CN114989257A - Amantadine antigen mimic epitope and application thereof in magnetic particle enzymatic chemiluminescence homogeneous immunoassay method - Google Patents
Amantadine antigen mimic epitope and application thereof in magnetic particle enzymatic chemiluminescence homogeneous immunoassay method Download PDFInfo
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Abstract
The invention relates to an amantadine antigen mimic epitope and application thereof in a magnetic particle enzymatic chemiluminescence homogeneous immunoassay method, wherein the amino acid sequence is as follows: SVYNALYLAASE, performing fusion expression on the antigen mimotope polypeptide and luciferase, biosynthesizing polypeptide-luciferase fusion protein, applying the fusion protein as a substitute of the traditional chemically synthesized amantadine hapten-luciferase conjugate to immunoassay, and establishing the method for magnetic particle enzymatic chemiluminescence homogeneous immunoassay of amantadine.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an amantadine antigen mimic epitope and application thereof in a magnetic particle enzymatic chemiluminescence homogeneous immunoassay method.
Background
Amantadine (AMD), which is a tricyclic amine, was the first human antiviral drug to inhibit influenza virus, but is also used in the breeding industry to prevent and treat diseases such as avian influenza and swine influenza. Due to its accumulation in the human body and the characteristic of causing viruses to generate drug resistance, the department of agriculture in China has already made the prohibition of amantadine as a veterinary drug. The currently common amantadine detection method mainly comprises instrument detection methods such as high performance liquid chromatography, gas chromatography, liquid chromatography-mass spectrometry combined method and the like. Although these methods have advantages in terms of detection sensitivity, specificity, stability, etc., they also have disadvantages of time consumption, complexity, high test cost, etc. The immunological analysis method has the advantages of strong specificity, high sensitivity, suitability for large-scale screening and the like, and plays an important role in the field of rapid detection of pesticide and veterinary drug residues. The existing immunoassay methods comprise ELISA, colloidal gold immunochromatography test strips, FRET homogeneous phase detection methods and the like.
The magnetic particle enzymatic chemiluminescence homogeneous immunoassay method is an analysis method combining a magnetic separation technology, an enzymatic chemiluminescence technology and an immunoassay technology, and the principle of detecting small molecular substances is as follows: coupling hapten and enzyme into hapten-enzyme, preparing conjugates of antibody and magnetic particles, and mixing the two conjugates with a sample to be detected for incubation reaction. When the sample to be detected does not contain a target substance, a magnetic particle-antibody-hapten-enzyme compound is formed in the solution, the magnetic particle-antibody-hapten-enzyme compound is adsorbed on the wall of a reaction tube after the reaction solution is placed in a magnetic field, the solution in the tube is discarded, then a substrate solution is added, the enzyme triggers the substrate to emit light, and the light emitting value is maximum; when a sample to be detected contains a target substance, the target substance competes for binding with the magnetic particle-antibody, so that the formed magnetic particle-antibody-hapten-enzyme complex is reduced, the formed magnetic particle-antibody-target complex is increased, the reactant is placed in a magnetic field to act, the magnetic particle-antibody-hapten-enzyme complex is fixed on the wall of a reaction tube, the free magnetic particle-antibody-target complex in the solution is discarded, after a substrate solution is added, the luminous value of the magnetic particle-antibody-hapten-enzyme complex is reduced due to the reduction of the amount of the magnetic particle-antibody-hapten-enzyme complex, and the reduced amount of the magnetic particle-antibody-hapten-enzyme complex and the added amount of the target substance present an inverse linear relationship, so that quantitative detection is realized. The method has the advantages of no need of plate washing, short detection time, wide linear range and the like.
The preparation of hapten-enzyme conjugate in the magnetic particle enzymatic chemiluminescence homogeneous immunoassay method is very important. However, the conventional hapten-enzyme conjugate is prepared by directly chemically modifying hapten and enzyme mainly through a chemical synthesis method, and has the problems of large batch error, complicated steps, high cost, easy environmental pollution and the like. In addition, since the hapten-enzyme conjugate directly uses the hapten to be detected as a raw material, the competitive power of the hapten-enzyme conjugate with that of a natural hapten and an antibody is the same in the competitive immunoassay process, and the sensitivity and the linear range of a reaction system are influenced.
