CN113248597A - Tofenamic acid artificial antigen, antibody, and synthetic method and application thereof - Google Patents
Tofenamic acid artificial antigen, antibody, and synthetic method and application thereof Download PDFInfo
- Publication number
- CN113248597A CN113248597A CN202110753256.XA CN202110753256A CN113248597A CN 113248597 A CN113248597 A CN 113248597A CN 202110753256 A CN202110753256 A CN 202110753256A CN 113248597 A CN113248597 A CN 113248597A
- Authority
- CN
- China
- Prior art keywords
- tolfenamic acid
- artificial antigen
- solution
- antibody
- emulsifying
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- 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
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- 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
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- 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
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
-
- 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
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9486—Analgesics, e.g. opiates, aspirine
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Hematology (AREA)
- Genetics & Genomics (AREA)
- Urology & Nephrology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Emergency Medicine (AREA)
- Pain & Pain Management (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmacology & Pharmacy (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a tolfenamic acid artificial antigen, a tolfenamic acid artificial antibody, a synthetic method and application thereof. The structure formula of the tolfenamic acid artificial antigen is shown as a formula I;
Description
Technical Field
The invention relates to the technical field of immunochemical analysis, in particular to artificial antigen and antibody of tolfenamic acid, and a synthetic method and application thereof.
Background
Tolfenamic acid (TLF) is a non-steroidal anti-inflammatory drug shared by human and livestock, has antipyretic, analgesic and anti-inflammatory effects, is often used in combination with antibiotics in animal husbandry and breeding industry, and is used for treating animal diseases. The unreasonable use of the veterinary drug such as overuse and abuse can cause the veterinary drug residue in the livestock and poultry to exceed standard, thereby causing potential harm to human health. With the increasing demand of people on meat, eggs, milk and the like, the problem of veterinary drug residue in animal-derived food is continuously attracting attention of people. In order to strengthen the supervision of the residue of tolfenamic acid in animal-derived food and ensure the quality safety of animal products, the European Committee stipulates that the maximum residue limit of tolfenamic acid in milk is 50 mug/kg; the maximum residual limits in the muscle, liver and kidney tissues of pigs and cattle were 50, 400 and 100. mu.g/kg, respectively.
At present, the liquid chromatography-mass spectrometry/mass spectrometry method is mainly adopted for detecting the residue of tolfenamic acid in animal-derived food in national industrial standards. Although the method has strong specificity and high sensitivity, the sample pretreatment is complicated, the detection time is long, the cost is high, and the popularization and the use are limited. The immunoassay method is a qualitative and quantitative analysis method based on antigen-antibody specific reaction, has strong specificity, high sensitivity, simple operation and quick reaction, and is suitable for real-time detection of a large number of samples on site. The antigen determines the specificity and sensitivity of the antibody, so that the preparation of the high-quality tolfenamic acid artificial antigen and the development of the anti-tolfenamic acid antibody with strong specificity and high sensitivity are very important for realizing the rapid detection of the tolfenamic acid residue.
Disclosure of Invention
The invention provides a tolfenamic acid artificial antigen, an antibody, a synthetic method and application thereof.
The technical problem to be solved by the invention is realized by the following technical scheme:
in the first aspect, the structure formula of the tolfenamic acid artificial antigen is shown as formula I.
In the formula I, n is a natural number of 1-100, and K represents carrier protein.
As a preferred embodiment of the artificial antigen of tolfenamic acid provided by the present invention, the carrier protein is bovine serum albumin, human serum albumin, ovalbumin or hemocyanin.
As a preferred embodiment of the tolfenamic acid artificial antigen provided by the invention, when K is hemocyanin, the tolfenamic acid artificial antigen is used as an immunogen; when K is bovine serum albumin, it is used as coating antigen.
In a second aspect, the method for synthesizing the artificial antigen of tolfenamic acid is obtained by using tolfenamic acid as a hapten and carrying out chemical coupling on the tolfenamic acid and a carrier protein.
As a preferred embodiment of the method for synthesizing the artificial antigen of tolfenamic acid provided by the invention, the method comprises the following steps:
(1) dissolving tolfenamic acid, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in N, N-dimethylformamide, and reacting for 4-48 hours to obtain solution A;
(2) dissolving carrier protein in phosphate buffer solution to obtain solution B;
(3) and mixing the solution A and the solution B, and reacting at room temperature for 6-48 hours to obtain the tolfenamic acid artificial antigen shown in the formula I.
