CN110229227B - Polypeptide for preparing ELISA mouse monoclonal coating antibody and rabbit polyclonal detection antibody and application thereof - Google Patents
Polypeptide for preparing ELISA mouse monoclonal coating antibody and rabbit polyclonal detection antibody and application thereof Download PDFInfo
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
Abstract
The invention discloses a polypeptide for preparing an ELISA mouse monoclonal envelope antibody and a rabbit polyclonal detection antibody and application thereof, belonging to the technical field of biomedicine. The polypeptide is a polypeptide with immunogenicity to anaphylactoid reaction specific receptor MRGPRX2 protein, and the amino acid sequence of the polypeptide is shown as SEQ ID NO: 1 is shown. The polypeptide is an immunogenic polypeptide of an anaphylactoid reaction specific receptor MRGPRX2 protein, can be used for preparing an ELISA mouse monoclonal coating antibody and a rabbit polyclonal detection antibody, successfully prepares a double-antibody sandwich ELISA method, wherein the monoclonal antibody is the coating antibody, and the polyclonal antibody is the detection antibody, and verifies the clinical application of the double-antibody sandwich ELISA method. The method is helpful for the research work of the anaphylactoid reaction specific receptor MRGPRX2, and has important significance for guiding the safety of medication in clinic.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and relates to a polypeptide for preparing an ELISA mouse monoclonal coating antibody and a rabbit polyclonal detection antibody and application thereof.
Background
Drug-like allergic reactions are serious and potentially life-threatening allergic reactions. In 1920, Hanzlik and Karsner first reported that phenomena similar to allergic symptoms were induced after intravenous injection of gels in humans or animals as "anaphylactoid reactions". In 1947-1949, Selye et al also called "anaphylactoid reaction" the phenomena of cyanosis and edema of skin around tongue, face and paw after injecting colloidal substances such as ovalbumin into rat vein for the first time. During 1986 and 1988, Toman et al found that typical allergic reactions such as itching, edema, restlessness and sialorrhea occurred after the first injection of the oil vaccine to animals, but the allergic reactions were not mediated by pre-existing IgE but were caused by the stimulation of Tween-80 contained in the vaccine. This is why such allergic reactions are only confirmed. Subsequently, a series of substances such as radiocontrast agents, liposome drugs, non-steroidal anti-inflammatory drugs, analgesics, and Chinese medicinal injections have been found to induce an anaphylactoid reaction.
G Protein Coupled Receptors (GPCRs) are the largest seven-transmembrane receptors, can transmit extracellular signals into cells to generate biological reaction, regulate cell proliferation, survival and metabolism, and play an important role in nerve signal transmission. The Mas-related gene (MRG) family is a recent newly discovered GPCRs, and about 50 MRGs, which are divided into several subtypes, are found in mice, rats, humans, rhesus monkeys, and rhesus monkeys, respectively. In humans, there are four MRGPRX (MRGPRX1-X4) subtypes, and in mice there are MrgprA (a 1-a 10), MrgprB (B1-B5, B8), MrgprC (C11), MrgprD, MrgprE, MrgprF, MrgprG, and MrgprH genes.
MRGPRX2 is a novel class of GPCRs that are activated by antimicrobial peptide peptides (HDPs), neuropeptide SP, FDA approved cationic drugs on the market, and opioids. MRGPRX2 is mainly at human skin MCTCHas higher expression in lung and visceral MCTIs hardly expressed. And, MCTCExpressing MRGPRX2 but not MRGPRX3 and MRGPRX 4.
The research proves that MRGPRX2 is the action target of many small molecule drugs for causing anaphylactoid and pseudo-anaphylaxis. There are gaps in the current antibodies and proteins for laboratory studies against MRGPRX2, and more so clinical test kits and related targeted drugs against MRGPRX2 are in the blank.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a polypeptide for preparing an ELISA mouse monoclonal coating antibody and a rabbit polyclonal detection antibody and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:
the invention discloses a polypeptide for preparing an ELISA mouse monoclonal coating antibody and a rabbit polyclonal detection antibody, wherein the polypeptide is a polypeptide with immunogenicity to an anaphylactoid reaction specific receptor MRGPRX2 protein, and the amino acid sequence of the polypeptide is shown as SEQ ID NO: 1 is shown.