Based on the phage display polypeptide technology, the antibody is used as a ligand in a biopanning mode, and a phage display polypeptide library is put into to obtain a polypeptide molecule specifically bound with the antibody, wherein the polypeptide molecule has the property of competing with natural hapten for binding the corresponding antibody and can be applied to the field of immunoassay, and the polypeptide molecule is an antigen mimic epitope.
Disclosure of Invention
The invention takes amantadine monoclonal antibody as target molecule, binds the target molecule to the hole of enzyme-labeled plate, puts phage random display dodecapeptide library, carries on liquid phase affinity panning, combines with the mode of amantadine competitive elution, obtains a polypeptide molecule which can compete with amantadine to bind amantadine monoclonal antibody, its amino acid sequence is: SVYNALYLAASE are provided.
In the molecular structure of the polypeptide, capital English letters respectively represent known natural L-type amino acid residues or one of D-type isomers thereof, namely S represents a serine residue, V represents a valine residue, Y represents a tyrosine residue, N represents an aspartic acid residue, A represents an alanine residue, L represents a leucine residue, and E represents a glutamic acid residue.
The invention also relates to nucleotide for coding the amino acid sequence of the polypeptide molecule, and specifically, the sequence is as follows:
AGCGTGTATAACGCGCTGTATCTGGCGGCGAGCGAA
the polypeptide molecules of the present invention can be prepared in large quantities by means of phage amplification, chemical synthesis or recombinant expression by genetic engineering. Phage amplification refers to the mass propagation and production of phage particles displaying polypeptide molecules by using phage displaying polypeptide molecules through a biological amplification mode. Chemical synthesis refers to polypeptide synthesis by chemically synthesizing polypeptides according to the published amino acid sequences of mimotopes. The mode of genetic engineering recombinant expression refers to the mass production of polypeptide molecules in the form of polypeptide-fusion proteins by cloning genes encoding the polypeptide molecules into expression vectors.
The invention specifically adopts a genetic engineering technology to express amantadine antigen mimic epitope, namely the polypeptide molecule and luciferase are recombined into polypeptide-luciferase fusion protein, and the polypeptide-luciferase fusion protein is applied to the amantadine magnetic particle enzymatic chemiluminescence homogeneous immunoassay method as a substitute of the traditional amantadine hapten-luciferase conjugate.
The beneficial effects of the invention are: the amantadine antigen mimotope provided by the invention can replace the traditional amantadine hapten, is used as a mimic of the amantadine hapten and is fused and expressed with luciferase, and is applied to homogeneous immunodetection analysis. Compared with the traditional chemically synthesized amantadine hapten-luciferase conjugate, the biologically synthesized polypeptide-luciferase fusion protein has the advantages of low preparation cost, good uniformity, environmental friendliness and the like, the selected luciferase has the characteristics of strong luminescence signal, wide linear range, high sensitivity and the like, and the established magnetic particle enzymatic chemiluminescence homogeneous immunoassay method has the advantages of simplicity in operation, few reaction steps, high sensitivity and low cost.
Drawings
FIG. 1 standard curve of indirect competition ELISA based on amantadine for phage display of polypeptides
FIG. 2 is a standard curve for detecting amantadine by using an antigen-mimic epitope-based magnetic particle enzymatic chemiluminescence homogeneous immunoassay method
Detailed Description
Example 1: panning and identification of amantadine antigen mimotope
1) Affinity panning of amantadine antigen mimotope: the specific method comprises the following steps: the amantadine monoclonal antibody was diluted with 10mM PBS (pH 7.2) and coated to wells of an enzyme-labeled plate (100. mu.L/well) at a final concentration of 5. mu.g/mL, and incubated at 15 ℃ for 20 h. Washed 5 times with PBST (10mM PBS pH 7.2 containing 0.1% Tween-20(v/v)), and incubated for 1h at 37 ℃ with the addition of 300. mu.L of blocking solution (3% skim milk). Discard blocking solution and wash 5 times with PBST.
