CN113881638B - De-himbine hapten, monoclonal antibody, hybridoma cell strain and application - Google Patents
De-himbine hapten, monoclonal antibody, hybridoma cell strain and application Download PDFInfo
- Publication number
- CN113881638B CN113881638B CN202111091568.5A CN202111091568A CN113881638B CN 113881638 B CN113881638 B CN 113881638B CN 202111091568 A CN202111091568 A CN 202111091568A CN 113881638 B CN113881638 B CN 113881638B
- Authority
- CN
- China
- Prior art keywords
- norlinderane
- monoclonal antibody
- hapten
- cell strain
- hybridoma cell
- 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.)
- Active
Links
Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D217/00—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems
- C07D217/12—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with radicals, substituted by hetero atoms, attached to carbon atoms of the nitrogen-containing ring
- C07D217/18—Aralkyl radicals
- C07D217/20—Aralkyl radicals with oxygen atoms directly attached to the aromatic ring of said aralkyl radical, e.g. papaverine
-
- 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
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Pathology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a kind of artificial antigen of norlinderane, monoclonal antibody, hybridoma cell strain and application, it is the field of food safety immunity detection, take the product with carboxyl that the reaction of norlinderane and succinic anhydride gets as hapten, couple hapten and carrier protein with carbodiimide method, get complete antigen, measure the coupling ratio of conjugate with ultraviolet method, and get a monoclonal antibody hybridoma cell strain, monoclonal antibody that this cell strain secretes, have better specificity and detection sensitivity to norlinderane, IC 50 The value is 10ng/mL, can realize the detection of the residual quantity of the norlinderane in the traditional Chinese medicine and the functional food, provides raw materials for the immunodetection of the norlinderane residual in the functional food, and has practical application value. The invention also successfully synthesizes the norlinderane artificial antigen, has simple and effective synthesis steps, can be completely used in immunoassay, and provides necessary artificial antigen for the later study of people.
Description
Technical Field
The invention belongs to the field of food safety immunodetection, and in particular relates to a norlinderamine hapten, a monoclonal antibody, a hybridoma cell strain and application.
Background
The norlinderane (HG) is a plant benzyl tetrahydroisoquinoline alkaloid, and the natural compound is extracted from various Asian medicinal plants and is a heart-strengthening active ingredient in Chinese medicinal materials such as aconite, lotus plumule and the like, so that the norlinderane (HG) can be processed into functional foods and additives. HG is, however, a beta-2 adrenergic agonist with remarkable antioxidant, anti-inflammatory, analgesic and antitumor effects, and is capable of stimulating beta receptor by reducing platelet adhesion, inhibiting iNOS and up-regulating HO -1 Is positive and positive for cardiovascular diseases. Because athletes taking HG-containing products may result in an accelerated increase in heart rate and oxygen intake, bringing their exercise level above normal. It was listed as a class S3 permanently disabled drug by the world' S anti-perphenating agency (WADA) since 2020. WADA takes the HG content of more than or equal to 10ng/mL in the urine sample of the athlete as the detection limit of a positive result, so that a rapid, accurate and reliable detection method is needed to quantitatively detect HG in food or medicine.
In the related art, typical detection methods for HG include high performance liquid chromatography-ultraviolet (HPLC-UV) method, high performance liquid chromatography-fluorescence (HPLC-FLD) method, high performance liquid chromatography-mass spectrometry (HPLC-MS) method, and the like. Wherein the sensitivity of the conventional HPLC-UV method is low; the pre-column derived fluorescence method improves the sensitivity but increases the error step, but has complex equipment, high requirements on professional operators, high organic solvent consumption and severely limits the application of the method in operations outside a laboratory.
The ELISA is a very efficient, sensitive and rapid detection method, and has the advantages of simple pretreatment of samples during detection, few purification steps, large analysis capacity, low detection cost and simple and convenient operation, and is suitable for the on-site rapid detection of a large number of samples, thus being widely applied to the analysis of drug residues. The premise of detecting the norlinderane by using an enzyme-linked immunosorbent assay is to obtain the monoclonal antibody with high specificity and high sensitivity to the norlinderane, so that the method for preparing the monoclonal antibody with high specificity and high sensitivity to the norlinderane is very critical.
