CN108508216A - The method and kit of anti-human bFGF nano antibodies detection bFGF - Google Patents
The method and kit of anti-human bFGF nano antibodies detection bFGF Download PDFInfo
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- CN108508216A CN108508216A CN201810574975.3A CN201810574975A CN108508216A CN 108508216 A CN108508216 A CN 108508216A CN 201810574975 A CN201810574975 A CN 201810574975A CN 108508216 A CN108508216 A CN 108508216A
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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
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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
- C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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- 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
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
Abstract
The invention discloses methods and kit that anti-human bFGF nano antibodies detect bFGF.The present invention provides the kits of the detection bFGF comprising the anti-human bFGF nano antibodies, especially detect the double-antibody sandwich elisa kit of bFGF；And it has been successfully established the double-antibodies sandwich ELISA of the non-diagnosis for disease or treatment detection bFGF, high specificity, detection result is good.The anti-human bFGF nano antibodies of the present invention can be expressed by prokaryotic system, easy to operate, reduced production cost, be easy to industrial production；The anti-human bFGF nano antibodies are other than possessing the features such as small molecular mass, high specificity, affinity are high, antigen binding capacity is strong, compared to traditional bFGF antibody, it also has the characteristics that good stability, high temperature resistant, easy to maintain, it is more suitable for the exploitation of detection reagent product, the detection reagent product of gained is more suitable for preserving, transport and using, and has a good application prospect.
Description
Technical field
The invention belongs to biotechnologies, more particularly to the method using anti-human bFGF nano antibodies detection bFGF and examination
Agent box.
Background technology
Antibody (Antibody) is also immunoglobulin (Immunoglobulin), is a kind of energy molecule of the antigen binding
Glycoprotein.Conventional antibodies exist with one or more " Y " font monomer, and each " Y " font monomer is by 4 polypeptide chain groups
At including two identical heavy chains and two identical light chains.Conventional antibodies molecular weight is big, complicated, causes its stability
It is poor with penetrability.And research and development and production cost are high, cause its cost of use high, limit it and further apply.Therefore, one
The research and development of a little new small molecule antibody become research emphasis, such as：Fab antibody, F (ab)2Antibody, is received single-chain antibody (scFv)
Meter Kang Ti etc..
1993, Hamers Casterman etc. reported in camel blood that there is the peculiar anti-of natural deletions light chain for the first time
Body, i.e. heavy chain antibody (Heavy-Chain Antibody, HCAb).Its variable region is cloned by technique for gene engineering, can be obtained
Contain only the antibody fragment of heavy chain variable region composition, i.e. single domain heavy chain antibody VHH (Variable Domain of Heavy
Chain of Heavy-Chain Antibody).Since its molecular weight only has 1/10th of conventional antibodies, and length is even more
In several nanometers of rank, therefore also referred to as nano antibody (Nanobody, Nb).It has molecular mass is small, stability is high,
Easily expression, cell/penetrability of vessels is strong, antigen binding capacity is strong, the features such as being expressed in prokaryotic expression system, have both
The advantage of conventional antibodies and small-molecule drug, the almost ideal development cycle for overcoming conventional antibodies is long, stability is relatively low, protects
The defects of condition is harsh is deposited, also the easily mutual adhesion unlike the small molecular antibodies such as single-chain antibody (scFv), or even aggregation is blocking,
Therefore as the novel antibody molecules of antibody drug and reagent research field, there is good development prospect.Currently, nano antibody
Gradually it is applied to the fields such as disease treatment, medical diagnosis on disease, radiological imaging.
Basic fibroblast growth factor (basic Fibroblast Growth Factor, bFGF), also known as at
Fibroblast growth factor (Fibroblast Growth Factor-2, FGF-2), be 1974 by Gospodarowiz etc. from
The fibroblastic mitogen purified in the extract of bovine brain hypophysis and brain tissue, it is by 146 amino acid groups
At, because its isoelectric point is 9.6, therefore referred to as basic fibroblast growth factor.The biological action of bFGF is extremely extensive, it
Vascularization promotes wound healing and tissue repair, promotes to play very in regeneration and nerve fiber growth and development process
Important role.
In some tumor diseases, unconventionality expression and tumor development, metastases, angiogenesis and the prognosis of bFGF
Closely related, this is primarily due to bFGF and not only promotees tumor by local angiogenesis by approach such as paracrine and autocrines, moreover it is possible to
Directly effect tumour cell promotes it to be proliferated and accelerate tumor-infiltrated and transfer.And internal and external test shows that bFGF can promote
The secretion of a variety of growth factors has synergistic effect with other angiogenesis promoting factor Ⅴs EGF, PDGF etc., collectively promotes tumour hair
It is raw.In addition, kinds of tumors can be overexpressed by adjusting bFGF, changes bFGF/FGFR signal paths, reach promotion tumour growth.
BFGF participates in the growth of a variety of (such as glioma, rhabdomyoma, leukaemia, kidney, pneumonocyte cancer, melanoma, lung cancer),
There is substantial portion of tumour the high expression of bFGF and bFGF receptors occur.Therefore, it fast and accurately detects in tumour cell
The content of bFGF all has the diagnosing and treating of disease certain necessity.
Presently commercially available bFGF reagent box for detecting content (ELISA method) is mostly tradition IgG class antibody.Traditional IgG classes antibody
Production rely on mammalian cell, the production cycle is long, of high cost, expensive, needs Cord blood, these features limit its
Application in detection kit.
Invention content
The primary purpose of the present invention is that the shortcomings that overcoming the prior art and deficiency, provide a kind of reagent of detection bFGF
Box, the kit contain anti-human bFGF nano antibodies, can be in the detection of bFGF.
It is another object of the present invention to provide the method that above-mentioned anti-human bFGF nano antibodies detect bFGF, described is anti-
People bFGF nano antibodies can specifically bind people bFGF (i.e. FGF-2), and double-antibody sandwich elisa side is established based on the nano antibody
Method is detected antigen bFGF.The heavy chain antibody found from camellid i.e. nano antibody, because of its structural particularity, tool
Have high specificity, affinity are high, stability is good, high temperature resistant, it is easy to maintain the features such as, and since prokaryotic system expression can be used very in it
Big degree reduces production cost, is solved well in the application of detection kit more than existing for traditional IgG classes antibody
Problem.
The purpose of the invention is achieved by the following technical solution：
A kind of kit of detection bFGF, the kit contain anti-human bFGF nano antibodies, described anti-human bFGF nanometers
Antibody includes the Variable domain being made of framework region FR and complementary determining region CDR, and the Variable domain includes choosing
From CDR1, CDR2 and CDR3 below：
(1) CDR1 shown in any amino acid sequence in No.1~4 SEQ ID,
(2) CDR2 shown in any amino acid sequence in No.5~8 SEQ ID；
(3) CDR3 shown in any amino acid sequence in No.9~12 SEQ ID.