As a preferred embodiment of the method for synthesizing the artificial antigen of tolfenamic acid provided by the invention, the method comprises the following steps:
(1) dissolving 10-60mg of tolfenamic acid, 10-60mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 10-40mg of N-hydroxysuccinimide in 0.2-2mL of N, N-dimethylformamide, and reacting for 4-48 hours under stirring to obtain a solution A;
(2) dissolving 10-70mg of carrier protein in 10mL of phosphate buffer solution with the concentration of 0.01M and the pH value of 7.4 to obtain solution B;
(3) dropwise adding the solution A into the solution B, stirring at room temperature, and reacting overnight;
(4) and (3) purification: dialyzing the reaction solution in PBS for 1-3 days to obtain the artificial antigen of tolfenamic acid shown in formula I.
In a third aspect, a tolfenamic acid antibody, which is produced by immunizing an animal with the tolfenamic acid immunizing antigen described above.
The tolfenamic acid antibody provided by the invention comprises a polyclonal antibody, a monoclonal antibody and a recombinant antibody.
In a fourth aspect, the method for preparing the tolfenamic acid antibody comprises the following steps:
(1) mixing tolfenamic acid immunogen with Freund's adjuvant in equal volume;
(2) fully emulsifying the immunogen and the adjuvant by using an automatic emulsifying instrument, wherein the emulsifying speed is set to 280 revolutions per minute, the total emulsifying is carried out for 3 times, each time of emulsifying is 5 minutes, and the immunogen and the adjuvant are placed for 3 minutes at the temperature of-20 ℃ after each time of emulsifying;
(3) the Freund complete adjuvant is used for primary immunization, the Freund incomplete adjuvant is used for boosting immunization, the immunization dose is 250 mu g/mouse, and the immunization interval is 60 days; the number of immunizations was 3. On the 7 th day after each immunization, tail vein blood was collected, centrifuged at 4000rpm for 10min, and the supernatant was collected as antiserum, which was stored at-20 ℃.
The emulsification and immunization procedure provided by the invention improves the efficient emulsification effect, increases the immunization dose, can effectively prolong the immune cycle, allows the antibody to perform more sufficient affinity maturation in vivo, and obtains the high-affinity tolfenamic acid antiserum.
In a fifth aspect, any one of the following uses of the tolfenamic acid artificial antigen described above, or the tolfenamic acid antibody described above:
(1) use in the detection of tolfenamic acid;
(2) the application in the preparation of the detection kit of tolfenamic acid;
(3) the application in preparing an immunochromatographic test strip of tolfenamic acid.
The invention has the following beneficial effects:
the invention discloses a new artificial antigen of tolfenamic acid and a preparation method thereof for the first time, the artificial antigen is used for immunizing animals to obtain specific antibodies with high titer and high sensitivity, the preparation process is simple and economic, and the detection sensitivity of the antibodies can reach 23.8 ng/mL. The method is high in practicability and has great value for residue detection of tolfenamic acid. The invention prepares the artificial antigen of tolfenamic acid for the first time, fills the gap at home and abroad, and can meet the needs of research at home and abroad.
Drawings
FIG. 1 is a matrix-assisted laser desorption tandem time of flight mass spectrometry (MALDI-TOF-MS) spectrum of an artificial antigen of tolfenamic acid.
FIG. 2 is a graph of the standard inhibition of indirect competition ELISA with a polyclonal antibody to tolfenamic acid.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are, unless otherwise specified, conventional procedures well known to those skilled in the art. The test materials used in the following examples were purchased from conventional biochemical manufacturers unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. Phosphate buffer solutions (abbreviated as PBS) used in the following examples were all phosphate buffer solutions of pH 7.4 and 0.01M, and carbonate buffer solutions (abbreviated as CB) were all sodium carbonate buffer solutions of pH 9.6 and 0.05M. Bovine serum albumin is abbreviated as BSA, and hemocyanin is abbreviated as KLH.
Example 1 preparation and characterization of artificial antigen of tolfenamic acid
Preparation of mono-and tolfenamic acid immunogens
(1) 13mg of TLF (tolfenamic acid) was dissolved in 0.5mL of DMF (N, N-dimethylformamide), and 15mg of EDC.HCL (1-ethyl- (3-dimethylaminopropyl) carbodiimides hydrochloride) and 8mg of NHS (N-hydroxysuccinimide) were added thereto, and the reaction was magnetically stirred at room temperature for 10 hours to obtain a solution A.
