CN109280647A - One plant of Nicarbazin monoclonal antibody hybridoma cell strain and its application - Google Patents
One plant of Nicarbazin monoclonal antibody hybridoma cell strain and its application Download PDFInfo
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- CN109280647A CN109280647A CN201811363303.4A CN201811363303A CN109280647A CN 109280647 A CN109280647 A CN 109280647A CN 201811363303 A CN201811363303 A CN 201811363303A CN 109280647 A CN109280647 A CN 109280647A
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- nicarbazin
- monoclonal antibody
- cell strain
- dnc
- bsa
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- UKHWDRMMMYWSFL-UHFFFAOYSA-N Nicarbazin Chemical compound CC=1C=C(C)NC(=O)N=1.C1=CC([N+](=O)[O-])=CC=C1NC(=O)NC1=CC=C([N+]([O-])=O)C=C1 UKHWDRMMMYWSFL-UHFFFAOYSA-N 0.000 title claims abstract description 49
- 229940073485 nicarbazin Drugs 0.000 title claims abstract description 49
- 210000004408 hybridoma Anatomy 0.000 title claims abstract description 18
- 210000004027 cell Anatomy 0.000 claims abstract description 31
- 238000001514 detection method Methods 0.000 claims abstract description 19
- 238000004321 preservation Methods 0.000 claims abstract description 8
- 235000013305 food Nutrition 0.000 claims abstract description 7
- 244000005700 microbiome Species 0.000 claims abstract description 6
- 238000004458 analytical method Methods 0.000 claims abstract description 3
- 239000007788 liquid Substances 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 19
- 239000000243 solution Substances 0.000 claims description 19
- 239000000427 antigen Substances 0.000 claims description 13
- 102000036639 antigens Human genes 0.000 claims description 13
- 108091007433 antigens Proteins 0.000 claims description 13
- 239000005457 ice water Substances 0.000 claims description 12
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 6
- 235000010288 sodium nitrite Nutrition 0.000 claims description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 5
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 5
- GLUUGHFHXGJENI-UHFFFAOYSA-N Piperazine Chemical compound C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 claims description 4
- 238000004090 dissolution Methods 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 4
- 238000010521 absorption reaction Methods 0.000 claims description 3
- 238000005303 weighing Methods 0.000 claims description 3
- 229940098773 bovine serum albumin Drugs 0.000 claims description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 2
- 150000001412 amines Chemical class 0.000 claims 1
- 230000020477 pH reduction Effects 0.000 claims 1
- 241000287828 Gallus gallus Species 0.000 abstract description 7
- 230000028327 secretion Effects 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 210000004185 liver Anatomy 0.000 abstract description 3
- 239000002994 raw material Substances 0.000 abstract description 2
- 238000002649 immunization Methods 0.000 description 16
- 238000002965 ELISA Methods 0.000 description 13
- 241000699666 Mus <mouse, genus> Species 0.000 description 13
- 239000002671 adjuvant Substances 0.000 description 12
- 230000003053 immunization Effects 0.000 description 11
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 10
- 239000001963 growth medium Substances 0.000 description 9
- 239000012980 RPMI-1640 medium Substances 0.000 description 8
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 241000224483 Coccidia Species 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 230000004927 fusion Effects 0.000 description 5
- 230000005764 inhibitory process Effects 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 238000011725 BALB/c mouse Methods 0.000 description 4
- 241000223932 Eimeria tenella Species 0.000 description 4
- 230000007910 cell fusion Effects 0.000 description 4
- 230000001900 immune effect Effects 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 229920001223 polyethylene glycol Polymers 0.000 description 4
- 206010003445 Ascites Diseases 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 230000001804 emulsifying effect Effects 0.000 description 3
- 239000012894 fetal calf serum Substances 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 244000144977 poultry Species 0.000 description 3
- 239000006152 selective media Substances 0.000 description 3
- 210000004988 splenocyte Anatomy 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- HJCUTNIGJHJGCF-UHFFFAOYSA-N 9,10-dihydroacridine Chemical compound C1=CC=C2CC3=CC=CC=C3NC2=C1 HJCUTNIGJHJGCF-UHFFFAOYSA-N 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 239000003636 conditioned culture medium Substances 0.000 description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