Preferably, the epitope of the polypeptide is the 286-330 amino acid sequence of the Mrgprx2 protein.
The invention also discloses application of the polypeptide for preparing the ELISA mouse monoclonal coating antibody and the rabbit polyclonal detection antibody in preparing an antibody for detecting Mrgprx2 protein.
The invention also discloses a mouse monoclonal antibody containing the polypeptide.
The invention also discloses a rabbit polyclonal antibody containing the polypeptide.
The invention discloses a detection kit containing the polypeptide for preparing an ELISA mouse monoclonal coated antibody and a rabbit polyclonal detection antibody, and the detection kit is used for detecting Mrgprx2 protein.
Preferably, when detecting the Mrgprx2 protein, a monoclonal antibody specifically recognizing the polypeptide of claim 1 is used as a coating antibody, a biotin-labeled polyclonal antibody specifically recognizing the polypeptide of claim 1 is used as a detection antibody, and the coating antibody and the detection antibody can be screened as ELISA-paired antibodies and can be recognized by a double antibody sandwich method.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a polypeptide for preparing an ELISA mouse monoclonal coating antibody and a rabbit polyclonal detection antibody, the epitope of the polypeptide is the 286-330 amino acid sequences of the Mrgprx2 protein, the polypeptide is an immunogenic polypeptide of an anaphylactoid reaction specific receptor MRGPRX2 protein, and the polypeptide is found to have strong immunogenicity through research, so the polypeptide can be used for preparing the ELISA mouse monoclonal coating antibody and the rabbit polyclonal detection antibody, which is favorable for the research work of the anaphylactoid reaction specific receptor MRGPRX2, and the mouse monoclonal antibody and the rabbit polyclonal antibody are prepared through a monoclonal technology and a polyclonal technology.
The invention successfully prepares the double-antibody sandwich ELISA kit, provides scientific research ideas for developing MRGPRX2 clinical detection kits of patients having anaphylactoid reaction risks in the medication process, and has important significance for clinically guiding the safety of medication.
Drawings
FIG. 1 is a DNAstar software analysis diagram;
FIG. 2 is a graph showing the results of the antibody titer of the purified monoclonal antibody identified by the indirect ELISA method;
FIG. 3 is a graph showing the results of the analysis of the titer of a purified polyclonal antibody by the Dot blot method;
FIG. 4 is a standard curve for establishing a double antibody sandwich method;
fig. 5 is a clinical application of the established method.
Detailed Description
In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
The invention discloses a polypeptide for preparing ELISA mouse monoclonal coating antibody and rabbit polyclonal detecting antibody, which is a polypeptide with immunogenicity of anaphylactoid reaction specificity receptor MRGPRX2 protein, and is composed of 45 amino acids, the polypeptide sequence is:
RKQWRLQQPILKLALQRALQDIAEVDHSEGCFRQGTPEMSRSSLV, as shown in SEQ ID NO: 1, the epitope of the polypeptide is 286-330 amino acid sequences of the MRGPRX2 protein. It has strong immunogenicity.
Through polypeptide synthesis, coupling and animal immunization, monoclonal antibodies and polyclonal antibodies are prepared by using monoclonal and polyclonal technologies, the titer of the mouse monoclonal antibody is detected by using an indirect ELISA method, and the result shows that the affinity capacity of the antibody is better; the Dot blot method is used for detecting the titer of the rabbit polyclonal antibody, and the result titer is good and can reach 1/200000. The double-antibody sandwich ELISA method which uses a mouse monoclonal antibody as a coating antibody and a biotin-labeled rabbit polyclonal antibody as a detection antibody is successfully established by using a double-antibody sandwich method, and a standard curve R of the established method2The value reaches above 0.99.