Disclosure of Invention
In order to solve the problems in the related art, a detection means with high specificity and high sensitivity on the norlindrine is found, and one purpose of the invention is to provide a hybridoma cell strain secreting the norlindrine monoclonal antibody, which is preserved in China general microbiological culture Collection center (LNTD) 2E 8) with the preservation name of CGMCC No.20793 and the preservation address of North Chengxi Lu No. 1 and No. 3 in the Korean region of Beijing in the 09 month 27 of 2020.
The invention also aims to provide a preparation method of the norlinderane hapten and the complete antigen, which is simple and effective for immunoassay and provides a convenient way for subsequent research.
It is still another object of the present invention to provide a monoclonal antibody of norlinderamine, which has good specificity and detection sensitivity (IC 50 value is 10 ng/mL) for norlinderamine when applied in detection of norlinderamine.
The invention also aims to provide a kit which comprises the hybridoma cell strain or the norlindrine monoclonal antibody and can realize the detection of the norlindrine residue in traditional Chinese medicines and functional foods.
The specific technical scheme is as follows:
as a first aspect of the present invention, there is provided a hybridoma cell strain secreting the norlindrine monoclonal antibody, which has been deposited at China general microbiological culture Collection center under the accession number of LNTD 2E8, with the accession number of CGMCC No.20793, and with the accession number of North Silu No. 1, no. 3 in the Korean region of Beijing, at the year 09 month 27 of 2020.
As a second aspect of the present invention, there is provided a hapten for use in the preparation of said hybridoma cell line, said hapten having two molecular structures of the following formulas (1) and (2):
as a third aspect of the present invention, there is provided a norlinderamine monoclonal antibody, wherein the hybridoma cell strain with a preservation number of CGMCC No.20793 is secreted; or obtained after immunization of mice with said hapten conjugated to BSA or KLH protein.
In an alternative embodiment, the hapten is obtained by the following method:
dissolving norlinderane and 4-dimethylaminopyridine in anhydrous pyridine, then adding succinic anhydride, and refluxing the mixture at 40-60 ℃ to obtain a precipitate;
drying the precipitate in a rotary evaporator and dissolving it in methanol/water;
extracting with ethyl acetate, collecting organic phase, adding glacial hydrochloric acid for reaction, and obtaining clear white precipitate;
drying the precipitate to obtain hapten with two molecular structures.
In a preferred embodiment of the invention, the hapten is obtained by the following method:
dissolving 5-50 mg of norlinderane (HG) and 1.5-5 mg of 4-Dimethylaminopyridine (DMAP) in 2mL of anhydrous pyridine, then adding 10-25 mg of succinic anhydride, and refluxing the mixture at 60 ℃ for 12 hours to obtain a precipitate;
the precipitate was dried in a rotary evaporator and dissolved in 2ml of 50% methanol/water;
after 3 times extraction with ethyl acetate, the organic phase was collected, 10% glacial hydrochloric acid (v/v) was added and reacted at 4℃for 2h, a clear white precipitate was seen;
transferring the precipitate into an oven, and heating to dryness at 60 ℃ to obtain two hapten HG with carboxyl structure.
As a fourth aspect of the present invention, there is provided the use of said hybridoma cell line or norlindrine monoclonal antibody for detecting norlindrine.
In an alternative embodiment, the hybridoma cell line or norlindrine monoclonal antibody is used for analyzing and detecting norlindrine residues in traditional Chinese medicinal materials and functional foods.
In an alternative embodiment of the invention, the norlinderane complete antigen is obtained by the following method:
1) Dissolving the norlinderane hapten, 1-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide with anhydrous N, N-dimethylformamide, and stirring at room temperature for 12h to obtain solution A;
2) Diluting hemocyanin (KLH) or Bovine Serum Albumin (BSA) with boric acid buffer solution to obtain a solution B;
3) Under the room temperature condition, dropwise adding the solution A into the solution B, and reacting at room temperature to obtain solution C; and then, dialyzing with PBS solution to remove unreacted small molecule hapten, thus obtaining KLH norlinderamine complete antigen (HG-HS-KLH) or BSA norlinderamine complete antigen (HG-HS-BSA).
