CN112094816B - Hybridoma cell strain secreting 2-mercaptobenzimidazole monoclonal antibody, monoclonal antibody and application - Google Patents
Hybridoma cell strain secreting 2-mercaptobenzimidazole monoclonal antibody, monoclonal antibody and application Download PDFInfo
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- CN112094816B CN112094816B CN202011007610.6A CN202011007610A CN112094816B CN 112094816 B CN112094816 B CN 112094816B CN 202011007610 A CN202011007610 A CN 202011007610A CN 112094816 B CN112094816 B CN 112094816B
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- 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
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- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Abstract
The invention discloses a hybridoma cell strain secreting a 2-mercaptobenzimidazole monoclonal antibody, the monoclonal antibody and application. The preservation number of the hybridoma cell strain is CCTCC NO: C2020176. the hybridoma cell strain can produce a 2-mercaptobenzimidazole monoclonal antibody, and the antibody has the capacity of specifically recognizing and combining a 2-mercaptobenzimidazole antigen with high affinity and can be used for detecting the 2-mercaptobenzimidazole.
Description
Technical Field
The invention relates to the technical field of detection, and particularly relates to a hybridoma cell strain secreting a 2-mercaptobenzimidazole monoclonal antibody, the monoclonal antibody and application.
Background
2-Mercaptobenzimidazole (2-Mercaptobenzimidazole) is an imidazole-based antithyroid compound. The action mechanism is to inhibit the peroxidase in the thyroid gland, thereby preventing the oxidation of iodide absorbed into the thyroid gland and the coupling of tyrosine, and preventing the synthesis of thyroxine (T4) and triiodothyronine (T3). Animal experiments show that the compound can inhibit B lymphocyte to synthesize antibody, reduce the level of thyroid stimulating antibody in blood circulation and restore the function of inhibiting T cell to normal.
The 2-mercaptobenzimidazole can increase the water absorption of subcutaneous tissues, muscles and intestines and stomach of animals by inhibiting the secretion of thyroid hormone, thereby achieving the purpose of weight increment. However, thyroid antagonists have teratogenic and carcinogenic effects, and there is a great safety risk in eating food with such drugs left behind. Therefore, the european union banned the use of such drugs for animal products as early as 1981.
In recent years, cases of illegal addition of 2-mercaptobenzimidazole discovered or investigated in China still occur, the development of animal husbandry in China is seriously influenced, the reputation of the industry is damaged, and potential harm is caused to the health of people. With the increasing importance of the country on food safety and the increasing attack of illegal acts, the illegal addition of 2-mercaptobenzimidazole is greatly converged, and the treatment effect is obvious.
At present, the authoritative detection method of 2-mercaptobenzimidazole specified by national and industrial standards is a liquid chromatography-mass spectrometry technology. The method has high sensitivity, strong specificity and good reliability; but the method depends on large-scale instruments, has high requirement on professional knowledge storage of operators, is complicated and time-consuming to operate, has high cost, and cannot be widely popularized and used. The immunological analysis technology based on antigen-antibody specific binding, such as the detection methods of colloidal gold immunochromatography, fluorescence immunochromatography and the like based on antigen-antibody specific binding, has the characteristics of good sensitivity and specificity, no dependence on large instruments, convenient operation, short time and low cost, and is a detection technology which is easy to use on site and in real time. If the immunochromatography detection method and the product for detecting the 2-mercaptobenzimidazole can be developed, the wide popularization and the use of the detection of the 2-mercaptobenzimidazole are facilitated.
Disclosure of Invention
The core of the detection method based on the immunoreaction is a monoclonal antibody for specifically recognizing 2-mercaptobenzimidazole, and the binding sensitivity and specificity of the antibody determine the accuracy of the detection method, so that the development of a good monoclonal antibody for resisting 2-mercaptobenzimidazole is a key precondition for the development of the detection method.
The invention provides a hybridoma cell strain capable of secreting a monoclonal antibody which specifically recognizes 2-mercaptobenzimidazole and is combined with 2-mercaptobenzimidazole with high affinity, and a 2-mercaptobenzimidazole monoclonal antibody secreted by the hybridoma cell strain.
The invention also provides application of the antibody secreted by the hybridoma cell strain, and the antibody can be used for detecting 2-mercaptobenzimidazole.