The invention is described in further detail below with reference to the accompanying drawings:
example 1 Synthesis of antigen peptide of allergy-specific receptor MRGPRX2
Using DNAstar analysis software to carry out prediction analysis on antigen epitopes such as protein hydrophilicity, sequence flexibility, protein surface accessibility, protein antigen index and the like of the human MRGPRX2 amino acid sequence, and finally determining that the 286 th and 330 th position are targets, wherein the amino acid sequence is as follows:
RKQWRLQQPILKLALQRALQDIAEVDHSEGCFRQGTPEMSRSSLV。
and synthesizing from the C end to the N end by adopting a manual solid phase Fmoc method to obtain a crude product of the target polypeptide. The target polypeptide is purified by a reversed phase High Performance Liquid Chromatography (HPLC) method according to the principle that different polypeptide molecules are separated according to the difference of hydrophobicity. The solvent is lyophilized to obtain a fluffy polypeptide pure product, the chemical structure of which is characterized by MALDI-TOF mass spectrum, and the purity of which is identified by an analytical high performance liquid chromatograph (Agela C18-10X 250mm, flow rate: 1ml per minute). .
The results are shown in figure 1, and show that the selected peptide fragment is positioned in the segment with stronger antigenicity, immunogenicity and hydrophilicity of the anaphylactoid reaction specific receptor MRGPRX2 protein.
EXAMPLE 2 preparation of anti-polypeptide murine monoclonal antibodies
Preparing coupled KLH-polypeptide into emulsion injection for immunization, spraying alcohol on the dorsal midline of a mouse in a mode of subcutaneous split-point injection to avoid a part with immune nodules, and injecting one injection into 4 different points by four times. After five times of immunization, the tail vein of the mice is collected and subjected to indirect ELISA to detect the titer of the antiserum. The ELISA antiserum titer reaches 1: 50000, which indicates that the immunity is qualified. And selecting the mouse with the highest titer for fusion screening, then subcloning, and screening the pure monoclonal cell strain according to the result of subcloning. And (3) injecting the supernatant of the monoclonal cell strain into a mouse to prepare ascites, and performing protein G affinity purification on the ascites to obtain a purified monoclonal antibody.
Example 3 antibody identification monoclonal antibody titers were detected using an indirect ELISA method
1) Preparing an enzyme label plate: after the microplate is assembled, antigen coating is prepared.
2) Antigen dilution and coating: adding the prepared antigen working solution into a concave sample adding groove, taking 100 mu L of the antigen working solution by using a 300 mu L range 8-hole liquid shifter, adding the antigen working solution to the bottom of a hole of an enzyme-labeled plate, covering aluminum foil paper after sample adding is finished, and incubating for 2h or overnight at 4 ℃ in a 37 ℃ oven.
3) Washing the plate: taking out the ELISA plate coated with the antigen, reversely buckling to remove liquid in the hole, and then placing on a clean water absorption towel for draining; adding 200ml TBST into each well with a discharging gun until the well is full without overflowing, throwing TBST out after the whole plate is added and standing for 3min, throwing the TBST for 3 times, putting the plate on a clean water-absorbing towel for draining, and beating each plate for 6 times (until no obvious liquid residue exists). This set of operations was repeated 4 times.
4) And (3) sealing: after the plate washing was completed, 200. mu.L of the prepared blocking solution (3% skim milk) was added to each well with a 300. mu.L range 8-well pipette, covered with aluminum foil paper, and allowed to stand in an incubator at 37 ℃ for 1 hour.
5) Primary antibody incubation: adding the diluted primary antibodies (positive serum and negative serum) into an enzyme label plate, and standing and incubating for 2h at 37 ℃ or overnight at 4 ℃.
6) Washing the plate: the operation is the same as step 3).
7) And (3) secondary antibody incubation: the secondary antibody was diluted with TBST at a ratio of 1:5000, and then 100. mu.L of the secondary antibody was added to each well with a line gun, followed by incubation at 37 ℃ for 1 hour in an incubator.