In an alternative embodiment of the invention, the hybridoma cell line secreting the norlinderamine monoclonal antibody is obtained mainly by the following method:
s1, mixing and emulsifying the norlinderane complete antigen and equivalent Freund' S adjuvant, and performing subcutaneous multipoint injection immunization on the back and neck of a BALB/c mouse, and performing primary immunization and multiple boosting immunization: the first immunization was performed with complete Freund's adjuvant at a dose of 100 ug/dose; multiple boosting with incomplete Freund's adjuvant and halving the dose to 50 ug/dose, one month interval between the first and second boosting, 21 days interval between multiple boosting;
s2, taking blood of the mice subjected to the immunization process, detecting serum immune titer and immune suppression capacity of the mice by indirect ELISA, and screening the immunized mice with high norlindrine antibody content in the serum;
s3, carrying out final boosting immunization on the screened mice by using incomplete Freund 'S adjuvant, and then carrying out sprint immunization by intraperitoneal injection, wherein the sprint immunization is carried out by adopting norlinderane complete antigen without Freund' S adjuvant;
s4, fusing spleen cells and myeloma cells of the BALB/c mice subjected to sprint immunization, culturing the fused cells through a culture medium, and screening positive cell holes by using an indirect ELISA method;
s5, selecting norlinderane as a standard substance, further utilizing an indirect ELISA method to determine the inhibition effect of the positive cell holes screened in the S4, carrying out three subcloning on the positive cell holes with the best inhibition by a limiting dilution method, and finally screening to obtain the hybridoma cell strain capable of secreting the norlinderane monoclonal antibody.
The invention has the beneficial effects that: the invention successfully synthesizes the artificial antigen of the norlinderane, comprises hapten, complete antigen, coating antigen and the like, has simple and effective synthesis steps, can be completely used in immunoassay, and provides a convenient way for the research of people in the future; the monoclonal antibody secreted by the hybridoma cell strain has good specificity and detection sensitivity (IC 50 value is 10 ng/mL) to the norlindrine, can realize the detection of the norlindrine residue in traditional Chinese medicine materials and functional foods, provides raw materials for the immunodetection of the norlindrine residue in the functional foods, and has practical application value.
Preservation of biological material samples: the norlinderamine monoclonal antibody hybridoma cell strain 1C11 is preserved in China general microbiological culture collection center (CGMCC) with the following addresses: the preservation name of the monoclonal cell strain LNTD 2E8 is the CGMCC No. 3 of North West Lu No. 1 in the Korean region of Beijing, and the preservation number is CGMCC No.20793 in the year 09 and 27 of 2020.
Drawings
FIG. 1 NMR chart of the identification of norlinderamine hapten.
FIG. 2 LC-MS identification of norlinderane hapten.
FIG. 3 UV identification of immunogens of HG-KLH antigen.
FIG. 4 Standard inhibition curve of monoclonal antibody 1C11 against norlinderane.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The following examples relate to the following media:
RPMI-1640 medium (mg/L): l-arginine 290, L-asparagine 50, L-aspartic acid 20, L-cystine dihydrochloride 65.15, L-glutamic acid 20, glycine 10, L-histidine 15, L-hydroxyproline 20, L-isoleucine 50, L-leucine 50, L-lysine hydrochloride 40, L-methionine 15, L-phenylalanine 15, L-proline 20, L-serine 30, L-threonine 20, L-tryptophan 5, L-tyrosine 23.19, L-valine 20, p-aminobenzoic acid 1, calcium nitrate 100, anhydrous magnesium sulfate 48.84, anhydrous sodium dihydrogen phosphate 676.13, potassium chloride 400, sodium chloride 6000, glucose 2000, reduced glutathione 1, phenol red 5, L-glutamine 300, biotin 0.2, D-calcium pantothenate 0.25, folic acid 1, i-inositol 35, nicotinamide 1, choline chloride 3, pyridoxine hydrochloride 1, riboflavin 0.2, thiamine hydrochloride 1, vitamin B12.005, sodium bicarbonate 2000.
The reagents involved in the following examples were as follows:
carbonate Buffer (CBS): weighing Na 2 CO 3 1.59g，NaHCO 3 2.93g, respectively dissolving in a small amount of double distilled water, mixing, adding double distilled water to about 800mL, mixing, adjusting pH to 9.6, adding double distilled water to 1000mL, and storing at 4deg.C for use.
