CN108508216B - Method and kit for detecting bFGF by using anti-human bFGF nano antibody - Google Patents
Method and kit for detecting bFGF by using anti-human bFGF nano antibody Download PDFInfo
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Abstract
The invention discloses a method for detecting bFGF by an anti-human bFGF nano antibody and a kit, and provides the kit for detecting bFGF by the anti-human bFGF nano antibody, in particular to a double-antibody sandwich E L ISA kit for detecting bFGF, and successfully establishes a double-antibody sandwich E L ISA method for detecting bFGF, which is not used for diagnosing or treating diseases, and has strong specificity and good detection effect.
Description
Technical Field
The invention belongs to the field of biotechnology, and particularly relates to a method and a kit for detecting bFGF by using an anti-human bFGF nano antibody.
Background
Antibodies (antibodies), also called immunoglobulins (immunoglobulins), are glycoproteins that specifically bind to antigens. Traditional antibodies exist as one or more "Y" shaped monomers, each consisting of 4 polypeptide chains, comprising two identical heavy chains and two identical light chains. The traditional antibody has large molecular weight and complex structure, and the stability and penetrability of the antibody are poor. And the development and production cost is high, so that the use cost is high, and the further application of the composite material is limited. Therefore, the development of some novel small molecule antibodies is an important research point, such as: fab antibody, F (ab)2Antibodies, single chain antibodies (scFv), nanobodies, and the like.
In 1993, Hamers Casterman et al first reported the presence of peculiar antibodies that naturally lack light chains in camelid blood, i.e., Heavy Chain antibodies (HCAb). The Variable region is cloned by genetic engineering technology to obtain an Antibody fragment consisting of only Heavy Chain Variable regions, namely a Heavy Chain single Domain Antibody VHH (Variable Domain of Heavy Chain of Heavy-Chain Antibody). It is also called Nanobody (Nb) because its molecular weight is only one tenth of that of traditional antibodies, and its length is more on the order of a few nanometers. The novel antibody molecule has the characteristics of small molecular mass, high stability, easy expression, strong cell/blood vessel penetrability, strong antigen binding capacity, capability of expressing in a prokaryotic expression system and the like, has the advantages of the traditional antibody and the micromolecule medicine, almost perfectly overcomes the defects of long development period, low stability, harsh storage conditions and the like of the traditional antibody, is not easy to stick to each other and even to aggregate into a block like the micromolecule antibody such as a single chain antibody (scFv) and the like, thereby becoming a novel antibody molecule in the field of antibody medicine and reagent research and having good development prospect. At present, nano antibodies have been gradually applied to the fields of disease treatment, disease diagnosis, radiological imaging, and the like.
Basic Fibroblast Growth Factor (bFGF), also known as Fibroblast Growth Factor (FGF-2), is a mitogen for fibroblasts purified in 1974 from extracts of bovine pituitary and brain tissue by Gospodalozi et al, and is composed of 146 amino acids and is called basic Fibroblast Growth Factor because its isoelectric point is 9.6. bFGF has very wide biological action and plays an important role in the processes of angiogenesis, wound healing and tissue repair promotion, tissue regeneration promotion and growth and development of nerve tissues.
In some tumor diseases, the abnormal expression of bFGF is closely related to tumorigenesis and development, tumor metastasis, angiogenesis and prognosis, mainly because bFGF not only promotes local tumor angiogenesis through paracrine and autocrine and other ways, but also directly acts on tumor cells to promote proliferation and accelerate tumor infiltration and metastasis. In-vitro and in-vivo experiments show that bFGF can promote the secretion of various growth factors, has synergistic effect with other angiogenesis promoting factors VEGF, PDGF and the like, and promotes tumorigenesis together. In addition, various tumors can change bFGF/FGFR signal path by regulating bFGF overexpression, so that the aim of promoting the growth of the tumors is achieved. bFGF is involved in the growth of various tumors (such as glioma, rhabdomyoma, leukemia, renal carcinoma, lung cell carcinoma, melanoma and lung cancer), and a considerable part of tumors show high expression of bFGF and bFGF receptors. Therefore, the rapid and accurate detection of bFGF content in tumor cells has certain necessity for diagnosis and treatment of diseases.
The conventional IgG antibody production depends on mammalian cells, the production period is long, the cost is high, the price is high, and the traditional IgG antibody needs to be stored at low temperature, and the characteristics limit the application of the traditional IgG antibody in the detection kit.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a kit for detecting bFGF.
The heavy chain antibody found from camelids, namely the nano antibody, has the characteristics of strong specificity, high affinity, good stability, high temperature resistance, easiness in storage and the like due to the structural specificity, and the production cost is greatly reduced due to the fact that the heavy chain antibody can be expressed by a prokaryotic system, so that the problems of the traditional IgG antibody are well solved in the application of a detection kit.
The purpose of the invention is realized by the following technical scheme:
a kit for detecting bFGF, the kit comprising an anti-human bFGF nanobody comprising a variable region domain consisting of framework region FRs and complementarity determining region CDRs, the variable region domain comprising CDRs 1, CDR2 and CDR3 selected from the group consisting of:
(1) CDR1 shown in any amino acid sequence of SEQ ID No. 1-4,
(2) CDR2 shown by any amino acid sequence of SEQ ID No. 5-8;
(3) CDR3 shown in any amino acid sequence of SEQ ID Nos. 9-12.
