fragment selected from the group consisting of Fab, F (ab ') 2, Fab', scFv, and di-scFv that binds to PF 4.

A second aspect of the application provides a DNA molecule encoding the antibody of

claim

1 or 2 or 3.

In a third aspect, the application provides an application of any one of the above antibody pairs in preparation of a kit for detecting the content of VEGF in serum.

The application has the advantages and positive effects that: the VEGF antigen is divided into two sequences of an N end (SEQ ID NO:2) and a C end (SEQ ID NO:3), mice are immunized by the two sequences respectively, antibody pairs with high specificity and high sensitivity, namely AntiVEGF _ N and AntiVEGF _ C are screened, and the content of VEGF in serum can be effectively determined by the antibody pairs, so that the broad-spectrum early-stage screening of tumors is facilitated, and the accuracy of early-stage screening of tumors is greatly improved.

In addition to the technical problems addressed by the present application, the technical features constituting the technical solutions, and the advantages brought by the technical features of the technical solutions described above, other technical problems solved by the present application, other technical features included in the technical solutions, and advantages brought by the technical features will be described in further detail below.

Drawings

FIG. 1 is a schematic diagram of the detection of the pair of antibodies AntiVEGF _ N and AntiVEGF _ C provided in the examples herein;

FIG. 2 is an electrophoretogram of pcDNA3.1-NL-LFc vector constructed as provided in the examples of the present application;

FIG. 3 is an electrophoretogram of pcDNA3.1-NH-HFc vector constructed as provided in the examples of the present application;

FIG. 4 is the electrophoresis picture of the recombinant AntiVEGF _ N protein purification provided by the embodiment of the present application;

FIG. 5 is an electrophoretogram of pcDNA3.1-CL-LFc vector constructed as provided in the examples of the present application;

FIG. 6 is an electrophoretogram of the pcDNA3.1-CL-HFc vector constructed as provided in the examples of the present application;

FIG. 7 is an electrophoretogram of recombinant AntiVEGF _ C protein purification provided in the examples of the present application;

FIG. 8 is a schematic diagram of the validation process of the kit prepared by the antibodies AntiVEGF _ N and AntiVEGF _ C provided in the examples of the present application;

FIG. 9 is a first measurement of the linearity of the dose-response curve provided in example four of the present application;

FIG. 10 is a second measurement of the dose-response curve in a linear test provided in example four of the present application;

FIG. 11 is a third measurement curve in a linear assay of the dose-response curve provided in example four of the present application;

FIG. 12 is a graph showing the dose-response curves of VEGF detection by the antibody preparation kit and the control kit of the present application in clinical diagnosis provided in the examples of the present application.

Detailed Description

The present application will be described in further detail with reference to the following drawings and examples. It is to be understood that the specific embodiments described herein are merely illustrative of the relevant invention and not restrictive of the invention. It should be noted that, for convenience of description, only the portions related to the present invention are shown in the drawings.

It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict. The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.

The first embodiment is as follows: preparation of monoclonal antibodies AntiVEGF _ N and AntiVEGF _ C

1. The antigenic analysis of VEGF (SEQ ID NO:1) protein is carried out, the VEGF antigen is divided into two sequences of N end (SEQ ID NO:2) and C end (SEQ ID NO:3), the N end epitope (SEQ ID NO:2) of VEGF and the C end epitope (SEQ ID NO:3) of VEGF are respectively synthesized, and KLH is respectively coupled to obtain N end immunogen and C end immunogen.

2. Animal immunization: an N-terminal immunogen and a C-terminal immunogen are adopted to immunize female Balb/C mice with age of 8 weeks respectively, and 100ug of antigen is used to immunize the mice 4 times with an interval of 4 weeks each time.

3. Cell fusion: killing the mouse by breaking the neck, placing the mouse in a 75% ethanol solution for 5-10min, taking out the spleen under aseptic conditions, extruding and slightly grinding spleen cells by using a sterilized ground glass slide, counting the spleen cells, mixing the immunized mouse spleen cells and mouse myeloma cells according to the ratio of 1:1, washing the mixture for 1 time by using a serum-free incomplete culture solution in a 50ml centrifuge tube, centrifuging the mixture for 10min at 1000rpm/min, discarding supernatant, sucking residual liquid by using a pipette (in order to avoid influencing the concentration of PEG), and slightly flicking the bottom of the centrifuge tube to ensure that cell precipitation is slightly loosened.