Phosphate Buffer (PBS): 8.00g NaCl,0.2g KCl,0.2g KH 2 PO 4 ，2.9g Na 2 HPO 4 ·12H 2 O is dissolved in 800mL of pure water, pH is regulated to 7.2-7.4 by NaOH or HCl, and volume is regulated to 1000mL;
PBST: PBS containing 0.05% Tween 20;
TMB color development liquid: and (3) solution A: na (Na) 2 HPO 4 . 12H 2 18.43g of O, 9.33g of citric acid and pure water to 1000mL; and (2) liquid B: 60mg of TMB was dissolved in 100mL of ethylene glycol. A. The solution B is prepared from the following components in percentage by weight: 1 to obtain TMB color development liquid, and mixing immediately.
The detection method involved in the following examples is as follows:
the method for detecting the inhibition rate of the norlinderane comprises the following steps: the most appropriate antigen and antibody concentrations in the ic-ELISA were selected by a checkerboard assay. The antigen was diluted to 0.01,0.03,0.1 and 0.3 μg/mL with Carbonate Buffer (CBS) and the antibody diluted to 0.03,0.1,0.3 and 1 μg/mL with antibody dilution. After selecting the optimal working point, the standard norlinderane is diluted to 6 concentrations (2.5, 5, 10, 25, 50, 100 ng/mL), and the standard norlinderane inhibition curve is obtained by plotting OrigingPro 8.5 (the result is shown in FIG. 4) according to the IC-ELISA operation steps, and IC is calculated 50 。
Example 1 preparation of norlinderane hapten:
5 mg-50 mg of norlinderane and 1.5 mg-5 mg of 4-Dimethylaminopyridine (DMAP) are dissolved in 2mL of anhydrous pyridine, 10 mg-25 mg of succinic anhydride is added, and the mixture is refluxed at 60 ℃ for 12h. Subsequently, the precipitate was dried in a rotary evaporator and dissolved in 2ml of 50% methanol/water. After 3 extractions with ethyl acetate, the organic phase was collected, 10% glacial hydrochloric acid (v/v) was added and reacted at 4℃for 2h, a clear white precipitate was seen. Transferring the precipitate into an oven, and heating to dryness at 60 ℃ to obtain two hapten with carboxyl structure:
example 2 preparation of HG-KLH complete antigen:
the hapten prepared in the example 1 is coupled with KLH to obtain conjugate complete antigen HG-KLH, and the preparation method is specifically as follows:
a. weighing 5-25 mg of norlinderane hapten, 8-20 mg of 1-ethylcarbodiimide hydrochloride and 4-15 mg of N-hydroxysuccinimide, dissolving with 600 mu L of anhydrous N, N-dimethylformamide, and stirring at room temperature for 12 hours to obtain solution A; weighing 10mg of hemocyanin KLH (the molar ratio of the norlinderane hapten to the KLH is 2000:1), adding 3ml of boric acid buffer solution to obtain solution B, dropwise adding solution A into solution B at room temperature, reacting at room temperature, and standing for 8-12 hours to obtain a conjugate HG-KLH mixed solution;
b. and (3) dialysis: boiling the dialysis bag in boiling water for 3min, washing with deionized water for 3min, and storing in deionized water at 4deg.C; and (3) putting the conjugate HG-KLH mixed solution into a dialysis bag, dialyzing for 3 days in 0.01mol/L PBS, and changing the solution once every 6-8 hours to obtain the complete antigen HG-KLH.
Example 3 preparation of HG-BSA complete antigen:
the hapten prepared in example 1 is coupled with BSA to obtain conjugate complete antigen HG-BSA, and the preparation method is specifically as follows:
hapten and BSA were coupled with N, N' -Carbonyldiimidazole (CDI) as heterogeneous coating antigen. A mixture of 2.5-5 mg hapten and 2-3 mg CDI was dissolved in 0.3mL anhydrous N, N-Dimethylformamide (DMF), stirred at 37℃for 1h, then 10mg BSA (molecular weight 67000 Da) was added to 3mL Carbonate Buffer (CBS), and stirred at room temperature for 12h. Finally, the solution was dialyzed against PBS for 2 days and stored at-20 ℃.
