CN113005098A - Hybridoma cell strain secreting hyoscyamine monoclonal antibody and application thereof - Google Patents

Hybridoma cell strain secreting hyoscyamine monoclonal antibody and application thereof Download PDF

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CN113005098A
CN113005098A CN202110286115.1A CN202110286115A CN113005098A CN 113005098 A CN113005098 A CN 113005098A CN 202110286115 A CN202110286115 A CN 202110286115A CN 113005098 A CN113005098 A CN 113005098A
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hyoscyamine
solution
reaction
hapten
monoclonal antibody
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胥传来
郭鑫
匡华
刘丽强
宋珊珊
胡拥明
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Jiangnan University
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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D451/00—Heterocyclic compounds containing 8-azabicyclo [3.2.1] octane, 9-azabicyclo [3.3.1] nonane, or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane or granatane alkaloids, scopolamine; Cyclic acetals thereof
    • C07D451/02—Heterocyclic compounds containing 8-azabicyclo [3.2.1] octane, 9-azabicyclo [3.3.1] nonane, or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane or granatane alkaloids, scopolamine; Cyclic acetals thereof containing not further condensed 8-azabicyclo [3.2.1] octane or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane; Cyclic acetals thereof
    • C07D451/04—Heterocyclic compounds containing 8-azabicyclo [3.2.1] octane, 9-azabicyclo [3.3.1] nonane, or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane or granatane alkaloids, scopolamine; Cyclic acetals thereof containing not further condensed 8-azabicyclo [3.2.1] octane or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane; Cyclic acetals thereof with hetero atoms directly attached in position 3 of the 8-azabicyclo [3.2.1] octane or in position 7 of the 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring system
    • C07D451/06—Oxygen atoms
    • C07D451/10—Oxygen atoms acylated by aliphatic or araliphatic carboxylic acids, e.g. atropine, scopolamine
    • 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
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • 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

Abstract

A hybridoma cell strain secreting a hyoscyamine monoclonal antibody and application thereof belong to the field of food safety immunodetection. The hybridoma cell strain Pba secreting the hyoscyamine monoclonal antibody has been preserved in a Chinese microbial strain preservation tubeThe general microbiological center CGMCC of the committee is classified and named as a monoclonal cell strain, the preservation date is 2020, 4 and 23 days, and the preservation number is CGMCC No. 19678. Immunizing BALB/c mouse with complete antigen of hyoscyamine to obtain high titer and low IC50The spleen cells of the mouse are fused with myeloma cells of the mouse by a PEG method, and the hybrid cells after the fusion of the two cells are screened out by adopting a selective culture medium; and screening cells by an indirect competitive enzyme-linked immunosorbent assay and subcloning for three times to finally obtain a hybridoma cell strain Pba. The monoclonal antibody secreted by Pba has better detection sensitivity (IC) to hyoscyamine50The value is 1.03ng/mL), can be used for detecting the residue of the hyoscyamine.

Description

Hybridoma cell strain secreting hyoscyamine monoclonal antibody and application thereof
Technical Field
The invention relates to a hybridoma cell strain secreting a hyoscyamine monoclonal antibody and application thereof, and belongs to the field of food safety immunodetection.
Background
The hyoscyamine is one of belladonna alkaloids separated from semen hyoscyami and flos Daturae Metelis, and has structure of ester formed by condensation of hyoscyamine and hyoscyamine acid. Hyoscyamine is parasympathetic nerve inhibitor, has similar pharmacological action to atropine, but has higher toxicity and less clinical application. Hyoscyamine has analgesic and spasmolytic effects, and can be used for treating sciatica, epilepsy, and seasickness. It is mainly used for biochemical research, anticholinergic and gold detecting reagent.
At present, the detection method of hyoscyamine mainly adopts an instrument for detection, and high performance liquid chromatography, gas chromatography-mass spectrometry and the like are commonly used. Although these chromatographic-based methods have high sensitivity and specificity, they suffer from drawbacks such as the need for thorough sample purification, high solvent consumption, expensive equipment and skilled technicians. Therefore, a fast and simple method for analyzing scopolamine residues is needed.
The enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive and rapid detection method, has the advantages of simple pretreatment of a sample during detection, few purification steps, large analysis capacity, low detection cost and simple and convenient operation, and is suitable for field rapid detection of a large number of samples, thereby being widely applied. The precondition of detecting hyoscyamine by using an enzyme-linked immunosorbent assay is to obtain a monoclonal antibody with high specificity and high sensitivity to hyoscyamine, so that the method for preparing the monoclonal antibody with high specificity and high sensitivity to hyoscyamine is very key. The inventor tries to prepare a hyoscyamine monoclonal antibody through hybridoma cells, but in the process of preparing hybridoma cell strains capable of secreting the hyoscyamine monoclonal antibody, further research is needed on how to prepare a hyoscyamine hapten and a hyoscyamine complete antigen and how to make mice generate strong immunity; how to make the prepared hybridoma cell strain successfully secrete the hyoscyamine monoclonal antibody needs further research; how to make the secreted hyoscyamine monoclonal antibody have strong specificity and high sensitivity also needs further research.
Disclosure of Invention
The invention aims to provide a hybridoma cell strain secreting a hyoscyamine monoclonal antibody and application thereof, wherein the monoclonal antibody prepared from the cell strain has good affinity and sensitivity (IC) to hyoscyamine50The value is 1.03ng/mL), can be used for establishing an immunological detection method of the hyoscyamine and detecting the residue of the hyoscyamine.
The technical scheme of the invention is that a hybridoma cell strain Pba secreting hyoscyamine monoclonal antibody has been preserved in China general microbiological culture Collection center (CGMCC), China academy of sciences, 3, of the West Lu 1 Hospital, 3, of the Chaoyang district, Beijing, and is classified and named as monoclonal cell strain, the preservation date is 2020, 4 months and 23 days, and the preservation number is CGMCC No. 19678.
The hyoscyamine monoclonal antibody is secreted and generated by the hybridoma cell strain Pba with the preservation number of CGMCC No. 19678.
A preparation method of a hybridoma cell strain secreting a hyoscyamine monoclonal antibody comprises the following steps:
(1) designing a hyoscyamine hapten and preparing a hyoscyamine complete antigen, and preparing a Freund's adjuvant and an incomplete Freund's adjuvant from the obtained hyoscyamine complete antigen;
(2) injecting the Freund's adjuvant into BALB/c mouse via back subcutaneous injection for multiple times of immunization, wherein complete Freund's adjuvant is adopted for the first immunization, and incomplete Freund's adjuvant is adopted for boosting immunization;
(3) collecting blood from the mice subjected to the immunization process, detecting the serum immune titer and the immune suppression capacity of the mice through indirect ELISA, and screening the mice with high content of hyoscyamine antibody in the serum to obtain immunized mice;
(4) performing last boosting immunization on the screened mice by using incomplete Freund's adjuvant, and performing thrust immunization by intraperitoneal injection, wherein the thrust immunization is performed by using a hyoscyamine complete antigen without Freund's adjuvant;
(5) fusing splenocytes and myeloma cells of the BALB/c mice after the sprint immunization, culturing the fused cells through a culture medium, detecting positive cell holes by using indirect ELISA, further determining the inhibition effect of the positive cell holes by using an indirect competitive ELISA method, subcloning the positive cell holes with the best inhibition by using a limiting dilution method, and finally screening to obtain a hybridoma cell strain capable of secreting a hyoscyamine monoclonal antibody.
In one embodiment of the present invention, the first immunization and the booster immunization in the steps (2) and (4) are separated by one month, the booster immunization is separated by 21 days, and the booster immunization and the sprint immunization are separated by 18-21 days.
In one embodiment of the present invention, the primary immune dose in the steps (2) and (4) is 100 μ g/mouse, the booster immune dose is 50 μ g/mouse, and the sprint immune dose is 25 μ g/mouse.
In one embodiment of the present invention, the immunization process in the steps (2) and (4) comprises 1 first immunization, 4 booster immunizations and 1 sprint immunizations;
in one embodiment of the present invention, the blood collection in step (3) is performed on day 7 after the 3 rd immunization course.
In one embodiment of the present invention, the cell fusion in step (5) is performed 3 days after the completion of the sprint immunization.
In one embodiment of the present invention, the cell fusion in step (5) is performed by a polyethylene glycol (PEG4000) method.
In one embodiment of the present invention, the medium in the step (5) is RPMI-1640 medium.
In one embodiment of the present invention, the number of subclones in step (5) is 3.