(2) 10mg of KLH was dissolved in 10mL of PBS buffer to obtain solution B.
(3) And dropwise adding the solution A into the solution B, stirring at room temperature, and reacting overnight to obtain a reaction product, namely tolfenamic acid immunogen. The reaction product was dialyzed against PBS for 72 hours with 6 water changes in between. After dialysis, the reaction product was dispensed into 2mL centrifuge tubes and stored at-20 ℃ for further use.
Preparation and characterization of tolfenamic acid coating antigen
1. Preparation of toffenamic acid coating antigen
(1) 13mg of TLF was dissolved in 0.5mL of DMF, and 15mg of EDC.HCL and 8mg of NHS were added and the reaction was magnetically stirred at room temperature for 10 hours to obtain solution A.
(2) 20mg BSA was dissolved in 10mL PBS buffer to obtain solution B.
(3) And dropwise adding the solution A into the solution B, stirring at room temperature, and reacting overnight to obtain a reaction product, namely tolfenamic acid coating antigen (TLF-BSA). The reaction product was dialyzed against PBS for 72 hours with 6 water changes in between. After dialysis, the reaction product was dispensed into 2mL centrifuge tubes and stored at-20 ℃ for further use.
2. Determination of coupling ratio of tolfenamic acid coating source
The product synthesized above was identified by MALDI-TOF-MS and the coupling ratio was calculated. The results are shown in figure 1, where BSA has a molecular weight of 64720.259 and the synthesized product has a molecular weight of 68126.637, indicating successful synthesis of the coating source with a coupling ratio of (68126.637-64720.259)/261 ═ 13, i.e. an average of 13 tolfenamic acid haptens per BSA molecule.
Example 2 preparation of tolfenamic acid antiserum
Fully emulsifying the tolfenamic acid immunogen obtained in the example 1 and an equivalent amount of Freund's adjuvant by using an automatic emulsifying machine, wherein the emulsifying speed is set to 280 revolutions per minute, the emulsifying is carried out for 3 times, each time is 5 minutes, and the tolfenamic acid immunogen is placed at the temperature of-20 ℃ for 3 minutes after each time of emulsifying; and immunizing female Balb/c mice with 6-8 weeks old and 8-20g of body weight by using the emulsified immunogen, wherein Freund's complete adjuvant is used for primary immunization, and Freund's incomplete adjuvant is used for boosting immunization. The immunization mode is subcutaneous multipoint injection of neck and back, the immunization dose is 250 mug/mouse, the immunization interval is 60 days, and 3 times of immunization are carried out totally. On the 7 th day after each immunization, tail vein blood was collected, centrifuged at 4000rpm for 10min, and the supernatant was collected as antiserum, which was stored at-20 ℃.
Example 3 identification of Tofenamic acid antiserum
The antiserum obtained in example 2 was identified by indirect competition ELISA. The specific operation steps are as follows:
(1) coating: diluting the tolfenamic acid coating source to 1 mu g/mL by using CB solution, adding 100 mu L of tolfenamic acid coating source to an ELISA plate in each hole, and incubating for 2 hours at 37 ℃;
(2) washing: pouring out liquid in the holes, washing for 3 times with washing liquid for 1min each time, and patting dry on absorbent paper;
(3) and (3) sealing: adding 150 mu L of confining liquid into each hole, incubating for 1 hour at 37 ℃, and washing;
(4) sample adding: adding 50 μ L of tolfenamic acid standard substance with serial concentration diluted by PBS and 50 μ L of antiserum with working concentration into each well, incubating at 37 deg.C for 30min, and washing;
(5) adding a secondary antibody: adding 100 mu L of HRP-goat anti-mouse IgG into each hole, incubating for 30min at 37 ℃, and washing;
(6) color development: adding 100 μ L of freshly prepared TMB solution into each well, and developing at 37 deg.C in dark for 15 min;
(7) and (4) terminating: 50 μ L of 2mol/L H was added to each well2Stopping the reaction by using SO4 solution;
(8) reading: read OD of each well with microplate reader450nmA value;
(9) establishing a standard curve: taking the logarithm value of the concentration of the CPF standard substance as an abscissa, taking the OD value corresponding to each concentration standard substance as an ordinate, drawing a standard inhibition curve, and calculating IC50The value is obtained.