- 229910000397 disodium phosphate Inorganic materials 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 238000009629 microbiological culture Methods 0.000 description 2
- 201000000050 myeloid neoplasm Diseases 0.000 description 2
- WWZKQHOCKIZLMA-UHFFFAOYSA-N octanoic acid Chemical compound CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 1
- 241000252983 Caecum Species 0.000 description 1
- 239000005635 Caprylic acid (CAS 124-07-2) Substances 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 239000005662 Paraffin oil Substances 0.000 description 1
- 206010034133 Pathogen resistance Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 235000021120 animal protein Nutrition 0.000 description 1
- 238000011091 antibody purification Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 210000005252 bulbus oculi Anatomy 0.000 description 1
- 210000004534 cecum Anatomy 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 239000000890 drug combination Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229920001971 elastomer Polymers 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 238000007499 fusion processing Methods 0.000 description 1
- 210000003128 head Anatomy 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 229960002446 octanoic acid Drugs 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 210000001563 schizont Anatomy 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- 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
-
- 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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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
One plant of Nicarbazin monoclonal antibody hybridoma cell strain and its application, belong to food safety field of immunodetection.One plant of Nicarbazin monoclonal antibody hybridoma cell strain SS0713 of the present invention, it has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, abbreviation CGMCC, deposit number CGMCC No.14691, preservation address Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, preservation date on September 5th, 2017, classification naming monoclonal cell strain.The Nicarbazin monoclonal antibody generated by SS0713 secretion, for the remaining analysis detection of Nicarbazin in food safety detection.The monoclonal antibody of this cell strain secretion has preferable specificity and detection sensitivity (IC to Nicarbazin50It is worth for 0.05 ng/mL) detection, it can be achieved that Nicarbazin residual quantity in the liver and feed of chicken, chicken, provides raw material for the remaining immune detection of Nicarbazin in food, there is practical application value.
Description
Technical field
The present invention relates to one plant of Nicarbazin monoclonal antibody hybridoma cell strain and its applications, and it is immune to belong to food safety
Detection field.
Background technique
Nicarbazin (Nicarbazin, DNC) is a kind of feed addictive, and vermifuge has various coccidias relatively strong
Inhibition and killing effect, be not likely to produce drug resistance and cross-resistance, rapidly, residual quantity is extremely low for excretion.Nicarbazin is to chicken
Caecum coccidia and heap shape eimeria tenella, huge eimeria tenella, murder by poisoning eimeria tenella, Podbielniak eimeria tenella have well
Preventive effect, have many advantages, such as efficiently, low toxicity, performance is stable, drug resistance is small, with coccidia ester drug combination better effect.Buddhist nun
Carbazine is effective to coccidia second generation schizont, and recommended amounts do not influence chicken and generate immunity to coccidia, and safety is higher, high temperature
Season is used with caution, laying period disabling.Two kinds of ingredients of Nicarbazin can be absorbed by poultry alimentary canal respectively, and be distributed widely in tissue
And in body fluid.
Nicarbazin has been widely used in the fields such as agricultural, livestock and poultry and feed.Therefore Nicarbazin is in animal tissue, flesh
Generally have certain residual in meat and feed, and human body intake Nicarbazin excessively can the organs such as liver kidney to the mankind cause generation
Thank to burden, if having Nicarbazin residual in animal protein (chicken, egg, milk), being then harmful to human health.
Standard GB/T/T 29691-2013 by Nicarbazin (DNC) as allow using feed addictive, simultaneously
Alsoing specify its highest allows using limitation (0.02mg/kg) and highest maximum permission quantity (0.1mg/kg).Chinese people's republicanism
The residual quantity standard of Nicarbazin in outlet poultry is alsied specify in state import-export commodity inspection professional standard SN0216-1993,
Provide its maximum permission quantity (0.03 mg/kg-1.20 mg/kg).