The invention provides the following specific technical scheme:
a hybridoma cell strain with a preservation number of CCTCC NO: C2020176. the cell strain is named as a hybridoma cell strain 15D2, is preserved in China Center for Type Culture Collection (CCTCC) in 09 months and 10 days in 2020, addresses Wuhan Hubei, Wuhan university, and has a preservation number of CCTCC NO: C2020176.
the 2-mercaptobenzimidazole monoclonal antibody is secreted and produced by the hybridoma cell strain.
The 2-mercaptobenzimidazole monoclonal antibody can specifically recognize 2-mercaptobenzimidazole and is combined with 2-mercaptobenzimidazole with high affinity, and can be used for detecting 2-mercaptobenzimidazole.
The 2-mercaptobenzimidazole monoclonal antibody can be used for preparing a 2-mercaptobenzimidazole detection kit for qualitative and/or quantitative determination of 2-mercaptobenzimidazole.
Compared with the prior art, the invention has the beneficial effects that:
the monoclonal antibody efficiently combined with the 2-mercaptobenzimidazole antigen and the hybridoma cell strain secreting the monoclonal antibody are obtained for the first time, and a foundation is provided for the application of a subsequent antibody reagent to the detection of the 2-mercaptobenzimidazole.
The 2-mercaptobenzimidazole monoclonal antibody can specifically recognize 2-mercaptobenzimidazole and is combined with 2-mercaptobenzimidazole with high affinity, and can be used for detecting 2-mercaptobenzimidazole.
Drawings
FIG. 1 is a graph showing the results of ELISA potency assay of anti-2-mercaptobenzimidazole monoclonal antibodies; wherein, the abscissa conc represents the concentration, the ordinate represents the OD value of 450nm, BSA represents the blank control group, and 2M-BSA represents the 2-mercaptobenzimidazole monoclonal antibody group.
Detailed Description
The invention is described in further detail below with reference to the figures and examples.
The following are the specific preparation processes of the hybridoma cell strain 15D2 and the 2-mercaptobenzimidazole monoclonal antibody. Unless otherwise specified, the reagents and materials used in the experimental procedures of the present invention are conventional commercial reagents and materials, and the standard experimental methods (refer to "antibody technology experimental guidelines" (e. harlo, science publishers, 2005) and "antibody preparation and use experimental guidelines" (g.c. houghard, science publishers, 2010) are used in the methods of the "national science and technology noun examination committee" in terms of "immunologic noun" (science publishers, 2008).
2-mercaptobenzimidazole was purchased from Beijing Solibao. Mouse myeloma cells (SP2/0) were purchased from a cell bank of a Chinese academy of sciences.
Example 1 hapten conjugation
2-mercaptobenzimidazole protein conjugates, namely antigens (2M-BSA for short) formed by chemically coupling 2-mercaptobenzimidazole and Bovine Serum Albumin (BSA).
Coupling hapten (2-mercaptobenzimidazole) and BSA protein were coupled using succinimide-4- (N-maleimide) cyclohexane-1-1-hydroxy acid ester (SMCC) as a crosslinker. SMCC is a bifunctional coupling agent containing N-hydroxysuccinimide (NHS) active ester and maleimide, and can bond compounds respectively containing sulfydryl and amino. Firstly, the protein containing amino group reacts with several times of coupling agent, after the reaction is finished, the unreacted SMCC is removed by a desalting column or a dialysis method, and then the protein containing sulfhydryl group reacts.
(1) 10mg of SMCC was dissolved in 2ml of Dimethylformamide (DMF) to obtain an SMCC solution.
(2) 0.4ml of BSA was added to a 25ml round-bottom flask, supplemented with 1 XPBS (pH 7.2) buffer to give a final protein concentration of 15mg/ml, giving a BSA protein system.
(3) The dissolved SMCC solution is slowly added into a 60mgBSA protein system in a dropwise manner, and the reaction is stirred at room temperature for 1 h.
(4) The reaction in step (3) was dialyzed against 1L of 1 XPBS (pH 7.4) buffer at 4 ℃ for 6 hours to remove free SMCC, and a dialyzed BSA-SMCC solution was obtained.
(5) The dialyzed BSA-SMCC solution was poured into a 50ml centrifuge tube, the volume of which was determined by the scale of the centrifuge tube, the concentration of dialyzed protein was calculated from the amount of BSA protein added before the reaction, and 1.5mg of BSA-SMCC solution was transferred to a 5ml centrifuge tube according to the concentration.
(6) 1.0mg of 2-mercaptobenzimidazole was dissolved in 0.5ml of 1 XPBS (pH 7.2) buffer to obtain a 2-mercaptobenzimidazole solution.
(7) And dropwise adding the 2-mercaptobenzimidazole solution into 1.5mg of BSA-SMCC solution, and uniformly mixing the solution by using a vertical mixer at room temperature for 4 hours to react to obtain 2M-BSA.
Example 2 animal immunization
Experimental animals female 6-week-old mice of Balb/c strain were used. An immunization process: 2M-BSA was mixed with Freund's complete adjuvant or Freund's incomplete adjuvant, emulsified, and injected subcutaneously at multiple sites for immunization. The dose of 2M-BSA was 0.1 mg/dose. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the second immunization. The interval time of each immunization was 3 weeks, and 3 immunizations were performed. After the third immunization, the mice were reminiscent stimulated before cell fusion, and 0.1mg of 2M-BSA was dissolved in 0.5ml of PBS buffer and injected intraperitoneally. Cell fusion and hybridoma construction were performed 3 days after recall stimulation.
Example 3 hybridoma cell line construction
Feeder cell suspension was prepared the day before cell fusion: one mouse can obtain 5X 1068 x 106Peritoneal macrophages, when mouse thymocytes were used as feeder cells, cell concentration was 5X 106Each ml of the mouse spleen cell is 1 multiplied by 106Mouse fibroblast cells (3T3) 1X 10 cells/ml5Each of the cells was 100. mu.l/well.
(1) Logarithmic growth mouse myeloma cells (SP2/0) were collected, centrifuged at 1000rpm for 5 minutes, the supernatant was discarded, cells were suspended in incomplete culture medium (RPMI1640) and counted, and the desired number of cells were collected and washed 2 times with incomplete culture medium (RPMI 1640).
(2) At the same time, immune splenocyte suspension was prepared and washed 2 times with incomplete medium.
(3) Myeloma cells and spleen cells were mixed at a ratio of 1:10 or 1: 5 in terms of the number of cells, and washed 1 time with an incomplete culture medium at 1200rpm for 8 minutes in a 50ml plastic centrifuge tube.
(4) The supernatant was discarded and the residual liquid was aspirated off with a dropper to avoid affecting the subsequent polyethylene glycol (PEG) concentration.
(5) Lightly flick the bottom of the tube to loosen the cell pellet.
(6) Fusion at room temperature: to a centrifuge tube containing cells, 1ml of a 45 mass% aqueous solution of PEG (Merck, molecular weight 4000) containing 5 mass% dimethyl sulfoxide (DMSO) was added with stirring within 30 seconds. The reaction was continued for 90 seconds. The PEG action was stopped by adding the preheated incomplete culture medium, and 1ml, 2ml, 3ml, 4ml, 5ml and 10ml of RPMI1640 were added at 2 minute intervals.
(7) Centrifuge, 800rpm, 6 minutes.
(8) The supernatant was discarded, and the suspension was gently suspended in 6ml of 20% (mass percent) calf serum RPMI1640, and the cells were not blown by force to avoid scattering the fused cells.
(9) According to the number of 96-well culture plates, complete culture solution is added, and a 96-well plate with 10ml is used, so that fused cell suspension is obtained.
(10) Adding the fused cell suspension into 96-well plate containing feeder cell suspension (feeder cells are selected from abdominal cavity macrophage) at 100 μ l/well, 37 deg.C and 5% (volume percentage) CO2And (5) incubator culture.
(11) HAT selection medium was added 24 hours after fusion. When used, 1ml of the extract is added into 50ml of 20% calf serum complete culture solution. 37 ℃ and 5% (volume percent) CO2Culturing for 3-7 days.
EXAMPLE 4 Mono-cloning of hybridoma cell lines (limiting dilution method)
(1) Preparation of feeder cell suspension: one mouse can obtain 5X 1068 x 106Peritoneal macrophages, when mouse thymocytes were used as feeder cells, cell concentration was 5X 106Each ml of the mouse spleen cell is 1 multiplied by 106Mouse fibroblast cells (3T3) 1X 10 cells/ml5Each of the cells was 100. mu.l/well.
(2) Counting of positive well cells and conditioning of cellsThe number is 1 × 103one/ml-5X 103One per ml.
