CN114200142A - ELISA kit for detecting exosome Wnt5a protein - Google Patents
ELISA kit for detecting exosome Wnt5a protein Download PDFInfo
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Abstract
The invention provides an ELISA kit for detecting an exosome Wnt5a protein, belonging to the technical field of biological medicines; the ELISA kit for detecting the exosome Wnt5a protein can quantitatively detect the content of the Wnt5a protein in serum exosome; the minimum detection limit of the kit is 156pg/ml, a good linear relation exists between 156pg/ml and 10000pg/ml, and the kit has high sensitivity, accuracy, simplicity, convenience and rapidness.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an ELISA kit for detecting an exosome Wnt5a protein.
Background
Exosomes are nano-scale vesicles with the diameter of about 30-150nm and rich in nucleic acid, protein, lipid and other components, are secreted and released by various cells and exist in body fluids such as serum, plasma, urine, cerebrospinal fluid and the like, are important mediators of intercellular signal transmission, and are widely involved in the development and control of tumorigenesis. The tumor cell exosome is highly enriched in non-coding RNA, protein and the like abnormally expressed by tumor cells, and can remotely remodel the tumor microenvironment, transfer malignant phenotype, drug resistance and the like and promote the malignant progression of tumors. Therefore, the detection of the tumor-related molecules in the serum exosomes becomes an important way for the noninvasive detection of tumors, and tumor diagnosis, curative effect monitoring and prognosis judgment can be performed by detecting the tumor-related molecules in the serum exosomes.
Wnt5a is a highly evolutionarily conserved, atypical Wnt ligand that plays a key role in the regulation of Planar Cell Polarity (PCP), Convergence Elongation (CE), and epithelial-mesenchymal interactions during embryonic morphological development. Wnt5a is closely related to various malignant tumors, and can regulate and control the biological behavior of tumor cells through regulating and controlling multiple aspects of cell proliferation, migration, invasion, drug resistance, immune environment and the like, for example, Wnt5a plays a role in inhibiting oncogenes in thyroid and colorectal cancer and the like, and plays a role in oncogenes in melanoma, gastric cancer and the like, and even research shows that Wnt5a plays a quite opposite regulation and control role in the same type of tumor. Aberrant activation or inhibition of Wnt5a signaling has been shown to be an important event in tumorigenesis.
It has been proved that the Wnt family members are highly enriched in exosomes, Wnt5a is often abnormally highly expressed in tumor cells and stromal cells, and the exosomes secreted by the cells can selectively enrich Wnt5a and secrete and release into the circulating blood, so that the Wnt5a protein in the exosomes needs to be detected. At present, the detection of Wnt5a at home and abroad is mainly limited to tissue and cell detection, and the detection method also adopts detection at the level of quantitative PCR genes and detection at the level of Western blot and immunohistochemical proteins, so that the clinical requirements of rapidness, high sensitivity and large-scale screening cannot be met.
Serum is the most commonly used clinical screening sample, Wnt5a is proved to be widely involved in tumor development and regulation and highly enriched in exosome, and the detection of Wnt5a in serum exosome becomes the currently ideal screening sample, but the detection of Wnt5a protein by the western blot method in the method is qualitative semi-quantitative detection, is complicated to operate, cannot meet the requirement of detecting clinical specimens in large scale, and PCR is a transcription level detection target gene, cannot reflect the protein level, and cannot meet the detection requirement of Wnt5a in serum exosome.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides an ELISA kit for detecting an exosome Wnt5a protein, and the ELISA kit for detecting the exosome Wnt5a protein can quantitatively detect the Wnt5a protein content in serum exosome; the minimum detection limit of the kit can reach 156pg/mL, a good linear relation exists between 156pg/mL and 10000pg/mL, and the kit has high sensitivity, accuracy, simplicity, convenience and rapidness.
The invention provides an ELISA kit for detecting an exosome Wnt5a protein, which comprises a Wnt5a coated antibody, a Wnt5a detection antibody, a biotin-labeled goat anti-mouse polyclonal antibody, HRP enzyme-labeled streptavidin and a Wnt5a protein standard substance;
the Wnt5a coating antibody is a mouse anti-human monoclonal Wnt5a capture antibody, and the concentration is 1-10 mug/mL;
the Wnt5a detection antibody is a mouse anti-human monoclonal Wnt5a antibody, and the concentration is 1-10 mug/mL;
the biotin-labeled goat anti-mouse polyclonal antibody is diluted to a concentration of 1: 80000-1: 4000 by a sample diluent.
Preferably, the mouse anti-human monoclonal Wnt5a capture antibody is 5 μ g/mL; the concentration of the mouse anti-human monoclonal Wnt5a antibody is 2.5 mug/mL; the concentration of the biotin-labeled goat anti-mouse polyclonal antibody is diluted according to 1: 24000.
Further, the HRP enzyme-labeled streptavidin is diluted to a concentration of 1:6000 by a sample diluent.
Further, the concentration of the Wnt5a protein standard is 0.156-10 ng/mL.
Further, the kit also comprises DuoSet ELISA auxiliary reagents.
Furthermore, the DuoSet ELISA auxiliary reagent comprises a 96-well ELISA plate, a sealing plate film, a color developing agent A, a color developing agent B, a stop solution, a coating buffer solution, a sample diluent, a sealing solution and a washing buffer solution.
The invention also provides application of the ELISA kit in detection of an exosome Wnt5a protein.
The invention also provides a detection method of an exosome Wnt5a protein, which is carried out by adopting the ELISA kit and specifically comprises the following steps:
(1) determining the OD value of Wnt5a standard using the ELISA kit of claim 1 and plotting a fitted standard curve;
(2) separating and extracting serum exosomes, and determining the OD value in the serum exosomes by adopting an ELISA kit;
(3) and calculating the content of the exosome Wnt5a protein according to the fitted standard curve.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides an ELISA kit for detecting exosome Wnt5a protein, which takes human serum as a detection object, detects the Wnt5a protein in exosome based on a double-antibody sandwich method, avoids the detection of traditional tissue and cell Wnt5a protein which is complex in operation, low in sensitivity and incapable of meeting the clinical rapid and large-scale screening requirements, and realizes the detection of the content of exosome Wnt5a protein from a clinical common screening serum sample.
The ELISA kit for detecting the exosome Wnt5a protein has the lowest detection limit of 156pg/mL, has a good linear relation between 156 and 10000pg/mL, has a recovery rate of 80-120 percent and an optimal recovery rate of 103.61 percent, has high sensitivity and accuracy, is simple, convenient and quick to operate, and can realize large-scale detection of a sample within two days.
The invention establishes an ELISA kit which has high detection sensitivity, high accuracy and wide linear range and can be used for detecting the exosome Wnt5a protein, realizes the bottleneck that the existing Wnt5a protein cannot be quickly detected and has high cost by using the reagent optimization combination of the existing manufacturers, and also establishes a method for detecting the serum exosome Wnt5a protein at first.
Drawings
FIG. 1 is a standard fit graph.
FIG. 2 is an identification chart of a serum exosome, wherein A is a Nanosight particle size analysis chart, and B is a chart of detecting CD63 and CD9 by Western blot.
FIG. 3 shows the result of the detection of Wnt5a protein as a serum exosome of healthy people and patients with gastric cancer.
FIG. 4 shows that the value of serum exosome Wnt5a in gastric cancer diagnosis is analyzed by a ROC curve.
Detailed Description
The invention will be further described with reference to the following figures and specific examples, but the scope of the invention is not limited thereto.
The formula and sources of the reagent of the invention are as follows:
the Wnt5a coated antibody is a mouse anti-human monoclonal Wnt5a capture antibody of R & D Systems, cat number MAB 6452;
the Wnt5a detection antibody is a mouse anti-human monoclonal Wnt5a antibody of R & D Systems company, with the code of MAB 6451; the biotin-labeled secondary Goat anti-mouse antibody is Goat anti-mouse IgG (H + L) biotin conjugate of Proteintech company, and has a product number of SA 00004-1;
HRP enzyme-labeled Streptavidin is Streptavidin HRP conjugate of Proteitech company, and the product number is SA 00001-0;
the Wnt5a Protein standard is Recombinant Human/Mouse Wnt-5a Protein of R & D Systems company, and the product number is 645-WN;
the DuoSet ELISA auxiliary Reagent is DuoSet ELISA Ancilllary Reagent Kit 2 of R & D Systems company, the product number is DY008, and the DuoSet ELISA auxiliary Reagent specifically comprises a 96-hole ELISA plate, a plate sealing film, a color developing agent A, a color developing agent B, a stop solution, a coating buffer solution, a sample diluent and a washing buffer solution.
Developer a was purchased from R & D Systems, specifically hydrogen peroxide;
developer B was purchased from R & D Systems, Inc., specifically tetramethyl benzidine;
stop solutions were purchased from R & D Systems, specifically 2N sulfate;
coating buffer was purchased from R & D Systems, specifically sterile filtered 1 x PBS;
sample dilutions were purchased from R & D Systems, specifically containing 10% BSA;
the wash buffer was purchased from R & D Systems, inc, and specifically was a buffered surfactant concentrated solution containing a preservative.
Example 1: preparation and condition optimization of ELISA kit for detecting exosome Wnt5a protein
The ELISA kit for detecting the exosome Wnt5a protein comprises the following components:
(1) wnt5 a-coated antibody, mouse anti-human monoclonal Wnt5a capture antibody from R & D Systems, cat 6452.
(2) The Wnt5a detection antibody is a mouse anti-human monoclonal Wnt5a antibody from R & D Systems, Inc., cat 6451.
(3) The biotin-labeled Goat anti-mouse polyclonal antibody is Goat anti-mouse IgG (H + L) biotin conj mu gate of Proteitech company, and has a product number of SA 00004-1.
(4) The HRP enzyme labeled Streptavidin is Streptavidin HRP conjugate from Proteitech company, and the product number is SA 00001-0.
(6) The Wnt5a Protein standard is Recombinant Human/Mouse Wnt-5a Protein of R & D Systems company with a trade name of 645-WN.
(7) The DuoSet ELISA auxiliary Reagent is DuoSet ELISA Ancilllary Reagent Kit 2 of R & D Systems company, has a product number of DY008, and comprises a 96-hole ELISA plate, a plate sealing film, a color developing agent A, a color developing agent B, a stop solution, a coating buffer solution, a diluent, a confining solution and a washing solution.
The ELISA kit for detecting the exosome Wnt5a protein is optimally prepared by the following method:
(1) balancing the ELISA plate to room temperature for later use; diluting Wnt5 a-coated antibody with coating buffer solution to make the concentration of the coated antibody respectively 10 mug/mL, 5.0 mug/mL, 2.5 mug/mL and 1.0 mug/mL, adding 100 mug L of diluted Wnt5 a-coated antibody with different concentrations into each well, setting two wells for each well, and staying overnight at 4 ℃; and (3) completely throwing liquid in the holes the next day, adding 100 mu L of washing liquid into each hole, soaking for 3 minutes, spin-drying, beating the absorbent paper dry, and repeating for three times.
(2) Adding 100 mu L of confining liquid into each hole after washing and spin-drying to seal the ELISA plate, incubating for 2h at 37 ℃, and adding washing liquid to wash for 3 times; adding 100 mu L of negative control (a healthy human serum sample with an OD value close to the OD value of the diluent) and positive control (Wnt 5a standard is added in advance in healthy human serum so that the final concentration of the Wnt5a standard is 500pg/mL) in sequence into a transverse coated hole in an ELISA plate, incubating for 2h at 37 ℃, and adding a washing solution for washing for 3 times.
(3) And adding 100 mu L of Wnt5a detection antibody into each hole of the washed ELISA plate, wherein the Wnt5a detection antibody is the Wnt5a detection antibody which is diluted into 10 mu g/mL, 5.0 mu g/mL, 2.5 mu g/mL and 1.0 mu g/mL by using a sample diluent, respectively, incubating at 37 ℃ for 1h, discarding the liquid in each hole, adding a washing solution, washing for 3 times, and drying by spinning.
(4) Adding biotin-labeled goat-anti-mouse secondary antibodies (diluted by the sample diluent) diluted by 1:4000, 1:8000, 24000, 1:48000 and 80000 into each coated longitudinal hole, incubating for 1h at 37 ℃, throwing off liquid in the holes, adding 100 mu L of washing liquid into each hole, soaking for 3 minutes, drying, beating water-absorbent paper, and repeating for three times.
(5) Adding HRP-labeled streptavidin diluted by 1:1000, 1:2000, 1:6000, 12000 and 20000 (diluted by the sample diluent) in the longitudinal direction of each coating concentration, incubating for 1h at 37 ℃, draining off liquid in the wells, adding 100 mu L of washing liquid into each well, soaking for 3 min, drying, beating up absorbent paper, and repeating for three times.
(6) Preparing a developing solution by the developing solution A and the developing solution B according to the volume ratio of 1:1, adding 100 mu L of developing solution into each hole, developing for 10-15 minutes in a dark place at 37 ℃, adding 50 mu L of a stop solution to stop the reaction, and reading the absorbance (OD) value at 450nm of a BioTek 800TS enzyme-linked immunosorbent assay.
(7) The positive control (P) OD value/negative control (N) OD value, i.e., the combination of the maximum values of P/N, was determined as the most desirable combination, and the specific data are shown in Table 1.
TABLE 1 optimization of ELISA assay kit preparation conditions
The method comprises the steps of searching for appropriate determination conditions in a range of a selected coating antibody of 1-10 mu g/mL, a detected antibody of 1-10 mu g/mL and a dilution ratio of a secondary antibody of 1: 80000-1: 4000, wherein Table 1 shows optimization results of preparation conditions of an ELISA detection kit, and selecting a coating antibody of 5 mu g/mL, a detected antibody of 2.5 mu g/mL, a biotin-labeled goat-anti-mouse secondary antibody of 1:24000 and an HRP-labeled streptavidin of 1:6000 as working concentrations of the kit according to the results in the table.
Example 2: wnt5a standard detection and fitting standard curve drawing
In this example, the Wnt5a standard was tested, and the equation of the Wnt5a protein standard curve is given according to the test data, and the specific test steps are as follows:
(1) and (3) taking out the required lath from the aluminum foil bag which is balanced at room temperature, adding 100 mu l of 5 mu g/mL Wnt5a coating antibody into each hole, sealing the lath by using a sealing film, standing overnight at 4 ℃, drying the coating liquid the next day, filling the coating liquid with a washing liquid, standing for 3 minutes, throwing off the washing liquid, beating the water-absorbing paper to dry, and repeatedly washing the lath for 3 times in the way.
(2) Adding 100 mul of sealing liquid into each hole, placing the hole in a thermostat at 37 ℃ for sealing for 2h, drying the sealing liquid, filling the sealing liquid with washing liquid, standing for 3 minutes, removing the washing liquid, drying the water-absorbing paper, and repeatedly washing the laths for 3 times in the way. And melting the sample to be detected and the standard substance on ice. And (3) arranging standard substance holes on the lath, adding 100 mu L of standard substances with different concentrations (0ng/mL, 0.156ng/mL, 0.312ng/mL, 0.625ng/mL, 1.25ng/mL, 2.5ng/mL, 5ng/mL and 10ng/mL) into each standard substance hole, marking as 1-8 of the standard substance, arranging two repeated holes at each concentration, placing the standard substances in a thermostat, incubating for 2h at 37 ℃, discarding the liquid, filling the washing liquid, standing for 3 min, throwing off the washing liquid, beating the water-absorbing paper dry, and repeating the step of washing the lath for 3 times.