4. 1ml of 50% PEG (pH 8.0) preheated to 40 ℃ was added to the mixture in 60 seconds by means of a pipette while gently stirring.

5. Adding 20-30ml of preheated incomplete culture medium in 90s with a 10ml pipette (terminating PEG action); standing at 20-27 deg.C for 10 min.

6. Centrifuging at 1000r/min for 5 min, discarding the supernatant

7. Adding 5ml HAT culture medium, gently sucking the precipitated cells, suspending and mixing, and supplementing HAT culture medium containing peritoneal macrophages to 80-100 ml.

8. Subpackaging 96-well cell culture plate (with feeder cell layer in the well plate) 0.1-0.15ml per well (or subpackaging 24-well plate with 1.0-1.5ml per well), and culturing at 37 deg.C in 6% CO2 incubator.

9. After 5 days, 1/2 medium was replaced with HAT medium

10. Changing out HAT culture medium after 7-10 days;

11. and (3) frequently observing the growth condition of the hybridoma cells, sucking out supernatant when the hybridoma cells grow to a pore bottom area of 1/10 or more, detecting the activity (positive clone) of the hybridoma cells by ELISA, screening the hybridoma cells with high antibody titer, carrying out 3-4 times of subcloning to ensure that the hybridoma cells are subjected to amplification culture when the monoclonal antibody is formed, injecting ascites cells, and purifying the antibody.

Example two: the monoclonal antibody obtained in example 1 was verified

(1) Respectively coating VEGF complete antigen (SEQ ID NO:1), VEGF N-terminal antigen (SEQ ID NO:2) and VEGF C-terminal antigen (SEQ ID NO:3), wherein the coating concentration is 2ug/ml, the temperature is 2 hours at 4 ℃, washing the plate for 3 times by using 0.9% NaCl washing solution, patting the plate dry, adding 200 mu l of sealing solution (0.5mol PBS + 1% BSA + 2.5% sucrose), sealing the plate at 37 ℃ for 2 hours, pouring the liquid in the plate hole completely, and patting the plate dry;

(2) adding different volumes of AntiVEGF _ N and AntiVEGF _ C obtained in example 1 (as shown in Table 1), and reacting at 37 ℃ for 1 hour;

(3) washing the plate with 0.9% NaCl washing solution for 3 times, and drying;

(4) adding a secondary antibody of goat anti-mouse IgG marked by HRP, and reacting for 1 hour at 37 ℃;

(5) washing the plate with 0.9% NaCl washing solution for 5 times, and drying;

(6) finally, adding substrate solution luminol, measuring the luminous value of the luminol, and measuring the result as shown in table 1:

TABLE 1

As can be seen from Table 1, AntiVEGF _ N specifically binds to the N-terminal antigen of VEGF (SEQ ID NO:2) and does not bind to the C-terminal antigen of VEGF (SEQ ID NO: 3); AntiVEGF _ C specifically binds to the C-terminal antigen of VEGF (SEQ ID NO:3) but not to the N-terminal antigen of VEGF (SEQ ID NO: 2).

Example three: sequencing of monoclonal antibodies AntiPF4_ N and AntiPF4_ C and preparation of corresponding antibodies by hybridoma cells

1. Total RNA of the anti VEGF _ N hybridoma in the embodiment 2 is extracted, a cDNA synthesis reverse transcription kit is adopted, RNA is taken as a template to synthesize first-strand cDNA, and then cDNA is taken as a template to amplify the variable region gene of the monoclonal antibody anti VEGF _ N. And (3) carrying out T/A cloning on the variable region PCR product sequence, then selecting a positive bacterial colony for sequencing, and carrying out amino acid translation analysis on the sequencing result.

The results show that the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region of AntiVEGF _ N monoclonal antibody are respectively listed in SEQ ID NO. 4, 5 and 6. In addition, the light chain variable region gene containing the variable region and the coded amino acid sequence are shown as SEQ ID NO: 12 and 7.

The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the antibody VEGF _ N single-antibody heavy chain variable region are respectively shown in SEQ ID NO:8, 9 and 10. In addition, the heavy chain variable region gene comprising the above variable region and the encoded amino acid sequence are shown in SEQ ID N0:13 and 11.