The hyoscyamine hapten has the following structural formula:
Figure BDA0002980539170000021
a preparation method of the hyoscyamine hapten is characterized by comprising the following steps: weighing hyoscyamine technical product, succinic anhydride and triethanolamine, dissolving in dichloromethane, stirring at room temperature for reaction, stopping the reaction with hydrochloric acid, adjusting pH, removing impurities from the mixed solution with dichloromethane, purifying the water layer by preparative high performance liquid chromatography to obtain high-purity target substance, concentrating and enriching to obtain white solid which is hyoscyamine hapten;
the technical formula of the hyoscyamine is as follows:
Figure BDA0002980539170000031
further, the method comprises the following specific steps: weighing 1.73mmol of hyoscyamine technical product, 2.6mmol of succinic anhydride and 2.6mmol of triethanolamine, dissolving in 10mL of dichloromethane, stirring at room temperature for reaction for 4h, stopping the reaction with 1M hydrochloric acid, adjusting the pH value to 5-6, removing impurities from the mixed solution with dichloromethane, purifying the water layer by preparative high performance liquid chromatography to obtain a high-purity target substance, and concentrating and enriching to obtain the hyoscyamine hapten.
The hyoscyamine complete antigen has the following structural formula:
Figure BDA0002980539170000032
the preparation method of the hyoscyamine complete antigen comprises the following steps: dissolving hyoscyamine hapten, N-hydroxysuccinimide NHS and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC in N, N-dimethylformamide DMF, and stirring for reaction to obtain hyoscyamine hapten solution A; diluting hemocyanin KLH with carbonate buffer solution CBS to obtain solution B; adding the solution A into the solution B for reaction to obtain a reaction solution; the reaction solution was dialyzed with phosphate buffered saline PBS to obtain complete antigen.
Further, the method comprises the following specific steps: weighing 2.92mg of hyoscyamine hapten and 2.6mg of N-hydroxysuccinimide NHS, dissolving in 300 mu L N of N-dimethylformamide DMF, and stirring at room temperature for reaction for 5 min; then adding 4.3mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC, stirring and reacting at room temperature for 6-8h to obtain solution A; taking 10mgKLH, diluting to 5mg/mL with 0.01M carbonate buffer CBS, and obtaining solution B; slowly adding the solution A into the solution B drop by drop, and reacting at room temperature for 12 h; then dialyzing with 0.01M PBS solution to remove unreacted small molecule hapten to obtain the hyoscyamine complete antigen.
A method for preparing hyoscyamine coating antigen comprises dissolving hyoscyamine original drug and N, N' -carbonyldiimidazole CDI in anhydrous N, N-dimethylformamide DMF, stirring at room temperature, and reacting to obtain hyoscyamine hapten solution (solution A); diluting chicken egg albumin OVA with carbonate buffer CBS to obtain solution B; slowly adding the solution A into the solution B dropwise for reaction, stirring at room temperature for 12h to obtain a reaction solution, dialyzing the reaction solution with phosphate buffer solution PBS to remove unreacted micromolecules to obtain a hyoscyamine coated antigen;
the technical formula of the hyoscyamine is as follows:
Figure BDA0002980539170000033
further, the method comprises the following specific steps: dissolving 2.0mg of technical hyoscyamine and 11.20mg of N, N' -carbonyldiimidazole CDI in 300 μ L of anhydrous N, N-dimethylformamide DMF, and reacting at 37 deg.C under stirring for 8 hr to obtain hyoscyamine hapten solution (solution A); diluting 5mg of chicken egg albumin OVA with 1mL of carbonate buffer CBS with the concentration of 0.01mol/L to obtain solution B; and (3) dropwise adding the solution A into the solution B slowly for reaction, stirring at room temperature for 12 hours to obtain a reaction solution, dialyzing the reaction solution by using 0.01mol/L phosphate buffer solution PBS for 3 days to remove unreacted small molecules, and thus obtaining the hyoscyamine coated antigen.
The application of the hyoscyamine monoclonal antibody is characterized in that: can be used for detecting scopolamine residue in food.
Further, an immunoassay kit for preparing hyoscyamine by adopting the hyoscyamine monoclonal antibody and a colloidal gold test strip are used for detection.
The invention has the beneficial effects that: the monoclonal antibody of hyoscyamine has high detection sensitivity (IC) to hyoscyamine50A value of 1.03 ng/mL); the hyoscyamine monoclonal antibody cell strain obtained by the invention can be used for immunoassay detection.
Biological material sample preservation: a hybridoma cell strain Pba secreting hyoscyamine monoclonal antibody has been deposited in China general microbiological culture Collection center (CGMCC), China academy of sciences, institute of microbiology, 3, national institute of sciences, West Lu 1, North Cheng, Chaoyang, Beijing, and is classified and named as monoclonal cell strain with the preservation date of 2020, 4 months and 23 days and the preservation number of CGMCC No. 19678.
Drawings
FIG. 1 Standard inhibition curves for monoclonal antibodies to hyoscyamine.
Detailed Description
The following examples are provided as further illustration of the invention and are not to be construed as limitations or limitations of the invention. The invention is further illustrated by the following examples.
The media involved in the following examples are as follows:
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, calcium D-pantothenate 0.25, Folic acid 1, i-inositol 35, nicotinamide 1, choline chloride 3, pyridoxine hydrochloride 1, riboflavin 0.2, thiamine hydrochloride 1, vitamin B120.005, and sodium bicarbonate 2000.
The reagents involved in the following examples are as follows:
carbonate Buffer (CBS): weighing Na2CO3 1.59g，NaHCO32.93g of the mixture is respectively dissolved in a small amount of double distilled water and then mixed, the double distilled water is added to about 800mL and mixed evenly, the pH value is adjusted to 9.6, the double distilled water is added to the volume of 1000mL, and the mixture is stored for later use at 4 ℃;
phosphate Buffered Saline (PBS): 8.00g NaCl, 0.2g KCl, 0.2g KH2PO4，2.9g Na2HPO4·12H2Dissolving O in 800mL of pure water, adjusting the pH value to 7.2-7.4 by using NaOH or HCl, and fixing the volume to 1000 mL;
PBST: PBS containing 0.05% tween 20;
antibody dilution: PBS containing 0.1% gelatin;
TMB color development liquid: solution A: na (Na)2HPO4 .12H218.43g of O, 9.33g of citric acid and pure water to reach the constant volume of 1000 mL; and B, liquid B: 60mg of TMB was dissolved in 100mL of ethylene glycol. A. And the volume ratio of the solution B to the solution B is 5: 1 to obtain the TMB color developing solution which is mixed at present.
The detection methods referred to in the following examples are as follows:
the detection method of the hyoscyamine inhibition rate comprises the following steps: the most suitable 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. mu.g/mL with Carbonate Buffer (CBS) and the antibody was diluted to 0.01,0.03,0.1 and 0.3. mu.g/mL with antibody diluent. After selecting the optimal working point, the hyoscyamine standard was diluted to 8 concentrations (0, 0.03,0.1, 0.3, 1, 3, 9, 27ng/mL), and plotted according to the IC-ELISA protocol using originPro 8.5 (results are shown in FIG. 1), to obtain the hyoscyamine standard inhibition curve, and IC was calculated50。
Example 1: synthesis of hyoscyamine hapten
Because the small molecule of hyoscyamine has no immunogenicity, and can not stimulate mice to generate immune response so as to generate antibodies, the hyoscyamine is coupled to protein by a protein connection technology so as to obtain the immunogenicity; in order to better stimulate the immune response of mice, a hyoscyamine hapten is designed and derived by the following process:
weighing hyoscyamine technical material (500mg, 1.73mmol), succinic anhydride (259mg, 2.6mmol) and triethanolamine (260mg, 2.6mmol) and dissolving in 10ml dichloromethane, stirring at room temperature for reaction for 4h, stopping the reaction with 1M hydrochloric acid, adjusting pH to 5-6, removing impurities from the mixed solution with dichloromethane, taking a water layer, purifying by preparative high performance liquid chromatography to obtain a high-purity target substance, and concentrating and enriching to obtain 250mg of white solid, namely hyoscyamine hapten.
Example 2: synthesis of complete antigen of hyoscyamine
Weighing 2.92mg of hyoscyamine hapten and 2.6mg of N-hydroxysuccinimide (NHS), dissolving in 300 mu L N of N-Dimethylformamide (DMF), and stirring at room temperature for 5 min; 4.3mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) was added thereto, and the reaction was stirred at room temperature for 6 to 8 hours (referred to as solution A). Taking 10mg of KLH, diluting the KLH to 5mg/mL (called as solution B) by using 0.01M Carbonate Buffer Solution (CBS), then slowly adding the solution A into the solution B dropwise, and reacting for 12 hours at room temperature; then dialyzing with 0.01M PBS solution to remove unreacted small molecule hapten to obtain the hyoscyamine complete antigen.