(3) Adding 100 mu L of 2.5 mu g/mL Wnt5a detection antibody into each standard sample hole, sealing the standard sample holes by using a sealing plate membrane, placing the standard sample holes in a thermostat, incubating for 1h at 37 ℃, then removing liquid, patting the standard sample holes dry on absorbent paper, filling the standard sample holes with washing liquid, standing for 3 minutes, throwing off the washing liquid, patting the standard sample holes dry on the absorbent paper, and repeating the step of washing the laths for 3 times.
(4) Adding 100 μ L of biotin-labeled goat anti-mouse IgG (H + L) diluted at a ratio of 1:24000 into the wells, sealing the standard wells with a sealing plate membrane, placing in a thermostat, incubating at 37 ℃ for 1H, removing the liquid, patting dry on absorbent paper, adding a washing solution into the standard wells, standing for 3 minutes, throwing off the washing solution, patting dry on the absorbent paper, and washing the laths repeatedly for 3 times.
(5) Adding 100 mu L of HRP-labeled streptavidin diluted according to a ratio of 1:6000 into standard product holes respectively, sealing the standard product holes by using a sealing plate membrane, placing the standard product holes in a thermostat at 37 ℃ for incubation for 1h, then removing liquid, patting the standard product holes dry on absorbent paper, adding washing liquid into the standard product holes, standing for 3 minutes, throwing off the washing liquid, patting the standard product holes dry on the absorbent paper, and repeatedly washing the laths for 3 times in the way.
(6) Respectively adding 100 μ L of color development solution into the standard wells, sealing the standard wells with sealing plate membrane, incubating in thermostat at 37 deg.C in dark for 15 min, adding 50 μ L of stop solution into the standard wells, and measuring OD values of standard wells 1-8 at 450nm wavelength within 15 min, as shown in Table 2
TABLE 2 OD values of Standard holes 1-8
| |
|
Standard hole 3 | |
|
|
Standard hole 7 | |
|
| Concentration (ng/mL) | 0 | 0.156 | 0.312 | 0.625 | 1.25 | 2.5 | 5 | 10 |
| OD1 | 0.067 | 0.127 | 0.147 | 0.170 | 0.211 | 0.283 | 0.418 | 0.676 |
| OD2 | 0.053 | 0.122 | 0.149 | 0.173 | 0.208 | 0.2985 | 0.415 | 0.685 |
| Average OD value | 0.060 | 0.1245 | 0.148 | 0.1715 | 0.2095 | 0.284 | 0.4165 | 0.6805 |
Table 2 shows the OD values of standard wells 1-8, FIG. 1 shows a standard curve of Wnt5a protein according to the data in Table 2, and the equation of the standard curve of Wnt5a protein is Y ═ a + b × rx+ cx where a is 0.1437, b is-0.0832, c is 0.0539, R is 0.0014, R2＝0.9997，R2X is standard photometry and Y is standard concentration, 0.9997. The standard curve shows that when the concentration of the standard substance is 0.156ng/mL, the standard substance is statistically different from a blank control, which indicates that the lowest detection limit of the kit is 156pg/mL, and a good linear relationship exists between 156-10000 pg/mL.
Example 3: wnt5a recovery assay
In this example, the recovery rate of the ELISA methodology was calculated according to the DuoSet ELISA auxiliary reagent, as follows:
three groups of experiments are set, namely an unlabeled group, a labeled group and a control group, wherein the unlabeled group is 1mL serum, and the labeled group is 980 muL serum +20 muL (500ng/mL) Wnt5a standard; control groups were 980 μ L of dilution +20 μ L (500ng/mL) of Wnt5a standard, 3 duplicate wells were set for each group, and concentration means were determined and calculated as described in example 2.