2) mu.L of phage display peptide library (phage display dodecapeptide library, from NEB, Inc., 10-fold dilution of phage stock with PBS, about 2.0X 10 11 pfu), adding the mixture into an enzyme-labeled plate hole coated with the amantadine antibody, and carrying out shaking reaction at 20 ℃ for 5 h. Free unbound phage in the liquid was discarded and washed 5 times with PBST. Bound phage were competitively eluted with 400ng/mL of amantadine standard in PBS, 10. mu.L of eachPhage were eluted to titer and the remainder was used to infect 20mL of e.coli ER2738 strain grown to early log. The phage were purified by PEG/NaCl precipitation and the titer of the amplified phage was determined.
3) In the second and third rounds of panning, the concentration of the amantadine monoclonal antibody was 2. mu.g/mL and 1. mu.g/mL, respectively, and the amantadine standards used for elution were 200ng/mL and 100ng/mL, respectively, and the rest of the procedure was the same as in the first round.
4) Identification of positive phage clones: randomly picking 100 phage plaques from a flat plate for determining the phage titer after the third round of panning, amplifying the phage, and identifying positive phage clones by adopting an indirect enzyme-linked immunosorbent assay method, wherein the specific method comprises the following steps: first, amantadine monoclonal antibody was diluted to 1. mu.g/mL with 10mM PBS (pH 7.4), coated on an enzyme plate, and incubated overnight at 4 ℃. The following day after 3 washes with PBST (10mM PBS, 0.05% Tween-20(v/v)), blocked with PBS containing 5% skim milk powder, and incubated at 37 ℃ for 1 h; mu.l of phage plaque amplification solution (1.0X 10) was added 11 pfu), taking the original phage peptide library as a negative control, and incubating at 37 ℃ for 0.5 h; adding 100 mu L of HRP-labeled anti-M13 phage secondary antibody diluted by 1:5000 times, and incubating at 37 ℃ for 0.5 h; adding 100 μ L TMB substrate solution, incubating at 37 deg.C for 7min, and reading OD with enzyme-labeling instrument 450 . Selection of OD 450 Phage clones 2 times larger than the negative control were positive clones.
5) Identification of amantadine mimotope: the binding property of the screened positive clones to the amantadine monoclonal antibody is determined by adopting an indirect ELISA method, which comprises the following steps: respectively coating amantadine monoclonal antibody, malachite green monoclonal antibody, chloramphenicol monoclonal antibody and uniconazole monoclonal antibody at the concentration of 1 mu g/mL, coating 200 mu L/pore of the amantadine monoclonal antibody and the malachite green monoclonal antibody on an ELISA plate, and incubating overnight at 4 ℃; the following day after 3 washes with PBST (10mM PBS, 0.05% Tween-20(v/v)), blocked with PBS containing 5% skim milk powder, and incubated at 37 ℃ for 0.5 h; adding 100 μ L phage spot amplification solution (1.0 × 1011 pfu), incubating at 37 deg.C for 0.5h with original phage peptide library as negative control; adding 100 mu L of HRP-labeled anti-M13 phage secondary antibody diluted by 1:5000 times, and incubating at 37 ℃ for 0.5 h; adding 100 mu LTMB substrate solution, and incubating at 37 DEG C7min, reading OD by a microplate reader 450 。
The method for identifying the amantadine antigen mimic epitope by adopting an indirect competitive ELISA method comprises the following steps: diluting the anti-amantadine monoclonal antibody with 10mM PBS (pH 7.4), coating the ELISA plate with 1 mug/mL, and incubating overnight at 4 ℃; the following day after 3 washes with PBST (10mM PBS, 0.05% Tween-20(v/v)), blocked with PBS containing 5% nonfat dry milk, incubated for 0.5h at 37 ℃; throwing 50 mu L of phage clone (1.0 multiplied by 1011 pfu) which is identified as positive by indirect ELISA and is not combined with other monoclonal antibody and 50 mu L of amantadine standard substance (concentration range is 0-50ng/mL), incubating for 0.5h at 37 ℃, adding 100 mu L of anti-M13 phage secondary antibody labeled by 1:5000 dilution HRP after PBST washing, and incubating for 0.5h at 37 ℃; after washing the plate with PBST, adding 100. mu.L of TMB substrate solution, developing for 5min in the dark, and reading OD 450 。
The indirect ELISA results show: among the screened positive clones, there was a phage clone (amino acid sequence is SVYNALYLAASE), the binding OD value of which with amantadine monoclonal antibody is 1.5, and the binding OD values with other malachite green monoclonal antibody, chloramphenicol monoclonal antibody and uniconazole monoclonal antibody are all less than 0.1, which indicates that the phage display polypeptide has good binding property with amantadine monoclonal antibody.
Further adopting indirect competition ELISA to identify the phage clones, and displaying the results: the phage clone (amino acid sequence: SVYNALYLAASE) can be competitively combined with the amantadine standard substance to form the amantadine monoclonal antibody coated on the ELISA plate, and has linear competitive blocking relation, the standard curve is S-shaped, the linear correlation is good, and IC 50 At 4.3ng/mL (FIG. 1), the phage-displayed polypeptide was identified as an antigenic mimotope of amantadine.
Example 2 sequencing of the Gene encoding the antigenic mimotope of amantadine and determination of the amino acid sequence thereof
Amplifying the phage which is identified and displayed with the amantadine antigen mimic epitope by indirect competition ELISA, and extracting a DNA sequencing template of the phage. The brief procedure is as follows: phage amplification was performed and after the first centrifugation step 800. mu.L of phage-containing supernatant was transferred to a new centrifuge tube. Add 200 u L PEG/NaCl precipitation phage. After centrifugation, the pellet was resuspended in 100. mu.L of iodide buffer (10mM Tris-HCl (pH 8.0),1mM EDTA,4M NaI), and after centrifugation, the pellet was washed with 70% ethanol (DNA sequencing template). The precipitate was finally resuspended in 20. mu.L of sterile water, 2. mu.L of the precipitate was analyzed by agarose gel electrophoresis; taking 5 mu L of phage template for DNA sequencing, wherein-96 gIII sequencing primers are as follows: 5 '-HOCCCTCATATAGTTAGGTAGCGTACG-3'. The DNA sequencing results were: AGCGTGTATAACGCGCTGTATCTGGCGGCGAGCGAA, the amino acid sequence of the amantadine antigen mimotope obtained according to the codon table is: SVYNALYLAASE is added.
Example 3 biosynthesis of amantadine antigen mimotope polypeptide and luciferase fusion proteins
Synthesis of A Gene of interest
The gene sequence of the mimotope polypeptide is passed through a flexible linker ((GGGGS) 3 ) Is connected with a Nano luciferase (Nano luciferase, Nluc) gene, enzyme cutting sites Nco I and Xho I are designed at two ends of a fusion protein gene, general biosynthesis is carried out, and the gene sequence is as follows: accatgggcAGCGTGTATAACGCGCTGTATCTGGCGGCGAGCGAAGGTGGAGGTTCGGGATCCGGCGGTGGCGGTAGCGGCGGTGGCGGTTCTGTCTTCACTCTTGAAGATTTCGTGGGAGACTGGCGCCAGACGGCGGGTTACAACTTGGATCAGGTCCTTGAGCAGGGGGGGGTGTCGAGCTTGTTTCAAAACCTGGGTGTCAGCGTCACCCCTATCCAACGTATTGTCTTGTCAGGAGAGAATGGCCTGAAGATAGACATTCACGTCATCATTCCGTATGAAGGTCTGAGTGGCGACCAGATGGGACAAATAGAGAAGATTTTTAAGGTCGTTTATCCCGTGGACGATCATCATTTCAAGGTTATACTTCATTACGGTACCCTGGTTATTGATGGCGTAACCCCGAACATGATTGACTATTTCGGAAGACCGTATGAGGGTATTGCGGTCTTCGATGGCAAAAAAATCACGGTCACCGGAACATTGTGGAACGGAAACAAGATAATTGATGAGCGTCTGATCAATCCTGATGGATCGTTGTTGTTCCGCGTCACCATCAATGGAGTAACTGGGTGGCGGCTGTGCGAACGCATTCTGGCG ctcgag are provided.
B. Double enzyme digestion of target gene and expression vector
Restriction enzymes Nco I and Xho I were selected to perform double digestion on the target gene and expression vector pET28 a.
C. Ligation and transformation of products after enzyme digestion
Plasmid pET28a and the target fragment were mixed together at a molar ratio of 1: 10, ligated in a T4 DNA ligase system at 16 ℃ for 16 hours in a metal bath, 5. mu.L of the ligation product was added to 100. mu.L of competent cell DH 5. alpha. and mixed well. After ice-bath for 35min, 42
And (3) thermally shocking the mixture in a water bath for 90s, immediately carrying out ice bath for 3min, supplementing 700 mu L of LB liquid culture solution preheated at 37 ℃, culturing the mixture at 37 ℃ and 200rpm for 45min, taking about 100 mu L of a conversion product, coating the conversion product in an LB-A solid (Amp) culture medium, and culturing the conversion product at 37 ℃ overnight to obtain a positive subclone.
D. Clonal transformation
Extracting the objective plasmid from the subclone obtained by the above steps by using a Tiangen kit, taking 10 mu L to 100 mu L of competent cells Rosseta, and fully mixing the competent cells Rosseta and the Rosseta. The remaining transformation steps were the same as the previous step C, resulting in the positive clone Rosseta pET28a amantadine mimotope polypeptide-luciferase.
E. Expression of polypeptide-luciferase fusion protein-
Selecting the positive clone obtained above on a plate, inoculating the positive clone into 5mL of LB-A, 0.5% sucrose, culturing at 37 ℃ at 220r/min under shaking for 12h, inoculating the culture into 50mL of LB-A, 0.2% sucrose culture medium according to 1% inoculum size (v/v), culturing at 37 ℃ at 220r/min under shaking until culture bacterial concentration OD 600 When the concentration reaches 0.6, adding 0.4mM IPTG, carrying out shaking culture at 25 ℃ and 180r/min for 16h, centrifuging the induced culture at 4 ℃ and 5000g for 15min, collecting thalli sediment, and removing the supernatant. Resuspend the cells in 10mM PBS, pH 7.4(80mL/g wet weight of cells), ultrasonication (200w, 8min), 8000g, centrifugation at 4 ℃ for 15min, collect the supernatant, purify with nickel column, and dialyze with 10mM PBS (pH 7.4) to obtain the amantadine mimotope polypeptide-luciferase fusion protein.
Example 4 detection of amantadine by magnetic particle enzymatic chemiluminescence homogeneous immunoassay based on antigen mimotope
1) Coupling of magnetic microparticles with amantadine antibodies
mu.L of 10mg/mL carboxylated modified magnetic particles (200 nm in diameter) was placed in a 2mL centrifuge tube containing 800. mu.L of 50mM MES (pH6.0, containing 0.005% SDS), 20. mu.L of freshly prepared 20mg/mL EDC and 40. mu.L of freshly prepared NHS solution were added, and the mixture was rapidly mixed and activated at room temperature for 30min using a vertical mixer. After activation, centrifugation is carried out at 10000rpm for 15min, supernatant is removed, 1mL of 0.005% SDS is used for washing for 1 time, then 10mM PB (pH7.0) is used for supplementing to 1mL, ultrasonic treatment is carried out for 10min until the mixture is fully dispersed, 200 mu g of amantadine antibody is added and fully mixed, a rotary mixer is used for coupling at room temperature for 2h, 110 mu L of 10% sodium caseinate is added for sealing for 1h, centrifugation is carried out at 10000rpm for 15min, supernatant is removed, 1mL of preserving fluid (20mM Tris-HCl, 1% sodium caseinate, 0.5% Tween-20, 0.02% sodium azide and pH 8.0) is used for washing once, then 1mL of preserving fluid is used for supplementing to 1mL, and 4 ℃ is used for standby.
2) Establishment of detection method
Mixing PBS, magnetic particles-amantadine antibody and polypeptide-luciferase fusion protein (1mg/mL) according to a volume ratio of 400:2:1, adding 100 mu L of each hole into a reaction tube, adding a series of 100 mu L of samples to be detected with different concentrations, shaking and incubating for 15min at 37 ℃, carrying out magnetic separation for 5min, removing supernatant, washing for 3 times by PBST, adding 100 mu L of chemiluminescent substrate solution (5 mu g/mL coelenterazine, 10mM PBS, 0.25mg/mLBSA, 10mM EDTA disodium), and measuring the chemiluminescence value by a chemiluminescence apparatus within 2 min. With RLU as ordinate and amantadine standard solution concentration as abscissa, drawing standard curve, IC 50 It was 0.4 ng/mL.
By applying the standard curve, the amantadine content of a subsequent sample to be detected can be determined.
Sequence listing
<110> Nanchang Futao detection application System Co., Ltd
Lidelinuo Biotechnology (Beijing) Co., Ltd.
<120> amantadine antigen mimic epitope and application thereof in magnetic particle enzymatic chemiluminescence homogeneous immunoassay method
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Ser Val Tyr Asn Ala Leu Tyr Leu Ala Ala Ser Glu
1 5 10
<210> 2
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
agcgtgtata acgcgctgta tctggcggcg agcgaa 36
Claims (7)
1. An amantadine antigen mimotope based on polypeptide, the amino acid sequence of which is: SVYNALYLAASE are provided.
2. A nucleotide sequence encoding the antigenic mimotope of claim 1.
3. A nucleotide sequence encoding the antigenic mimotope of claim 1, 5 '-3': AGCGTGTATAACGCGCTGTATCTGGCGGCGAGCGAA is added.
4. The use of the antigenic mimic epitope in an amantadine magnetic microparticle enzymatic chemiluminescence homogeneous immunoassay according to claim 1, wherein: the method comprises the following steps:
coupling the antigen mimic epitope polypeptide with luciferase to prepare a polypeptide-luciferase conjugate;
coupling the amantadine monoclonal antibody and the magnetic particles to prepare a magnetic particle-antibody conjugate;
placing the polypeptide-enzyme conjugate and the magnetic particle-antibody conjugate in a PBS buffer solution to serve as core detection elements;
the specific detection steps are as follows: adding a polypeptide-enzyme conjugate, a magnetic particle-antibody conjugate and a sample extracting solution to be detected into a reaction tube, incubating at 37 ℃, adsorbing magnetic particles on the tube wall by using a magnetic frame, removing the solution in the reaction tube, and adding a substrate solution of luciferase into the tube to measure the luminescence value; if the sample does not contain amantadine, the magnetic particle-antibody conjugate is completely combined with the polypeptide-enzyme conjugate to form a magnetic particle-antibody-polypeptide-enzyme complex, and the luminous value of the magnetic particle-antibody-polypeptide-enzyme complex is the maximum after a substrate is added; if the sample contains amantadine, the magnetic particle-antibody adsorbs the amantadine in the sample to be detected to form a part of magnetic particle-antibody-amantadine compound, the formation amount of the magnetic particle-antibody-polypeptide-enzyme compound is reduced, the polypeptide-enzyme compound which is dissociated in the liquid phase is increased and then discarded from the reaction tube, so that the luminous value in the tube is reduced, the luminous value in the tube is gradually reduced along with the increase of the content of the added amantadine, a quantitative reaction curve is formed, and the amantadine content in the specific sample to be detected can be known.
5. The polypeptide-luciferase conjugate for use in the detection method according to claim 4, wherein: is a fusion protein of amantadine antigen mimic polypeptide and luciferase.