The Variable domain includes selected from any one of following CDR1, CDR2 and CDR3：
(1) shown in CDR2 and SEQ ID No.9 shown in CDR1 shown in SEQ ID No.1, SEQ ID No.5
CDR3；
(2) CDR2 and SEQ ID shown in CDR1 shown in SEQ ID No.2, SEQ ID No.5 or SEQ ID No.6
CDR3 shown in No.9 or SEQ ID No.10；
(3) shown in CDR2 and SEQ ID No.11 shown in CDR1 shown in SEQ ID No.3, SEQ ID No.7
CDR3；
(4) shown in CDR2 and SEQ ID No.12 shown in CDR1 shown in SEQ ID No.4, SEQ ID No.8
CDR3。
Described framework region FR FR1, FR2, FR3 and the FR4 selected from the group below：
(1) FR1 shown in any amino acid sequence in No.13~15 SEQ ID；
(2) FR2 shown in any amino acid sequence in No.16~19 SEQ ID；
(3) FR3 shown in any amino acid sequence in No.20~24 SEQ ID；
(4) FR4 shown in any amino acid sequence in No.25~27 SEQ ID.
The anti-recombination human basic fibroblast growth factor nano antibody, amino acid sequence are selected from as follows
Amino acid sequence in any one (NO.28~33 SEQ ID)；It is specific as follows：
The nucleotide sequence of the amino acid sequence as shown in NO.28~33 SEQ ID is encoded successively such as SEQ ID NO.34
Shown in~39.
A kind of double-antibody sandwich elisa kit of detection bFGF, including：
(1) using the anti-human bFGF nano antibodies with following Variable domain as primary antibody (capturing antibody)：
1. shown in CDR2 and SEQ ID No.9 shown in CDR1 shown in SEQ ID No.1, SEQ ID No.5
CDR3；
2. CDR2 and SEQ ID shown in CDR1 shown in SEQ ID No.2, SEQ ID No.5 or SEQ ID No.6
CDR3 shown in No.9 or SEQ ID No.10；
3. shown in CDR2 and SEQ ID No.12 shown in CDR1 shown in SEQ ID No.4, SEQ ID No.8
CDR3。
(2) using the anti-human bFGF nano antibodies with following Variable domain as secondary antibody (detecting antibody)：
The CDR1 as shown in SEQ ID No.3, the CDR2 as shown in SEQ ID No.7 and as shown in SEQ ID No.11
CDR3。
The amino acid sequence of the anti-human bFGF nano antibodies as primary antibody preferably as No.28~30 SEQ ID,
Amino acid sequence shown in any one of SEQ ID No.32 or SEQ ID No.33；Further preferably such as SEQ ID No.33
Shown in amino acid sequence.
The amino acid sequence of the anti-human bFGF nano antibodies as secondary antibody is preferably as shown in SEQ ID No.31
Amino acid sequence.
The kit is preferably cancer diagnosing kit；The further preferably cancer diagnosing agent of detection people bFGF
Box.
The nano antibody by 342 bases, 336 bases or 369 base compositions, 114 amino acid of coding,
112 amino acid or 123 amino acid are made of 4 framework regions (FR) of antibody and 3 complementary determining regions (CDR)：Nano antibody
CDR1 encode 10 amino acid, CDR2 encode 7 amino acid, CDR3 encode 11 amino acid；Or CDR1 encodes 10 amino
Acid, CDR2 encode 7 amino acid, and CDR3 encodes 9 amino acid；Or CDR1 encodes 10 amino acid, CDR2 encodes 8 amino
Acid, CDR3 encode 19 amino acid；3 CDR regions are specific sequence.
The nano antibody is made of 4 framework regions (FR) and 3 complementary determining regions (CDR).The nano antibody
Function is to determine specific nucleotide sequences in race CDR1, CDR2, CDR3 (being the functional activity area of the present invention) by antibody antigen
It determines, corresponding amino acid sequence constitutes the antigen binding domains antibody specificity bFGF.
It is a kind of detection bFGF double-antibodies sandwich ELISA, with its amino acid sequence such as No.28~30 SEQ ID,
Anti-human bFGF nano antibodies are as primary antibody shown in any one of SEQ ID No.32 or SEQ ID No.33；With its amino acid sequence
The anti-human bFGF nano antibodies of the amino acid sequence as shown in SEQ ID No.31 are arranged as secondary antibody；This method is not intended to disease
Diagnosis or treatment.
The double-antibodies sandwich ELISA of the detection bFGF specifically comprises the following steps：
(1) it is coated with：Coating plate be coated with overnight with the solution containing primary antibody；
(2) it closes：After washing is coated with the coating plate of primary antibody, closed；
(3) add antigen：The coating plate closed is washed, sample to be tested is added, is incubated；
(4) add secondary antibody：Washing is incubated the coating plate after sample to be tested, and secondary antibody is added, and is incubated；
(5) enzyme labeling antibody：Washing is incubated the coating plate after secondary antibody, and HRP labelled antibodies are added, and is incubated；
(6) after washing coating plate, TMB developing solutions are added, is protected from light incubation, absorbance value is measured at 450nm wavelength.
The concentration proportioning of the primary antibody and secondary antibody is preferably 5:2.
The cloning expression method of the anti-human bFGF nano antibodies, includes the following steps：
The nucleotide sequence of the coding anti-human bFGF nano antibodies is cloned into expression vector and builds recombinant expression
Carrier；The recombinant expression carrier is transferred in expression system again and carries out heterogenous expression；It is finally purified to obtain described resist
People's bFGF nano antibodies.
The expression vector is preferably pMECS or pNCS.
The nano antibody gene or the nano antibody gene complementation that contains based on this gene after reconstruction determines
Any gene in area can express in prokaryotic cell, yeast cells, insect cell and eukaryocyte, and obtaining has in conjunction with people BFGF
Antibody products, can be applied to：(1) diagnostic products of tumour are prepared；(2) antigen concentration detects.
Compared with prior art, the present invention having the following advantages that and advantageous effect：
(1) anti-human bFGF nano antibodies are in addition to possessing small molecular mass, high specificity, affinity height, antigen binding capacity
Outside the features such as strong, compared to tradition bFGF antibody, it also has the characteristics that good stability, high temperature resistant, easy to maintain, growth cost is low, more
Suitable for the exploitation of detection reagent product, the detection reagent product of gained is more suitable for preserving, transport and using.
(2) present invention has good detection result, and anti-human bFGF nano antibodies are combined especially with antibody conjugates
NbbFGF6 and NbbFGFWhen 4 pairs of samples carry out double crush syndrome detection, there are no its ranges of linearity of addition signal amplification molecule
10~400ng/mL can be reached, lowest detection is limited to 7.81ng/mL.