According to the measurement, the obtained average concentration of the non-standard group is 0.325ng/mL, the obtained average concentration of the standard group is 3.572ng/mL, the obtained average concentration of the control group is 3.134ng/mL, and the recovery rate of the ELISA Kit for detecting the exosome Wnt5a protein is 103.61% according to the formula of (standard group concentration mean-non-standard group concentration mean)/control group concentration mean multiplied by 100%, which meets the requirement of the DuoSet ELISA anal Reagent Kit 2 of R & D Systems company that the recovery rate is 80-120%.
Example 4: detection and comparison of serum exosome Wnt5a content of gastric cancer patient and healthy patient
(1) Separation, identification and detection pretreatment of serum exosomes:
approved by ethical committees of Jiangsu university and Nantong city tumor hospitals, patients were informed, 37 preoperative serum samples of patients with gastric cancer confirmed diagnosis were collected in Nantong city tumor hospitals, 2mL of the serum samples were collected, and 14 serum samples of healthy volunteers of contemporary age-matched physical examination centers, 2mL of the serum samples were collected.
Centrifuging the serum specimen to remove fragments, subpackaging with 200 mu L/tube, and freezing at-80 ℃ for later use; taking the subpackaged serum to be completely melted, adding an ExoQuick exosome precipitation solution reagent (with the product number of EXOQ20A-1) of 63 mu LSBI into each tube after instantaneous separation, blowing, uniformly mixing, and incubating at 4 ℃ for overnight; centrifuging at 4 deg.C 1500g for 30 min, discarding the supernatant, centrifuging the obtained precipitate at 4 deg.C 1500g for 5 min, and carefully discarding the supernatant; then adding 40 mu l of DPBS (PBS without calcium and magnesium ions) for resuspension, and gently blowing, beating and mixing evenly to obtain the serum exosome.
The serum exosomes extracted by separation were detected by using a NanoSight NTA nanoparticle analyzer, and the detection results are shown in fig. 2A. FIG. 2A is a Nanosight particle size analysis diagram, wherein (i) serum exosome 1, serum exosome 2 and serum exosome 3 in (iii) three diagrams are respectively measured in parallel, and it can be seen from the diagram that the average particle size of the exosomes obtained by separation is about 100nm, which accords with the particle size characteristics of the exosomes.
Adding equal volume of RIPA lysate (purchased from Shanghai Bin Yuntian biotechnology limited company, with the product number of P0013B) into the obtained serum exosome, fully mixing the serum exosome and the lysate with equal volume, placing the mixture on ice for 5 minutes, oscillating the mixture for 1 minute, repeating the process for 5 times, centrifuging the mixture for 10 minutes at the temperature of 4 ℃ and the temperature of 12000g, taking supernatant, obtaining the total protein of the serum exosome, and determining the protein concentration by adopting a BCA protein quantification kit (purchased from Kangji for century, with the product number of CW 0014S). Western blot detection of the expression of the exosome marker proteins CD9 and CD63 is carried out on 30 mu g of total serum exosome protein, the result is shown in figure 2B, the extracted serum exosomes all express CD9 and CD63, CD9 and CD63 are exosome protein markers, and the separated exosomes are verified by combining particle size analysis.
(2) Detection of serum exosome Wnt5a content in healthy people and gastric cancer patients:
in this example, the ELISA kit for detecting exosome Wnt5a protein prepared in example 1 of the present invention was used to perform the processing in step (1) on the collected preoperative sera of 14 healthy persons and 37 gastric cancer patients, and then the OD values of the sample wells were determined by the method described in example 2, and the Wnt5a protein content was calculated according to the equation of standard curve fitting by reading the OD values with a microplate reader.