Example 4 identification of norlinderamine antigen:
(1) Hapten identification using nuclear magnetic resonance and liquid chromatography-mass spectrometry techniques is shown in figures 1 and 2.
(2) The artificial antigen adopts ultraviolet method to identify the coupling result, and uses the concentration of small molecule and protein in the conjugate to calculate the coupling ratio.
Coupling ratio determination: methods for estimating the ratio of two molecules coupled in a conjugate (coupling ratio) are based on the principle of detecting the content (or relative content) of the two molecules coupled in the conjugate, although the types of measurement methods are numerous. The ultraviolet method is to determine the coupling ratio according to the ratio of the concentration of small molecules to the concentration of protein in the synthesized artificial antigen.
Conjugate protein concentration determination: the protein content in the coupling can be obtained by dividing the mass of the protein before the reaction by the volume of the conjugate after the dialysis.
FIG. 3 shows a UV-identified chart of HG-KLH immunogens from example 2;
according to the coupling ratio formula: 1.2510-7.6.7510-8
The conjugate HG-KLH has a characteristic absorbance peak at 276nm, where the absorbance a2=1.12314, KLH has an absorbance a1=0.87341, HG has a characteristic absorbance peak a3=0.54127, HG has a relative molecular mass m1=271.31, KLH has a relative molecular mass m2=4000000, and x is 10 μg/mL according to the formula:
coupling ratio = [ (A2-A1) (X/M1)/A3 ]/(0.5/M2), coupling ratio of hapten HG and protein KLH in HG-KLH is 1003:1.
Example 5 preparation of hybridoma cell lines secreting norlindrine monoclonal antibodies:
1. animal immunization:
healthy Balb/C mice of 6-8 weeks of age were selected for immunization. After the norlinderane complete antigen is mixed and emulsified with the equivalent Freund's adjuvant, BALB/c mice are subjected to subcutaneous multipoint injection immunization (except sprint immunization) on the back of the neck. The first immunization was performed with complete Freund's adjuvant at a dose of 100 ug/dose; multiple boosting with incomplete Freund's adjuvant and halving the dose to 50 ug/dose; the sprint immunity is directly diluted by normal saline without adjuvant, and then the dosage is halved to 25 ug/patient. One month is separated from the first immunization and the second immunization, 21 days is separated from the multiple boosting, and 18-21 days is separated from the final boosting. The immune effect of the mice is observed by an indirect competition enzyme-linked immunosorbent assay (ic-ELISA), namely the titer and inhibition of the serum of the mice are detected.
2. Cell fusion and selection:
three days after the impact immunization, cell fusion was performed according to the conventional PEG (polyethylene glycol, molecular weight 4000) method, specifically as follows: (1) Taking a mouse spleen in a sterile mode, grinding and passing through a 200-mesh cell screen to obtain spleen cell suspension, and performing cell counting; (2) Collecting SP2/0 cells, suspending in RPMI-1640 basic culture solution, and performing cell count; (3) spleen cells and SP2/0 cells were isolated according to 1:10, centrifuging, fusing with 50% PEG for 1min, adding RPMI-1640 basal culture solution from slow to fast, centrifuging, suspending in RPMI-1640 screening culture solution containing 20% fetal bovine serum and 2% 50 XHAT, adding into 96-well cell culture plate, standing at 37deg.C, and 5% CO 2 Is cultured in an incubator of (a).
3. Cell screening and cell strain establishment:
half-changing the fused cells by HAT medium on the 3 rd day after cell fusion; full exchange with RPMI-1640 medium (HT medium) containing 20% fetal bovine serum, 1% 100×ht on day 5; cell supernatants were taken on day 7 for screening. Screening is carried out in two steps: the first step is to screen out positive cell holes by using an ic-ELISA method, and the second step is to select chlorpheniramine as a standard substance, and to measure the inhibition effect of positive cells by using the ic-ELISA method. Selecting cell holes with better inhibition on chlorpheniramine standard, subcloning by adopting a limiting dilution method, and detecting by adopting the same method after seven days. And performing subcloning for three times according to the method to finally obtain the norlinderamine monoclonal antibody cell strain 1C11.