(3) anti-human bFGF nano antibodies of the invention can be expressed by prokaryotic system, easy to operate, reduced and be produced into
This, is easy to industrial production and promotes.
Description of the drawings
Fig. 1 is gene electrophoretogram when building pMECS-Nbs (His-HA tag) recombinant plasmid.
Fig. 2 is gene electrophoretogram when building pNCS-Nbs (His-Flag tag) recombinant plasmid.
Fig. 3 is the SDS-PAGE analysis charts after six plants of bFGF nano antibodies (His-HA tag) expression.
Fig. 4 is the Western Blot analysis result figures of six plants of bFGF nano antibodies (His-HA tag) expression.
Fig. 5 is six plants of bFGF nano antibodies (His-HA tag) Ni-NTA affinitive layer purification figures.
Fig. 6 is six plants of bFGF nano antibodies (His-HA tag) the SDS-PAGE analyses of sample and Western after purification
Blot analysis result figures.
Fig. 7 is the SDS-PAGE analysis result figures of two plants of bFGF nano antibodies (His-Flag tag) expression of results.
Fig. 8 is the Western Blot analysis charts of two plants of bFGF nano antibodies (His-Flag tag) expression of results.
Fig. 9 is two plants of bFGF nano antibodies (His-Flag tag) Ni-NTA affinitive layer purification figures.
Figure 10 is two plants of bFGF nano antibodies (His-Flag tag) the SDS-PAGE analyses of sample and Western after purification
Blot analysis result figures.
Figure 11 is that bFGF nano antibodies specifically bind interpretation of result figure with bFGF；Wherein 1 is positive control, and 2~7 indicate
Different nano antibody clone strains, is followed successively by Nb1~Nb6.
Figure 12 is bFGF nano antibody thermostabilization analysis of experimental results figures；Wherein bFGF mAb be positive control, Nb1~
Nb6 indicates different nano antibody clone strains.
Figure 13 is bFGF2 nano antibodies and the binding site comparative analysis figure of FGFR2；Wherein Nb1~Nb6 indicates different
Nano antibody clone strain.
Figure 14 is bFGF nano antibodies and bFGF mAb binding site comparative analysis figures；Wherein Nb1~Nb6 indicates different
Nano antibody clone strain.
Figure 15 is bFGF nano antibodies pairing analysis of experimental results figure；Wherein figure A~F is followed successively by a concentration of 2 μ g/mL, 1 μ
The Nb of g/mL, 0.5 μ g/mL, 0.25 μ g/mL, 0.125 μ g/mL, 0.0625 μ g/mLbFGF4 (His-Flag tag) respectively with its
The pairing experimental result of remaining bFGF nano antibodies.
Figure 16 is the antigen bFGF Concentration Testing area Results analysis charts for the double sandwich-ELISA method established.
Figure 17 is the double sandwich-ELISA method detection antigen bFGF concentration standard curve figures established.
Figure 18 is the interpretation of result figure of specific test in embodiment 4.
Specific implementation mode
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
The construction method of embodiment 1 anti-recombination human basic fibroblast growth factor (bFGF) nano antibody
1, the structure of pMECS-Nbs (His-HA tag) recombinant plasmid
1.1 design of primers
NbbFGF 1、NbbFGF 2、NbbFGF 3、NbbFGF6 sense primer is as shown in SEQ ID No.40, and downstream primer is such as
Shown in SEQ ID No.41, NbbFGF4 upstream and downstream primer, NbbFGF5 upstream and downstream primer is successively such as No.42~45 SEQ ID
It is shown.
1.2PCR
Use the PrimeSTAR of TaKaRa companies@HS DNA Polymerase carry out PCR to nano antibody target gene
Reaction, electrophoresis result are shown in Fig. 1.
1.3 endonuclease reaction
Using the FastDigest Nco I and FastDigest Not I of Thermo companies respectively to carrier pMECS (purchases
From Chinese plasmid vector bacterium cell gene collection) and NbbFGF1~NbbFGF6 gene carries out double digestion；It uses
FastDigest Pst I and FastDigest the Not I of Thermo companies are to carrier pMECS and NbbFGF5 genes carry out double
Digestion.
1.4 connection reactions
Using the T4DNA Ligase of Thermo companies respectively to carrier pMECS and NbbFGF1 gene；Carrier pMECS and
NbbFGF2 genes；Carrier pMECS and NbbFGF3 genes；Carrier pMECS and NbbFGF4 genes；Carrier pMECS and NbbFGF5 bases
Cause；Carrier pMECS and NbbFGF6 genes are attached.
1.5 conversion
PMECS-Nbs electricity is transferred to WK6 competent cells.
Sequencing result shows recombinant plasmid pMECS-NbbFGF 1、pMECS-NbbFGF 2、pMECS-NbbFGF 3、pMECS-
NbbFGF 4、pMECS-NbbFGF 5、pMECS-NbbFGF6 build successfully.
2, the structure of pNCS-Nbs (His-Flag tag) recombinant plasmid
2.1 design of primers
NbbFGF2 upstream and downstream primer, NbbFGF4 upstream and downstream primer is successively such as SEQ ID No.46~SEQ ID
Shown in No.49.
2.2PCR
PCR reactions are carried out to nano antibody target gene using the Pfu Kit of Dongsheng biology, electrophoresis result is shown in Fig. 2.
2.3 endonuclease reaction
Using FastDigest BamH I and FastDigest the Xhol I of Thermo companies to carrier pNCS (in being purchased from
State's plasmid vector bacterium cell gene collection) and NbbFGF2 genes, NbbFGF4 genes carry out double digestion.
2.4 connection reactions
Using the T4DNA Ligase of Thermo companies to carrier pNCS and NbbFGF2 genes, carrier pNCS and NbbFGF 4
Gene is attached.
2.5 conversion
PNCS-Nbs is transformed into DH5a competent cells.
Sequencing result shows recombinant plasmid pNCS-NbbFGF2 and pNCS-NbbFGF4 build successfully.
3, the expression and purifying of bFGF nano antibodies
The expression and purifying of 3.1bFGF nano antibodies (His-HA tag)
(1) the strain plate streaking that will be frozen, to choose positive monoclonal.(2) positive monoclonal addition 5mL is contained
In the TB culture mediums of the final concentration of 100 μ g/mL of Amp, 37 DEG C, 220rpm shaken cultivations 11~13 hours are used as first order seed.
(3) first order seed bacterium solution is taken, is seeded in 0.1% ratio in the 30mLTB culture mediums of the final concentration of 100 μ g/mL of Amp, and is added
Enter 2mM MgCl2Solution and 0.1% (w/v) glucose solution, 37 DEG C, 220rpm shaken cultivations 8 hours are used as secondary seed.