The variable region domain comprises a CDR1, a CDR2, and a CDR3 selected from any one of:
(1) CDR1 shown in SEQ ID No.1, CDR2 shown in SEQ ID No.5, and CDR3 shown in SEQ ID No. 9;
(2) CDR1 shown in SEQ ID No.2, CDR2 shown in SEQ ID No.5 or SEQ ID No.6, and CDR3 shown in SEQ ID No.9 or SEQ ID No. 10;
(3) CDR1 shown in SEQ ID No.3, CDR2 shown in SEQ ID No.7, and CDR3 shown in SEQ ID No. 11;
(4) CDR1 shown in SEQ ID No.4, CDR2 shown in SEQ ID No.8, and CDR3 shown in SEQ ID No. 12.
The framework region FR is selected from the group consisting of FR1, FR2, FR3 and FR 4:
(1) FR1 represented by any one of SEQ ID Nos. 13-15;
(2) FR2 represented by any amino acid sequence of SEQ ID Nos. 16-19;
(3) FR3 represented by any amino acid sequence of SEQ ID Nos. 20-24;
(4) FR4 represented by any amino acid sequence of SEQ ID Nos. 25 to 27.
The amino acid sequence of the anti-recombinant human basic fibroblast growth factor nano antibody is selected from any one of the amino acid sequences shown in the specification (SEQ ID NO. 28-33); the method comprises the following specific steps:
nucleotide sequences of the amino acid sequences shown as SEQ ID NO. 28-33 are shown as SEQ ID NO. 34-39 in sequence.
A double antibody sandwich E L ISA kit for detecting bFGF, comprising:
(1) an anti-human bFGF nanobody with the following variable region domains is used as a primary antibody (namely a capture antibody):
① CDR1 shown in SEQ ID No.1, CDR2 shown in SEQ ID No.5, and CDR3 shown in SEQ ID No. 9;
② CDR1 shown in SEQ ID No.2, CDR2 shown in SEQ ID No.5 or SEQ ID No.6, and CDR3 shown in SEQ ID No.9 or SEQ ID No. 10;
③ CDR1 shown in SEQ ID No.4, CDR2 shown in SEQ ID No.8, and CDR3 shown in SEQ ID No. 12.
(2) An anti-human bFGF nano antibody with the following variable region structural domains is used as a secondary antibody (namely a detection antibody):
CDR1 as shown in SEQ ID No.3, CDR2 as shown in SEQ ID No.7 and CDR3 as shown in SEQ ID No. 11.
The amino acid sequence of the anti-human bFGF nano antibody as a primary antibody is preferably an amino acid sequence shown in any one of SEQ ID No. 28-30, SEQ ID No.32 or SEQ ID No. 33; further preferred is the amino acid sequence shown as SEQ ID No. 33.
The amino acid sequence of the anti-human bFGF nano antibody as the secondary antibody is preferably the amino acid sequence shown in SEQ ID No. 31.
The kit is preferably a cancer diagnosis kit; more preferably, it is a cancer diagnostic kit for detecting human bFGF.
The nanobody consists of 342 bases, 336 bases or 369 bases, codes 114 amino acids, 112 amino acids or 123 amino acids, and consists of 4 Framework Regions (FRs) and 3 Complementary Determining Regions (CDRs) of the antibody: the CDR1 of the nanobody encodes 10 amino acids, CDR2 encodes 7 amino acids, and CDR3 encodes 11 amino acids; or CDR1 encodes 10 amino acids, CDR2 encodes 7 amino acids, CDR3 encodes 9 amino acids; or CDR1 encodes 10 amino acids, CDR2 encodes 8 amino acids, CDR3 encodes 19 amino acids; 3 CDR regions are specific sequences.
The nanobody consists of 4 Framework Regions (FR) and 3 Complementarity Determining Regions (CDR). The function of the nano antibody is determined by specific nucleotide sequences in antibody antigen determinant families CDR1, CDR2 and CDR3 (which are functional active regions of the invention), and corresponding amino acid sequences form antibody specific bFGF antigen binding regions.
A double-antibody sandwich E L ISA method for detecting bFGF takes an anti-human bFGF nano antibody with an amino acid sequence shown as any one of SEQ ID No. 28-30, SEQ ID No.32 or SEQ ID No.33 as a primary antibody and takes an anti-human bFGF nano antibody with an amino acid sequence shown as SEQ ID No.31 as a secondary antibody, and the method is not used for diagnosis or treatment of diseases.
The method for detecting the double-antibody sandwich E L ISA of the bFGF specifically comprises the following steps:
(1) coating: coating the coated plate with a solution containing a primary antibody overnight;
(2) and (3) sealing: washing the coated plate coated with the primary antibody, and then sealing;
(3) adding an antigen: washing the sealed coating plate, adding a sample to be tested, and incubating;
(4) adding a secondary antibody: washing the coated plate after incubating the sample to be tested, adding a second antibody, and incubating;
(5) adding an enzyme-labeled antibody: washing the coated plate after incubation of the secondary antibody, adding an HRP labeled antibody, and incubating;
(6) washing the coated plate, adding TMB color development solution, incubating in dark, and measuring absorbance value at 450nm wavelength.
The concentration ratio of the primary antibody to the secondary antibody is preferably 5: 2.
The cloning expression method of the anti-human bFGF nano antibody comprises the following steps:
cloning the nucleotide sequence of the anti-human bFGF nano antibody into an expression vector to construct a recombinant expression vector; then transferring the recombinant expression vector into an expression system for heterologous expression; and finally, purifying to obtain the anti-human bFGF nano antibody.