Comparing the difference of serum exosome Wnt5a protein content between healthy people and gastric cancer patients by adopting Graphpad prism 8 drawing and Mann-Whitney U test analysis, as shown in figure 3, the serum exosome Wnt5a content of the healthy group is 0.6196 +/-0.07417 ng/mL, and the serum exosome Wnt5a content of the gastric cancer patient group is 2.774 +/-0.2249 ng/mL. Therefore, the content of the exosome Wnt5a in the serum of the gastric cancer patient is obviously higher than that in the healthy group (P <0.0001), so that the kit prepared by the invention can be used for rapidly detecting the content of the exosome Wnt5a in the serum.
An ROC curve is drawn and the diagnostic value of serum exosome Wnt5a in gastric cancer is analyzed, as shown in figure 4, the AUC area is 0.9859, P is less than 0.0001, the maximum value of York index 0.8983 is the critical point, the corresponding sensitivity is 96.97%, and the specificity is 92.86%.
The present invention is not limited to the above-described embodiments, and any obvious improvements, substitutions or modifications can be made by those skilled in the art without departing from the spirit of the present invention.
Claims (10)
1. An ELISA kit for detecting an exosome Wnt5a protein is characterized in that the serum exosome Wnt5a protein ELISA kit comprises a Wnt5a coating antibody, a Wnt5a detection antibody, biotin-labeled goat anti-mouse polyclonal antibody, HRP enzyme-labeled streptavidin and a Wnt5a protein standard substance;
the Wnt5a coating antibody is a mouse anti-human monoclonal Wnt5a capture antibody, and the concentration is 1-10 mug/mL;
the Wnt5a detection antibody is a mouse anti-human monoclonal Wnt5a antibody, and the concentration is 1-10 mug/mL;
the biotin-labeled goat anti-mouse polyclonal antibody is diluted to a concentration of 1: 80000-1: 4000 by a sample diluent.
2. The ELISA kit for detecting exosome Wnt5a protein according to claim 1, wherein the mouse anti-human monoclonal Wnt5a capture antibody concentration is 5 μ g/mL.
3. The ELISA kit for detecting exosome Wnt5a protein according to claim 1, wherein the mouse anti-human monoclonal Wnt5a antibody concentration is 2.5 μ g/mL.
4. The ELISA kit for detecting the exosome Wnt5a protein according to claim 1, wherein the concentration of the biotin-labeled goat anti-mouse polyclonal antibody is diluted 1: 24000.
5. The ELISA kit for detecting the exosome Wnt5a protein according to claim 1, wherein the HRP enzyme-labeled streptavidin is diluted to a concentration of 1:6000 by a sample diluent.
6. The ELISA kit for detecting exosome Wnt5a protein according to claim 1, wherein the concentration of the Wnt5a protein standard is 0.156-10 ng/mL.
7. The ELISA kit for detecting the exosome Wnt5a protein according to claim 1, wherein the kit further comprises DuoSet ELISA auxiliary reagents.
8. The ELISA kit for detecting the exosome Wnt5a protein according to claim 7, wherein the DuoSet ELISA auxiliary reagent comprises a 96-well ELISA plate, a sealing plate membrane, a color reagent A, a color reagent B, a stop solution, a coating buffer solution, a sample diluent, a sealing solution and a washing buffer solution.
9. Use of the ELISA kit of claim 1 to detect the exosome Wnt5a protein.
10. The method for detecting the exosome Wnt5a protein is characterized by being carried out by adopting the ELISA kit and specifically comprising the following steps of:
(1) determining the OD value of Wnt5a standard using the ELISA kit of claim 1 and plotting a fitted standard curve;
(2) separating and extracting serum exosomes, and determining the OD value in the serum exosomes by adopting an ELISA kit;
(3) and calculating the content of the exosome Wnt5a protein according to the fitted standard curve.
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