CN108640994A - A kind of VEGF-C monoclonal antibodies and kit - Google Patents
A kind of VEGF-C monoclonal antibodies and kit Download PDFInfo
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- CN108640994A CN108640994A CN201810683404.3A CN201810683404A CN108640994A CN 108640994 A CN108640994 A CN 108640994A CN 201810683404 A CN201810683404 A CN 201810683404A CN 108640994 A CN108640994 A CN 108640994A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/475—Assays involving growth factors
Abstract
The invention discloses a kind of VEGF C monoclonal antibodies, the amino acid sequence such as VH of heavy chain:Shown in CAA63907, the amino acid sequence such as VL of light chain:Shown in 2X1W_D.There is the VEGF C monoclonal antibodies higher affinity, high specificity to have good biological activity in vitro.The VEGF C protein detection kits of VEGF C monoclonal antibodies of the present invention and the how anti-foundation of VEGF C be can be used as into the generation of evaluation lymphangiogenesis and Lymph Node Metastasis index.
Description
Technical field
The invention belongs to biotechnologies, and in particular to a kind of VEGF-C monoclonal antibodies and the examination of VEGF-C Protein Detections
Agent box.
Background technology
Vascular endothelial growth factor C (VEGF-C) belongs to VEGF/PDGF family members, and people's VEGF-C genes are located at chromosome
On 4q34, coded product is the protein of 419 amino acid residues, belongs to secreted polypeptide, after protease hydrolytic is processed, is formed
With the homodimer that disulfide bond connects, molecular weight 46.9kD.VEGF-C receptors are VEGFR-2, VEGFR-3.VEGF passes through
VEGFR-2 adjusts blood vessel and vasculolymphatic hyperplasia, and VEGFR-3 expression is then confined to lymphatic endothelial, VEGF-C VEGFR-2
Combination, generate biological effect required concentration be far above VEGFR-C, therefore, VEGFR-3 is lymphatic vessel hyperplasia specificity tune
The factor is saved, with VEGF-C trangenic mices it is experimentally confirmed that the high expression of VEGF-C can selectively cause lymphatic vessel hyperplasia
[NeufeldG etc., FASEBJ, 1999,13：9-22], through research, the pass of VEGF-C expression and lymphangiogenesis hyperplasia and transfer
System, which is VEGF-C high expression, can promote the Lymph Node Metastasis of tumour, and mechanism is that VEGF-C is promoted around tumour and the lymph of interstitial
Lymphatic vessel hyperplasia in pipe growth and tumor tissues.
VEGF-C is the Vascular ar endothelial growth facor-c found earliest, more close with vasculolymphatic relationship, including to lymphatic vessel
The induction of hyperplasia and lymphocyte endothelium is effectively adjusted, and tumour is often shifted by lymphatic vessel at first, because
This, VEGF-C is closely related with metastases.VEGF-C can promote blood vessel to break up, grow in the embryo of development, because
This, compared with the factor of other promotion angiogenesis, the time to play a role is earlier.VEGF-C may act on VEGFR-2 and
VEGFR-3, inducing endothelial cell hyperplasia, especially microvessel cell hyperplasia.After VEGF-C is combined with VEGFR-3, activation
VEGFR-3 causes the phosphorylation of SHC, leads to the activation of ERK1 and ERK2, and then activating cell skelemin Paxillin promotes
The transfer of lymphatic vessel epithelial cell；Ras/MAPK signal transduction pathways or TNK accesses can be also activated simultaneously, induce lymphatic endothelial
The recombination of cell actin, stimulating endothelial cell hyperplasia.VEGF-C is combined with receptor VEGFR-2, passes through PI3K approach, activation
Antibody apoptotic proteins Akt/PKB, upregulation of apoptosis protein Bcl-2 inhibits the apoptosis of tumour cell, to promote the life of tumour cell
It is long, vascular endothelial proliferation and movement can be stimulated in vitro, can increase the permeability of blood vessel in vivo.And in same tissue,
The binding force of VEGF-C and VEGFR-3 is 3 times with VEGFR-2 binding forces, this makes the rush lymphatic vessel generation of VEGF-C act on
As dominating, only under certain condition (such as:The main VEGF expression R-2 of high concentration, local organization) just promote blood vessel generation.
As the growth factor of specific lymphatic endothelial cells, VEGF-C tumorigenesis different phase all
Play an important roll.Current clinical research statistics and experiment display, VEGF-C is acted on respectively in a manner of paracrine and autocrine
In blood vessel and lymphatic endothelial cells and its new life is induced, changes the adhesion properties of lymphatic endothelial, increases secrete cytokines
And chemotactic factor (CF), promote tumor cell proliferation.
One of the main reason for influencing cancer clinical curative effect is the transfer of tumour, moreover, lymph metastases generation is more early.
Therefore, the mechanism of lymph metastases, effective therapy of the research for lymph metastases process, it appears particularly heavy are explored
It wants.With the successive discovery of VEGF-C and its receptor, research lymphangiogenesis generates and the mechanism of Lymph Node Metastasis has become at present
The hot spot of tumor research.Although the specific mechanism that the lymphangiogenesis that VEGF-C is mediated generates is unclear.But by a large amount of
Research has proven to the high of VEGF-C and expresses phase related with clinical stage and node positive with lymphangiogenesis density
It closes, VEGF-C can promote lymphatic vessel generation, closely related with lymphatic metastasis situation, and the positive expression of VEGF-C and tumour blood
Row transfer is dangerous closely related, so the expression of monitoring VEGF-C is expected to the biomarker as lymph metastases.
The research of labelling immunoassay technology and application development are rapid nearly ten years, are widely used to biomedical basis
Theoretical research and each field of clinical disease diagnosis.Method for detecting serological index is mainly immune including radioactive isotope
Analysis, enzyme linked immunosorbent assay and chemiluminescence immune assay.These methods can both can also be used as really as Primary Screening Test
Recognize experiment, wherein chemoluminescence method has many advantages, such as to detect that the range of linearity is wide, detecting instrument is simple and convenient to operate.
Be directed in the prior art lymph metastases biomarker product it is domestic at present still belong to blank, similar product its
Specificity and affinity etc. are possible there is also further being promoted, and are based on this, we have developed VEGF-C assay kits
(chemoluminescence method) and preparation method thereof.
Invention content
In order to overcome it is existing in the above problem have higher the present invention provides a kind of VEGF-C monoclonal antibodies
Affinity has good biological activity in vitro；Development prospect is wide.
A kind of VEGF-C monoclonal antibodies, which is characterized in that the heavy chain variable amino acid sequence of the antibody is：Met
His Leu Leu Gly Phe Phe Ser Val Ala Cys Ser Leu Leu Ala Ala Ala Leu Leu Pro
Gly Pro Arg Glu Ala Pro Ala Ala Ala Ala Ala Phe Glu Ser Gly Leu Asp Leu Ser
Asp Ala Glu Pro Asp Ala Gly Glu Ala Thr Ala Tyr Ala Ser Lys Asp Leu Glu Glu
Gln Leu Arg Ser Val Ser Ser Val Asp Glu Leu Met Thr Val Leu Tyr Pro Glu Tyr
Trp Lys Met Tyr Lys Cys Gln Leu Arg Lys Gly Gly Trp Gln His Asn Arg Glu Gln
Ala Asn Leu Asn Ser Arg Thr Glu Glu Thr Ile Lys Phe Ala Ala Ala His Tyr Asn
Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu Trp Arg Lys Thr Gln Cys Met Pro Arg
Glu Val Cys Ile Asp Val Gly Lys Glu Phe Gly Val Ala Thr Asn Thr Phe Phe Lys
Pro Pro Cys Val Ser Val Tyr Arg Cys Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln
Cys Met Asn Thr Ser Thr Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro
Leu Ser Gln Gly Pro Lys Pro Val Thr Ile Ser Phe Ala Asn His Thr Ser Cys Arg
Cys Met Ser Lys Leu Asp Val Tyr Arg Gln Val His Ser Ile Ile Arg Arg Ser Leu
Pro Ala Thr Leu Pro Gln Cys Gln Ala Ala Asn Lys Thr Cys Pro Thr Asn Tyr Met
Trp Asn Asn His Ile Cys Arg Cys Leu Ala Gln Glu Asp Phe Met Phe Ser Ser Asp
Ala Gly Asp Asp Ser Thr Asp Gly Phe His Asp Ile Cys Gly Pro Asn Lys Glu Leu
Asp Glu Glu Thr Cys Gln Cys Val Cys Arg Ala Gly Leu Arg Pro Ala Ser Cys Gly
Pro His Lys Glu Leu Asp Arg Asn Ser Cys Gln Cys Val Cys Lys Asn Lys Leu Phe
Pro Ser Gln Cys Gly Ala Asn Arg Glu Phe Asp Glu Asn Thr Cys Gln Cys Val Cys
Lys Arg Thr Cys Pro Arg Asn Gln Pro Leu Asn Pro Gly Lys Cys Ala Cys Glu Cys
Thr Glu Ser Pro Gln Lys Cys Leu Leu Lys Gly Lys Lys Phe His His Gln Thr Cys
Ser Cys Tyr Arg Arg Pro Cys Thr Asn Arg Gln Lys Ala Cys Glu Pro Gly Phe Ser
Tyr Ser Glu Glu Val Cys Arg Cys Val Pro Ser Tyr Trp Lys Arg Pro Gln Met Ser；
Chain variable region amino acid sequence is：Ala His Tyr Asn Thr Glu Ile Leu Lys Ser Ile
Asp Asn Glu Trp Arg Lys Thr Gln Cys Met Pro Arg Glu Val Ala Ile Asp Val Gly
Lys Glu Phe Gly Val Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val Tyr
Arg Cys Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln Cys Met Asn Thr Ser Thr Ser
Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro Leu Ser Gln Gly Pro Lys Pro
Val Thr Ile Ser Phe Ala Asn His Thr Ser Cys Arg Cys Met Ser Lys Leu His His
His His His His。
Further, the present invention also provides the multinuclears for encoding above-mentioned light chain variable region and heavy chain variable amino acid sequence
Thuja acid.
Further, the present invention also provides the recombinant dna expression vector for including above-mentioned polynucleotides, the recombinant DNAs
Include coding VEGF-C monoclonal antibody heavies variable region, light chain variable region and the DNA sequences with antibody constant region in expression vector
Row.
Further, the present invention provides the detection kits for including above-mentioned VEGF-C monoclonal antibodies.The kit system
Preparation Method is：It is reaction support with Nunc chemiluminescence reaction plates, VEGF-C monoclonal antibodies is diluted to 0.5mPBS
2.5ug/ml is coated with the holes 100ul/ into chemiluminescence blank plate, at 4 DEG C after 24 hours, with 0.9%Nacl board-washings 3 times, is added
Enter the 0.5mol PBS closings containing 1%BSA, is dried after 12 hours at 4 DEG C.
The present invention has the following advantages：
(1) there is VEGF-C monoclonal antibodies of the invention higher affinity, high specificity to have in vitro good
Biological activity.
(2) include VEGF-C monoclonal antibodies detection kit detection method is easy, accuracy is strong, at low cost.
(3) VEGFc419 and the how anti-VEGF-C protein detection kits of establishing of VEGF-C be can be used as into VEGF/VEGFR accesses
The monitoring instrument that tumor-targeting drug moves.
Description of the drawings
Fig. 1 is VEGFc419 antibody dosages-reaction system schematic diagram.
Fig. 2 is VEGFc419 antibody quality controlled serums detection scatter plot.
Specific implementation mode
It is better understood the present invention by means of following specific implementation modes, however, these specific implementation modes are only used for
It illustrates the present invention, is not necessarily to be construed as limitation of the present invention.
Embodiment 1
One, the preparation of VEGF-C antigens：
(1) according to the sequence design pair of primers of people VEGF-C (X94216.1) in GenBank databases, sense primer is
vP：5 '-ATACCCCGCCACGGACCGGTCCCCCACCCCCGGTCC-3 ' introduce Xho I restriction enzyme sites；Downstream primer is vP：
5 '-TTCATATTGGAAAGACCACAAATGAGCTA-3 ' introduce EcoR I restriction enzyme sites, thin from people liver by RT-PCR method
Total serum IgE is extracted in born of the same parents, then reverse transcription is at cDNA, and using cDNA as template, VEGF-C bases are carried out with Pyrobest archaeal dna polymerases
Because of specific amplification.PCR product after amplification carries out gel recycling, and the VEGF-C genes of recycling carry out respectively with pPIC9K plasmids
Xho I and EcoR I digestions, the target fragment after the connection recycling of T4 ligases, connection product convert DH5 α competent cells, lead to
It crosses ammonia benzyl (1mg/mL) antibody screen and selects positive colony, body takes plasmid and identified through digestion and sequencing.
(2) Pichia pastoris GS115 of the pPIC9K-VEGF-C expression vectors through Stu I linearisations, electrotransformation to competence
(his4), the transformant of MD plate screening height copy of the coating containing G418, waits for that bacterium colony is grown, selects monoclonal bacterial strain and carry out PCR
PCR results are that positive clone is converted with plasmid pPIC9-Kal/GS115 (His4) to Pichia pastoris by identification,
High-density cells 3E3 ferments in 7.5L culture tanks, with the formaldehyde inducement VEGF expression-C of 1%-2%.Fermented supernatant fluid passes through
Phenyl Sephadex G-25 gel permeation chromatographies, Heparin Sepharose FF heparin affinity chromatographies, Sephacryl
S-100 gel permeation chromatographies obtain sufficient people VEGF-C albumen, and expression 50mg/L carries out SDS-PAGE electroresis appraisals,
VEGF-C purity of protein 98% after purification.
Two, mostly anti-VEGF-C preparation and label
(1) it uses male New Zealand rabbit as immune animal, first uses 10mg BCG vaccines injection stimulation animal, start after a week
It is immune.Using VEGF-C albumen as immunogene；Immune pattern is subcutaneously injected using foot injection and back 4-6 points, and assistant is immunized
Agent uses Freund's complete adjuvant and incomplete Freund's adjuvant, and animal is immunized in four times, takes VEGF-C antigen 1s mg by equivalent every time
Freund's complete adjuvant and antigenic solution suck respectively it is 30-60 minutes fully emulsified in two syringes after metapedes be subcutaneously injected, often
Minor tick two weeks.Ear vein blood examination is taken to survey potency, CLIA reaches 1:50000 carry out strength arterial blood letting, centrifuging and taking serum.It utilizes
DEAE ion-exchange purifications, gained antibody-solutions are up to 98% or more.
(2) how anti-VEGF-C is dilutes 1mg/ml with 0.5mol PBSPH7.5, configures EZ-LinkTMSulfo-NHS-LC-LC-
21338 biotin reagent 10mM of Biotin article No.s marks more anti-and biotins, molar ratio 1:20；Room temperature reaction 1 hour, PD10
Desalting column crosses column, collects and preserves.
Three, the preparation of VEGF-C monoclonal antibodies
(1) mouse immune：The 3E3 cells of expression are stablized in culture, are resuspended with PBS (pH7.4) after centrifugation, and 3 females are immunized
BALA/c mouse, every BALB/c mouse are subcutaneously injected 5 × 106Cell, continuous 3 times, every minor tick 2 weeks, after the 4th is immune
7d detects antiserum titre with CLIA methods, takes the highest mouse of potency, intrasplenic injection 1 × 105A cell booster immunization, after 3d
Mouse spleen is taken, is ground, and splenocyte is counted for use.
(2) cell fusion and Antibody preparation：Extracting spleen cell presses cell count 6 with bone marrow cell Sp2/0:1 ratio fusion,
PEG1200 is induced, and the 96 orifice plate cultures that the feeder layer completed is added to after fusion change liquid, CLIA with half amount of HAT culture mediums after a week
Method screens positive cell strain.Positive hybridoma cell strain is screened with indirect CLIA methods.After 3 effective dilution method clonings,
Select I plant 98% or more VEGF-CAb continuous releases positive rates hybridoma 6H2 and expand culture prepare be injected intraperitoneally it is small
Mouse.I weeks before inoculation hybridoma, mouse peritoneal injects 500 μ L atoleines.Hybridoma is collected by centrifugation when inoculation, uses
Endless full nutrient solution suspends and mixing, and cell number is adjusted to 1 × 109/ L, every mouse peritoneal inject 500 μ L, mouse abdomen after 1 week
Portion's obvious tumefaction after sterilizing lower abdomen skin, extracts ascites, and gained ascites 3000r/min collects supernatant.
(3) GE company proteinG are selected to purify column purification ascites；Purification column is balanced with 20mMPB buffer solutions, ascites is added
Loading is eluted with PH2.70.1mol glycine HCI buffers, is received with the EP pipes containing PH8.71moltris buffer solutions
Collection, 0.05mMPB dialysis, concentration freeze.
Four, monoclonal antibody application screening
(1) monoclonal antibody that coating preceding method prepares dilutes antibody to 1-5ug/ml with 0.5mPBS.100ul is every
Hole is coated with into chemiluminescence blank plate, and after 4 DEG C are stayed overnight, 0.9%Nacl is washed 3 times, is added and is sealed per hole 150ul containing 1%BSA
Close, 4 DEG C overnight after dry it is spare.
(2) optimal monoclonal antibody is screened：Dilution prepare antigen dilute in proportion 8 gradients (0,10,50,100,200,400,
1000,2000pg/m), sequentially add in the aforementioned Chemiluminescent plate prepared, per hole 50ul, add biotinylation it is mostly anti-and
It is marked with the Streptavidin of horseradish peroxidase, 37 DEG C incubate 1h, and PBST is washed 5 times, and Chemoluminescent substrate is added, keeps away
Light reaction detects luminous value；The linearly dependent coefficient R values for comparing calibration object under different monoclonal antibody coating Chemiluminescent plates, with VEGF-C
A concentration of abscissa X values, RLU values are ordinate Y value.Final to select with good linear, the best monoclonal of evaluation index is anti-
Body VEGFc419, as a result such as figure below.The grand antibody of monoclonal antibody answers S0RLU values ＜ as the evaluation criterion of sandwich method CLIA detection methods
100, S5/S0 (P/N) are maximum, and 30 quality controlled serum recall rates are more than the conditions such as 90%.
VEGFc419 antibody dosages-reaction system schematic diagram is as shown in Figure 1.
It is as shown in Figure 2 that VEGFc419 antibody quality controlled serums detect scatter plot.
Five, monoclonal antibody gene is transferred and is prepared in hybridoma
(1) Trizol reagents are used to extract 5 × 106Hybridoma 4C5 total serum IgE, then using OligodT as primer, AMV
37 DEG C of 15min of reverse transcriptase reverse transcription temperature, synthesis obtain cDNA.To synthesize cDNA as template, primer for RNA sequence and
Gene-specific primer GSP carries out nested PCR amplification, and the condition of PCR is：95 DEG C of denaturation 5min, 94 DEG C of 30s, 55 DEG C of 30s,
72 DEG C of 1min, totally 35 cycles, finally extend 10min then at 72 DEG C.
(2) PCR product is identified through 1% agarose gel electrophoresis, cuts and recycle purpose gel-tape, transfers purpose
Gene is connected in pGEM-T carriers, and the positive recombinant plasmid of EcoRI digestions identification, the measurement for carrying out DNA sequence dna obtains respectively
VL and VH gene orders.
VH (weight chain variabl area sequence)
LOCUS CAA63907 419aa
Met His Leu Leu Gly Phe Phe Ser Val Ala Cys Ser Leu Leu Ala Ala Ala
Leu Leu Pro Gly Pro Arg Glu Ala Pro Ala Ala Ala Ala Ala Phe Glu Ser Gly Leu
Asp Leu Ser Asp Ala Glu Pro Asp Ala Gly Glu Ala Thr Ala Tyr Ala Ser Lys Asp
Leu Glu Glu Gln Leu Arg Ser Val Ser Ser Val Asp Glu Leu Met Thr Val Leu Tyr
Pro Glu Tyr Trp Lys Met Tyr Lys Cys Gln Leu Arg Lys Gly Gly Trp Gln His Asn
Arg Glu Gln Ala Asn Leu Asn Ser Arg Thr Glu Glu Thr Ile Lys Phe Ala Ala Ala
His Tyr Asn Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu Trp Arg Lys Thr Gln Cys
Met Pro Arg Glu Val Cys Ile Asp Val Gly Lys Glu Phe Gly Val Ala Thr Asn Thr
Phe Phe Lys Pro Pro Cys Val Ser Val Tyr Arg Cys Gly Gly Cys Cys Asn Ser Glu
Gly Leu Gln Cys Met Asn Thr Ser Thr Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile
Thr Val Pro Leu Ser Gln Gly Pro Lys Pro Val Thr Ile Ser Phe Ala Asn His Thr
Ser Cys Arg Cys Met Ser Lys Leu Asp Val Tyr Arg Gln Val His Ser Ile Ile Arg
Arg Ser Leu Pro Ala Thr Leu Pro Gln Cys Gln Ala Ala Asn Lys Thr Cys Pro Thr
Asn Tyr Met Trp Asn Asn His Ile Cys Arg Cys Leu Ala Gln Glu Asp Phe Met Phe
Ser Ser Asp Ala Gly Asp Asp Ser Thr Asp Gly Phe His Asp Ile Cys Gly Pro Asn
Lys Glu Leu Asp Glu Glu Thr Cys Gln Cys Val Cys Arg Ala Gly Leu Arg Pro Ala
Ser Cys Gly Pro His Lys Glu Leu Asp Arg Asn Ser Cys Gln Cys Val Cys Lys Asn
Lys Leu Phe Pro Ser Gln Cys Gly Ala Asn Arg Glu Phe Asp Glu Asn Thr Cys Gln
Cys Val Cys Lys Arg Thr Cys Pro Arg Asn Gln Pro Leu Asn Pro Gly Lys Cys Ala
Cys Glu Cys Thr Glu Ser Pro Gln Lys Cys Leu Leu Lys Gly Lys Lys Phe His His
Gln Thr Cys Ser Cys Tyr Arg Arg Pro Cys Thr Asn Arg Gln Lys Ala Cys Glu Pro
Gly Phe Ser Tyr Ser Glu Glu Val Cys Arg Cys Val Pro Ser Tyr Trp Lys Arg Pro
Gln Met Ser；
VL (light-chain variable sequence)
LOCUS 2X1W_D 110aa
Ala His Tyr Asn Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu Trp Arg Lys
Thr Gln Cys Met Pro Arg Glu Val Ala Ile Asp Val Gly Lys Glu Phe Gly Val Ala
Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val Tyr Arg Cys Gly Gly Cys Cys
Asn Ser Glu Gly Leu Gln Cys Met Asn Thr Ser Thr Ser Tyr Leu Ser Lys Thr Leu
Phe Glu Ile Thr Val Pro Leu Ser Gln Gly Pro Lys Pro Val Thr Ile Ser Phe Ala
Asn His Thr Ser Cys Arg Cys Met Ser Lys Leu His His His His His His。
Heavy chain and chain variable region gene sequence will be obtained and the constant region of antibody carries out codon optimization and synthesizes together,
The light chain of acquisition and heavy chain gene are cloned into pMD18-T expression by the heavy chain and light chain for obtaining complete antibody gene respectively respectively
In carrier.Plasmid extracts the amount of 100-150ug respectively, and removes endotoxin (＜ 1EU/mg), by 2 plasmids 1 of acquisition:1 corotation
In the competence TG1 cells for contaminating the suspension of 50ml volumes, transfection reagent uses PEI, and the ratio of plasmid and PEI are 1:2.Transfection
After 3-7 days, cell conditioned medium is collected, whether SDS-PAGE detection antibody correctly expresses, and ProteinA column purifications are closed after concentration
The VEGF-C antibody VEGFc419 of lattice, purity are more than 98%.
Six, VEGF-C detects the preparation of CLIA kits
(1) monoclonal antibody is coated with：It is reaction support, the optimal monoclonal antibody that will be filtered out with Nunc chemiluminescence reaction plates
VEGFc419 is diluted to 2.5ug/ml with 0.5mPBS.Be coated with into chemiluminescence blank plate with the holes 100ul/, 4 degree 24 hours
Afterwards, with the holes 0.9%Nacl 300ul/ board-washing 3 times, the 0.5mol PBS/ hole 150ul closings containing 1%BSA are added, it is 4 degree 12 small
When dry it is spare.
(2) it is loaded：Dilution prepare VEGF-C antigens dilute in proportion 6 gradients (0,10,50,100,200,400,
800pg/m) and test serum sample, each holes 50ul/ are added in the coating plate of coating VEGFc419, and the holes 50ul/ add biology
Elementization VEGF-C it is mostly anti-and be marked with the Streptavidin of horseradish peroxidase (0.5mPBS, 1:10000 dilutions), 37 DEG C of temperature
1h is educated, PBST is washed 5 times, removes anti-and Streptavidin-horseradish peroxidase more than unbonded VEGF.
(3) luminol-holes peroxide chemical luminous substrate 50ul/ are added, utilize TZD-CL-200S chemiluminescence immunoassays
Analyzer detects chemiluminescence intensity (RLU).
Seven, the performance of VEGF-C detection kits
(1) analytical performance of VEGF-C detection kits
Repeatability：The variation within batch coefficient (CV) of VEGF-C detection kits is not more than 10%；Interassay coefficient of variation (CV)
No more than 15%；
Sensitivity for analysis：Kit minimum detectability is not more than 30pg/mL；
Analysis specificity：A certain concentration specificity substance (FGF2, EGF) testing result is not more than 50pg/mL；
The range of linearity：In 0-800pg/mL concentration ranges, linearly dependent coefficient r is not less than 0.9900.
(2) clinical performance of VEGF-C detection kits
Carry out 299 clinical sample detections, 156 normal persons, 143 breast cancer Lymph Node Metastasis patients.Kit is normal
Reference value 0-135pg/ml, kit testing result are as follows：
Coincidence rate and its confidence interval
Crosstab
It counts
Coincidence rate and its confidence interval
Symmetry magnitude
A. without assuming empty hypothesis.
B. assume empty hypothesis using asymptotic standard error.
Consistency coefficient Kappa (K) value is：0.980。
Vascular endothelial growth factor C (VEGF-C) chemiluminescence detection kit breast cancer Lymph Node Metastasis coincidence rate is
99%, the effective clinical of breast cancer Lymph Node Metastasis can be monitored.
SEQUENCE LISTING
<110>The Zhejiang bio tech ltd Zhong Yi
<120>A kind of VEGF-C monoclonal antibodies and kit
<130>
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 419
<212> PRT
<213>VEGF-C monoclonal antibody heavies variable region
<400> 1
Met His Leu Leu Gly Phe Phe Ser Val Ala Cys Ser Leu Leu Ala Ala
1 5 10 15
Ala Leu Leu Pro Gly Pro Arg Glu Ala Pro Ala Ala Ala Ala Ala Phe
20 25 30
Glu Ser Gly Leu Asp Leu Ser Asp Ala Glu Pro Asp Ala Gly Glu Ala
35 40 45
Thr Ala Tyr Ala Ser Lys Asp Leu Glu Glu Gln Leu Arg Ser Val Ser
50 55 60
Ser Val Asp Glu Leu Met Thr Val Leu Tyr Pro Glu Tyr Trp Lys Met
65 70 75 80
Tyr Lys Cys Gln Leu Arg Lys Gly Gly Trp Gln His Asn Arg Glu Gln
85 90 95
Ala Asn Leu Asn Ser Arg Thr Glu Glu Thr Ile Lys Phe Ala Ala Ala
100 105 110
His Tyr Asn Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu Trp Arg Lys
115 120 125
Thr Gln Cys Met Pro Arg Glu Val Cys Ile Asp Val Gly Lys Glu Phe
130 135 140
Gly Val Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val Tyr
145 150 155 160
Arg Cys Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln Cys Met Asn Thr
165 170 175
Ser Thr Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro Leu
180 185 190
Ser Gln Gly Pro Lys Pro Val Thr Ile Ser Phe Ala Asn His Thr Ser
195 200 205
Cys Arg Cys Met Ser Lys Leu Asp Val Tyr Arg Gln Val His Ser Ile
210 215 220
Ile Arg Arg Ser Leu Pro Ala Thr Leu Pro Gln Cys Gln Ala Ala Asn
225 230 235 240
Lys Thr Cys Pro Thr Asn Tyr Met Trp Asn Asn His Ile Cys Arg Cys
245 250 255
Leu Ala Gln Glu Asp Phe Met Phe Ser Ser Asp Ala Gly Asp Asp Ser
260 265 270
Thr Asp Gly Phe His Asp Ile Cys Gly Pro Asn Lys Glu Leu Asp Glu
275 280 285
Glu Thr Cys Gln Cys Val Cys Arg Ala Gly Leu Arg Pro Ala Ser Cys
290 295 300
Gly Pro His Lys Glu Leu Asp Arg Asn Ser Cys Gln Cys Val Cys Lys
305 310 315 320
Asn Lys Leu Phe Pro Ser Gln Cys Gly Ala Asn Arg Glu Phe Asp Glu
325 330 335
Asn Thr Cys Gln Cys Val Cys Lys Arg Thr Cys Pro Arg Asn Gln Pro
340 345 350
Leu Asn Pro Gly Lys Cys Ala Cys Glu Cys Thr Glu Ser Pro Gln Lys
355 360 365
Cys Leu Leu Lys Gly Lys Lys Phe His His Gln Thr Cys Ser Cys Tyr
370 375 380
Arg Arg Pro Cys Thr Asn Arg Gln Lys Ala Cys Glu Pro Gly Phe Ser
385 390 395 400
Tyr Ser Glu Glu Val Cys Arg Cys Val Pro Ser Tyr Trp Lys Arg Pro
405 410 415
Gln Met Ser
<210> 2
<211> 110
<212> PRT
<213>VEGF-C monoclonal antibody light chain variable regions
<400> 2
Ala His Tyr Asn Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu Trp Arg
1 5 10 15
Lys Thr Gln Cys Met Pro Arg Glu Val Ala Ile Asp Val Gly Lys Glu
20 25 30
Phe Gly Val Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val
35 40 45
Tyr Arg Cys Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln Cys Met Asn
50 55 60
Thr Ser Thr Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro
65 70 75 80
Leu Ser Gln Gly Pro Lys Pro Val Thr Ile Ser Phe Ala Asn His Thr
85 90 95
Ser Cys Arg Cys Met Ser Lys Leu His His His His His His
100 105 110
<210> 3
<211> 36
<212> DNA
<213>Artificial sequence
<400> 3
ataccccgcc acggaccggt cccccacccc cggtcc 36
<210> 4
<211> 29
<212> DNA
<213>Artificial sequence
<400> 4
ttcatattgg aaagaccaca aatgagcta 29
Claims (5)
1. a kind of VEGF-C monoclonal antibodies, which is characterized in that the heavy chain variable amino acid sequence of the antibody is：Met
His Leu Leu Gly Phe Phe Ser Val Ala Cys Ser Leu Leu Ala Ala Ala Leu Leu Pro
Gly Pro Arg Glu Ala Pro Ala Ala Ala Ala Ala Phe Glu Ser Gly Leu Asp Leu Ser
Asp Ala Glu Pro Asp Ala Gly Glu Ala Thr Ala Tyr Ala Ser Lys Asp Leu Glu Glu
Gln Leu Arg Ser Val Ser Ser Val Asp Glu Leu Met Thr Val Leu Tyr Pro Glu Tyr
Trp Lys Met Tyr Lys Cys Gln Leu Arg Lys Gly Gly Trp Gln His Asn Arg Glu Gln
Ala Asn Leu Asn Ser Arg Thr Glu Glu Thr Ile Lys Phe Ala Ala Ala His Tyr Asn
Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu Trp Arg Lys Thr Gln Cys Met Pro Arg
Glu Val Cys Ile Asp Val Gly Lys Glu Phe Gly Val Ala Thr Asn Thr Phe Phe Lys
Pro Pro Cys Val Ser Val Tyr Arg Cys Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln
Cys Met Asn Thr Ser Thr Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro
Leu Ser Gln Gly Pro Lys Pro Val Thr Ile Ser Phe Ala Asn His Thr Ser Cys Arg
Cys Met Ser Lys Leu Asp Val Tyr Arg Gln Val His Ser Ile Ile Arg Arg Ser Leu
Pro Ala Thr Leu Pro Gln Cys Gln Ala Ala Asn Lys Thr Cys Pro Thr Asn Tyr Met
Trp Asn Asn His Ile Cys Arg Cys Leu Ala Gln Glu Asp Phe Met Phe Ser Ser Asp
Ala Gly Asp Asp Ser Thr Asp Gly Phe His Asp Ile Cys Gly Pro Asn Lys Glu Leu
Asp Glu Glu Thr Cys Gln Cys Val Cys Arg Ala Gly Leu Arg Pro Ala Ser Cys Gly
Pro His Lys Glu Leu Asp Arg Asn Ser Cys Gln Cys Val Cys Lys Asn Lys Leu Phe
Pro Ser Gln Cys Gly Ala Asn Arg Glu Phe Asp Glu Asn Thr Cys Gln Cys Val Cys
Lys Arg Thr Cys Pro Arg Asn Gln Pro Leu Asn Pro Gly Lys Cys Ala Cys Glu Cys
Thr Glu Ser Pro Gln Lys Cys Leu Leu Lys Gly Lys Lys Phe His His Gln Thr Cys
Ser Cys Tyr Arg Arg Pro Cys Thr Asn Arg Gln Lys Ala Cys Glu Pro Gly Phe Ser
Tyr Ser Glu Glu Val Cys Arg Cys Val Pro Ser Tyr Trp Lys Arg Pro Gln Met Ser；
Chain variable region amino acid sequence is：Ala His Tyr Asn Thr Glu Ile Leu Lys Ser Ile Asp
Asn Glu Trp Arg Lys Thr Gln Cys Met Pro Arg Glu Val Ala Ile Asp Val Gly Lys
Glu Phe Gly Val Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val Tyr Arg
Cys Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln Cys Met Asn Thr Ser Thr Ser Tyr
Leu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro Leu Ser Gln Gly Pro Lys Pro Val
Thr Ile Ser Phe Ala Asn His Thr Ser Cys Arg Cys Met Ser Lys Leu His His His
His His His。
2. a kind of polynucleotides of coding light chain variable region and heavy chain variable amino acid sequence described in claim 1.
3. a kind of recombinant dna expression vector including polynucleotides as claimed in claim 2, the recombinant dna expression vector
In include coding VEGF-C monoclonal antibody heavies variable region, light chain variable region and the DNA sequence dna with antibody constant region.
4. a kind of detection kit including VEGF-C monoclonal antibodies described in claim 1.
5. the preparation method of detection kit described in claim 4, which is characterized in that with Nunc chemiluminescence reaction plates be reaction
VEGF-C monoclonal antibodies are diluted to 2.5ug/ml with 0.5mPBS, are coated with to chemiluminescence blank with the holes 100ul/ by support
In plate, at 4 DEG C after 24 hours, with 0.9%Nacl board-washings 3 times, the 0.5mol PBS closings containing 1%BSA are added, 12 at 4 DEG C
It is dried after hour.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810683404.3A CN108640994A (en) | 2018-06-28 | 2018-06-28 | A kind of VEGF-C monoclonal antibodies and kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810683404.3A CN108640994A (en) | 2018-06-28 | 2018-06-28 | A kind of VEGF-C monoclonal antibodies and kit |
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Family
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| CN201810683404.3A Withdrawn CN108640994A (en) | 2018-06-28 | 2018-06-28 | A kind of VEGF-C monoclonal antibodies and kit |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023016516A1 (en) * | 2021-08-13 | 2023-02-16 | 信达生物制药(苏州)有限公司 | Anti-vegf a and -vegf c bispecific antibody and use thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1338943A (en) * | 1998-10-26 | 2002-03-06 | 路德维格癌症研究院 | Use of VEGF-C or VEGF-D gene or protein to prevent resitenosis |
| CN105682675A (en) * | 2013-08-14 | 2016-06-15 | 拉伦蒂斯制药有限公司 | Therapeutic use of VEGF-C and CCBE1 |
-
2018
- 2018-06-28 CN CN201810683404.3A patent/CN108640994A/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1338943A (en) * | 1998-10-26 | 2002-03-06 | 路德维格癌症研究院 | Use of VEGF-C or VEGF-D gene or protein to prevent resitenosis |
| CN105682675A (en) * | 2013-08-14 | 2016-06-15 | 拉伦蒂斯制药有限公司 | Therapeutic use of VEGF-C and CCBE1 |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023016516A1 (en) * | 2021-08-13 | 2023-02-16 | 信达生物制药(苏州)有限公司 | Anti-vegf a and -vegf c bispecific antibody and use thereof |
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