Example 6 preparation and use of monoclonal antibodies:
taking 8-10 week old BALB/c mice, and injecting 1mL of sterile paraffin oil into the abdominal cavity of each mouse; 2X 10 per mouse was intraperitoneally injected 7 days later 6 Chlorpheniramine hybridoma cells, ascites was collected from the seventh day, and antibody purification was performed on the ascites by octanoic acid-saturated ammonium sulfate method. Under the condition of meta-acid, n-octanoic acid can precipitate other hetero proteins except IgG immunoglobulin in ascites, and then the hetero proteins are centrifuged and discardedPrecipitating; then the monoclonal antibody of IgG type is precipitated by an ammonium sulfate solution with equivalent saturation, centrifugated, the supernatant is discarded, dissolved by a PBS solution (pH 7.4) of 0.01M, and then dialyzed and desalted, and finally the purified monoclonal antibody is preserved at-20 ℃.
(1) Coating: the coating antigen HG-BSA was diluted 3-fold from 1. Mu.g/mL with 0.05M Carbonate Buffer (CBS) pH9.6, 100. Mu.L/well, and reacted at 37℃for 2 hours.
(2) Washing: the plate solution was poured off and washed 3 times with wash solution for 3min each.
(3) Closing: after drying, 200. Mu.L/well of blocking solution was added thereto and reacted at 37℃for 2 hours. And (5) drying for standby after washing.
(4) Sample adding: the antisera was diluted from 1:1000 in a double ratio and added to the coated wells at each dilution, 100. Mu.L/well, and reacted at 37℃for 30min; after extensive washing, HRP-goat anti-mouse IgG diluted 1:3000 was added, 100. Mu.L/well, and reacted at 37℃for 30 minutes.
(5) Color development: and taking out the ELISA plate, fully washing, adding 100 mu L of TMB color developing solution into each hole, and carrying out light-shielding reaction for 15min at 37 ℃.
(6) Termination and measurement: 50. Mu.L of stop solution was added to each well to terminate the reaction, and the OD 450 value of each well was measured by using a microplate reader.
IC for determining monoclonal antibody chlorpheniramine by IC-ELISA 50 The method comprises the following steps: 10ng/mL, the method has good sensitivity to the norlinderane, and can be effectively used for the immunoassay detection of the norlinderane.
According to the embodiment, the synthetic steps of the artificial hapten and the complete antigen of the norlinderane are simple and effective, the method can be effectively used in immunoassay, a convenient way is provided for subsequent research and analysis, the provided monoclonal antibody secreted by the cell strain 1C11 has good specificity and detection sensitivity (IC 50 value is 10 ng/mL) for the norlinderane, the detection of the norlinderane residue in traditional Chinese medicine materials and functional foods can be realized, raw materials are provided for the immunodetection of the norlinderane residue in the functional foods, and the method has practical application value.
It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered in the scope of the claims of the present invention.
Claims (6)
1. The hybridoma cell strain secreting the norlindrine monoclonal antibody is preserved in China general microbiological culture Collection center (LNTD 2E 8) on the 09 th month 27 of 2020, and has the preservation name of CGMCC No.20793 and the preservation address of North Chen West Lu No. 1 and No. 3 in the Korean region of Beijing city.
2. The norlinderamine monoclonal antibody secreting hybridoma cell line of claim 1, prepared from the following hapten having two molecular structures of formula (1) and formula (2):
3. the norlinderamine monoclonal antibody is secreted by the hybridoma cell strain with the preservation number of CGMCC No.20793 in claim 1.