(3) 130 cells were taken and placed in 6.5ml complete medium containing feeder cells, i.e. 20 cells/ml, 100. mu.l/well plus A, B, C triple rows of 2 cells per well. The remaining 2.9ml of cell suspension was supplemented with 2.9ml of complete medium containing feeder cells, the number of cells was 10 per ml, and D, E, F rows were added at 100. mu.l/well, for 1 cell per well. The remaining 2.2ml of cell suspension was supplemented with 2.2ml of complete medium containing feeder cells, 5 cells/ml, 100. mu.l/well, and G, H two rows of 0.5 cells per well.
(4) After 4-5 days of culture, small cell clones were visible on an inverted microscope and 200. mu.l/well of complete culture medium was added.
(5) And at 8-9 days, cell cloning can be seen by naked eyes, and antibody detection is carried out in time. Note that the primary cloned hybridoma cells require addition of HT medium to the complete medium.
(6) Positive hybridoma cells were cloned 3 times in succession using limiting dilution. And (3) constructing a stable cell strain, and performing expanded culture and cryopreservation.
The obtained hybridoma cell strain is named as hybridoma cell strain 15D2, belongs to mouse hybridoma cell strain (Mus musculus), is preserved in China Center for Type Culture Collection (CCTCC) in 2020, 09, 10 and has a preservation number of CCTCC NO: C2020176.
example 5 antibody production
(1) And (3) injecting 0.5ml of pristane into the abdominal cavity of the mouse, and planting hybridoma cells within 1-9 weeks after injection.
(2) Collecting hybridoma cells in logarithmic growth phase, washing with incomplete culture solution once, and centrifuging at 1000r/min for 10 min.
(3) Sampling, staining with trypan blue, counting viable cells, and preparing again with incomplete culture medium to 1.0 × 107Individual cells/ml suspension.
(4) Mice injected with pristane were inoculated with hybridoma cells by intraperitoneal injection of 1ml (containing 1.0X 10)7Individual cells/ml).
(5) The tumor volume is maximum about 10 days after inoculation, and ascites can be extracted from the abdominal cavity at the moment and taken 1 time every 1-3 days. Serum can be isolated from the axillary artery or from the heart after collection.
Example 6 antibody purification
(1) The ascites fluid of the mouse was diluted 4-fold with cold PBS buffer and then concentrated at 1X 105Centrifuging for 30min, and removing precipitate.
(2) Saturated ammonium sulfate solution was slowly added dropwise to the supernatant at 4 ℃ while stirring, so that the final concentration of the solution was 50% (volume percentage) ammonium sulfate.
(3) The 50% ammonium sulfate solution is placed in ice for 30 min-60 min, then centrifuged for 10min at 5000r/min, and the supernatant is removed.
(4) The pellet was dissolved in Tris-HCl buffer (40mMol/L NaCl) (solution may be cloudy).
(5) The mixture was put into a dialysis bag and dialyzed in Tris-HCl buffer (20mMol/L NaCl) to remove the salts.
(6) And centrifuging to remove the precipitate.
(7) After dilution of the solution (1:100 or more fold dilution), the protein content was measured at 280nm and estimated: 1a 280unit ═ 0.8mg protein.
Generally, about 25mg to 36mg of total protein is contained per ml of ascites.
(8) Passing through a DEAE-cellulose column: the cellulose column was 40cm high and equilibrated with 20mMol/L NaCl Tris-HCl buffer. Dialyzed samples were diluted in equal amounts with Tris-HCl buffer (20mMol/L NaCl). The speed of the sample entering the column bed is 1ml-2ml/min, and the sample is eluted by linear gradient of NaCl aqueous solution. Most of the monoclonal antibody IgG eluted at 40mMol/L and 80mMol/L NaCl in water, and few exceptions of the monoclonal antibody eluted at 120mMol/L-150mMol/L NaCl in water. Protein peaks were collected by measuring OD280nm and monoclonal antibody IgG was stored for further use. Obtaining the purified 2-mercaptobenzimidazole monoclonal antibody.
Example 7 detection of 2-Mercaptobenzimidazole antigen recognition (ELISA)
(1) Antigen coating: antigen 2M-BSA and BSA control are respectively prepared into coating solutions with certain concentration by using carbonate buffer solution with pH9.6, and the coating solutions are mixed evenly. 96 well ELISA assay plates were added, 100 ul/well. The mixture was placed in a refrigerator at 4 ℃ overnight.
(2) Sealing and washing the plate: blocking solution (BSA in PBS buffer, 0.5% BSA by mass) was added at 200. mu.l/well. Incubate at 37 ℃ for 2 hours.