(4) secondary seed bacterium solution is taken, is seeded in 1% ratio in the large volume TB culture mediums of the final concentration of 100 μ g/mL of Amp, simultaneously
2mM MgCl are added2Solution and 0.1% (w/v) glucose solution, 37 DEG C, 220rpm shaken cultivations to OD600In 0.8 to 1.0 models
In enclosing.(5) IPTG is added, makes its final concentration of 1mM, 28 DEG C, 220rpm continues shaken cultivation 12h.(6) 6,000rpm is centrifuged
30min.Take bacterial sediment.(7) use 1X PBS that simultaneously washing thalline precipitation, 8,000g centrifugation 20min are resuspended.Take bacterial sediment.It should
Step is in triplicate.(8) bacterial sediment is weighed, and according to l:1X PBS buffer solution is added in 10 ratio, and thalline is resuspended.
(9) under condition of ice bath, the ultrasonic probe of Ultrasonic Cell Disruptor is inserted under thalline suspension, setup parameter power：800W；Ultrasound
Time：3s；Intermittent time：5s.Ultrasound to thalline light transmittance obviously rises.10,000g centrifuges 90min under the conditions of 4 DEG C, takes supernatant
Affinitive layer purification is carried out with Ni-NTA columns, the destination protein solution after elution carries out de- miaow with SephadexG-25 molecular sieve columns
Azoles processing, is finally concentrated with 3K super filter tubes, and sample after concentration is measured its concentration by BCA methods.
Experimental result is as shown in figure 3, by SDS-PAGE electrophoresis, and compared with WK6 empty bacterium control groups, 6 plants bFGF nanometers anti-
Full bacterium of the body (His-HA tag) after being induced by IPTG, cellular lysate supernatant precipitation in, have at about 18kDa
Apparent band illustrates that 6 plants of bFGF nano antibodies (His-HA tag) have an a certain amount of expression, and with solubility expression with forgive
Two kinds of forms of body exist simultaneously.Each nano antibody expression Supernatant samples are taken to carry out Western Blot verifications, as a result such as Fig. 4 institutes
Show, further demonstrating 6 plants of bFGF nano antibodies (His-HA tag) has soluble-expression.
Affinitive layer purification is carried out to sample after expression, the results are shown in Figure 5.Will after purification sample carry out SDS-PAGE and
Western Blot analyses (see Fig. 6) as a result show and have obtained the Nb that purity is 95.24% after purificationbFGF 1(His-HA
tag)；The Nb that purity is 96.72%bFGF2(His-HA tag)；The Nb that purity is 98.87%bFGF3(His-HA tag)；It is pure
The Nb that degree is 97.51%bFGF4(His-HA tag)；The Nb that purity is 93.61%bFGF5 (His-HA tag) and purity are
96.19% NbbFGF 6(His-HA tag)。
The expression and purifying of 3.2bFGF nano antibodies (His-Flag tag)
(1) the strain plate streaking that will be frozen, to choose positive monoclonal.(2) 5mL LB+ are added in positive monoclonal
In Amp culture mediums, 37 DEG C, 200rpm 11~13h of shaken cultivation, it is used as first order seed.(3) first order seed bacterium solution is taken, by 1%
Ratio is seeded in the large volume LB culture mediums containing the final concentration of 100 μ g/mL of Amp, 37 DEG C, and 200rpm shaken cultivations 11~
13h.(4) 6,000rpm centrifuges 30min.Take bacterial sediment.(5) use 1X PBS that simultaneously washing thalline precipitation, 8,000g centrifugations are resuspended
20min.Take bacterial sediment.The step is in triplicate.(6) bacterial sediment is weighed, and according to l:1X is added in 10 ratio
Thalline is resuspended in PBS buffer solution.(7) under condition of ice bath, the ultrasonic probe of Ultrasonic Cell Disruptor is inserted under thalline suspension, if
Determine parameter power：800W；Ultrasonic time：4s；Intermittent time：4s；Ultrasound to thalline light transmittance obviously rises.Under the conditions of (8) 4 DEG C
10,000g centrifuges 90min.Take supernatant for protein purification.(9) the thalline ultrasonication after great expression, 10 under the conditions of 4 DEG C,
000g centrifuges 90min, and supernatant is taken to carry out affinitive layer purification with Ni-NTA columns, and the destination protein solution after elution is used
SephadexG-25 molecular sieve columns carry out de- imidazoles processing, are finally concentrated with 3K super filter tubes, and sample after concentration is passed through
BCA methods measure its concentration.
There are a certain amount of expression as shown in fig. 7, showing two plants of bFGF nano antibodies (His-Flag tag) for experimental result, and
Predominantly soluble-expression form.Two plants of bFGF nano antibodies (His-Flag tag) expression Supernatant samples are taken to carry out Western
Blot is verified, and the results are shown in Figure 8, and further demonstrating two plants of bFGF nano antibodies (His-HA tag) has soluble-expression.
Expression sample is subjected to affinitive layer purification, the results are shown in Figure 9.SDS-PAGE and Western will be carried out by sample after purification
Blot analyzes (see Figure 10), as a result shows and obtains the Nb that purity is 92.58% after purificationbFGF2 (His-Flag tag) and pure
The Nb that degree is 97.81%bFGF 4(His-Flag tag)。
2 anti-bFGF nano antibodies physicochemical property of embodiment and biological activity test
1, the specificity analysis of anti-bFGF nano antibodies
BFGF, aFGF, KGF, EGF, TNF α, the VEGF that the holes 50ng/ are added into 96 hole elisa Plates (are purchased from Beijing justice to stick up
Divine Land Bioisystech Co., Ltd), BSA, milk, in 4 DEG C coating overnight.Next day, with 5% skim milk closing ELISA Plate 2h
Afterwards, positive control bFGF mAb (being purchased from sigma companies), blank control PBS and the reality in a concentration of holes 100ng/ are separately added into
Apply anti-bFGF nano antibodies made from example 1,37 DEG C of incubation 1h.Anti- Flag (the being purchased from sigma companies) mark in the holes 100ng/ is added afterwards
Antibody is signed, 1h is incubated.The TMB developing solutions in 100 holes μ L/ are added, are incubated 10min.It is finally terminated and is reacted with 2.29% sulfuric acid, measured
Light absorption value at 450nm.With anti-bFGF nano antibodies antigen light absorption value 3 is compareed with other with the absorbance value of corresponding antigen bFGF
Times or more antibody cloning be defined as the positive.
As a result as Figure 11 is indicated, there are specific binding capacity is strong and other 7 by 6 plants of anti-bFGF nano antibodies and bFGF
The binding ability for compareing antigen is weaker.
2, the affinity determination of anti-bFGF nano antibodies
Using the affinity of the anti-bFGF nano antibodies of surface plasma resonance biosensor assay.By 0.4mol N-
With 20 μ L/min after ethyl-N '-(dimethylamino-propyl) carbodiimides and the isometric mixing of 0.1mol n-hydroxysuccinimides
Flow velocity be passed through in instrument chip surface activated.BFGF albumen is diluted to the 0.2mol acetate buffer solutions of pH 4.2
After 2mg/mL, chip surface is flowed through.Fixed amount is passed through PBS buffer solution after reaching aequum.It is passed through 1mol ethanol amines (pH 8.5)
Solution 7min inactivates remaining ester bond.The anti-bFGF of various concentration (3.125,6.25,12.5,25,50,100,200nM) is received
Meter Kang Ti is passed through with the flow velocity of 20 μ L/min in instrument.The affinity of anti-bFGF nano antibodies is calculated by instrument software.As a result
As shown in table 1, there is anti-bFGF nano antibodies high-affinity, highest affinity to reach 0.69nM.Remaining anti-bFGF nano antibody
Affinity constant is respectively 11.32nM, 317.60nM, 5.37nM, 12.37nM and 10.32nM.
1 anti-bFGF nano antibodies affinity constant of table
| Nb1 | Nb2 | Nb3 | Nb4 | Nb5 | Nb6 | |
| KD(nM) | 11.32 | 317.60 | 5.37 | 0.69 | 12.37 | 10.32 |
3, the measurement of thermal stability
The obtained nano antibody (i.e. Nb1~6) of screening is respectively placed in 25 DEG C, 37 DEG C, 60 DEG C, 90 DEG C of four kinds of different temperatures
Under each 10min, 30min, 60min, 120min and 180min；Made simultaneously with bFGF monoclonal antibodies (Sigma, mouse source antibody)
For positive control.Collect different temperatures, different time treated sample, with ELISA method detection they with bFGF associativities, i.e.,
After the processing of different temperatures different time, whether antibody also has the ability combined with bFGF.Testing result Figure 12 show with not
The nano antibody of processing is compared, and after 25 DEG C and 37 DEG C processing, nano antibody relative activity is held in 90% or more, and passes through
After crossing 60 DEG C of processing, nano antibody relative activity is declined, but remains at 80% or more.But after 90 DEG C of processing,
Nano antibody relative activity is remarkably decreased, and relative activity is reduced to 0% when wherein Nb5 processing 60min, and Nb6 processing
Relative activity is just down to 0% after 180min, other several groups of nano antibody relative activities drop to 0% in 120min.This with
BFGF monoclonal antibodies as positive control are compared to having a clear superiority, although bFGF monoclonal antibodies are at 25 DEG C, 37 DEG C, 60
Relative activity under DEG C three kinds of different temperatures is 90% or more, but after 90 DEG C of processing, its relative activity is in 10min
Inside just have fallen to 0%.Therefore, illustrate the relatively common mouse monoclonal antibody of nano antibody, the stability in high-temperature process
It is better, advantage is had more as detection reagent antibody, can be extended the shelf life.
4, competion experiment
(1) nano antibody (Nb of the Inhibition ELISA detection present invention is usedbFGF) with FGFR2 (be purchased from Divine Land Yi Qiao, Beijing
Bioisystech Co., Ltd) competitive binding bFGF (be purchased from Sino Biological Inc.) ability.This experiment
The FGFR2 of the nano antibody of quantitative 100ng and variable (0~1 μ g) is added in the tablet of coating 50ng bFGF simultaneously and is incubated,
If with the raising of FGFR2 concentration, the content that nano antibody detected can be fewer and fewer, illustrates that nano antibody is same with FGFR2
The consistent either close or nano antibody of combination epitope of bFGF and the affinity of bFGF are not as good as FGFR2 with the affinity of bFGF；
If FGFR2 concentration gradually rises, the content of nano antibody does not change, then illustrates nano antibody with FGFR2 and with bFGF's
It is higher than the tightness degree that FGFR2 is combined with bFGF in conjunction with the tightness degree that epitope is inconsistent or nano antibody is combined with bFGF.
As a result such as Figure 13, with the increase of FGFR2 contents, Nb2 and Nb4 contents are always maintained at steadily, can speculate they and bFGF's
The affinity that combination epitope in conjunction with epitope and FGFR2 and bFGF is inconsistent or they are with bFGF is higher than FGFR2's and bFGF
Affinity.The binding force of tetra- plants of nano antibodies of Nb1, Nb3, Nb5 and Nb6 and bFGF are showed with the increase of FGFR2 concentration
The trend successively decreased illustrates that the combination epitope of four plants of nano antibodies and bFGF are consistent with the combination epitope of FGFR2 and bFGF or connect
Closely or their affinity with bFGF are less than the affinity of FGFR2 and bFGF.
(2) Inhibition ELISA is used to detect NbbFGF(it is purchased from Sigma with business mouse monoclonal antibody bFGF mAb (FB-8)
Company, article No. A8592-.2MG) competitive binding bFGF ability.Figure 14 shows increases of the Nb4 with bFGF mAb contents, contains
Amount is always maintained at steadily, can speculate that the combination epitope and the combination epitope of bFGF mAb and bFGF of it and bFGF are inconsistent, or
The affinity of person Nb4 and bFGF are higher than the affinity of bFGF mAb and bFGF.6 five plants of nanometers of Nb1, Nb2, Nb3, Nb5 and Nb are anti-
The binding force of body and bFGF show the trend successively decreased with the increase of bFGF mAb concentration, illustrate five plants of nano antibodies with
The combination epitope of bFGF is consistent or close with the combination epitope of bFGF mAb and bFGF or the affinity of they and bFGF are less than
The affinity of bFGF mAb and bFGF.
The structure of 3 double-antibodies sandwich ELISA of embodiment
1, the pairing experiment of bFGF nano antibodies
The double sandwich-ELISA method of antigen bFGF concentration can be detected for structure, we match bFGF nano antibodies
It is right, with the Nb of a concentration of 5 μ g/mLbFGF 1(His-HA tag)、NbbFGF 2(His-HA tag)、NbbFGF 3(His-HA
tag)、NbbFGF 5(His-HA tag)、NbbFGF6 (His-HA tag) wrapper sheets, after the antigen bFGF for adding 2 μ g/mL, in incubation
The Nb of a concentration of 2 μ g/mL, 1 μ g/mL, 0.5 μ g/mLbFGF4 (His-Flag tag), then with Anti-Flag tag-HRP
(being purchased from Sigma companies, article No. A8592-.2MG mouse are anti-) is finally developed the color with TMB, is surveyed at 450nm wavelength as detection antibody
Measure absorbance value.