8) Washing the plate: the operation is the same as step 3).
9) Color development: 30min before development, the ELISA substrate A, B was removed from the freezer and allowed to return to room temperature (when the room temperature was too low, the substrate was removed 2h earlier). The substrate A, B was formulated at 1:1 volume according to the required amount and mixed by vortexing on a vortex shaker for 10 s. Substrate mixture (100. mu.L) was added to each well of the ELISA plate, covered with a lid, and allowed to stand for 5 min. The volume of the developing solution is equal to the number of pores × 100 μ L +2 mL.
10) And (4) terminating: after developing for 5min, 100 mu L of stop solution is added into each hole, and the stop solution adding speed is high so as to avoid excessive samples and inconsistent developing reaction time.
The results are shown in FIG. 2, the titers of the monoclonal antibody and the polypeptide antigen prepared by ELISA detection are better, and Positive in the figure represents Positive control; sample represents the monoclonal antibody prepared; nagetive represents a negative control; blank represents a Blank control.
EXAMPLE 4 preparation of anti-polypeptide Rabbit polyclonal antibody
Preparing coupled KLH-polypeptide into emulsion injection for immunization, spraying alcohol on the dorsal midline of a New Zealand rabbit in a mode of subcutaneous split-point injection to avoid a part with immune nodule, and injecting one injection into 4 different points by four times. After five times of immunization, blood is collected from the ear vein of the rabbit, and the titer of the anti-rabbit serum is detected by an indirect Dot blot method. Results referring to figure 3, the antiserum titer reached 1: 100000, which indicates the immune is qualified. The antibodies were collected by carotid bleeding from rabbits and affinity purified for rabbit polyclonal antibodies.
FIG. 3 shows that the titer of the polyclonal antibody prepared by Dot blot assay is 1/200000 (generally, the titer is up to 1/50000, which is considered as acceptable, and the immunization is successful).
Example 5 antibody identification: detection of polyclonal antibody titer by Dot blot method
1) Preparation of the membrane: the Dot blot detection adopts an NC film, the NC film is cut into strips with the width of 6.5cm, square small grids with the length and the width of 1cm multiplied by 1cm are drawn on the NC film (the films (white) and blue paper are required to be completely overlapped when the grids are drawn on the blue paper, and the square small grids on the blue paper can leave correct scratches on the film). The two sides of the membrane are respectively provided with a distance of 0.25cm in width, and a corresponding number of square small lattices are drawn according to the condition that one antibody corresponds to one strip of 1cm multiplied by 6cm (when the small lattices are drawn, the force is not too small, scratches of each small lattice are left on the membrane, but the membrane cannot be scratched).
2) Antigen coating: the blue protective paper on the film is uncovered, 2 microliter of corresponding polypeptide diluent with the concentration of 50 ng/. mu.L is taken out from each square grid by a 2.5 microliter pipette in a reverse imbibition mode, and the polypeptide is spotted in the center of the square grid with the concentration of 1cm multiplied by 1cm on the film (white), namely the coating amount of the polypeptide is 100 ng. After spotting is complete, the NC film and the blue protective paper underneath the film are placed together in a 37 ℃ oven for 30 min.
3) And (3) sealing: to the container is provided with 1cm21mL of confining liquid is added into each hole of the small square grids; cutting off each 6 small squares (1 cm multiplied by 6 cm: A1-A6) according to the scratch, then cutting the 6 small squares into 6 independent small squares, putting each small square into a 24-hole plate according to the numbering sequence (6 holes in each 24-hole plate, 4 in total, A, B, C, D is marked on the left and right of the 24-hole plate), and enabling the surface of the membrane coated with the polypeptide to face upwards. And sealing for 1h.
4) Primary antibody incubation: after blocking, the blocking solution was poured off, 1mL of the corresponding primary antibody dilution was added to each well, and the 24-well plate was placed on a shaker and the primary antibody was incubated for 2 h.