(3) Adding primary antibody (sample to be tested), washing plate: the treated primary antibody solution was added to the wells at a volume of 100. mu.l/well, and the primary antibody solution was selected from a dilution of hybridoma cell culture supernatant (i.e., supernatant obtained by centrifuging the culture obtained in the step (11) of example 3) after cell fusion, or mouse serum for the immune antigen (mouse serum prepared by collecting blood from the tail vein after 7 days from the completion of the immunization procedure of example 2), or 2-mercaptobenzimidazole monoclonal antibody purified in example 6. Incubate at 37 ℃ for 2 hours.
(4) Adding a secondary antibody and washing a plate: diluted secondary antibodies (commercial goat anti-mouse Ig-HRP, e.g., ab6789, abcam, 1:2000 dilution) were added to the wells. 100 μ l/well. Incubate at 37 ℃ for 1 hour.
(5) Color development and plate washing: the TMB substrate (i.e., TMB developing solution) was prepared and added to the wells at 100. mu.l/well. Developing for 5-10 min. The reaction was stopped by adding 100. mu.l/well of 2M (mol/L) sulfuric acid.
(6) And (6) detecting. And detecting the absorbance OD value of the solution at 450nm by using a microplate reader. Data were obtained. The results are shown in FIG. 1.
ELISA detection results show that the 2-mercaptobenzimidazole monoclonal antibody has specific recognition activity on the 2-mercaptobenzimidazole antigen, can be combined with the 2-mercaptobenzimidazole antigen with high affinity, still has obvious recognition reaction on 2M-BSA when the antibody concentration is 15ng/mL, and has no recognition on the BSA. The 2-mercaptobenzimidazole monoclonal antibody can be used for detecting 2-mercaptobenzimidazole.
Claims (4)
1. A hybridoma cell strain is characterized in that the preservation number of the hybridoma cell strain is CCTCC NO: C2020176.
2. a2-mercaptobenzimidazole monoclonal antibody secreted by the hybridoma cell line of claim 1.
3. The use of the 2-mercaptobenzimidazole monoclonal antibody of claim 2 for detecting 2-mercaptobenzimidazole.
4. The use of the 2-mercaptobenzimidazole monoclonal antibody of claim 2 in the preparation of a 2-mercaptobenzimidazole assay kit.
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Citations (3)
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| WO2006110277A1 (en) * | 2005-04-11 | 2006-10-19 | Medarex, Inc. | Protein purification using hcic amd ion exchange chromatography |
| CN107413312A (en) * | 2017-07-18 | 2017-12-01 | 天津工业大学 | A kind of mixed mode protein adsorption film and preparation method for antibody purification |
| CN109374778A (en) * | 2018-12-14 | 2019-02-22 | 长沙理工大学 | A kind of method of organic impurities in measurement 2-mercaptobenzimidazole |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006110277A1 (en) * | 2005-04-11 | 2006-10-19 | Medarex, Inc. | Protein purification using hcic amd ion exchange chromatography |
| CN107413312A (en) * | 2017-07-18 | 2017-12-01 | 天津工业大学 | A kind of mixed mode protein adsorption film and preparation method for antibody purification |
| CN109374778A (en) * | 2018-12-14 | 2019-02-22 | 长沙理工大学 | A kind of method of organic impurities in measurement 2-mercaptobenzimidazole |
Non-Patent Citations (3)
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| 2-巯基苯并咪唑衍生物的设计、合成及抗肿瘤活性研究;李红侠 等人;《化学通报》;20191231;第82卷(第10期);第909-916页 * |
| Synthesis, biological activities, and molecular docking studies of 2-mercaptobenzimidazole based derivatives;Mumtaz Ali et al.;《Bioorganic Chemistry》;20181231;第80卷;第472-479页 * |
| USE OF HAPTEN-CARRIER COMPLEXES FOR BENZIMIDAZOLE PESTICIDES IMMUNOASSAYS DEVELOPMENT;Veronica TANASA et al.;《AgroLife Scientific Journal》;20161231;第5卷(第2期);第143-150页 * |
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Denomination of invention: Hybridoma cell lines secreting monoclonal antibodies against 2-mercaptobenzimidazole, monoclonal antibodies and their applications Effective date of registration: 20220617 Granted publication date: 20210604 Pledgee: Guangfa Bank Co.,Ltd. Hangzhou Baoshan sub branch Pledgor: GREENTOWN NONGKE DETECTION TECHNOLOGY Co.,Ltd. Registration number: Y2022330001013 |