Example 3: synthesis of scopolamine coatingen
Dissolving 2.0mg of technical hyoscyamine and 11.20mg of N, N' -Carbonyldiimidazole (CDI) in 300 μ L of anhydrous N, N-Dimethylformamide (DMF), and stirring at 37 deg.C for 8 hr to obtain hyoscyamine hapten solution (solution A); diluting 5mg of egg albumin (OVA) with 1mL of Carbonate Buffer Solution (CBS) with the concentration of 0.01mol/L to obtain solution B; and (3) dropwise adding the solution A into the solution B slowly for reaction, stirring at room temperature for 12 hours to obtain a reaction solution, dialyzing the reaction solution by using 0.01mol/L phosphate buffer solution PBS for 3 days to remove unreacted small molecules, and thus obtaining the hyoscyamine coated antigen.
The molecular structure of the technical product of hyoscyamine is shown in the figure:
Figure BDA0002980539170000061
example 4: preparation of hybridoma cell strain secreting hyoscyamine monoclonal antibody
(1) Obtaining animal immunity: mixing and emulsifying a hyoscyamine complete antigen and an equivalent amount of Freund's adjuvant, and performing neck and back subcutaneous multipoint injection immunization (except puncture immunization) on a BALB/c mouse; complete Freund adjuvant is used for the first immunization, and the dosage is 100 mug/mouse; incomplete Freund's adjuvant is used for multiple times of boosting immunization, and the dosage is reduced by half to be 50 mu g/mouse; the thorny immunity does not use an adjuvant, the thorny immunity is directly diluted by normal saline and then injected into the abdominal cavity, and the dosage is reduced by half to obtain 25 mu g/mouse; one month is separated between the first immunization and the second boosting immunization, 21 days are separated between the multiple boosting immunizations, and 18-21 days are separated between the sprint immunization and the last boosting immunization; the immune effect of the mouse is observed by an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), namely the titer and inhibition of the mouse serum are detected;
(2) cell fusion: after three days of the spurting immunization, cell fusion is carried out according to a conventional PEG (polyethylene glycol, molecular weight is 4000) method, and the specific steps are as follows:
a. cutting the tail, taking blood, immediately putting the mouse into 75% alcohol for disinfection after the mouse is killed by a cervical vertebra dislocation method, soaking for about 5min, taking out the spleen of the mouse through aseptic operation, properly grinding the spleen by using a rubber head of an injector, passing through a 200-mesh cell screen to obtain a spleen cell suspension, collecting, centrifuging (1200rpm, 8min), washing the spleen cells for three times by using an RPMI-1640 culture medium, diluting the spleen cells to a certain volume after the last centrifugation, and counting for later use;
b. collecting SP2/0 cells: 7-10 days before fusion, SP2/0 tumor cells were cultured in RPMI-1640 medium containing 10% FBS (fetal bovine serum) at 5% CO2Culturing in an incubator, wherein the number of SP2/0 tumor cells is required to reach (1-4) x 10 before fusion7Ensuring SP2/0 tumor cells to be in logarithmic growth phase before fusion, collecting the tumor cells during fusion, suspending the tumor cells in RPMI-1640 basic culture solution, and counting the cells;
c. the fusion process is 7 min: 1min, 1mL of PEG4000 was added to the cells dropwise from slow to fast; 2min, standing; dropping 1mL of RPMI-1640 culture medium within 1min at 3min and 4 min; dropping 2mL of RPMI-1640 culture medium within 1min at 5min and 6 min; at 7min, 1mL of RPMI-1640 culture medium is added dropwise every 10 s; then carrying out warm bath at 37 ℃ for 5 min; centrifuging (800rpm, 8min), discarding supernatant, resuspending in RPMI-1640 screening medium containing 20% fetal calf serum and 2% 50 XHAT, adding to 96-well cell plate at 200. mu.L/well, placing at 37 ℃ and 5% CO2Culturing in an incubator.