5) Incubation with secondary antibody
(1) Washing membrane
After the primary antibody incubation was completed, the primary antibody was poured into a waste tank, 2mL of TBST was added to each well to wash the membrane for 5min, and after the time was up, the TBST was poured into the waste tank. The membrane washing was repeated 5 times.
(2) Dilution with secondary antibody
During membrane washing, secondary antibodies from the same species as the primary antibody were diluted with TBST at 1:8000 as secondary antibody working solution. After membrane washing, 1mL of secondary antibody working solution was added to each well, and the mixture was placed in a shaker and incubated for 1h.
(3) Washing membrane
After the secondary antibody incubation was completed, the secondary antibody was poured into a waste liquid tank, 2mL of TBST was added to each well, and the membrane was washed for 5min, and at the end of the time, TBST was poured into the waste liquid tank. The membrane washing was repeated 5 times.
6) And (6) developing.
The results are shown in fig. 3, and the prepared polyclonal antibody titer is high and reaches 1: 200000, which indicates that the invention successfully prepares polyclonal antibody.
Example 6 double antibody Sandwich screening ELISA paired (murine monoclonal and Rabbit monoclonal) antibodies
1) Coating: coating the mouse monoclonal antibody 100-400 ng/well with coating solution (CBS coating solution: PH 9.66), 100 μ l/well, 4 deg.C overnight or 37 deg.C for 2 h;
2) washing the plate: washing solution (PBST) 300. mu.l/well, washing the plate 3 times;
3) and (3) sealing: blocking solution (1% -5% BSA), 200 μ l/well, 37 ℃ for 2 h;
4) washing the plate: washing solution (PBST) 300. mu.L/well, washing the plate 3 times;
5) filling: the MRGPRX2 protein (ab 165129, available from abcam) was diluted with a diluent (20 XPBS pH 7.2) at 100. mu.l/well for 2h at 37 ℃;
6) washing the plate: washing solution (PBST) 300. mu.L/well, washing the plate 3 times;
7) and (3) detection: dilution (20 × PBS PH 7.2) rabbit polyclonal antibody: 50-400 ng/well, 100 mul/well, 37 ℃ for 1 h;
8) washing the plate: washing solution (PBST) 300. mu.L/well, washing the plate 3 times;
9) enzyme labeling: HRP-streptavidin was diluted with a diluent (20 XPBS pH 7.2) (A21000, available from Abbkine) at 1:500 for 30min at 37 ℃;
10) washing the plate: washing solution (PBST) 300. mu.L/well, washing the plate 3 times;
11) color development: a single-component developing solution (PR1200, available from Solarbio corporation) was used at 100. mu.l/well for 10min at 37 ℃.
12) And (4) terminating: stop solution (2mol/L H)2SO4) 50 μ l/well.
The results are shown in FIG. 4, where FIG. 4 is a standard curve for establishing a double antibody sandwich method, R2The value reaches above 0.996, the linearity is good, and the linear range is 0-100 ng/ml.
In summary, the test results of the present invention show the following advantages:
1. coupling efficiency of polypeptide to carrier: the coupling efficiencies determined by the Ellman (DTNB) reagent method and the BCA method roughly correspond to each other, being greater than 90%.
2. Antibody titer: the antiserum titers of the mice were all 1: 32000 or above; the antiserum titers of three new zealand rabbits were all 1: over 200000.
3. Antibody affinity: the affinity is stronger.
4. Establishment of the double-antibody sandwich ELISA method: successfully matches a matched antibody using the double-antibody sandwich kit, wherein a mouse monoclonal antibody is used as a coating antibody, and a rabbit polyclonal antibody is used as a detection antibody. Preparing a standard curve with good linearity by condition optimization, R2The value reaches above 0.99.
5. The clinical application is as follows: the established double-antibody sandwich ELISA method is used for detecting blood samples of patients with clinically determined urticaria and normal physical examination people, and the result is shown in figure 5 and shows that the difference is significant (p is less than 0.05).