The antiserum after the three-immunization is identified as shown in figure 2, and the tolfenamic acid antiserum is obtained by calculating a standard curveIC of50It was 23.8 ng/mL.
The above-mentioned embodiments only express the embodiments of the present invention, and the description is more specific and detailed, but not understood as the limitation of the patent scope of the present invention, but all the technical solutions obtained by using the equivalent substitution or the equivalent transformation should fall within the protection scope of the present invention.
Claims (10)
1. The tolfenamic acid artificial antigen is characterized by having a structural formula shown as a formula I.
In the formula I, n is a natural number of 1-100, and K represents carrier protein.
2. The artificial antigen of tolfenamic acid of claim 1, wherein said carrier protein is bovine serum albumin, human serum albumin, ovalbumin, or hemocyanin.
3. The artificial antigen of tolfenamic acid as claimed in claim 2, wherein when K is hemocyanin, as an immunogen; when K is bovine serum albumin, it is used as coating antigen.
4. The method of synthesizing the artificial antigen of tolfenamic acid as claimed in claim 1, which is obtained by chemical coupling of tolfenamic acid as a hapten with a carrier protein.
5. The method for synthesizing artificial antigen of tolfenamic acid according to claim 4, characterized in that it comprises the following steps:
(1) dissolving tolfenamic acid, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in N, N-dimethylformamide, and reacting for 4-48 hours to obtain solution A;
(2) dissolving carrier protein in phosphate buffer solution to obtain solution B;
(3) and mixing the solution A and the solution B, and reacting at room temperature for 6-48 hours to obtain the tolfenamic acid artificial antigen shown in the formula I.
6. The method for synthesizing artificial antigen of tolfenamic acid according to claim 4, characterized in that it comprises the following steps:
(1) dissolving 10-60mg of tolfenamic acid, 10-60mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 10-40mg of N-hydroxysuccinimide in 0.2-2mL of N, N-dimethylformamide, and reacting for 4-48 hours under stirring to obtain a solution A;
(2) dissolving 10-70mg of carrier protein in 10mL of phosphate buffer solution with the concentration of 0.01M and the pH value of 7.4 to obtain solution B;
(3) dropwise adding the solution A into the solution B, stirring at room temperature, and reacting overnight;
(4) and (3) purification: dialyzing the reaction solution in PBS for 1-3 days to obtain the artificial antigen of tolfenamic acid shown in formula I.
7. A tolfenamic acid antibody, characterized in that it is obtained by immunizing an animal with the tolfenamic acid immunizing antigen of claim 3.
8. The tolfenamic acid antibody of claim 7, characterized in that it comprises polyclonal, monoclonal and recombinant antibodies.
9. A method of making the tolfenamic acid antibody of claim 7, comprising the steps of:
(1) mixing tolfenamic acid immunogen with Freund's adjuvant in equal volume;
(2) fully emulsifying the immunogen and the adjuvant by using an automatic emulsifying instrument, wherein the emulsifying speed is set to 280 revolutions per minute, the total emulsifying is carried out for 3 times, each time of emulsifying is 5 minutes, and the immunogen and the adjuvant are placed for 3 minutes at the temperature of-20 ℃ after each time of emulsifying;
(3) the Freund complete adjuvant is used for primary immunization, the Freund incomplete adjuvant is used for boosting immunization, the immunization dose is 250 mu g/mouse, and the immunization interval is 60 days; the number of immunizations was 3.