3. Cell screening and cell line establishment
Performing RPMI-1640 screening culture medium half-exchange on fused cells on the 3 rd day of cell fusion, performing total-exchange with RPMI-1640 transitional culture medium containing 20% fetal calf serum and 1% 100 XHT on the 5 th day, and taking cell supernatant on the 7 th day for screening;
the screening is divided into two steps: firstly, screening out positive cell holes by an ic-ELISA method, secondly, selecting hyoscyamine as a standard substance, and measuring the inhibition effect of positive cells by the ic-ELISA method;
selecting a cell hole with good inhibition on a hyoscyamine standard substance, carrying out subcloning by adopting a limiting dilution method, and carrying out detection by using the same method after seven days;
subcloning for three times according to the method to finally obtain the hyoscyamine monoclonal antibody cell strain Pba.
Example 5: preparation and identification of hyoscyamine monoclonal antibody
Taking BALB/c mice 8-10 weeks old, and injecting 1mL of sterile paraffin oil into the abdominal cavity of each mouse; 7 days later, each mouse was injected intraperitoneally with 1X 106Hyoscyamine hybridoma cells, ascites fluid was collected from the seventh day, and antibody purification was performed on the ascites fluid by the octanoic acid-saturated ammonium sulfate method.
Under the condition of partial acid, the caprylic acid can precipitate other hybrid proteins except IgG immunoglobulin in the ascites, then the centrifugation is carried out, and the precipitate is discarded; then, the IgG type monoclonal antibody was precipitated with an ammonium sulfate solution of the same saturation, centrifuged, the supernatant was discarded, and the supernatant was dissolved in a 0.01M PBS solution (pH7.4), dialyzed and desalted to finally obtain a purified monoclonal antibody, which was stored at-20 ℃.
Measurement of Seelongation Using Indirect competitive ELISAIC of monoclonal antibodies to scopolamine50The value is 1.03ng/mL, which shows that the reagent has good sensitivity to the hyoscyamine and can be used for immunoassay detection of the hyoscyamine.
Example 6: application of hyoscyamine monoclonal antibody
The monoclonal antibody prepared from hybridoma cell strain Pba through in vivo ascites is applied to an ELISA detection test of hyoscyamine, and the specific steps are as follows:
(1) coating a 96-well enzyme label plate with coating antigen with the concentration of 0.1 mu g/mL diluted by Carbonate Buffer Solution (CBS), coating 100 mu L of the enzyme label plate in each well at 37 ℃ for 2h, washing the plate with PBST washing liquor three times, wherein 200 mu L of the washing liquor is used in each well for 3min, and drying by beating;
(2) sealing with CBS containing 0.2% gelatin, sealing at 37 deg.C for 2 hr with 200 μ L per well, washing the plate with PBST lotion for three times, each time with 200 μ L per well, each time for 3min, and drying;
(3) preparing 0, 0.03,0.1, 0.3, 1, 3, 9, 27ng/mL of a hyoscyamine standard solution by using Phosphate Buffer Solution (PBS), adding the standard solution and a sample extracting solution to be detected into an enzyme label plate which is sealed, wherein each hole is 50 mu L, 3 holes are repeated for each sample, 50 mu L of an anti-hyoscyamine monoclonal antibody diluted to 0.1 mu g/mL is added into each hole, reacting at 37 ℃ for 0.5h, and then washing and drying the plate;
(4) adding 100 μ L of HRP-labeled goat anti-mouse IgG secondary antibody diluted with PBS containing 0.1% gelatin at a ratio of 1:3000 into each well, reacting at 37 ℃ for 0.5h, washing and drying;
(5) adding 100 μ L of TMB developing solution into each well, developing at 37 deg.C for 15min, and adding 50 μ L of H2M into each well2SO4Stopping solution, measuring the light absorption value at 450 nm;
determination of IC of monoclonal antibodies against hyoscyamine by IC-ELISA50Is 1.03ng/mL, which shows that the product has good sensitivity to hyoscyamine and can be used for hyoscyamine immunoassay detection.

Claims (10)

1. A hybridoma cell strain Pba secreting hyoscyamine monoclonal antibody has been deposited in China general microbiological culture Collection center (CGMCC), China academy of sciences, institute of microbiology, 3, national institute of sciences, West Lu 1, North Cheng, Chaoyang, Beijing, and is classified and named as monoclonal cell strain with the preservation date of 2020, 4 months and 23 days and the preservation number of CGMCC No. 19678.
2. A hyoscyamine monoclonal antibody characterized by: the hybridoma cell strain Pba with the preservation number of CGMCC No.19678 as defined in claim 1 is secreted.