The expression vector is preferably pMECS or pNCS.
The nano antibody gene or any gene containing the complementary determining region of the nano antibody gene reconstructed based on the gene can be expressed in prokaryotic cells, yeast cells, insect cells and eukaryotic cells to obtain an antibody product capable of combining with human BFGF, and the antibody product can be applied to: (1) preparing a diagnostic product for tumors; (2) and (5) detecting the concentration of the antigen.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) besides the characteristics of small molecular mass, strong specificity, high affinity, strong antigen binding capacity and the like, the anti-human bFGF nano antibody also has the characteristics of good stability, high temperature resistance, easy storage, low growth cost and the like compared with the traditional bFGF antibody, and is more suitable for development of detection reagent products, and the obtained detection reagent products are more suitable for storage, transportation and use.
(2) The invention has good detection effect, and particularly utilizes the antibody pairing combination to resist the human bFGF nano antibody Nb bFGF6 and Nb bFGF4 when the sample is subjected to double-antibody sandwich E L ISA detection, the linear range of the sample can reach 10-400 ng/m L without adding signal amplification molecules, and the lowest detection limit is 7.81ng/m L.
(3) The anti-human bFGF nano antibody can be expressed by a prokaryotic system, is simple to operate, reduces the production cost, and is easy for industrial production and popularization.
Drawings
FIG. 1 is a gene electrophoresis chart for constructing pMECS-Nbs (His-HA tag) recombinant plasmid.
FIG. 2 is a gene electrophoresis chart of the construction of pNCS-Nbs (His-Flag tag) recombinant plasmid.
FIG. 3 is an SDS-PAGE analysis of six bFGF nanobodies (His-HA tag) after expression.
FIG. 4 is a graph showing the results of Western Blot analysis of six bFGF nanobodies (His-HA tag) expression.
FIG. 5 is a six-strain bFGF nanobody (His-HA tag) Ni-NTA affinity chromatography purification diagram.
FIG. 6 is a graph showing the results of SDS-PAGE analysis and WesternBlot analysis of a sample after purification of six strains of bFGF nanobody (His-HA tag).
FIG. 7 is a SDS-PAGE analysis result of the expression results of two strains of bFGF nanobodies (His-Flag tag).
FIG. 8 is a Western Blot analysis chart showing the expression results of two strains of bFGF nanobodies (His-Flag tag).
FIG. 9 is a Ni-NTA affinity chromatography purification chart of two strains of bFGF nanobodies (His-Flag tag).
FIG. 10 is a graph showing the results of SDS-PAGE analysis and WesternBlot analysis of a sample after purification of two strains of bFGF nanobodies (His-Flag tag).
FIG. 11 is a graph showing the analysis of the specific binding between bFGF nanobody and bFGF; wherein 1 is a positive control, 2-7 represent different nano antibody clone strains, and Nb 1-Nb 6 are sequentially adopted.
FIG. 12 is an analysis chart of the results of the thermal stability experiment of bFGF nanobody; wherein bFGF mAb is a positive control, and Nb 1-Nb 6 represent different nano antibody clone strains.
FIG. 13 is a comparison analysis chart of binding sites of bFGF2 nanobody and FGFR 2; wherein Nb 1-Nb 6 represent different nano antibody clone strains.
FIG. 14 is a graph of the comparative analysis of bFGF nanobodies and bFGF mAb binding sites; wherein Nb 1-Nb 6 represent different nano antibody clone strains.
FIG. 15 is an analysis chart of bFGF nanobody pairing experiment results, in which graphs A-F are Nb concentrations of 2 μ g/m L, 1 μ g/m L, 0.5 μ g/m L, 0.25 μ g/m L, 0.125 μ g/m L, 0.0625 μ g/m LbFGF4(His-Flag tag) and the other bFGF nano-antibodies respectively.
FIG. 16 is a graph showing the result of analyzing the detection range of bFGF concentration by the established double-sandwich E L ISA method.
FIG. 17 is a standard curve diagram of the established double-sandwich E L ISA method for detecting the concentration of bFGF antigen.
FIG. 18 is a graph showing the analysis results of the specificity test in example 4.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1 construction method of anti-recombinant human basic fibroblast growth factor (bFGF) Nanobody
1. Construction of pMECS-Nbs (His-HA tag) recombinant plasmid
1.1 primer design
1.2PCR
PrimeStar from TaKaRa was used@HS DNA Polymerase performs PCR reaction on target gene of nano antibody, and the electrophoresis result is shown in figure 1.
1.3 enzyme digestion
Vectors pMECS (purchased from China plasmid vector cell Gene Collection) and Nb were treated with FastDigest Nco I and FastDigest Not I from Thermo, respectivelybFGF1～Nb bFGF6, carrying out double enzyme digestion on the gene; vectors pMECS and Nb were paired using FastDigest Pst I and FastDigest Not I from Thermo bFGF5, double enzyme digestion of the gene.
1.4 ligation reactions
Vectors pMECS and Nb were separately ligated with T4DNA L igase from Thermo bFGF1 gene; vectors pMECS and Nb bFGF2 gene; vectors pMECS and Nb bFGF3 gene; vectors pMECS and Nb bFGF4 gene; vectors pMECS and Nb bFGF5 gene; vectors pMECS and Nb bFGF6 genes are linked.