10. The artificial antigen of tolfenamic acid of claim 1, or the use of any one of the following of the tolfenamic acid antibody of claim 7:
(1) use in the detection of tolfenamic acid;
(2) the application in the preparation of the detection kit of tolfenamic acid;
(3) the application in preparing an immunochromatographic test strip of tolfenamic acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110753256.XA CN113248597A (en) | 2021-07-02 | 2021-07-02 | Tofenamic acid artificial antigen, antibody, and synthetic method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110753256.XA CN113248597A (en) | 2021-07-02 | 2021-07-02 | Tofenamic acid artificial antigen, antibody, and synthetic method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113248597A true CN113248597A (en) | 2021-08-13 |
Family
ID=77190671
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110753256.XA Pending CN113248597A (en) | 2021-07-02 | 2021-07-02 | Tofenamic acid artificial antigen, antibody, and synthetic method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113248597A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109369435A (en) * | 2018-10-26 | 2019-02-22 | 北京勤邦生物技术有限公司 | Tolfenamic Acid haptens, artificial antigen and antibody and its preparation method and application |
| CN110294762A (en) * | 2019-06-14 | 2019-10-01 | 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所） | Toxoflavin haptens, artificial antigen, antibody and its synthetic method and application |
| CN112939873A (en) * | 2021-02-08 | 2021-06-11 | 华南农业大学 | Trimethoprim hapten TMPH, artificial antigen, antibody and preparation method and application thereof |
-
2021
- 2021-07-02 CN CN202110753256.XA patent/CN113248597A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109369435A (en) * | 2018-10-26 | 2019-02-22 | 北京勤邦生物技术有限公司 | Tolfenamic Acid haptens, artificial antigen and antibody and its preparation method and application |
| CN110294762A (en) * | 2019-06-14 | 2019-10-01 | 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所） | Toxoflavin haptens, artificial antigen, antibody and its synthetic method and application |
| CN112939873A (en) * | 2021-02-08 | 2021-06-11 | 华南农业大学 | Trimethoprim hapten TMPH, artificial antigen, antibody and preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| CD CREATIVE DIAGNOSTICS: "《Anti-Tolfenamic acid polyclonal antibody（DPAB-DC4829）》", 18 November 2019, HTTPS://WWW.CREATIVE-DIAGNOSTICS.COM/ANTI-TA-PAB-193009-147.HTM * |
| 熊艳华 等: ""卡洛芬单克隆抗体的制备"", 《中国免疫学杂志》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106867971B (en) | Flunixin meglumine monoclonal antibody hybridoma cell strain YY and application thereof | |
| CN114316027B (en) | Fluoronii Xin Rengong antigen and preparation method and application thereof | |
| CN108680755B (en) | Sulfonamide synergist hapten and holoantigen as well as preparation method and application thereof | |
| CN108558718B (en) | Florfenicol, florfenicol amine antigen and antibody and simultaneous detection enzyme-linked immunoassay method thereof | |
| CN110713986B (en) | Vitamin B strain 1 Monoclonal antibody hybridoma cell strain CBDD and application thereof | |
| CN109212200A (en) | A kind of Nicarbazin haptens, artificial antigen and the preparation method and application thereof | |
| CN105131121A (en) | Monoclonal antibody for detecting furazolidone metabolites, ELISA method, and kit | |
| CN113968853B (en) | Hapten and artificial antigen of atropine alkaloid, and preparation method and application thereof | |
| CN112375744A (en) | Dihydropyridine monoclonal antibody hybridoma cell strain and application thereof | |
| CN110642743B (en) | Nifuroxazide hapten and artificial antigen as well as preparation methods and application thereof | |
| CN110343669B (en) | Hybridoma cell strain DNC secreting anti-triclabendazole monoclonal antibody and application thereof | |
| CN111334479A (en) | Chlorhydroxypyridine monoclonal antibody hybridoma cell strain TYL and application thereof | |
| CN114031528B (en) | Florfenicol hapten, artificial antigen, antibody and synthetic method and application thereof | |
| CN113248597A (en) | Tofenamic acid artificial antigen, antibody, and synthetic method and application thereof | |
| CN113046325B (en) | Vitamin K 3 Monoclonal antibody hybridoma cell strain and application thereof | |
| CN110683965A (en) | Nifurtida hapten and artificial antigen as well as preparation methods and application thereof | |
| CN113582923B (en) | Quinaldine hapten, artificial antigen and antibody as well as preparation methods and application thereof | |
| CN111454912B (en) | Cyperazine monoclonal antibody hybridoma cell strain and application thereof | |
| CN113444166A (en) | Carprofen artificial antigen, antibody, and synthetic method and application thereof | |
| CN109187948B (en) | Paroxarsone and nitrophenylarsonic acid duplex detection test paper | |
| CN112813036B (en) | Triamcinolone acetonide monoclonal antibody hybridoma cell strain and application thereof | |
| CN112094816B (en) | Hybridoma cell strain secreting 2-mercaptobenzimidazole monoclonal antibody, monoclonal antibody and application | |
| CN110616194A (en) | Aconitine monoclonal antibody cell strain SJ and application thereof | |
| CN110746506A (en) | Preparation method of rabbit anti-human myoglobin polyclonal antibody serum | |
| CN114315817A (en) | Meloxicam hapten and artificial antigen as well as preparation methods and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210813 |
|
| RJ01 | Rejection of invention patent application after publication |