3. A hyoscyamine hapten is characterized by the following structural formula:
Figure FDA0002980539160000011
4. a preparation method of the hyoscyamine hapten is characterized by comprising the following steps: weighing hyoscyamine technical product, succinic anhydride and triethanolamine, dissolving in dichloromethane, stirring at room temperature for reaction, stopping the reaction with hydrochloric acid, adjusting pH, removing impurities from the mixed solution with dichloromethane, purifying the water layer by preparative high performance liquid chromatography to obtain high-purity target substance, concentrating and enriching to obtain white solid which is hyoscyamine hapten;
the technical formula of the hyoscyamine is as follows:
Figure FDA0002980539160000012
5. a hyoscyamine complete antigen is characterized by having a structural formula as follows:
Figure FDA0002980539160000013
6. a preparation method of a hyoscyamine complete antigen is characterized by comprising the following steps: dissolving hyoscyamine hapten, N-hydroxysuccinimide NHS and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC in N, N-dimethylformamide DMF, and stirring for reaction to obtain hyoscyamine hapten solution A; diluting hemocyanin KLH with carbonate buffer solution CBS to obtain solution B; adding the solution A into the solution B for reaction to obtain a reaction solution; the reaction solution was dialyzed with phosphate buffered saline PBS to obtain complete antigen.
7. The method for preparing the complete antigen of hyoscyamine according to claim 6, which comprises the following steps: weighing 2.92mg of hyoscyamine hapten and 2.6mg of N-hydroxysuccinimide NHS, dissolving in 300 mu LN, N-dimethylformamide DMF, and stirring at room temperature for 5 min; then adding 4.3mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC, stirring and reacting at room temperature for 6-8h to obtain solution A; taking 10mg KLH, diluting to 5mg/mL by using 0.01M carbonate buffer CBS, and obtaining solution B; slowly adding the solution A into the solution B drop by drop, and reacting at room temperature for 12 h; then dialyzing with 0.01M PBS solution to remove unreacted small molecule hapten to obtain the hyoscyamine complete antigen.
8. The preparation method of the hyoscyamine coating antigen is characterized by comprising the following steps: dissolving hyoscyamine technical material and N, N' -carbonyldiimidazole CDI in anhydrous N, N-dimethylformamide DMF, and stirring at normal temperature to react to obtain hyoscyamine hapten solution (solution A); diluting chicken egg albumin OVA with carbonate buffer CBS to obtain solution B; slowly adding the solution A into the solution B dropwise for reaction, stirring at room temperature for 12h to obtain a reaction solution, dialyzing the reaction solution with phosphate buffer solution PBS to remove unreacted micromolecules to obtain a hyoscyamine coated antigen;
the technical formula of the hyoscyamine is as follows:
Figure FDA0002980539160000021
9. the method for preparing the hyoscyamine-coated antigen as claimed in claim 8, which comprises the following steps: dissolving 2.0mg of technical hyoscyamine and 11.20mg of N, N' -carbonyldiimidazole CDI in 300 μ L of anhydrous N, N-dimethylformamide DMF, and reacting at 37 deg.C under stirring for 8 hr to obtain hyoscyamine hapten solution (solution A); diluting 5mg of chicken egg albumin OVA with 1mL of carbonate buffer CBS with the concentration of 0.01mol/L to obtain solution B; and (3) dropwise adding the solution A into the solution B slowly for reaction, stirring at room temperature for 12 hours to obtain a reaction solution, dialyzing the reaction solution by using 0.01mol/L phosphate buffer solution PBS for 3 days to remove unreacted small molecules, and thus obtaining the hyoscyamine coated antigen.
10. The use of the monoclonal antibody against hyoscyamine according to claim 2, characterized in that: can be used for detecting scopolamine residue in food.
CN202110286115.1A 2021-03-17 2021-03-17 Hybridoma cell strain secreting hyoscyamine monoclonal antibody and application thereof Pending CN113005098A (en)

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
VIRTANEN R ET AL.: "Radioimmunoassay for atropine and l‐hyoscyamine", 《ACTA PHARMACOLOGICA ET TOXICOLOGICA》 *
WANG Z ET AL.: "Indirect competitive enzyme-linked immunosorbent assay based on a broad-spectrum monoclonal antibody for tropane alkaloids detection in pig urine, pork and cereal flours", 《FOOD CHEMISTRY》 *

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