As a result as shown in figure 15, the Nb of various concentrationbFGF4 (His-Flag tag) match with remaining bFGF nano antibody
3 times or more of the absorbance value of control group 1X PBS are all higher than to the absorbance value of experiment, and wherein absorbance value is highest is
NbbFGF4 (His-Flag tag) and NbbFGFThe pairing of 6 (His-HA tag).Therefore we determined that, by NbbFGF 6(His-HA
Tag) as the capture antibody in the double sandwich-ELISA method of detection antigen bFGF concentration, NbbFGF4 (His-Flag tag) make
For the detection antibody of antigen.
2, the determination of double sandwich-ELISA method detection antigen bFGF concentration ranges
After determining two plants of nano antibodies needed for the double sandwich-ELISA method of detection antigen bFGF concentration, pass through addition
The antigen of various concentration measures detection range of this method to antigen bFGF.With the Nb of a concentration of 5 μ g/mLbFGF 6(His-HA
Tag) wrapper sheet, and a concentration of 2000ng/mL, 1000ng/mL, 500ng/mL, 250ng/mL, 125ng/mL, 62.5ng/mL,
The antigen of 31.25ng/mL, 15.63ng/mL, 7.81ng/mL, 3.91ng/mL, 1.95ng/mL, 0.98ng/mL, 0.49ng/mL
After bFGF, the Nb of a concentration of 2 μ g/mL in incubationbFGF4 (His-Flag tag), then be added Anti-Flag tag-HRP into
Row is incubated, and is finally developed the color with TMB, and absorbance value is measured at 450nm wavelength.
As a result as shown in figure 16, show under the detection method, it is anti-more than or equal to 7.81ng/mL to be only able to detect concentration
Former (Figure 16 A)；By result using antigen concentration as abscissa, absorbance value is ordinate, is depicted as scatter plot, is found when antigen is dense
When degree is between 7.81ng/mL to 500ng/mL, absorbance value variation is apparent (Figure 16 B).
Adjustment antigen concentration makes it fall in the sections 10ng/mL to 400ng/mL, then carries out double sandwich-ELISA again
Experiment, with the Nb of a concentration of 5 μ g/mLbFGF6 (His-HA tag) wrapper sheets, in addition a concentration of 400ng/mL, 300ng/mL,
After the antigen bFGF of 200ng/mL, 100ng/mL, 80ng/mL, 40ng/mL, 20ng/mL, 10ng/mL, a concentration of 2 μ in incubation
The Nb of g/mLbFGF4 (His-Flag tag) are finally developed the color with TMB then using Anti-Flag tag-HRP as detection antibody,
Absorbance value is measured at 450nm wavelength.
By result using antigen concentration as abscissa, absorbance value is ordinate, is depicted as scatter plot and solves its straight line side
Journey and related coefficient：Equation is Y=0.0045X-0.005, coefficient R2=0.98977.Above the experimental results showed that, with
The Nb of a concentration of 5 μ g/mLbFGF6 (His-HA tag) are as capture antibody, with the Nb of a concentration of 2 μ g/mLbFGF 4(His-Flag
Tag) as under the double sandwich-ELISA method of the detection antibody of antigen, antigen bFGF can be diluted to 400ng/mL, 300ng/
Eight concentration such as mL, 200ng/mL, 100ng/mL, 80ng/mL, 40ng/mL, 20ng/mL, 10ng/mL, and be with antigen concentration
Abscissa, absorbance value are ordinate, are depicted as the standard curve (Figure 17) of sample detection.
4 anti-human bFGF nano antibodies double sandwich-ELISA standard measure of embodiment detects antigen bFGF performance evaluations
1, specific test
Using the double sandwich-ELISA method based on anti-human bFGF nano antibodies in embodiment 3, detectable concentration is 125ng/mL
BFGF, aFGF, KGF, EGF, TNF α, VEGF (being purchased from Sino Biological Inc.), BSA, milk, with
Examine the specificity of the double sandwich-ELISA method.When the absorbance value of detection control antigen is less than the absorbance value of detection bFGF
When one third, it is defined as negative reaction.
As a result as shown in figure 18, the absorbance value under the double sandwich-ELISA detection method when detection control antigen is obviously low
The one third of absorbance value when detecting bFGF, it was demonstrated that constructed based on anti-human bFGF nano antibodies in embodiment 3
Double sandwich-ELISA method specificity is good.
2, sensitivity analysis
The bFGF minimum concentrations that anti-human bFGF nano antibodies double sandwich-ELISA method is able to detect that, for the detection method
Detection sensitivity.It can be seen that from 3 standard curve of embodiment when containing 7.8ng and the above albumen in every milliliter of sample, it can
It is detected by this detection method, sensitivity is higher.
3, repeated experiment
(1) it repeats to test in criticizing
Using the same batch of anti-human bFGF nano antibody prepared, using the anti-human bFGF nano antibodies double fastener heart in embodiment 3
ELISA method carries out 6 detections respectively in different time to the bFGF of various concentration.As a result such as table 2, repeatability is good in batch.
Experimental result is repeated in 2 anti-human bFGF nano antibodies double sandwich-ELISA method of table batch
(2) it repeats to test between criticizing
The anti-human bFGF nano antibodies prepared using three batches, using the anti-human bFGF nano antibodies double fastener in embodiment 3
Heart ELISA method carries out 6 detections respectively in different time to the bFGF of various concentration.As a result such as table 3, repeatability is good between batch.
Experimental result is repeated between 3 anti-human bFGF nano antibodies double sandwich-ELISA method of table batch
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications,
Equivalent substitute mode is should be, is included within the scope of the present invention.