4. Use of the norlindrine monoclonal antibody of claim 3 for detecting norlindrine.
5. A kit comprising the norlinderamine monoclonal antibody of claim 3.
6. Use of the kit according to claim 5 for detecting norlinderane.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111091568.5A CN113881638B (en) | 2021-09-16 | 2021-09-16 | De-himbine hapten, monoclonal antibody, hybridoma cell strain and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111091568.5A CN113881638B (en) | 2021-09-16 | 2021-09-16 | De-himbine hapten, monoclonal antibody, hybridoma cell strain and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113881638A CN113881638A (en) | 2022-01-04 |
| CN113881638B true CN113881638B (en) | 2023-10-13 |
Family
ID=79009511
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111091568.5A Active CN113881638B (en) | 2021-09-16 | 2021-09-16 | De-himbine hapten, monoclonal antibody, hybridoma cell strain and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113881638B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105272915A (en) * | 2014-07-14 | 2016-01-27 | 上海医药工业研究院 | Semiantigen compound, polyclonal antibody and kit thereof, and application of polyclonal antibody and kit |
| CN110616194A (en) * | 2019-10-11 | 2019-12-27 | 江南大学 | Aconitine monoclonal antibody cell strain SJ and application thereof |
-
2021
- 2021-09-16 CN CN202111091568.5A patent/CN113881638B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105272915A (en) * | 2014-07-14 | 2016-01-27 | 上海医药工业研究院 | Semiantigen compound, polyclonal antibody and kit thereof, and application of polyclonal antibody and kit |
| CN110616194A (en) * | 2019-10-11 | 2019-12-27 | 江南大学 | Aconitine monoclonal antibody cell strain SJ and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| ELISA for the Detection of the Prohibited Doping Agent Higenamine;Poomraphie Nuntawong et al.;《Planta Med》;第1-7页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113881638A (en) | 2022-01-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113684187B (en) | Hybridoma cell strain secreting fluazinam monoclonal antibody as well as preparation method and application thereof | |
| CN113736744B (en) | Digitoxin monoclonal antibody hybridoma cell strain and application thereof | |
| CN110343669B (en) | Hybridoma cell strain DNC secreting anti-triclabendazole monoclonal antibody and application thereof | |
| CN110713986B (en) | Vitamin B strain 1 Monoclonal antibody hybridoma cell strain CBDD and application thereof | |
| CN111763658A (en) | Hybridoma cell strain secreting anti-dinitrotolamine monoclonal antibody and application thereof | |
| CN113881638B (en) | De-himbine hapten, monoclonal antibody, hybridoma cell strain and application | |
| CN114752568B (en) | Furosemide monoclonal antibody, hybridoma cell strain and application | |
| CN114181911B (en) | Hybridoma cell strain secreting spirolactone and metabolite monoclonal antibody thereof and application of hybridoma cell strain | |
| CN106929479B (en) | Vitamin B2 monoclonal antibody hybridoma cell strain GZ-4 and application thereof | |
| CN113005097B (en) | Hybridoma cell strain secreting anti-carbamazepine monoclonal antibody and application thereof | |
| CN113637642B (en) | Hybridoma cell strain secreting chlorfenapyr monoclonal antibody and application thereof | |
| CN114031528B (en) | Florfenicol hapten, artificial antigen, antibody and synthetic method and application thereof | |
| CN113717950B (en) | Hybridoma cell strain secreting penconazole monoclonal antibody and application thereof | |
| CN112939873B (en) | Trimethoprim hapten TMPH, artificial antigen, antibody and preparation method and application thereof | |
| CN109022366A (en) | One plant of hybridoma cell strain for secreting anti-levamisol monoclonal antibody and its application | |
| CN111454912B (en) | Cyperazine monoclonal antibody hybridoma cell strain and application thereof | |
| CN111499637B (en) | Yohimbine hapten YHA, artificial antigen and antibody thereof, and preparation and application thereof | |
| CN110616193B (en) | Hybridoma cell strain BCB secreting anti-sodium cyclamate monoclonal antibody and application thereof | |
| CN114317449B (en) | Ergot ethylenediamine antigen, ergot ethylenediamine monoclonal antibody, hybridoma cell strain and application | |
| CN111748528A (en) | Hybridoma cell strain secreting monoclonal antibody against fipronil and metabolite thereof and application of hybridoma cell strain | |
| CN113980909B (en) | Alpha-amanitin artificial antigen, monoclonal antibody, hybridoma cell strain and application | |
| CN111961010A (en) | Saccharin sodium hapten Ri, artificial antigen, antibody and preparation method and application thereof | |
| CN110616194A (en) | Aconitine monoclonal antibody cell strain SJ and application thereof | |
| CN114891752B (en) | Hybridoma cell strain of anti-S-adenosyl homocysteine monoclonal antibody and application | |
| CN114774368B (en) | Hybridoma cell strain secreting anti-flumioxazin monoclonal antibody 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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |