CN111925992B - Hybridoma cell strain secreting anti-Wnt-7 a monoclonal antibody, monoclonal antibody and application thereof - Google Patents
Hybridoma cell strain secreting anti-Wnt-7 a monoclonal antibody, monoclonal antibody and application thereof Download PDFInfo
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Abstract
The invention provides a hybridoma cell strain secreting anti-Wnt-7 a monoclonal antibody, wherein the hybridoma cell strain is 1B8A2 and/or 4G6F 3. The invention also provides the monoclonal antibody secreted by the anti-Wnt-7 a monoclonal antibody hybridoma cell strain, the epitope of the monoclonal antibody is positioned at the C end of the Wnt-7a protein or the M section of the Wnt-7a protein, and the amino acid sequence positioned at the C end of the Wnt-7a protein is shown as SEQ ID NO. 5; the amino acid sequence of the M segment of the Wnt-7a protein is shown as SEQ ID NO. 4. The kit has strong specificity and high sensitivity, can detect the Wnt-7a protein content in body fluid and tissues through matching, and is applied to the application of products for detecting the Wnt-7a protein overexpression or diagnosing diseases characterized by the abnormal expression of the Wnt-7a protein.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a Wnt-7a monoclonal antibody hybridoma cell strain.
Background
The Wnt protein family consists of 19 secreted glycoproteins, which are involved in a variety of biological processes, mainly by binding to frizzled (fzd) receptors, such as cell proliferation, differentiation, and tumor growth. Among them, Wnt-7a (i.e. Wnt7 a) belongs to the canonical Wnt signaling molecule in Wnt family, and participates in the development of various malignant tumors through the canonical Wnt/beta-catenin signaling process. Wnt-7a has a molecular weight of 39kDa, 349 amino acids, and is cleaved before secretion into a mature protein of 318 amino acids with a molecular weight of 48 kD. At the amino acid level, human Wnt-7a protein is 98% similar to mouse Wnt-7a protein. Wnt-7a is usually expressed in lung, testis, lymph node and brain, and participates in the regulation of cell life activity through Wnt/beta-catenin signaling pathway.
The Wnt signaling pathway plays an important regulatory role in cell proliferation and differentiation in a variety of normal and cancer tissues. Wnt signaling molecules are effective targets for cancer diagnosis, treatment and prevention, regenerative medicine and tissue engineering. Published data show that Wnt-7a exhibits different expression patterns in different types of malignancies. In serous ovarian cancer, in the case of CTNNB1 non-mutated, Wnt-7a promotes ovarian cancer progression through CTNNB1 (13-catenin)/TCF signaling pathway; in bladder cancer, the over-expression of Wnt-7a promotes the metastasis of tumor, and the total survival time of bladder cancer patients with high expression of Wnt-7a protein is short; in breast cancer, Wnt-7a, a key tumor cell secretion factor, promotes tumor cell metastasis in vivo by inducing fibroblast activation, resulting in poor patient prognosis. In contrast, in cervical cancer, Wnt-7a can inhibit the proliferation and migration of cancer cells. In mice, Wnt-7a induces cellular senescence by inactivating S-phase kinase-associated protein, an important alternative regulator of cellular senescence, thereby inhibiting the occurrence of lung cancer; in leukemia, Wnt-7a has also been identified as a tumor suppressor gene. Therefore, Wnt-7a has an important significance for the indication of ovarian cancer, and the existing detection kit in the market mostly adopts polyclonal antibodies, has poor antibody specificity, cannot distinguish Wnt-7a highly homologous family proteins, has limited clinical utility for Wnt-7a detection due to the titer or specificity of the antibodies, reduces the specificity of sample detection, and has certain influence on sensitivity. Therefore, monoclonal antibodies for specifically recognizing Wnt-7a protein are urgently needed to be obtained, the requirements of clinical sample detection are met, and the detection sensitivity and specificity are improved.
Polyclonal antibodies are mostly adopted in the existing detection kit system; the antibody has poor specificity, can not distinguish Wnt-7a highly homologous family proteins, and has limited clinical utility on Wnt-7a detection due to the titer or specificity of the antibody, so that the specificity of sample detection is reduced, and the sensitivity is influenced to a certain extent. The invention uses the principle of double antibody sandwich, uses high affinity high specificity pairing monoclonal antibody to prepare the detection kit, and adopts a full-automatic chemiluminescence apparatus to detect the concentration of the sample.
Disclosure of Invention
In order to solve the above problems, it is an object of the present invention to provide a hybridoma cell line secreting anti-Wnt-7 a monoclonal antibody; the second purpose of the invention is to provide a monoclonal antibody secreted by a hybridoma cell strain secreting anti-Wnt-7 a monoclonal antibody; the third purpose of the invention is to provide a kit containing the monoclonal antibody; the fourth purpose of the invention is to provide the application of the monoclonal antibody in preparing a reagent for detecting the Wnt-7a protein. In one aspect of the technical scheme, the invention provides a hybridoma cell strain secreting anti-Wnt-7 a monoclonal antibody, wherein the hybridoma cell strain is 1B8A2 and/or 4G6F3 and is preserved in China center for type culture Collection (the address of the preservation center is Wuhan university, Wuhan, China, 027) -68752319), the preservation number of 1B8A2 is CCTCC NO: C202095, and the preservation number of 4G6F3 is CCTCC NO: C202096.
On one aspect of the technical scheme of the invention, the monoclonal antibody secreted by the anti-Wnt-7 a monoclonal antibody hybridoma cell strain is provided, the epitope of the monoclonal antibody is positioned at the C end of the Wnt-7a protein or the M section of the Wnt-7a protein, and the amino acid sequence positioned at the C end of the Wnt-7a protein is shown as SEQ ID No. 5; the amino acid sequence of the M segment of the Wnt-7a protein is shown as SEQ ID NO. 4.
In one aspect of the technical scheme of the invention, the Wnt-7a protein detection kit is provided, and the Wnt-7a protein detection kit comprises the solid phase carrier coated by the monoclonal antibody. In some embodiments the solid support may be a magnetic particle.
In one aspect of the technical scheme, the invention provides a Wnt-7a protein detection kit, and the Wnt-7a protein detection kit further comprises a calibrator Wnt-7a protein, a detection antibody marked by alkaline phosphatase and a chemiluminescent substrate.
On one aspect of the technical scheme, the Wnt-7a protein detection kit is provided, wherein a chemiluminescent substrate is AMPPD, an antibody in a solid phase carrier coated by the monoclonal antibody is a monoclonal antibody secreted by a hybridoma cell strain 1B8A2, and a detection antibody is a monoclonal antibody secreted by a hybridoma cell strain 4G6F 3.
In one aspect of the technical scheme, the invention provides application of the monoclonal antibody, and the monoclonal antibody is used for preparing a reagent for detecting the over-expression of the Wnt-7a protein or diagnosing the abnormal expression of the Wnt-7a protein.
In one aspect of the technical scheme of the invention, the application of the monoclonal antibody is provided, and the reagent is used for preparing a Wnt-7a protein detection kit.
In one aspect of the technical scheme of the invention, the preparation method of the hybridoma cell strain secreting the anti-Wnt-7 a monoclonal antibody is provided, and the primers are as follows: wnt-7 a-F: 5'-TACACGTGCAAGTGA-3' (SEQ ID NO. 1); wnt-7 a-R: 5'-CGCTTTCCGGTTCAT-3') (SEQ ID NO. 2).
The two monoclonal antibodies provided by the invention can be used for detecting the Wnt-7a protein in a sample, and have strong specificity and high sensitivity; the two monoclonal antibodies specifically recognize different segments of Wnt-7a, and the specificity is better. Can be used for detecting the content of human Wnt-7a in body fluid and tissues through matching and applied to the detection of the over-expression of the Wnt-7a protein or the diagnosis of the diseases characterized by the abnormal expression of the Wnt-7a protein.
The invention has the beneficial effects that: the invention obtains 2 monoclonal antibodies aiming at different epitopes of Wnt-7a by obtaining the hybridoma cell strain secreting anti-Wnt-7 a monoclonal antibody, has strong specificity and high sensitivity, can detect the Wnt-7a protein content in body fluid and tissues through pairing, and is applied to the application of products for detecting the over-expression of the Wnt-7a protein or diagnosing diseases characterized by the abnormal expression of the Wnt-7a protein.
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FIG. 1 is an SDS-PAGE electrophoresis of recombinant Wnt-7a protein;
FIG. 2 is an SDS-PAGE of 1B8A2, 3G5G2 and 4G6F3 antibodies;
FIG. 3 shows the result of WB detection of Wnt-7a truncated protein immunoblot;
FIG. 4 is a schematic view of a reagent strip;
FIG. 5 is a ROC graph.
Detailed Description
The following examples are intended to further illustrate some, but not all, preferred embodiments of the present invention. Other embodiments of the invention based on the present invention, which can be made by a person skilled in the art without inventive step, belong to the scope of protection of the present invention. The invention will be further described with reference to the accompanying drawings.
The experimental procedures, for which specific conditions are not indicated in the examples, are generally carried out according to conventional conditions, for example those described in the molecular cloning protocols (third edition, J. SammBrooks et al), or according to the manufacturer's recommendations.
Example 1 recombinant protein expression and purification of Wnt-7a
Extracting RNA from ovarian cancer tissues, then using cDNA obtained by random primer RT-PCR as a template, designing a primer for cloning Wnt-7a gene, wherein the primer specifically comprises the following components: wnt-7 a-F: 5'-TACACGTGCAAGTGA-3' (SEQ ID NO. 1); wnt-7 a-R: 5'-CGCTTTCCGGTTCAT-3') (SEQ ID NO. 2). The amplified Wnt-7a gene is inserted into pcDNA3.1 vector containing 6 XHis tag to obtain Wnt-7 a-pcDNA3.1. Then the Wnt-7a-pcDNA3.1 is transformed into DH5 alpha, after positive clones are picked up and cultured in a large quantity, the recombinant plasmid Wnt-7a-pcDNA3.1 is extracted by a high-purity plasmid extraction kit. The recombinant plasmid is transferred into 293T cell, and pcDNA3.1 empty vector is simultaneously transfected as negative control, respectively in DMEM culture medium containing 10% fetal calf serum at 37 ℃ and 5% CO2After culturing for 72 hours under the conditions, the supernatant was collected and filtered through a 0.22 μm filter.
The 500mL filtrate was subjected to Ni-NTA affinity chromatography under non-denaturing conditions, with 50mM PBS, 10mM imidazole, 150mM NaCl in equilibrium buffer, pH 7.6. After the sample loading is finished, washing 10 mL; eluted with 50mM PBS, 250mM imidazole, 150mM NaCl, pH7.6 and the eluate was collected. The protein solution was concentrated using a 15kD ultrafiltration tube and the protein was stored in PBS buffer at pH 7.450 mM and at-80 ℃. The purity of the purified protein is identified by SDS-PAGE electrophoresis, the molecular weight is about 39kD, and the grey analysis shows that the purity of the protein reaches more than 95 percent, which is shown in figure 1.
Example 2 preparation of Wnt-7a monoclonal antibody hybridoma cell line
Mouse immunization: 3 batches of BALB/c mice were immunized with Wnt-7a protein, 3 at a time. Each mouse was immunized with 50. mu.g of antigen per time. Mixing the first immune Wnt-7a antigen with Freund's complete adjuvant 1:1, then mixing the Wnt-7a antigen with Freund's incomplete adjuvant 1:1, immunizing once every two weeks for 3 times, cutting off the tail after immunizing for ten days for the third time, collecting blood, and detecting the serum titer.