1.5 transformation
pMECS-Nbs were electroporated into WK6 competent cells.
The sequencing result shows that the recombinant plasmid pMECS-Nb bFGF1、pMECS-Nb bFGF2、pMECS-Nb bFGF3、pMECS-Nb bFGF4、pMECS-Nb bFGF5、pMECS-Nb bFGF6 the construction was successful.
2. Construction of pNCS-Nbs (His-Flag tag) recombinant plasmid
2.1 primer design
2.2PCR
PCR reaction was performed on the target gene of the nanobody using Pfu Kit of Dongsheng organism, and the electrophoresis results are shown in FIG. 2.
2.3 enzyme digestion
Vectors pNCS (purchased from the China plasmid vector cell Gene Collection) and Nb were paired with FastDigest BamH I and FastDigest Xhol I from Thermo bFGF2 Gene, Nb bFGF4, double enzyme digestion of the gene.
2.4 ligation reaction
The vectors pNCS and Nb were ligated with T4DNA L igase from Thermo bFGF2 Gene, vectors pNCS and Nb bFGF4 genes are linked.
2.5 transformation
pNCS-Nbs were transformed into DH5a competent cells.
The sequencing result shows that the recombinant plasmid pNCS-Nb bFGF2 and pNCS-Nb bFGF4 the construction was successful.
3. Expression and purification of bFGF nanobody
3.1 expression and purification of bFGF Nanobody (His-HA tag)
(1) Streaking frozen strains by using a plate to pick out positive monoclonals (2) adding the positive monoclonals into a TB culture medium with the concentration of 5m L and the final Amp concentration of 100 mu g/m L, carrying out shake culture at 37 ℃ and 220rpm for 11-13 hours, using the positive monoclonals as first-class seeds (3) taking a first-class seed bacterial liquid, inoculating the first-class seed bacterial liquid into a 30m L TB culture medium with the final Amp concentration of 100 mu g/m L according to the proportion of 0.1%, and adding 2mM MgCl2(4) taking a secondary seed bacterial liquid, inoculating the secondary seed bacterial liquid into a large-volume TB culture medium with the final Amp concentration of 100 mu g/m L according to the proportion of 1%, and simultaneously adding 2mM MgCl into the mixed solution and 0.1% (w/v) glucose solution, culturing the mixed solution at 37 ℃ and 220rpm for 8 hours by shaking2The solution and 0.1% (w/v) glucose solution were incubated at 37 ℃ and 220rpm with shaking to OD600In the range of 0.8 to 1.0. (5) IPTG was added to a final concentration of 1mM, and shaking culture was continued at 28 ℃ and 220rpm for 12 hours. (6) Centrifuge at 6,000rpm for 30 min. Taking the thalli to precipitate. (7) The pellet was resuspended and washed with 1XPBS and centrifuged at 8,000g for 20 min. Taking the thalli to precipitate. This step was repeated three times. (8) The pellet was weighed and 1XPBS buffer was added at a ratio of l:10 to resuspend the cells. (9) Under the condition of ice bathInserting an ultrasonic probe of an ultrasonic crusher into the thallus suspension, and setting parameter power: 800W; ultrasonic time: 3 s; intermittent time: and 5 s. The transmittance of the bacteria is obviously increased by ultrasonic treatment. Centrifuging at 4 deg.C for 90min at 10,000g, collecting supernatant, purifying by affinity chromatography with Ni-NTA column, removing imidazole from the eluted target protein solution with SephadexG-25 molecular sieve column, concentrating with 3K ultrafiltration tube, and measuring the concentration of the concentrated sample by BCA method.
The experimental result is shown in fig. 3, and through SDS-PAGE electrophoresis, compared with the WK6 empty bacteria control group, the 6 bFGF nano antibody (His-HA tag) HAs an obvious band at about 18kDa in the whole bacteria induced by IPTG, the supernatant and the precipitate of the lysed bacteria, which indicates that the 6 bFGF nano antibody (His-HA tag) HAs a certain amount of expression and exists in both soluble expression and inclusion body. Western Blot verification is carried out on each nano antibody expression supernatant sample, the result is shown in figure 4, and the soluble expression of 6 bFGF nano antibodies (His-HA tag) is further verified.
The sample after expression was purified by affinity chromatography, and the results are shown in FIG. 5. The purified sample was subjected to SDS-PAGE and Western Blot analysis (see FIG. 6), and it was revealed that Nb with a purity of 95.24% was obtained after purificationbFGF1 (His-HAtag); nb with purity of 96.72%bFGF2(His-HA tag); nb with purity of 98.87%bFGF3(His-HA tag); nb with purity of 97.51%bFGF4(His-HA tag); nb with a purity of 93.61%bFGF5(His-HA tag) and Nb with a purity of 96.19%bFGF6(His-HA tag)。
3.2 expression and purification of bFGF Nanobody (His-Flag tag)
(1) Streaking frozen strains by using a flat plate, picking out positive monoclonals, (2) adding the positive monoclonals into a 5m L L B + Amp culture medium, carrying out shaking culture at 37 ℃ and 200rpm for 11-13 h, using the positive monoclonals as first-stage seeds, (3) taking first-stage seed bacteria liquid, inoculating the first-stage seed bacteria liquid into a large-volume L B culture medium containing 100 mu g/m L of Amp according to the proportion of 1%, carrying out shaking culture at 37 ℃ and 200rpm for 11-13 h, (4) centrifuging at 6,000rpm for 30min, taking thalli precipitates, (5) carrying out heavy suspension and washing the thalli precipitates by using 1 XPPBS, carrying out centrifugation at 8,000g for 20min, taking the thalli precipitates, repeating the steps for three times, (6) weighing the thalli precipitates, adding 1XPBS buffer solution according to the proportion of l:10, carrying out centrifugation at ice bath, inserting ultrasonic waves of an ultrasonic crusher into a probe suspension, setting the power of 800W, carrying out ultrasonic time: 4s, adding a 1XPBS buffer solution according to the proportion of l:10, carrying out obvious agarose column chromatography, carrying out heavy suspension, carrying out ultrafiltration on a large-10 ℃ and carrying out purification on a supernatant protein by using an ultrasonic crusher under the conditions of a supernatant fluid of a supernatant obtained by using a supernatant fluid of a supernatant obtained by using a supernatant obtained by a centrifugal chromatography, and carrying out ultrafiltration method, wherein the supernatant obtained by using a supernatant obtained by.
The experimental results are shown in fig. 7, which indicates that two strains of bFGF nanobodies (His-Flag tag) have a certain amount of expression and are mainly in soluble expression forms. WesternBlot verification is carried out on two bFGF nano-antibody (His-Flag tag) expression supernatant samples, and the results are shown in FIG. 8, which further proves that the two bFGF nano-antibodies (His-HA tag) are soluble and expressed. The expression sample was purified by affinity chromatography, and the results are shown in FIG. 9. The purified sample was subjected to SDS-PAGE and WesternBlot analysis (see FIG. 10), and the results showed that Nb with a purity of 92.58% was obtained after purificationbFGF2(His-Flag tag) and Nb with a purity of 97.81%bFGF4(His-Flag tag)。
Example 2 test of physicochemical Properties and biological Activity of anti-bFGF Nanobody
1. Specificity analysis of anti-bFGF Nanobody
Adding 50 ng/well of bFGF, aFGF, KGF, EGF, TNF α, VEGF (all purchased from Beijing Yiqiao Shenzhou biotechnology Co., Ltd.), BSA and milk into a 96-well enzyme label plate, coating the plate overnight at 4 ℃, sealing the plate for 2h by using 5% skim milk the next day, respectively adding 100 ng/well of positive control bFGF mAb (purchased from sigma), blank control PBS and the anti-bFGF nano antibody prepared in example 1, incubating the plate for 1h at 37 ℃, then adding 100 ng/well of anti-Flag (purchased from sigma) label antibody, incubating the plate for 1h, adding 100 mu L/well of TMB developing solution, incubating the plate for 10min, finally terminating the reaction by using 2.29% sulfuric acid, and determining the light absorption value at 450 nm.
The results are shown in FIG. 11, in which 6 strains of anti-bFGF nanobody have strong specific binding ability with bFGF, and weak binding ability with 7 other control antigens.
2. Affinity assay for anti-bFGF Nanobodies
The affinity of the anti-bFGF nanobody is measured by adopting a surface plasma resonance biosensor, 0.4mol of N-ethyl-N' - (dimethylaminopropyl) carbodiimide and 0.1mol of N-hydroxysuccinimide are mixed in equal volume and then are introduced into an instrument at a flow rate of 20 mu L/min to activate the surface of a chip, bFGF protein is diluted to 2mg/m L by 0.2mol of acetic acid buffer solution with pH 4.2, and then flows through the surface of the chip, PBS buffer solution is introduced after the fixed amount reaches the required amount, 1mol of ethanolamine (pH 8.5) solution is introduced for 7min to inactivate the rest ester bonds, anti-bFGF nanobody with different concentrations (3.125, 6.25, 12.5, 25, 50, 100, 200nM) is introduced into the instrument at a flow rate of 20 mu L/min, the affinity of the anti-bFGF nanobody is calculated by instrument software, the results of the affinity of the anti-bFGF nanobody are shown in Table 1, the anti-bFGF nanobody has high affinity, and the highest affinity of the anti-bFGF nanobody reaching 0.69 nM. nM and the other affinity is respectively 11.32nM, 11.37, 53932 nM and 12.37nM respectively.
TABLE 1 affinity constants for anti-bFGF Nanobodies
| Nb1 | Nb2 | Nb3 | Nb4 | Nb5 | Nb6 | |
| KD(nM) | 11.32 | 317.60 | 5.37 | 0.69 | 12.37 | 10.32 |
3. Determination of thermal stability
The screening method comprises the steps of placing screened nano antibodies (namely Nb 1-6) at four different temperatures of 25 ℃, 37 ℃, 60 ℃ and 90 ℃ for 10min, 30min, 60min, 120min and 180min respectively, and simultaneously taking bFGF monoclonal antibodies (Sigma, murine antibodies) as positive controls, collecting samples treated at different temperatures and different times, and detecting the binding property of the samples and bFGF by using an E L ISA method, namely whether the antibodies have the capacity of binding with bFGF after being treated at different temperatures and different times, wherein the detection result is shown in a figure 12, compared with untreated nano antibodies, the relative activity of the nano antibodies is kept above 90% after being treated at 25 ℃ and 37 ℃, the relative activity of the nano antibodies is reduced after being treated at 60 ℃, but the relative dominant activity of the nano antibodies is still above 80%, the relative dominant activity of the nano antibodies is remarkably reduced after being treated at 90 ℃, wherein the relative activity of the nano antibodies is reduced to 0% after being treated at 60 ℃, the relative activity of Nb5 is reduced to 0% after being treated at 180min, the relative activity of other groups is reduced to 0% after being treated at 120min, and the monoclonal antibodies have the comparative dominant activity of the nano antibodies at 25 ℃ and the nano antibodies is prolonged to 0%, thus the monoclonal antibodies have the stability of the nano antibodies which is generally proved that the monoclonal antibodies under the high temperature, the comparative dominant temperature, the comparative stability of the monoclonal antibodies is improved when the monoclonal antibodies is 10%, and the monoclonal antibodies is improved when the nano antibodies are treated at 60 ℃.
4. Competitive binding assays
(1) Detection of Nanobodies (Nb) of the invention by competitive E L ISA methodbFGF) Competed with FGFR2 (purchased from beijing yinqiao shenzhou biotechnology limited) for the ability to bind bFGF (purchased from beijing yinqiao shenzhou biotechnology limited). In the experiment, a quantitative 100ng of nano antibody and a variable (0-1 mu g) of FGFR2 are added into a flat plate coated with 50ng of bFGF at the same time for incubation, if the content detected by the nano antibody is less and less along with the increase of the concentration of FGFR2, the nano antibody is consistent or similar to the binding epitope of FGFR2 and the bFGF, or the affinity of the nano antibody and the bFGF is different from that of FGFR2 and the bFGF; if the concentration of the FGFR2 is gradually increased and the content of the nano antibody is not changed, the situation that the binding epitopes of the nano antibody and the FGFR2 and the bFGF are inconsistent or the binding tightness of the nano antibody and the bFGF is higher than the binding tightness of the FGFR2 and the bFGF is shown. As a result, as shown in fig. 13, as the content of FGFR2 increases, the content of Nb2 and Nb4 remains stable all the time, and it can be presumed that their binding epitopes to bFGF do not coincide with the binding epitopes of FGFR2 and bFGF, or that their affinity to bFGF is higher than that of FGFR 2. The binding force of four nano antibodies Nb1, Nb3, Nb5 and Nb6 to bFGF shows a descending trend along with the increase of the concentration of FGFR2, which indicates that the binding epitope of the four nano antibodies to bFGF is consistent with or close to that of FGFR2 to bFGF, or the affinity of the four nano antibodies to bFGF is lower than that of FGFR2 to bFGF.
(2) Nb detection by adopting competitive E L ISA methodbFGFCompetes with the commercial murine monoclonal antibody bFGF mAb (FB-8) (purchased from Sigma, cat # A8592-.2MG) for the ability to bind bFGF. FIG. 14 shows that Nb4 levels off with increasing bFGF mAb levels, presumably by not matching the binding epitope for bFGF to that of bFGF mAb, or that Nb4 has higher affinity for bFGF than bFGF mAb. The binding force of five nano-antibodies Nb1, Nb2, Nb3, Nb5 and Nb6 to bFGF shows a descending trend along with the increase of the concentration of bFGF mAb, which indicates that the binding epitopes of the five nano-antibodies to bFGF and bFGF mAbs are identical or close to the binding epitope of bFGF, or their affinity for bFGF is lower than that of bFGF mAb.
Example 3 construction of the double antibody Sandwich E L ISA method
1. Pairing experiment of bFGF Nanobody
To construct a double sandwich E L ISA method capable of detecting the concentration of antigen bFGF, we paired the bFGF nanobodies with Nb with the concentration of 5 μ g/m LbFGF1(His-HA tag)、NbbFGF2(His-HA tag)、NbbFGF3(His-HAtag)、NbbFGF5(His-HA tag)、NbbFGF6(His-HA tag) plate, after adding 2. mu.g/m L antigen bFGF, incubating Nb at concentrations of 2. mu.g/m L, 1. mu.g/m L, 0.5. mu.g/m LbFGF4(His-Flag tag), followed by Anti-Flag tag-HRP (purchased from Sigma, cat # A8592-.2MG mouse antibody) as a detection antibody, and finally developed with TMB, and absorbance values were measured at a wavelength of 450 nm.
The results are shown in FIG. 15, with different concentrations of NbbFGF4(His-Flag tag) and the rest bFGF nanobodies have the absorbance values which are more than 3 times of the absorbance values of the 1X PBS of the control group, and the highest absorbance value is NbbFGF4(His-Flag tag) and NbbFGF6(His-HA tag). Therefore, we determined thatbFGF6(His-HAtag) as a capture antibody in the double-sandwich E L ISA method for detecting the concentration of antigen bFGF, NbbFGF4(His-Flag tag) as a detection antibody for the antigen.
2. Determination of concentration range of bFGF (basic fibroblast growth factor) of antigen detected by double-sandwich E L ISA (enzyme linked immunosorbent assay) method
After determining two strains of nano antibodies required by a double-sandwich E L ISA method for detecting the concentration of the antigen bFGF, adding antigens with different concentrations to determine the detection range of the method on the antigen bFGF, and taking Nb with the concentration of 5 mu g/m LbFGF6(His-HAtag) coated plate, antigen bFGF at a concentration of 2000ng/m L, 1000ng/m L, 500ng/m L0, 250ng/m L1, 125ng/m L2, 62.5ng/m L3, 31.25ng/m L, 15.63ng/m L, 7.81ng/m L, 3.91ng/m L, 1.95ng/m L, 0.98ng/m L, 0.49ng/m L was added, and Nb at a concentration of 2. mu.g/m L was incubatedbFGF4(His-Flag tag), then adding Anti-Flag tag-HRP for incubation, and finally developing with TMBThe absorbance values were measured at a wavelength of 450 nm.
As shown in FIG. 16, it was revealed that only an antigen having a concentration of 7.81ng/m L or more was detected by this detection method (FIG. 16A), and the results were plotted as a scattergram with the antigen concentration as the abscissa and the absorbance value as the ordinate, and it was found that the change in absorbance was significant when the antigen concentration was between 7.81ng/m L and 500ng/m L (FIG. 16B).
Antigen concentration was adjusted to fall within the interval of 10ng/m L to 400ng/m L, and then a second double sandwich E L ISA experiment was performed to obtain Nb concentration of 5. mu.g/m LbFGF6(His-HA tag) coated plates, antigen bFGF at concentrations of 400ng/m L, 300ng/m L, 200ng/m L, 100ng/m L, 80ng/m L, 40ng/m L, 20ng/m L and 10ng/m L, and then incubated with Nb at a concentration of 2 μ g/m LbFGF4(His-Flag tag), followed by Anti-Flag tag-HRP as detection antibody, and finally developed with TMB, and absorbance value was measured at a wavelength of 450 nm.
And (3) drawing a scatter diagram of the result by taking the antigen concentration as an abscissa and the absorbance value as an ordinate, and solving a linear equation and a correlation coefficient of the scatter diagram: the equation is that Y is 0.0045X-0.005 and the correlation coefficient R is2The above experimental results show that at Nb concentration of 5 μ g/m L, Nb concentration is not higher than 0.98977bFGF6(His-HA tag) as a capture antibody at a concentration of 2. mu.g/m L of NbbFGF4 (His-flag) as antigen, the antigen bFGF may be diluted to eight concentrations of 400ng/m L, 300ng/m L, 200ng/m L, 100ng/m L, 80ng/m L, 40ng/m L, 20ng/m L, 10ng/m L, and a standard curve for sample detection may be plotted with the antigen concentration as abscissa and the absorbance value as ordinate (fig. 17).
Example 4 evaluation of antigen bFGF Performance by anti-human bFGF Nanobody double-sandwich E L ISA method
1. Specificity test
The double sandwich E L ISA method based on anti-human bFGF nano-antibody in example 3 is adopted to detect bFGF, aFGF, KGF, EGF, TNF α, VEGF (all purchased from Beijing Yiqiao Shenzhou biotechnology Co., Ltd.), BSA and milk with the concentration of 125ng/m L so as to check the specificity of the double sandwich E L ISA method, and when the absorbance value of the detected control antigen is less than one third of the absorbance value of the detected bFGF, the negative reaction is defined.
As shown in fig. 18, the absorbance value of the control antigen detected by the double-sandwich E L ISA method is significantly lower than one third of the absorbance value of the bFGF detected, which proves that the double-sandwich E L ISA method based on the anti-human bFGF nanobody constructed in example 3 has good specificity.
2. Sensitivity analysis
The lowest bFGF concentration which can be detected by the anti-human bFGF nano antibody double-sandwich E L ISA method is the detection sensitivity of the detection method, and the standard curve in the example 3 shows that when each milliliter of sample contains 7.8ng or more of protein, the detection method can detect the protein, and the sensitivity is higher.
3. Repeatability test
(1) In-batch repeat experiments
The same batch of anti-human bFGF nanobody is used, the anti-human bFGF nanobody double-sandwich E L ISA method in the embodiment 3 is adopted to respectively carry out 6 times of detection on bFGF with different concentrations at different time, the results are shown in the table 2, and the batch repeatability is good.
TABLE 2 results of the anti-human bFGF Nanobody double-sandwich E L ISA method batch-to-batch repeated experiments
(2) Repeated experiments between batches
Three batches of prepared anti-human bFGF nano-antibodies are used, the anti-human bFGF nano-antibody double-sandwich E L ISA method in the embodiment 3 is adopted to carry out 6 times of detection on bFGF with different concentrations at different time, the results are shown in the table 3, and the batch-to-batch repeatability is good.
TABLE 3 results of batch-to-batch repeated experiments of anti-human bFGF Nanobody double-sandwich E L ISA method
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> river-south university
Method and kit for detecting bFGF by using anti-human bFGF nano antibody
<160>49
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Gly Ser Asn Phe Ser Asn Asn Asp Met Gly
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Gly Ser Asn Phe Ser Ile Asn Asp Met Gly
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Arg Asn Ile Phe Ser Val Asn His Met Gly
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Gly Arg Thr Phe Ser Ser Gly Ala Met Gly
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Ile Ser Ser Val Gly Arg Thr
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Ile Ser Ser Val Gly Arg Ala
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His Leu Tyr Gly Asp Tyr Arg Gly Thr Gly Phe
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Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg
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Ala Lys Asn Met Val Tyr Leu Gln Met Asn Arg Leu Lys Pro Glu Asp
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Asn Tyr Ala Asn Ser Val Lys Gly Arg Phe Thr Ile Ser Lys Asp Asn
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Met Tyr Gly Asp Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
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Ser Gly Gln Gly Thr Gln Val Thr Val Ser Thr
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Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
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Leu Gln Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly Ser Leu Arg
1 5 10 15
Leu Ser Cys Glu Val Ser Gly Ser Asn Phe Ser Asn Asn Asp Met Gly
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Ser Ser Val Gly Arg Thr Met Tyr Gly Asp Pro Val Lys Gly Arg Phe
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Arg Leu Lys Ala Lys Asp Thr Ala Val Tyr Tyr Cys His Leu Tyr Gly
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Ser Ser
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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
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Trp Tyr Arg Gln Ala Pro Gly Lys Arg Arg Glu Val Val Ala Gly Ile
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Ser Ser Val Gly Arg Ala Met Tyr Ala Asp Pro Val Lys Gly Arg Phe
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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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Ser Ser
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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
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Ser Ser Val Gly Arg Thr Met Tyr Ala Asp Pro Val Lys Gly Arg Phe
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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 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
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Leu Gln Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg
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Leu Ser Cys Lys Ala Ser Arg Asn Ile Phe Ser Val Asn His Met Gly
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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 Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Gly Ala Met Gly
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Trp Phe Arg Gln Ala Pro Gly Lys Glu Pro Glu Phe Val Ala Thr Ile
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Thr Trp Asp Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg
50 55 60
Tyr Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met
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Asn Asp Leu Lys Pro Glu Asp Thr Ala Val Tyr Ser Cys Ala Ala Arg
85 90 95
Ser Tyr Ser Glu Ala Tyr Tyr Leu Ile Gly Ser Ser Asp Tyr Asn Tyr
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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
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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 (8)
1. A kit for detecting bFGF, the kit comprising an anti-human bFGF nanobody comprising a variable region domain consisting of framework region FRs and complementarity determining region CDRs, wherein the variable region domain comprises CDRs 1, CDR2 and CDR3 selected from the group consisting of:
(1) CDR1 shown in SEQ ID No.1, CDR2 shown in SEQ ID No.5, and CDR3 shown in SEQ ID No. 9;
(2) CDR1 shown in SEQ ID No.2, CDR2 shown in SEQ ID No.6, and CDR3 shown in SEQ ID No. 10;
(3) CDR1 shown in SEQ ID No.2, CDR2 shown in SEQ ID No.5, and CDR3 shown in SEQ ID No. 9;
(4) CDR1 shown in SEQ ID No.4, CDR2 shown in SEQ ID No.8, and CDR3 shown in SEQ ID No. 12;
(5) CDR1 shown in SEQ ID No.3, CDR2 shown in SEQ ID No.7 and CDR3 shown in SEQ ID No. 11;
the kit takes an anti-human bFGF nano antibody with a variable region structure shown in any one of (1), (2), (3) and (4) as a primary antibody; an anti-human bFGF nanobody having a variable domain represented by (5) is used as a secondary antibody.
2. The bFGF detection kit according to claim 1, wherein: the framework region FR is selected from the group consisting of FR1, FR2, FR3 and FR 4:
(1) FR1 represented by any one of SEQ ID Nos. 13-15;
(2) FR2 represented by any amino acid sequence of SEQ ID Nos. 16-19;
(3) FR3 represented by any amino acid sequence of SEQ ID Nos. 20-24;
(4) FR4 represented by any amino acid sequence of SEQ ID Nos. 25 to 27.
3. The bFGF detection kit according to claim 1, wherein:
the amino acid sequence of the anti-human bFGF nano antibody as a primary antibody is the amino acid sequence shown in any one of SEQ ID No. 28-30, SEQ ID No.32 or SEQ ID No. 33.
4. The bFGF detection kit according to claim 1, wherein:
the amino acid sequence of the anti-human bFGF nano antibody as a primary antibody is shown as SEQ ID No. 33.
5. The bFGF detection kit according to any one of claims 1 to 4, wherein:
the amino acid sequence of the anti-human bFGF nano antibody as a second antibody is shown as SEQ ID No. 31.
6. The bFGF detection kit according to claim 1, wherein:
the kit is a cancer diagnosis kit.
7. A double-antibody sandwich E L ISA method for detecting bFGF, which is characterized in that:
an anti-human bFGF nano antibody with an amino acid sequence shown as any one of SEQ ID No. 28-30, SEQ ID No.32 or SEQ ID No.33 is used as a primary antibody; an anti-human bFGF nano antibody with an amino acid sequence shown as SEQ ID No.31 is used as a second antibody; this method is not used for the diagnosis or treatment of diseases.
8. The method of claim 7, wherein the method comprises the following steps:
(1) coating: coating the coated plate with a solution containing a primary antibody overnight;
(2) and (3) sealing: washing the coated plate coated with the primary antibody, and then sealing;
(3) adding an antigen: washing the sealed coating plate, adding a sample to be tested, and incubating;
(4) adding a secondary antibody: washing the coated plate after incubating the sample to be tested, adding a second antibody, and incubating;
(5) adding an enzyme-labeled antibody: washing the coated plate after incubation of the secondary antibody, adding an HRP labeled antibody, and incubating;
(6) washing the coated plate, adding TMB color development solution, incubating in dark, and measuring absorbance value at 450nm wavelength.
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| CN201810574975.3A 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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| CN201810574975.3A 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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| CN108508216B true CN108508216B (en) | 2020-08-07 |
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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 |
|---|
| 人源性bFGF Fab抗体体外亲和力成熟的研究。;陶俊。;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20080315(第03期);全文 * |
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