But these standards all use efficient liquid-phase chromatography method to detect, detection method is relatively complicated, complicated, in order to safeguard
The interests of the majority of consumers, it is necessary to establish a kind of efficient, quick detection method for DNC, and enzyme-linked immunization
(ELISA) pre-treatment is simple, it is at low cost, it can be achieved that a large amount of samples quick detection, and when detecting to the purity requirement of sample not
It is high.Therefore, it is necessary to establish efficient immunological detection method, and an important prerequisite for establishing the method need to filter out
For the high-specificity monoclonal monomer of Nicarbazin.
Summary of the invention
The purpose of the present invention is overcoming above-mentioned shortcoming, one plant of Nicarbazin monoclonal antibody hybridoma cell strain is provided
SS0713 and its application, the antibody prepared by the cell strain have preferably specificity and detection sensitivity to Nicarbazin, can be with
For establishing the immunological detection method of Nicarbazin.
Technical solution of the present invention, one plant of Nicarbazin monoclonal antibody hybridoma cell strain SS0713 have been preserved in
State's Microbiological Culture Collection administration committee common micro-organisms center, abbreviation CGMCC, deposit number CGMCC No.14691 are protected
Hide address Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, preservation date September 5 in 2017
Day, classification naming monoclonal cell strain.
Nicarbazin monoclonal antibody, it is resisted by the Nicarbazin monoclonal that the deposit number is CGMCC No.14691
Body hybridoma cell strain SS0713 secretion generates.
The application of the Nicarbazin monoclonal antibody, for the remaining analysis inspection of Nicarbazin in food safety detection
It surveys.
The preparation basic step of Nicarbazin monoclonal antibody hybridoma cell strain SS0713 provided by the invention are as follows:
(1) derivative of haptens:
Nicarbazin raw medicine are as follows:
Haptens: amino is derived with Nicarbazin raw medicine.
(2) preparation of comlete antigen DNC-BSA: weighing 4.38mg DNC derivative, is dissolved in 300 μ L n,N-Dimethylformamide
(DMF) in, 10min is stirred to react under the conditions of ice-water bath；Under conditions of ice-water bath, the HCL that 48 μ L 1mol/L d are added dropwise is carried out
It is acidified 0.5h；150mg sodium nitrite (DIA) is weighed again, after completely dissolution with 500 μ L pure water, 5.56 μ L sodium nitrites is added
Solution reacts 0.5-1 h (referred to as A liquid) under the conditions of ice-water bath into DNC derivative solution.Take 6mg BSA(DNC and cow's serum
Albumin BSA molar ratio is 60:1), (referred to as B liquid) is dissolved with 2mL 0.01M carbonate buffer solution (CB, pH=9.0), then
A liquid is slowly added into B liquid dropwise, 4h is reacted under the conditions of ice-water bath；Then it is dialysed, is removed not anti-with 0.01M PBS solution
The small haptens answered obtain comlete antigen DNC-BSA, and are identified by UV absorption scan method；
(3) mouse is immune: after comlete antigen DNC-BSA and equivalent Freund's adjuvant mixing and emulsifying, carrying out to BALB/c mouse
The subcutaneous multi-point injection of the nape of the neck is immune (except spurt is immune).First immunisation complete Freund's adjuvant, dosage are 100 μ g/；It is more
Secondary booster immunization cannots be used up full Freund's adjuvant and dosage halves as 50 μ g/ only；Spurt is immune not to have to adjuvant, directly uses physiology salt
Water dilution after be injected intraperitoneally, dosage halve again as 25 μ g/only.One is spaced between first immunisation and second of booster immunization
Month, it is spaced 21 days between multiple booster immunization, spurt is immune to be spaced 18-21 days between last time booster immunization.By
Connect potency and inhibition that competitive enzyme-linked immune method (ic-ELISA) observation mouse immune effect detects mice serum；
(4) cell fusion and cell strain are established: by polyethylene glycol (PEG 4000) method by mouse boosting cell and mouse myeloma
Cell is merged, and filters out hybridoma using selective medium (HAT culture medium), and carry out cell with HT culture medium
Culture.Fusion detects positive cell hole using ic-ELISA method after a week, and further positive thin using the measurement of ic-ELISA method
The inhibitory effect of hilum, by limiting dilution assay to inhibiting preferable positive cell hole to be subcloned, detect again after a week,
Choose hole, subclone.The strain of Nicarbazin monoclonal antibody hybridoma cell is obtained after being subcloned three times according to the above method
SS0713；
(5) identification of hybridoma cell strain property: pass through ic-ELISA measurement sensitivity and specificity.
Comlete antigen DNC-BSA and equivalent Freund's adjuvant mixing and emulsifying are complete, immune by the subcutaneous multi-point injection of the nape of the neck
BALB/c mouse.First immunisation (100 μ g/ are only) complete Freund's adjuvant, multiple booster immunization (50 μ g/ are only) cannot be used up full Freund
Adjuvant, last time, which makes a spurt to be immunized, carries out mouse peritoneal injection with DNC comlete antigen (25 μ g/ only, are free of adjuvant).Take specificity
Height, IC50Low mouse boosting cell is merged by PEG method with murine myeloma cell, by ic-ELISA method screening cell and
It is subcloned three times, obtains the hybridoma cell strain of plant height secretion specific antibody.
Beneficial effects of the present invention: the monoclonal antibody of cell strain SS0713 provided by the invention secretion, to DNC have compared with
Good specificity and detection sensitivity (IC50Value is 0.05ng/mL), it can be achieved that being remained to DNC in the liver of chicken and chicken, feed
The detection of amount provides raw material for the remaining immune detection of DNC in food, has practical application value.
Biological material specimens preservation: one plant of Nicarbazin monoclonal antibody hybridoma cell strain SS0713 has been preserved in
State's Microbiological Culture Collection administration committee common micro-organisms center, abbreviation CGMCC, deposit number CGMCC No.14691 are protected
Hide address Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, preservation date September 5 in 2017
Day, classification naming monoclonal cell strain.
Detailed description of the invention
Fig. 1 is the inhibition standard curve of Nicarbazin monoclonal antibody.
Specific embodiment
The following examples of the present invention are only used as the further explanation of the content of present invention, not as a limitation of the present invention interior
Perhaps range.Below by embodiment, the invention will be further described.
The present invention passes through cell fusion, the training of HAT selective medium by the way that mouse is immunized in Nicarbazin comlete antigen
It supports, cell conditioned medium is screened by ic-ELISA, has finally obtained the hybridization that there is hypersecretion specific antibody for Nicarbazin
Tumor cell strain.
The preparation of 1 hybridoma cell strain SS0713 of embodiment
(1) synthesis of comlete antigen: weighing 4.38mg DNC derivative, is dissolved in 300 μ L n,N-Dimethylformamide (DMF)
In, 10min is stirred to react under the conditions of ice-water bath；Under conditions of ice-water bath, 48 μ L 1mol/L HCL are added dropwise and are acidified
0.5h；150mg sodium nitrite (DIA) is weighed again, after completely dissolution with 500 μ L pure water, 5.56 μ L sodium nitrite solutions is added
Into DNC derivative solution, 0.5-1 h (referred to as A liquid) is reacted under the conditions of ice-water bath.Take 6mg BSA(DNC and bovine serum albumin
White (BSA) molar ratio is 60:1), (referred to as B liquid) is dissolved with 2mL 0.01M carbonate buffer solution (CB, pH=9.0), then by
A liquid is slowly added into B liquid by drop, and 4h is reacted under the conditions of ice-water bath；Then it is dialysed with 0.01M PBS solution, removes unreacted
Small haptens, obtain comlete antigen DNC-BSA, and identified by UV absorption scan method；
(2) after comlete antigen DNC-BSA and equivalent Freund's adjuvant mixing and emulsifying, neck animal immune: is carried out to BALB/c mouse
Dorsal sc multi-point injection is immune (except spurt is immune).First immunisation complete Freund's adjuvant, dosage are 100 μ g/；Repeatedly
Booster immunization cannots be used up full Freund's adjuvant and dosage halves as 50 μ g/ only；Spurt is immune not to have to adjuvant, directly uses physiological saline
It is injected intraperitoneally after dilution, dosage halves again as 25 μ g/ only.It is spaced one month between first immunisation and second of booster immunization,
It is spaced 21 days between multiple booster immunization, spurt is immune to be spaced 18-21 days between last time booster immunization.By indirectly competing
Strive potency and inhibition that enzyme-linked immunization (ic-ELISA) observation mouse immune effect detects mice serum；
(3) cell fusion: after spurt is three days immune, according to conventional PEG(polyethylene glycol, molecular weight 4000) method progress is carefully
Born of the same parents' fusion, the specific steps are as follows:
A, mouse plucks eyeball and takes blood, after cervical dislocation puts to death mouse, is immediately placed in 75% alcohol and sterilizes, and it is left to impregnate 5min
The spleen of mouse is taken out in the right side, sterile working, is moderately ground with syringe rubber head and obtains splenocyte by 200 mesh cell screen clothes and hanged
Liquid is collected, and is centrifuged (1200rpm, 8min), is washed splenocyte three times with RPMI-1640 culture medium, will after last time is centrifuged
Splenocyte is diluted to certain volume, counts, spare；
B, collect SP2/0 cell: 7-10 days before fusion, SP2/0 oncocyte being used and contains 10% FBS(fetal calf serum) RPMI-
1640 culture mediums are in 5% CO2It is cultivated in incubator.SP2/0 oncocyte quantity is required to reach (1-4) × 10 before fusion7, guarantee
SP2/0 oncocyte is in logarithmic growth phase before merging.When fusion, oncocyte is collected, RPMI-1640 basic culture solution is suspended in
In, carry out cell count；
C, the PEG 4000 of 1mL is added drop-wise in cell by the 7min: the 1min of fusion process from slow to fast；2min is stood.
1mL RPMI-1640 culture medium is added dropwise in 3min and 4min in 1min；5min and 6min is added dropwise in 1min
2mL RPMI-1640 culture medium；The RPMI-1640 culture medium of 1mL is added dropwise in 7min, every 10s.Then 37 DEG C of warm bath 5
min.It being centrifuged (800rpm, 10min), abandons supernatant, cell gently strikes scattered, and it is added into it and contains 20% fetal calf serum, 2% 50 ×
The RPMI-1640 selective medium (HAT culture medium) of HAT is added to 96 porocyte plates according to 200 holes μ L/, is placed in 37 DEG C,
5% CO2It is cultivated in incubator；
(4) cell screening and cell strain are established: partly being changed with HAT culture medium fused cell within the 3rd day after cell fusion
Liquid；It is changed entirely with the RPMI-1640 transition culture solution (HT culture medium) of 100 × HT containing 20% fetal calf serum, 1% within 5th day
Liquid；Cell conditioned medium is taken to be screened within 7th day.Screen in two steps: the first step first filters out positive cell hole with ic-ELISA method, the
It is standard items that two steps, which select Nicarbazin, carries out inhibitory effect measurement to positive cell with ic-ELISA method.Selection is to Buddhist nun's kappa
Piperazine standard items have the cell hole preferably inhibited, are subcloned using limiting dilution assay, are examined after seven days with same method
It surveys.It is subcloned three times according to the above method, it is final to obtain Nicarbazin cell strain of monoclonal antibody SS0713.