Hybridoma cell fusion: after the serum titer of the mice reaches 1:100000, the mice are subjected to intraperitoneal boosting immunization (30 mu g) by using Wnt-7a antigen (without adjuvant), spleen cells of the immunized mice are aseptically taken and mixed with SP2/0 mouse myeloma cells in a 50mL centrifuge tube according to a ratio of 4:1 after 3 days, the mixture is washed twice by using culture medium, supernatant is discarded, and then 1mL of preheated 50% PEG-1450 is added for action.
After 1min, slowly shaking for 90s in a water bath, immediately and slowly dripping 15mL of serum-free DMEM medium preheated at 37 ℃, carrying out 5min in the water bath at 37 ℃, then supplementing the serum-free DMEM medium to 40mL, centrifuging at 1000 rpm/mm for 10min, discarding supernatant, adding 40mL of preheated HAT medium, gently blowing, uniformly mixing, transferring to a 96-well culture plate with laid feeder cells, placing 100 mu L of each well, and culturing in an incubator. And identifying cell supernatant, and carrying out 3 times of clone screening on the positive hybridoma to obtain hybridoma cell 3 strains secreting specific monoclonal antibodies, namely 1B8A2, 3G5G2 and 4G6F 3.
Example 3 Wnt-7a monoclonal antibody preparation, purification, potency assay and specificity analysis
Preparing monoclonal antibody ascites: culturing the 3 selected hybridoma cell strains in culture medium to 90% density, collecting cells, diluting to 3 × 106one/mL. After 10-12 weeks BALB/c mice are adaptively raised for one week, the abdominal cavity is injected with immunosuppressant liquid paraffin, 0.5 mL/mouse, 7d later, the abdominal cavity is inoculated with hybridoma cells with stable secretion of antibody and better state, and the hybridoma cells are about (1-2) multiplied by 106After 7-10 days, after the abdomen of the mouse is expanded, the ascites is extracted, and the abdominal water supernatant is collected.
Antibody purification: ascites from mice was collected, the solution was filtered through a 0.45 μm filter, ammonium sulfate solid was added to a final concentration of 50%, and after stirring well at 4 ℃, the precipitate was collected by standing at 12000rpm and re-dissolved in 50mM PBS. The resulting crude purified antibody was applied to a Protein A-Sepharose affinity column at a flow rate of 1mL/min, washed with 5 column volumes of binding buffer (50mM PBS, pH7.0), and then the antibody was eluted with 0.1M glycine-hydrochloric acid solution, pH2.7 (1M Tris buffer pH 9.0 was added to each collection tube in advance for neutralization) to give the objective antibody, and dialyzed 3 times against 50mM PBS solution. The dialyzed antibodies were subjected to 10% SDS-PAGE, and the results showed that 3 antibodies had bands at 55kD and 20kD, and had greater than 90% purity in grayscale analysis, and the SDS-PAGE of the 1B8A2, 3G5G2 and 4G6F3 antibodies is shown in FIG. 2.
Detecting the titer of the monoclonal antibody: the microplate was coated with 1. mu.g/ml of recombinant Wnt-7a protein in carbonate buffer (pH9.5) in a volume of 100. mu.l overnight at 4 ℃, each antibody (1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000) was diluted in a gradient, goat anti-mouse IgG-AP (50ng/ml) was added, and the titers of the purified monoclonal antibodies (S/N >2.1) and 1B8A2, 4G6F3 and 3G5G2 were all 1: 128000.
Monoclonal antibody specificity analysis: microplates were coated overnight at 4 ℃ in a volume of 100. mu.l of CBS buffer containing Wnt-7a, Wnt7b and Wnt8a proteins at 1. mu.g/ml, respectively, and each antibody was diluted in multiples (500ng/ml, 250ng/ml, 125ng/ml, 62.5ng/ml, 31.25ng/ml, 15.625ng/ml, 7.8125ng/ml and 0ng/ml) and then added to microwell reaction plates followed by goat anti-mouse IgG-AP (50ng/ml) with the results shown in Table 1. The result confirms that 1B8A2 can specifically recognize Wnt-7a protein, 4G6F3 and 3G5G2 can react with Wnt7B and Wnt8a proteins, so 1B8A2 can be used as a capture antibody of the kit for specifically recognizing Wnt-7 a.
TABLE 1 analysis of monoclonal antibody specificity
Example 4 validation of Wnt-7a monoclonal antibody pairing
A microwell reaction plate is coated with 3 mu G/mL 1B8A2 antibody, 100 mu L of Wnt-7a calibrator with different concentrations (10-1600pg/mL) is added, the plate is incubated at 37 ℃ for 60min, 100 mu L of AP-labeled 4G6F3 antibody with the concentration of 100ng/mL and 3G5G2 antibody are respectively added after washing, the plate is incubated at 37 ℃ for 60min, and an AMPPD chemiluminescent substrate is added after washing to measure the chemiluminescence value of the plate in an instrument, and the results are shown in Table 2. From the results, 1B8A2 was better matched with 4G6F3, and the sensitivity and the light emission value were higher.
TABLE 2 pairing results for Wnt-7a monoclonal antibodies
| Calibrator (pg/ml) | 4G6F3 | 3G5G2 |
| 0 | 10 | 18 |
| 50 | 246.18 | 615.90 |
| 100 | 490.53 | 293.75 |
| 200 | 989.17 | 516.82 |
| 400 | 1981.15 | 474.35 |
| 800 | 4012.79 | 1148.72 |
| 1600 | 8317.98 | 2871.92 |
Example 5 in-situ identification of Wnt-7a monoclonal antibodies
Constructing and expressing recombinant truncated Wnt-7a protein: according to the sequence of the Wnt-7a protein, an N, M, C terminal sequence and a Wnt-7a full-length protein (marked as Wnt-7a-F) are designed, an expression vector pET30a is connected, and the protein is transferred into an expression strain BL21(DE 3). In LB culture medium, IPTG 0.1 mmol/L25 deg.C overnight induces Wnt-7a truncated recombinant protein soluble expression. Prokaryotic recombinant expression is subjected to affinity purification by 6 × His tags, and the purified protein is subjected to SDS-PAGE electrophoresis, and the purity is over 90 percent.
Wnt-7a-N fragments (32-58) (SEQ ID NO. 3): LGASIICNKIPGLAPRQRAICQSRPDA, respectively;
wnt-7a-M fragment (59-125) (SEQ ID NO.4):
IIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQ；
fragment Wnt-7a-C (308-349) (SEQ NO. 5):
CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK；
wnt-7a-F fragment (32-349) (SEQ ID NO. 6):
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK。
western blotting WB confirmed antibody recognition epitope: recombinant protein Wnt-7a-N/M/C, Wnt-7a full-length 4 proteins were sampled at 5. mu.g and subjected to 15% SDS-PAGE. After the electrophoresis, the protein was converted to 0.45 pg mVDF under a constant pressure of 150V. After the membrane transfer was completed, blocking was performed for two hours at 37 ℃ using PBST solution containing 5% BSA. Each was incubated with 5. mu.g/mL of antibody (1B8A2/4G6F3) solution at 37 ℃ for 1 hour. After the incubation was complete, 50ng/ml rabbit anti-mouse IgG-HRP was added and incubated for 1 hour. And after the cleaning is finished, adding DAB color developing solution for color development. The full-length protein of Wnt-7a is used as a positive control, and brown precipitate substances at the positions of the Wnt-7a-N/M/C fragments indicate that the antibody recognizes the target fragment. The results showed that the 1B8A2 antibody recognized fragment C of Wnt-7a protein and the 4G6F3 antibody recognized fragment M of Wnt-7a protein, as shown in FIG. 3.
Example 6 Wnt-7a detection kit
The embodiment provides a chemiluminescent immunoassay reagent strip, which is provided with a sample hole 4 and a detection hole 3, and nine reagent holes 2 and two reaction holes 1 are sequentially arranged from the sample hole 4, as shown in fig. 4. The reagent strip is characterized in that except that the sample in the sample hole is added by an external source, the reagent in the reagent hole is packaged in the reagent strip in advance, and the reagent is sealed by an aluminum-plastic composite material, and comprises reagents such as magnetic bead preservation solution coated with capture antibody 1B8A2, cleaning solution, AP-labeled 4G6F3 detection antibody preservation solution, substrate and the like.
(I) sample-magnetic bead incubation
50 μ L of plasma sample is added into the sample well 4 of the reagent strip, the reagent strip is added into a matched analysis instrument, a pipetting module in the instrument transfers 50 μ L of the sample into the reaction well 1, then 100 μ L of magnetic bead preservation solution containing the capture antibody 1B8A2 in the reagent well 2 is transferred into the reaction well 1 where the sample is located, and the incubation structure of the instrument maintains the reaction well 1 in a constant temperature state at 37 ℃ for 30 min.
(II) magnetic bead transfer washing
At the end of the previous incubation step, the magnetic separation module of the instrument adsorbs and enriches the magnetic particles in the reaction well 1, and then the pipetting module sucks the waste liquid without the magnetic particles. Then, the liquid-transfering module adds the cleaning liquid in the reagent hole 2 into the reaction hole 1, meanwhile, the magnetic separation module is closed, the liquid-transfering module blows, beats and mixes the reaction hole 1, stands for 30 seconds, the magnetic separation module is started to adsorb magnetic particles, and the liquid-transfering module sucks the cleaning liquid to complete one-time cleaning. The above washing step was carried out three times.
(III) detection of antibody binding
When the wash was complete, the instrument module transferred 200 μ L of AP-labeled 4G6F3 antibody in reagent well 2 to reaction well 1, blown up, mixed the magnetic particles and antibody, and the incubation structure of the instrument maintained the reaction well 1 at a constant temperature of 37 degrees for 30 min.
(IV) magnetic bead transfer washing
At the end of the previous incubation step, the magnetic separation module of the instrument adsorbs and enriches the magnetic particles in the reaction well 1, and then the pipetting module sucks the waste liquid without the magnetic particles. Then, the liquid-transfering module adds the cleaning liquid in the reagent hole 2 into the reaction hole 1, meanwhile, the magnetic separation module is closed, the liquid-transfering module blows, beats and mixes the reaction hole 1, stands for 30 seconds, the magnetic separation module is started to adsorb magnetic particles, and the liquid-transfering module sucks the cleaning liquid to complete one-time cleaning. The above washing step was carried out three times.
(V) Signal detection
After the previous step of cleaning is completed, the liquid transfer module transfers the reaction substrate from the reagent hole 2 to the reaction hole 1, then, the reaction substrate is uniformly blown and beaten, the mixed liquid is sucked and transferred to the detection hole 3, and the signal measurement module of the instrument performs signal acquisition and analysis on the signal of the detection hole 3 to obtain a corresponding concentration value.
(VI) Wnt-7a detection kit for ovarian cancer diagnosis
Pre-operative serum was collected from a hospital for 100 ovarian cancer patients; serum was collected from 100 healthy persons at the same time. And (3) detecting the concentration of Wnt-7a in the serum of the ovarian cancer and the normal human by using a Wnt-7a detection kit. The ROC curve statistical result is shown in FIG. 5, the area under the curve is 0.914, 627ng/mL is used as a detection reference value, the specificity of the Wnt-7a detection kit is 96%, and the sensitivity is 91%.
In conclusion, the 2 monoclonal antibodies provided by the invention can be used for detecting the Wnt-7a protein in a sample, and have strong specificity and high sensitivity. The 2 monoclonal antibodies specifically identify different sections of the Wnt-7a, can detect the content of the human Wnt-7a in body fluid and tissues through matching, and are applied to the application of products for detecting the over-expression of the Wnt-7a protein or diagnosing diseases characterized by the abnormal expression of the Wnt-7a protein.