Optimizing the light chain variable region gene and the heavy chain variable region gene of the AntiVEGF _ N monoclonal antibody, cloning the light chain variable region gene into a pcDNA3.1-LFc vector, and constructing the pcDNA3.1-NL-LFc vector, as shown in figure 2; cloning the heavy chain variable region gene into pcDNA3.1-HFc vector, constructing pcDNA3.1-NH-HFc vector as shown in FIG. 3, in this example, pcDNA3.1-LFc and pcDNA3.1-HFc already contain human light chain constant region and heavy chain constant region, co-transfecting pcDNA3.1-NL-LFc and pcDNA3.1-NH-HFc into CHO-S cell at 1:1, and recombinantly expressing anti-VEGF N-terminal antibody anti-VEGF _ N as shown in FIG. 4.

2. Total RNA of the anti VEGF _ C hybridoma in the embodiment 2 is extracted, a cDNA synthesis reverse transcription kit is adopted, RNA is taken as a template to synthesize first-strand cDNA, and then cDNA is taken as a template to amplify the variable region gene of the monoclonal antibody anti VEGF _ C. And (3) carrying out T/A cloning on the variable region PCR product sequence, then selecting a positive bacterial colony for sequencing, and carrying out amino acid translation analysis on the sequencing result.

The results show that the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region of AntiVEGF _ C monoclonal antibody are respectively listed in SEQ ID NO:14, 15 and 16. In addition, the light chain variable region gene containing the variable region and the coded amino acid sequence are shown as SEQ ID NO: 22 and 17.

The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the antibody VEGF _ C single-antibody heavy chain variable region are respectively shown in SEQ ID NO:18, 19 and 20. In addition, the heavy chain variable region gene comprising the above variable region and the encoded amino acid sequence are shown in SEQ ID Nos. 0:23 and 21.

Optimizing the light chain variable region gene and the heavy chain variable region gene of the AntiVEGF _ C monoclonal antibody, cloning the light chain variable region gene into a pcDNA3.1-LFc vector, and constructing the pcDNA3.1-CL-LFc vector, as shown in figure 5; the heavy chain variable region gene was cloned into pcDNA3.1-HFc vector, pcDNA3.1-CH-HFc vector was constructed as shown in FIG. 6, in this example, pcDNA3.1-LFc and pcDNA3.1-HFc already contained human light chain constant region and heavy chain constant region, pcDNA3.1-CL-LFc and pcDNA3.1-NH-CFc were co-transfected into CHO-S cells at 1:1 to recombinantly express anti-VEGF C-terminal antibody AntiVEGF _ C as shown in FIG. 7.

Example four: application of AntiVEGF _ N and AntiVEGF _ C antibody pair in chemiluminescence method diagnostic kit

According to the existing chemiluminescence method diagnostic reagent production process, the AntiVEGF _ C is used as a capture antibody to be coated to prepare a VEGF coated plate, the AntiVEGF _ N mark is used as a binding antibody to be prepared into a VEGF enzyme conjugate, and VEGF antigen is prepared into a series of calibrators (0pg/ml, 50pg/ml, 200pg/ml, 800pg/ml, 1600pg/ml and 3200pg/ml) to form a complete kit and carry out a series of verifications.

1. Performance verification index

Referring to the industrial standard of YY/T1175-2010 tumor marker quantitative determination reagent (kit) chemiluminescence method, a performance verification index of the VEGF wash-free detection reagent is formulated

(1) Minimum limit of detection

Should not be higher than 40 pg/ml.

(2) Linearity of dose-response curve

Within the linear range of 0 pg/ml-3200 pg/ml, the linear correlation coefficient (r) of the dose-response curve should be not less than 0.9900.

(3) Accuracy of

The VEGF calibrator detection reagent is used for detecting the international standard substance, and the accuracy is within the range of 90-110%.

(4) Precision degree

Precision (CV) should be no greater than 10%.

(5) Specificity of

20 mu g/ml of human fibroblast growth factor 2(FGF2), 30ng/ml of Epidermal Growth Factor (EGF), measured at an apparent value of not more than 50 pg/ml.

2. Experimental configuration

(1) VEGF coated plate configuration

AntiVEGF _ C was added to the coating buffer (0.05mol PBS) as a capture antibody at a ratio of 1 ug/ml. Adding 100ul of the blank luminescent plate holes, standing overnight at 4 ℃, taking out, washing for 2 times by using a plate washing solution (0.9% NaCl), adding 200ul of a confining solution (0.5mol PBS + 1% BSA + 2.5% sucrose) into each hole, standing overnight at 4 ℃, taking out, discarding the liquid in the plate, and drying to prepare the VEGF coated plate.

(2) VEGF enzyme binder configuration

A. The enzyme combined with the detection antibody is one of horseradish peroxidase, alkaline phosphatase, luciferase, beta-galactosidase, glucose oxidase, lysozyme and malate dehydrogenase, and a corresponding detection method is selected according to the enzyme combined with the detection antibody.

B. In this example, the anti-vegf _ N antibody was labeled with HRP, 1mg/ml of anti-vegf _ N antibody was taken, and 1mgHRP was activated with acetic acid solution for use, and the antibody and HRP were mixed in a ratio of 1:1, reversing and uniformly mixing, and adding sodium borohydride to terminate the reaction after 2h to obtain the AntiVEGF _ N-HRP conjugated antibody.

AntiVEGF _ N-HRP was added as a detection antibody to an enzyme diluent (50Mmol Tris + 1% BSA) at 1/1000.

(3) Calibrator arrangement

VEGF antigen (Tokuntau, 20190306, 0.15mg/ml) was diluted 6 gradients (0pg/ml, 50pg/ml, 200pg/ml, 800pg/ml, 1600pg/ml, 3200pg/ml) with antigen diluent (0.5mol PBS + 1% BSA).

(4) International Standard article (NIBSC 02/286) configuration

The international standard was diluted 5 gradients (50pg/ml, 200pg/ml, 800pg/ml, 1600pg/ml, 3200pg/ml) with antigen diluent (0.5mol PBS + 1% BSA).

(5) Quality control product configuration

VEGF antigen (purchased from Touchonguda, 20190306, 0.15mg/ml) was diluted with antigen diluent (0.5mol PBS + 1% BSA) to QC1(200 pg/ml. + -. 10%), QC2(1750 pg/ml. + -. 10%).

(6) Specific sample configuration

FGF2(NIBSC 91/550) and EGF (NIBSC 90/712) were diluted to 20. mu.g/ml and 30ng/ml, respectively, with diluent (0.5mol PBS + 1% BSA).

3. Sample application operation, as shown in FIG. 8

(1) And (3) respectively taking 50 mu L of each of the prepared VEGF calibrator (3) to (6), the international standard, the quality control product and the specific sample, adding into the corresponding coated plate hole of the prepared VEGF coated plate (1), then taking 50 mu L of the prepared VEGF enzyme conjugate (2), adding into each hole oscillator, and oscillating for 30 seconds to fully and uniformly mix the VEGF calibrator, the international standard, the quality control product and the specific sample.

(2) Cover the plate and incubate at 37 ℃ for 60 minutes.

(3) The plate was removed and washed 5 times with application wash (0.5mol PBS + 0.025% T-20).

(4) 100ul of substrate solution (purchased from ThermoFisher T2142) was added to each well.

(5) Chemiluminescence intensity (RLU) was measured using a Zhongshengbuck BHP9504 chemiluminescence apparatus.

4. Performance index assessment

(1) Minimum limit of detection

Parallel assay of 20-well zero calibrator (0pg/ml)Calculating the average value of the luminescence values with respect to the luminescence intensity

And standard deviation SD, calculating the luminescence value by using the dose-response curve

The corresponding concentration value was the lowest detection limit, and the results are shown in table 2.

TABLE 2

As can be seen from Table 2, the minimum detection limit was 4.73pg/ml, which was not higher than 40pg/ml, and the results were found to meet the criteria.

(2) Linearity of dose-response curve

The reference products (S0-S5, concentration of 0pg/ml, 50pg/ml, 200pg/ml, 800pg/ml, 1600pg/ml and 3200pg/ml) of the multi-well assay kit are determined in parallel three times, and are fitted by four parameters, and the dose-response curves of the three times of determination are shown in FIG. 9, FIG. 10 and FIG. 11 (which need to be shown in the attached drawings). Measured dose-response curve linear correlation coefficient (R)²) As shown in table 3.

TABLE 3

	First measurement	Second measurement	The third measurement
				Linear correlation coefficient R²	0.9999	0.9997	0.9997

As can be seen from FIGS. 7, 8 and 9, and Table 3, the linear correlation coefficient R of the VEGF antigen curves obtained by three consecutive determinations in the concentration range of 0-3200pg/ml²Are all larger than 0.9990, which meets the index.

(3) Accuracy of

The international standard (13 ug/count) was diluted into samples of theoretical concentrations of 50pg/ml, 200pg/ml, 800pg/ml, 1600pg/ml, 3200pg/m, and the ratio of the measured concentration to the theoretical concentration was calculated 3 times, and the results are shown in table 4.

TABLE 4

As can be seen from Table 4, the accuracy of the accuracy specimen prepared by the VEGF international standard is continuously detected for three times, the result accuracy is within the range of 95-105%, and within the requirement of 90-110%, and the accuracy meets the index.

(4) Precision degree

Each 10 wells of the quality control Q1 and the quality control Q2 were measured in parallel, and CV was calculated according to the formula, and the results are shown in Table 5.

In the formula:

average value of concentration values determined by quality control

SD-Standard deviation of concentration value measured by quality control Material

TABLE 5

As can be seen from Table 5, the results of three consecutive parallel determinations of QC1 and QC2 are not more than 10% and meet the index.

(4) Specificity of

Human fibroblast growth factor 2(FGF2) and Epidermal Growth Factor (EGF) were added to the diluted solutions, respectively, to prepare 20g/ml of FGF2 sample and 30ng/ml of EGF sample, and the results were measured as shown in Table 6:

TABLE 6

Item	Luminous value	Concentration value (pg/mL)
			Human fibroblast growth factor 2(20 ug/ml)	14858	11.265
Epidermal growth factor (30ng/ml)	8774	6.571

As can be seen from Table 6, the human fibroblast growth factor 2 (20. mu.g/ml) and epidermal growth factor (30ng/ml) were found to have apparent values of 10.496pg/ml and 7.235pg/ml, respectively, not greater than 50pg/ml, and were satisfactory.

In the embodiment, the minimum detection limit, the dose-response curve, the accuracy, the precision and the specificity performance indexes of the VEGF detection kit all meet the requirements of the prior art and are superior to the existing methodology reagents in the market.

Example five: clinical testing

Selecting 40 parts of healthy human serum specimen and 10 parts of diagnosed malignant tumor case serum, and detecting the 40 parts of healthy human serum specimen and 10 parts of diagnosed malignant tumor case serum by using the kit and the VEGF kit by an electrochemical luminescence method, wherein the clinical luminescence value of the healthy human serum is less than 200; the serum luminescence values of sexual tumor cases are all higher than 400.

1) The luminescence intensity of 40 samples of healthy human serum is shown in table 7:

TABLE 7

(2) The luminescence intensity of 10 malignant tumor serum samples is shown in table 8:

TABLE 8

Serum dose-response curves were drawn for healthy human serum and for sexual tumor cases according to tables 7 and 8, as shown in fig. 12.

The results are shown in tables 7 and 8, and the clinical diagnosis result coincidence rate of the VEGF kit based on the kit and the existing electrochemical luminescence method is 100%.

As shown in FIG. 10, based on the correlation R2 between the detection concentration result of the kit and the detection concentration result of the VEGF kit in the existing electrochemical luminescence method, the kit is 0.9930, and meets the industrial standard of the quantitative determination reagent (kit) for the YY/T1175-2010 tumor marker in the chemiluminescence method.

In the application, the detection antibody can be combined with various detection marker proteins, and the detection method for detecting the antibody comprises an enzyme-linked immunosorbent assay (ELISA) detection method, a chemiluminescence detection method, a western blot detection method, an Immunohistochemistry (IHC) detection method and an immunochromatography detection method, so that the appropriate detection marker protein can be conveniently selected according to needs, and the convenience of the prepared kit by the antibody is greatly improved.

The embodiments of the present invention have been described in detail, but the description is only for the preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made within the scope of the application of the present invention should fall within the scope of the patent coverage of the present invention.

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<400>7

Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Phe Ser Val Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Ile Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Arg Leu Leu Ile

35 40 45

Ser Gly Ala Thr Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Tyr Gly Thr Asn Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Arg Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210>8

<211>8

<212>PRT

<213> mouse (Mus musculus)

<400>8

Gly Phe Thr Phe Ser Ala Tyr Tyr

1 5

<210>9

<211>10

<212>PRT

<213> mouse (Mus musculus)

<400>9

Ile Arg Leu Arg Ser Asn Asn Tyr Thr Asn

1 5 10

<210>10

<211>12

<212>PRT

<213> mouse (Mus musculus)

<400>10

Ala Arg Tyr Ser Asn Tyr Ala Tyr Ala Leu Asp Tyr

1 5 10

<210>11

<211>121

<212>PRT

<213> mouse (Mus musculus)

<400>11

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr

20 25 30

Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Tyr Ile Arg Leu Arg Ser Asn Asn Tyr Thr Asn Asn Tyr Asn Pro

50 55 60

Ser Leu Lys Asn Arg Leu Ser Ile Thr Arg Asp Asn Ala Lys Asn Asn

65 70 75 80

Leu Tyr Leu His Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr

85 90 95

Tyr Cys Ala Arg Tyr Ser Asn Tyr Ala Tyr Ala Leu Asp Tyr Trp Gly

100 105 110

Gln GlyThr Thr Val Thr Val Ser Ser

115 120

<210>12

<211>321

<212>DNA

<213> mouse (Mus musculus)

<400>12

gacattcaga tgacccagtc aagctcctct ttttcagtct ctctggggga ccgggttaca 60

atcacatgca aggccagtga agacatctac atccgcctcg cttggtacca gcaaaaacca 120

ggtaatgccc caaggcttct gattagcggt gctacaaact tggagactgg agtgcccagc 180

agattttccg gatctggata cggtaccaac tattctttga ctattagcaa tcttgagcag 240

gaggacatcg ccacatactt ctgccaacag agaaacacat tgccatacac ctttggcggc 300

gggaccaagc tcgaaatcaa a 321

<210>13

<211>363

<212>DNA

<213> mouse (Mus musculus)

<400>13

gaagttcagc tggttgaaag cggtggcgat cttgtgaaac ctggaggcag tttgaagttg 60

agctgcgccg catctgggtt tactttttct gcctattata tgtattgggt caggcagaca 120

ccagaaaaga ggctggaatg ggtggcatat attcgcctgc ggagtaataa ttatactaac 180

aactataacc ccagccttaa gaaccgactg tcaatcacac gggataacgc taagaataat 240

ctgtacttgc acatgtcctc cctcaggagc gaagataccg ccatgtacta ttgtgcccgc 300

tactccaatt acgcttacgc tctcgattac tgggggcagg gaaccaccgt gacagtgtcc 360

agc 363

<210>14

<211>5

<212>PRT

<213> mouse (Mus musculus)

<400>14

Ser Ser Val Ser Tyr

1 5

<210>15

<211>3

<212>PRT

<213> mouse (Mus musculus)

<400>15

Asp Thr Ser

1

<210>16

<211>10

<212>PRT

<213> mouse (Mus musculus)

<400>16

Gln Gln Trp Ser Arg His Pro Pro Ile Thr

1 5 10

<210>17

<211>107

<212>PRT

<213> mouse (Mus musculus)

<400>17

Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Arg Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Val Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Arg His Pro Pro Ile

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210>18

<211>8

<212>PRT

<213> mouse (Mus musculus)

<400>18

Gly Phe Thr Phe Ser Ser Phe Gly

1 5

<210>19

<211>8

<212>PRT

<213> mouse (Mus musculus)

<400>19

Ile Asn Ser Asp Ser Ser Ala Phe

1 5

<210>20

<211>12

<212>PRT

<213> mouse (Mus musculus)

<400>20

Ala Arg Ala Gly Asn His Tyr Tyr Gly Gly Ala Tyr

1 5 10

<210>21

<211>119

<212>PRT

<213> mouse (Mus musculus)

<400>21

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Phe Ile Asn Ser Asp Ser Ser Ala Phe Tyr Tyr Ala Asp Thr Val

50 55 60

Gln Asp Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Ala Gly Asn His Tyr Tyr Gly Gly Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210>22

<211>321

<212>DNA

<213> mouse (Mus musculus)

<400>22

cagatagtcc tttcacagag ccctgcaatc cttagcgcct ctcctggtga gaaggtcacc 60

atgacctgtc gggcatcatc atccgtctcc tacatgcatt ggtaccagca gagacctggc 120

agctccccta agccctggat atacgataca agcaacctgg caagcggtgt gcctgtgaga 180

tttagcggat caggcagcgg cacatcatat agcttgacta tttccagggt agaagtggaa 240

gatgccgcca catattactg ccagcagtgg tccaggcacc caccaatcac attcggatcc 300

ggcacaaagc tggagatcaa a 321

<210>23

<211>357

<212>DNA

<213> mouse (Mus musculus)