In conclusion, the invention discovers that the target polypeptide can cause strong immune response by performing predictive analysis on the epitope such as protein hydrophilicity, sequence flexibility, protein surface accessibility, protein antigen index and the like by using a bioinformatics method. The target polypeptide is synthesized by utilizing a manual solid phase Fmoc method, and the purity of the synthesized target polypeptide is analyzed by using a high performance liquid chromatography, wherein the purity is more than 90 percent, and the purity requirement of an immunized mouse is met. By usingThe animal immunization method prepares the mouse monoclonal antibody and the rabbit polyclonal antibody, performs affinity purification on the antibodies, and finally detects that the affinity of the mouse monoclonal antibody is better and the titer of the rabbit polyclonal antibody is better by using indirect ELISA kit. Thus, MRGPRX2 polypeptides can be used to make murine monoclonal antibodies and rabbit polyclonal antibodies. And finally, successfully pairing the paired antibodies using the double-antibody sandwich kit, wherein the mouse monoclonal antibody is used as a coating antibody, and the rabbit polyclonal antibody is used as a detection antibody. Preparing a standard curve with good linearity by condition optimization, R2The value reaches above 0.99, and the application of the established method in clinic is verified. The method is helpful for the research work of the anaphylactoid reaction specific receptor MRGPRX2, and has important significance for guiding the safety of medication in clinic.
The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.
Sequence listing
<120> polypeptide for preparing ELISA mouse monoclonal coating antibody and rabbit polyclonal detection antibody and application thereof
<140> university of west ampere transportation
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 45
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Arg Lys Gln Trp Arg Leu Gln Gln Pro Ile Leu Lys Leu Ala Leu Gln
1 5 10 15
Arg Ala Leu Gln Asp Ile Ala Glu Val Asp His Ser Glu Gly Cys Phe
20 25 30
Arg Gln Gly Thr Pro Glu Met Ser Arg Ser Ser Leu Val
35 40 45
Claims (5)
1. A polypeptide for preparing ELISA mouse monoclonal coating antibody and rabbit polyclonal detecting antibody, characterized in that the polypeptide is immunogenic polypeptide, and the amino acid sequence is shown in SEQ ID NO: 1 is shown.
2. The use of the polypeptide of claim 1 for preparing ELISA mouse monoclonal coated antibody and rabbit polyclonal detection antibody for preparing antibodies for detecting human Mrgprx2 protein.
3. A murine monoclonal antibody prepared using the polypeptide of claim 1 as an antigen.
4. A rabbit polyclonal antibody prepared by using the polypeptide of claim 1 as an antigen.
5. A detection kit, which is characterized in that when detecting human Mrgprx2 protein, a monoclonal antibody specifically recognizing the polypeptide of claim 1 is used as a coating antibody, a biotin-labeled polyclonal antibody specifically recognizing the polypeptide of claim 1 is used as a detection antibody, and the coating antibody and the detection antibody can be screened as ELISA pairing antibodies and can be recognized by a double antibody sandwich method;
wherein the monoclonal antibody and the polyclonal antibody are both prepared by using the polypeptide of claim 1 as an antigen.
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| CN106754731B (en) * | 2017-02-23 | 2019-10-11 | 西安交通大学 | The highly expressed recombinant cell of MrgprX2 and MrgprX2 high expression membrane receptor stationary phase and preparation method and application |
| CN107703312A (en) * | 2017-11-15 | 2018-02-16 | 西安交通大学 | The protein marker and method of examination medicine anaphylactoid reaction sensitive group |
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| EP1340979A2 (en) * | 2002-02-27 | 2003-09-03 | Pfizer Limited | Neuropeptide receptor and uses thereof |
| WO2004000999A3 (en) * | 2002-06-21 | 2005-06-23 | California Inst Of Techn | Identification of a receptor controlling migration and metastasis of skin cancer cells |
| CN107002087A (en) * | 2014-08-01 | 2017-08-01 | 约翰·霍普金斯大学 | Detect false allergic drug reaction and differentiate blocking agent to prevent the experiment based on MRGPRX2/MRGPRB2 expression cells of adverse reaction |
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