6. The method of producing the polypeptide-luciferase conjugate of claim 5, wherein: the gene sequence of the antigen mimic epitope polypeptide is connected with the nano luciferase gene through a flexible linker by adopting a gene engineering recombination expression mode, and is cloned to a prokaryotic expression vector to express fusion protein.
7. The magnetic particle used in the detection method according to claim 4, wherein: comprises a magnetic inner core and a shell coating the outer surface of the inner core, and the diameter range is 100-2000 nm.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116478242A (en) * | 2022-08-25 | 2023-07-25 | 南京大学 | Phage polypeptide for targeting binding to novel coronavirus receptor binding region and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080090814A1 (en) * | 2006-06-22 | 2008-04-17 | Anadys Pharmaceuticals, Inc. | Pyridazinone compounds |
| CN104480072A (en) * | 2014-11-17 | 2015-04-01 | 浙江省农业科学院 | Hybridoma cell strain secreting anti-amantadine monoclonal antibody and application of hybridoma cell strain |
| US20160223527A1 (en) * | 2013-08-29 | 2016-08-04 | Biomark Technologies Inc. | An Immunological Assay To Detect And Quantify Acetylamantadine |
| CN108152499A (en) * | 2017-12-06 | 2018-06-12 | 华南农业大学 | A kind of antigen of amantadine, antibody and its enzyme-linked immunologic detecting kit |
| CN113461782A (en) * | 2021-08-02 | 2021-10-01 | 力德力诺生物技术(北京)有限公司 | Malachite green antigen mimic epitope, preparation method and application thereof |
| CN113795500A (en) * | 2019-04-16 | 2021-12-14 | 华盛顿大学 | amantadine binding protein |
| CN114874310A (en) * | 2022-06-15 | 2022-08-09 | 澳优乳业（中国）有限公司 | IgG epitope peptide of whey allergen beta-lactoglobulin |
-
2022
- 2022-04-22 CN CN202210428999.4A patent/CN114989257A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080090814A1 (en) * | 2006-06-22 | 2008-04-17 | Anadys Pharmaceuticals, Inc. | Pyridazinone compounds |
| US20160223527A1 (en) * | 2013-08-29 | 2016-08-04 | Biomark Technologies Inc. | An Immunological Assay To Detect And Quantify Acetylamantadine |
| CN104480072A (en) * | 2014-11-17 | 2015-04-01 | 浙江省农业科学院 | Hybridoma cell strain secreting anti-amantadine monoclonal antibody and application of hybridoma cell strain |
| CN108152499A (en) * | 2017-12-06 | 2018-06-12 | 华南农业大学 | A kind of antigen of amantadine, antibody and its enzyme-linked immunologic detecting kit |
| CN113795500A (en) * | 2019-04-16 | 2021-12-14 | 华盛顿大学 | amantadine binding protein |
| CN113461782A (en) * | 2021-08-02 | 2021-10-01 | 力德力诺生物技术(北京)有限公司 | Malachite green antigen mimic epitope, preparation method and application thereof |
| CN114874310A (en) * | 2022-06-15 | 2022-08-09 | 澳优乳业（中国）有限公司 | IgG epitope peptide of whey allergen beta-lactoglobulin |
Non-Patent Citations (2)
| Title |
|---|
| DAPENG PENG等: "Preparation of a monoclonal antibody against amantadine and rimantadine and development of an indirect competitive enzyme-linked immunosorbent assay for detecting the same in chicken muscle and liver", 《JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS》, vol. 133, pages 56 - 63 * |
| 彭娟等: "食品中金刚烷胺残留检测方法新进展", 《食品科学》, vol. 42, no. 19, pages 325 - 333 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116478242A (en) * | 2022-08-25 | 2023-07-25 | 南京大学 | Phage polypeptide for targeting binding to novel coronavirus receptor binding region and application thereof |
| CN116478242B (en) * | 2022-08-25 | 2024-05-31 | 南京大学 | Phage polypeptide for targeting binding to novel coronavirus receptor binding region and application thereof |
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