<400>23

gaagttcagc ttgttgaatc cggaggtgga ttggttcagc ctggaggatc tcgtaaactg 60

tcttgtgccg ccagcggctt caccttcagt agtttcggaa tgcactgggt gcgtcaggca 120

cccgagaaag gcctcgaatg ggtggctttc ataaatagcg actcatccgc tttctactat 180

gccgatactg tccaggacag atttaccatc agtcgagaca accccaagaa tacactgttc 240

ctgcagatga cctccctgcg aagtgaggac actgccatgt actactgtgc acgagctggg 300

aatcattact atggaggcgc atactggggg cagggcacac ttgtgactgt cagcgct 357

Claims

1. An antibody pair for detecting the amount of VEGF in serum, comprising: the monoclonal antibody AntiVEGF-N is combined with an N-terminal epitope of VEGF, namely an amino acid sequence shown in SEQ ID NO. 2, and the monoclonal antibody AntiVEGF-C is combined with a C-terminal epitope of VEGF, namely an amino acid sequence shown in SEQ ID NO. 3, wherein the monoclonal antibody AntiVEGF-N comprises a light chain variable region of the amino acid sequence shown in SEQ ID NO. 7 and a heavy chain variable region of the amino acid sequence shown in SEQ ID NO. 11, and the AntiVEGF-C comprises a light chain variable region of the amino acid sequence shown in SEQ ID NO. 17 and a heavy chain variable region of the amino acid sequence shown in SEQ ID NO. 21.

2. The antibody pair for detecting the content of VEGF in serum according to claim 1, wherein the light chain amino acid sequence of monoclonal antibody AntiVEGF _ N comprises LCDR1 shown in SEQ ID NO. 4, LCDR2 shown in SEQ ID NO. 5 and LCDR3 shown in SEQ ID NO. 6; the heavy chain amino acid sequence of the monoclonal antibody AntiVEGF _ N comprises HCDR1 shown in SEQ ID NO. 8, HCDR2 shown in SEQ ID NO. 9 and HCDR3 shown in SEQ ID NO. 10.

3. The antibody pair for detecting the content of VEGF in serum according to claim 2, wherein the light chain amino acid sequence of monoclonal antibody AntiVEGF _ C comprises LCDR1 shown in SEQ ID NO. 14, LCDR2 shown in SEQ ID NO. 15 and LCDR3 shown in SEQ ID NO. 16; the heavy chain amino acid sequence of the monoclonal antibody AntiVEGF _ C comprises HCDR1 shown in SEQ ID NO. 18, HCDR2 shown in SEQ ID NO. 19 and HCDR3 shown in SEQ ID NO. 20.

4. The antibody pair for detecting the amount of VEGF in serum according to claim 3, wherein the monoclonal antibodies AntiVEGF _ N and AntiVEGF _ C are both IgG type monoclonal antibodies, one being a capture antibody and one being a detection antibody.

5. The antibody pair for detecting the amount of VEGF in serum according to claim 4, wherein said AntiVEGF _ N is a capture antibody and said AntiVEGF _ C is a detection antibody.

6. The antibody pair for detecting the content of VEGF in serum according to claim 4, wherein the detection marker of the detection antibody is one of a detection marker protein, a fluorescent group and a radioactive isotope.

7. The antibody pair for detecting the content of VEGF in serum according to claim 6, wherein the detection marker protein of the detection antibody comprises horseradish peroxidase, alkaline phosphatase, luciferase, beta-galactosidase, glucose oxidase, lysozyme and malate dehydrogenase, and the detection antibody is expressed by recombination with the detection marker protein.

8. The antibody pair of claim 7, wherein the detection method for detecting the level of PF4 in serum comprises enzyme-linked immunosorbent assay (ELISA), chemiluminescence detection, Western blot detection, Immunohistochemistry (IHC) detection, and immunochromatographic detection.

9. The antibody pair for detecting the level of PF4 in serum according to claim 1, wherein the antibody pair is an antibody fragment selected from the group consisting of Fab, F (ab ') 2, Fab', scFv, and di-scFv that binds to PF 4.

10. A DNA molecule encoding the antibody of claim 1 or 2 or 3.

11. Use of the antibody of claims 1-10 for the preparation of a kit for detecting VEGF levels in serum.