The foregoing examples are intended to further illustrate some preferred embodiments of the invention, not all embodiments. Other embodiments of the invention based on the present invention, which can be made by a person skilled in the art without inventive step, belong to the scope of protection of the present invention.
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Claims (8)
1. The hybridoma cell strain secreting the anti-Wnt-7 a monoclonal antibody is characterized by being two hybridoma cell strains, wherein the two hybridoma cell strains are 1B8A2 and 4G6F3 and are preserved in China center for type culture collection, the preservation number of the 1B8A2 is CCTCC NO: C202095, and the preservation number of the 4G6F3 is CCTCC NO: C202096.
2. The monoclonal antibody secreted by the anti-Wnt-7 a monoclonal antibody hybridoma cell strain of claim 1, wherein the epitope of the monoclonal antibody is positioned at the C end of the Wnt-7a protein and the M segment of the Wnt-7a protein, and the amino acid sequence positioned at the C end of the Wnt-7a protein is shown as SEQ ID No. 5; the amino acid sequence of the M segment of the Wnt-7a protein is shown as SEQ ID NO. 4.
A Wnt-7a protein detection kit, comprising the monoclonal antibody-coated solid phase carrier of claim 2.
4. The Wnt-7a protein detection kit according to claim 3, wherein the solid phase carrier is a magnetic particle.
5. The Wnt-7a protein detection kit according to claim 4, wherein the Wnt-7a protein detection kit further comprises a calibrator Wnt-7a protein, an alkaline phosphatase-labeled detection antibody, a washing solution, and a chemiluminescent substrate.
6. The kit for detecting the Wnt-7a protein according to claim 3, wherein the chemiluminescent substrate is AMPPD, the antibody in the solid phase carrier coated by the monoclonal antibody is the monoclonal antibody secreted by hybridoma cell line 1B8A2, and the detection antibody is the monoclonal antibody secreted by hybridoma cell line 4G6F 3.
7. The use of the monoclonal antibody of claim 2 for the preparation of a reagent for detecting the overexpression of Wnt-7a protein or for diagnosing aberrant expression of Wnt-7a protein.
8. The use of the monoclonal antibody of claim 7, wherein the reagent is used for preparing a Wnt-7a protein detection kit.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102906117A (en) * | 2010-03-24 | 2013-01-30 | 霍夫曼-拉罗奇有限公司 | Anti-LRP6 antibodies |
| CN103555668A (en) * | 2013-10-28 | 2014-02-05 | 中国人民解放军第三军医大学第一附属医院 | Hybridoma cell strain and anti-Wnt5a monoclonal antibody produced thereby as well as application thereof |
| EP2975047A1 (en) * | 2014-07-18 | 2016-01-20 | Université de Lausanne | Wnt7a polypeptides and their use |
| CN108287242A (en) * | 2017-11-30 | 2018-07-17 | 深圳市老年医学研究所 | The method of liver cancer TCM liver depression biomarker detection based on sialoprotein matter group |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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-
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102906117A (en) * | 2010-03-24 | 2013-01-30 | 霍夫曼-拉罗奇有限公司 | Anti-LRP6 antibodies |
| CN103555668A (en) * | 2013-10-28 | 2014-02-05 | 中国人民解放军第三军医大学第一附属医院 | Hybridoma cell strain and anti-Wnt5a monoclonal antibody produced thereby as well as application thereof |
| EP2975047A1 (en) * | 2014-07-18 | 2016-01-20 | Université de Lausanne | Wnt7a polypeptides and their use |
| CN108287242A (en) * | 2017-11-30 | 2018-07-17 | 深圳市老年医学研究所 | The method of liver cancer TCM liver depression biomarker detection based on sialoprotein matter group |
Non-Patent Citations (1)
| Title |
|---|
| circRNA_001275 upregulates Wnt7a expression by competitively sponging miR-370-3p to promote cisplatin resistance in esophageal cancer;Fang-Wen Zou等;《Int J Oncol》;20200422;第57卷(第1期);方法和材料部分，图9 * |
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