Sequence table
<110>Ji'nan University
<120>The method and kit of anti-human bFGF nano antibodies detection bFGF
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Tyr Tyr Ala Asp Ser Val Lys Gly Arg Tyr Thr Ile Ser Arg Asp Asn
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Leu Gln Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly Ser Leu Arg
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Ser Ser Val Gly Arg Ala Met Tyr Ala Asp Pro Val Lys Gly Arg Phe
50 55 60
Thr Ile Ser Arg Asp Asn Ala Lys Asn Met Val Tyr Leu Gln Met Asn
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Arg Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr Leu Tyr Gly
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Asp Tyr Arg Gly Thr Gly Phe Trp Gly Lys Gly Thr Gln Val Thr Val
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Leu Gln Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly Ser Leu Arg
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Thr Ile Ser Arg Asp Asn Ala Lys Asn Met Val Tyr Leu Gln Met Asn
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Leu Gln Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg
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Thr Pro Gly Gly Thr Arg Asn Tyr Ala Asn Ser Val Lys Gly Arg Phe
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Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn
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Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg
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50 55 60
Tyr Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met
65 70 75 80
Asn Asp Leu Lys Pro Glu Asp Thr Ala Val Tyr Ser Cys Ala Ala Arg
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100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
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Leu Gln Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly Ser Leu Arg
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Leu Ser Cys Glu Val Ser Gly Ser Asn Phe Ser Ile Asn Asp Met Gly
20 25 30
Trp Tyr Arg Gln Ala Pro Gly Lys Arg Arg Glu Val Val Ala Gly Ile
35 40 45
Ser Ser Val Gly Arg Thr Met Tyr Gly Asp Pro Val Lys Gly Arg Phe
50 55 60
Thr Ile Ser Arg Asp Asn Ala Lys Asn Met Val Tyr Leu Gln Met Asn
65 70 75 80
Arg Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys His Leu Tyr Gly
85 90 95
Asp Tyr Arg Gly Thr Gly Phe Trp Gly Lys Gly Thr Gln Val Thr Val
100 105 110
Ser Ser
<210> 34
<211> 342
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 34
ctgcaggagt ctgggggaga cttggtgcag cctggggggt ctctgagact ctcctgtgaa 60
gtttctggaa gcaacttcag taacaatgac atgggctggt accgccaggc tccagggaag 120
cagcgcgagg tggtcgcagg tattagtagt gttggacgta caatgtatgg agaccccgtg 180
aagggccgat tcaccatctc cagagacaac gccaagaaca tggtgtatct gcaaatgaac 240
agactgaaag ctaaagacac ggccgtctat tactgtcacc tttatggtga ctataggggg 300
actggtttct ggggcaaggg gacccaggtc accgtctcgt cg 342
<210> 35
<211> 342
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 35
ctgcaggagt ctgggggaga cttggtgcag cctggggggt ctctgagact ctcctgtgaa 60
gtttctggaa gcaacttcag tatcaatgac atgggctggt accgccaggc tccggggaag 120
cggcgcgagg tggtcgcagg tattagtagt gttggacgcg caatgtatgc agaccccgtg 180
aagggccgat tcaccatctc cagagacaac gccaagaaca tggtgtactt gcaaatgaac 240
agactgaaac ctgaggacac ggccgtttat tactgttacc tttatggtga ttataggggg 300
actggtttct ggggcaaggg gacccaggtc accgtctcgt cg 342
<210> 36
<211> 342
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 36
ctgcaggagt ctgggggaga cttggtgcag cctggggggt ctctgagact ctcctgtgaa 60
gtttccggaa gcaacttcag tatcaatgac atgggctggt accgccaggc tccagggaag 120
cggcgcgagg tggtcgcagg tattagtagt gttggacgca caatgtatgc agaccccgtg 180
aagggccgat tcaccatctc cagagacaac gccaagaaca tggtgtatct gcaaatgaac 240
agactgaaac ctgaggacac ggccgtctat tactgtcacc tttatggtga ctataggggg 300
actggtttct ggggcaaggg gacccaggtc accgtctcgt cg 342
<210> 37
<211> 336
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 37
ctgcagcagt ctgggggagg cttggtgcag cctggggggt ctctgagact ctcctgtaaa 60
gcctctagaa acatcttcag tgtcaatcac atgggctatt accgccaggc tccagggaag 120
gagcgcgagc tggtcgcgct tattactccc ggtggtacca gaaactatgc aaactccgtg 180
aagggccgat tcaccatctc caaagacaac gccaagaaca cggtgtatct gcagatgaac 240
agcctgcaac ctgaggacac ggccgtctat tactgtaata cctggccata tgagtctgcc 300
tattcgggcc aggggaccca ggtcaccgtc tccaca 336
<210> 38
<211> 369
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 38
ctgcaggagt ctgggggagg attggtgcag gctgggggct ctctgagact ctcctgtgca 60
gcctctggac gcaccttcag tagcggtgcc atgggctggt tccgccaggc tccagggaag 120
gagcctgagt ttgtggcaac tattacgtgg gatgggggta cgacatacta tgcagactcc 180
gtgaagggcc gatacaccat ctccagagac aacgccaaga atacggtata tctgcaaatg 240
aacgacctga aacctgagga cacggccgtt tattcctgtg cagcgagatc ttatagtgag 300
gcttactact taatcggctc gtccgattat aactactggg gtcaggggac ccaggtcacc 360
gtctcctca 369
<210> 39
<211> 342
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 39
ctgcaggagt ctgggggaga cttggtgcag cctggggggt ctctgagact ctcctgtgaa 60
gtttctggaa gcaacttcag tatcaatgac atgggctggt accgccaggc tccagggaag 120
cgacgcgagg tggtcgcagg tattagtagt gttggacgca caatgtatgg agaccccgtg 180
aagggccgat tcaccatctc cagagacaac gccaagaaca tggtgtatct gcaaatgaac 240
agactgaaac ctgaggacac ggccgtctat tactgtcacc tttatggtga ctataggggg 300
actggtttct ggggcaaggg gacccaggtc accgtctcgt cg 342
<210> 40
<211> 36
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 40
ataaaaccat ggctgcagga gtctggggga gacttg 36
<210> 41
<211> 38
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 41
ataaaagcgg ccgctggttt tggtgtcttg ggttccga 38
<210> 42
<211> 36
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 42
ataaaaccat ggctgcagca gtctggggga ggcttg 36
<210> 43
<211> 38
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 43
ataaaagcgg ccgctggttt tggtgtcttg ggttctgt 38
<210> 44
<211> 36
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 44
ataaaactgc aggagtctgg gggaggattg gtgcag 36
<210> 45
<211> 38
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 45
ataaaagcgg ccgctggttt tggtgtcttg ggttctga 38
<210> 46
<211> 31
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 46
ataaaaggat ccggagtctg ggggagactt g 31
<210> 47
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 47
ataaaactcg agcgacgaga cggtgacctg 30
<210> 48
<211> 31
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 48
ataaaaggat ccgcaggtca agctgcagca g 31
<210> 49
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 49
ataaaactcg agcgacgaga cggtgacctg 30
Claims (10)
1. a kind of kit of detection bFGF, the kit contain anti-human bFGF nano antibodies, described anti-human bFGF nanometers anti-
Body includes the Variable domain being made of framework region FR and complementary determining region CDR, which is characterized in that the variable region structure
Domain includes CDR1, CDR2 and CDR3 selected from the following：
(1) CDR1 shown in any amino acid sequence in No.1~4 SEQ ID,
(2) CDR2 shown in any amino acid sequence in No.5~8 SEQ ID；
(3) CDR3 shown in any amino acid sequence in No.9~12 SEQ ID.
2. the kit of detection bFGF according to claim 1, which is characterized in that the Variable domain includes choosing
From any one of following CDR1, CDR2 and CDR3：
(1) CDR3 shown in CDR2 and SEQ ID No.9 shown in CDR1 shown in SEQ ID No.1, SEQ ID No.5；
(2) CDR2 and SEQ ID No.9 shown in CDR1 shown in SEQ ID No.2, SEQ ID No.5 or SEQ ID No.6
Or CDR3 shown in SEQ ID No.10；
(3) CDR3 shown in CDR2 and SEQ ID No.11 shown in CDR1 shown in SEQ ID No.3, SEQ ID No.7；
(4) CDR3 shown in CDR2 and SEQ ID No.12 shown in CDR1 shown in SEQ ID No.4, SEQ ID No.8；
Described framework region FR FR1, FR2, FR3 and the FR4 selected from the group below：
(1) FR1 shown in any amino acid sequence in No.13~15 SEQ ID；
(2) FR2 shown in any amino acid sequence in No.16~19 SEQ ID；
(3) FR3 shown in any amino acid sequence in No.20~24 SEQ ID；
(4) FR4 shown in any amino acid sequence in No.25~27 SEQ ID.
3. the kit of detection bFGF according to claim 1, it is characterised in that：
The amino acid sequence of the anti-human bFGF nano antibodies is selected from any amino acid as shown in NO.28~33 SEQ ID
Sequence.
4. a kind of double-antibody sandwich elisa kit of detection bFGF, which is characterized in that include：
(1) using the anti-human bFGF nano antibodies with following Variable domain as primary antibody：
1. CDR3 shown in CDR2 and SEQ ID No.9 shown in CDR1 shown in SEQ ID No.1, SEQ ID No.5；
2. CDR2 and SEQ ID No.9 shown in CDR1 shown in SEQ ID No.2, SEQ ID No.5 or SEQ ID No.6
Or CDR3 shown in SEQ ID No.10；
3. CDR3 shown in CDR2 and SEQ ID No.12 shown in CDR1 shown in SEQ ID No.4, SEQ ID No.8.
(2) using the anti-human bFGF nano antibodies with following Variable domain as secondary antibody：
The CDR1 as shown in SEQ ID No.3, the CDR2 as shown in SEQ ID No.7 and as shown in SEQ ID No.11
CDR3。
5. the double-antibody sandwich elisa kit of detection bFGF according to claim 4, it is characterised in that：
The amino acid sequence of the anti-human bFGF nano antibodies as primary antibody is such as No.28~30 SEQ ID, SEQ ID
Amino acid sequence shown in any one of No.32 or SEQ ID No.33.
6. the double-antibody sandwich elisa kit of detection bFGF according to claim 4 or 5, it is characterised in that：
The amino acid sequence of the anti-human bFGF nano antibodies as primary antibody is as shown in SEQ ID No.33.
7. the double-antibody sandwich elisa kit of detection bFGF according to any one of claim 4 to 6, it is characterised in that：
The amino acid sequence of the anti-human bFGF nano antibodies as secondary antibody is as shown in SEQ ID No.31.
8. the double-antibody sandwich elisa for detecting the detection bFGF described in the kit or 4 of bFGF according to claim 1 tries
Agent box, it is characterised in that：
The kit is cancer diagnosing kit.
9. a kind of double-antibodies sandwich ELISA of detection bFGF, it is characterised in that：
With its amino acid sequence as No.28~30 SEQ ID, SEQ ID No.32 or SEQ ID No.33 any one shown in
Anti-human bFGF nano antibodies are as primary antibody；With the anti-human of its amino acid sequence amino acid sequence as shown in SEQ ID No.31
BFGF nano antibodies are as secondary antibody；This method is not intended to the diagnosis or treatment of disease.
10. it is according to claim 9 detection bFGF double-antibodies sandwich ELISA, which is characterized in that specifically include as
Lower step：
(1) it is coated with：Coating plate be coated with overnight with the solution containing primary antibody；
(2) it closes：After washing is coated with the coating plate of primary antibody, closed；
(3) add antigen：The coating plate closed is washed, sample to be tested is added, is incubated；
(4) add secondary antibody：Washing is incubated the coating plate after sample to be tested, and secondary antibody is added, and is incubated；
(5) enzyme labeling antibody：Washing is incubated the coating plate after secondary antibody, and HRP labelled antibodies are added, and is incubated；
(6) after washing coating plate, TMB developing solutions are added, is protected from light incubation, absorbance value is measured at 450nm wavelength.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810574975.3A CN108508216B (en) | 2018-06-06 | 2018-06-06 | Method and kit for detecting bFGF by using anti-human bFGF nano antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810574975.3A CN108508216B (en) | 2018-06-06 | 2018-06-06 | Method and kit for detecting bFGF by using anti-human bFGF nano antibody |
Publications (2)
| Publication Number | Publication Date |
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| CN108508216A true CN108508216A (en) | 2018-09-07 |
| CN108508216B CN108508216B (en) | 2020-08-07 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810574975.3A Active CN108508216B (en) | 2018-06-06 | 2018-06-06 | Method and kit for detecting bFGF by using anti-human bFGF nano antibody |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022268993A1 (en) * | 2021-06-23 | 2022-12-29 | Vib Vzw | Means and methods for selection of specific binders |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002083850A2 (en) * | 2001-04-13 | 2002-10-24 | Human Genome Sciences, Inc. | Vascular endothelial growth factor 2 |
| CN101092457A (en) * | 2007-06-08 | 2007-12-26 | 暨南大学 | Human source Fab antibody for anti recombined alkalescent fibroblast growth factor and application |
| CN101370525A (en) * | 2005-08-19 | 2009-02-18 | 艾博特公司 | Dual variable domain immunoglobin and uses thereof |
| CN102558351A (en) * | 2011-12-28 | 2012-07-11 | 暨南大学 | Anti to human alkaline fibroblast growth factor human s c F v antibody and application thereof |
-
2018
- 2018-06-06 CN CN201810574975.3A patent/CN108508216B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002083850A2 (en) * | 2001-04-13 | 2002-10-24 | Human Genome Sciences, Inc. | Vascular endothelial growth factor 2 |
| CN101370525A (en) * | 2005-08-19 | 2009-02-18 | 艾博特公司 | Dual variable domain immunoglobin and uses thereof |
| CN101092457A (en) * | 2007-06-08 | 2007-12-26 | 暨南大学 | Human source Fab antibody for anti recombined alkalescent fibroblast growth factor and application |
| CN102558351A (en) * | 2011-12-28 | 2012-07-11 | 暨南大学 | Anti to human alkaline fibroblast growth factor human s c F v antibody and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 陶俊。: "人源性bFGF Fab抗体体外亲和力成熟的研究。", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022268993A1 (en) * | 2021-06-23 | 2022-12-29 | Vib Vzw | Means and methods for selection of specific binders |
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| Publication number | Publication date |
|---|---|
| CN108508216B (en) | 2020-08-07 |
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