The preparation and identification of 2 monoclonal antibody of embodiment
The configuration of solution:
Carbonate buffer solution (CBS): Na is weighed2CO31.59 g, NaHCO32.93 g are mixed after being dissolved in a small amount of distilled water respectively
It closes, adds distilled water to mix to about 800mL, adjust pH value to 9.6, add distilled water to be settled to 1000mL, 4 DEG C of storages are spare；
Phosphate buffer (PBS): 8.00g NaCl, 0.2g KCl, 0.2g KH2PO4, 2.9g Na2HPO4·12 H2O, it is molten
In 800mL pure water, with NaOH or HCl tune pH to 7.2~7.4, it is settled to 1000mL；
PBST: the PBS containing 0.05 % polysorbas20；
TMB developing solution: A liquid: Na2HPO4 .12H2O 18.43g, citric acid 9.33g, pure water are settled to 1000mL；B liquid: 60mg
TMB is dissolved in 100mL ethylene glycol.A, B liquid 5:1 mixing by volume is TMB developing solution, current existing mixed.
8-10 week old BALB/c mouse is taken, every mouse peritoneal injects sterile paraffin oil 1mL；Every mouse peritoneal after 7 days
Injection 1 × 106 Nicarbazin hybridoma collected ascites since the 7th day, and ascites is passed through octanoic acid-saturated ammonium sulfate method
Carry out antibody purification.Under the conditions of meta-acid, caprylic acid, which can precipitate, removes other ultrawhite foreign proteins of IgG immune globulin in ascites,
It is then centrifuged for, abandons precipitating；Again with the monoclonal antibody of the ammonium sulfate precipitating IgG type of equivalent saturation degree, it is centrifuged, abandons supernatant,
After the dissolution of 0.01 M PBS solution (pH7.4), dialysis desalting, the monoclonal antibody finally obtained after purification is placed in -20 DEG C of guarantors
It deposits.
(1) be coated with: by coating antigen DNC-BSA, with 0.05M pH9.6 carbonate buffer solution, 3 multiple proportions since 1 μ g/mL are dilute
It releases, 100 holes μ L/, 37 DEG C of reaction 2h；
(2) it washs: solution in plate being inclined, and is washed 3 times with cleaning solution, each 3min；
(3) it closes: after patting dry, 200 hole μ L/ confining liquids, 37 DEG C of reaction 2h is added.It is dried for standby after washing；
(4) be loaded: by antiserum since 1:1000 doubling dilution, and be added in the coating hole of each dilution, 100 holes μ L/,
37 DEG C of reaction 30min；Sufficiently after washing, the diluted HRP- sheep anti-mouse igg of 1:3000,100 holes μ L/, 37 DEG C of reactions are added
30min；
(5) it develops the color: ELISA Plate is taken out, sufficiently after washing, the TMB developing solution of 100 μ L is added in every hole, and 37 DEG C are protected from light
15min；
(6) terminate and measure: 50 μ L terminate liquids are added to terminate reaction in every hole, and the OD 450 in each hole is then measured with microplate reader
Value.
With the IC of ic-ELISA measurement monoclonal antibody Nicarbazin50Are as follows: 0.05ng/mL illustrates have very to Nicarbazin
Good sensitivity can be used for the detection of Nicarbazin immunoassay.
The operating point for choosing monoclonal antibody Nicarbazin is measured with ic-ELISA, according to the antigen-antibody concentration of operating point
By gradient mark-on, then mapped to obtain the inhibition standard curve of Nicarbazin monoclonal antibody with Origin software, it is specific such as Fig. 1
It is shown.
Claims (4)
1. one plant of Nicarbazin monoclonal antibody hybridoma cell strain SS0713, has been preserved in Chinese microorganism strain preservation management
Committee's common micro-organisms center, abbreviation CGMCC, deposit number CGMCC No.14691, preservation address Chaoyang District, Beijing City north
No. 3 Institute of Microorganism, Academia Sinica of institute of occasion West Road 1, preservation date on September 5th, 2017, classification naming monoclonal cell
Strain.
2. Nicarbazin monoclonal antibody, it is characterised in that: its deposit number as described in claim 1 is CGMCC No.14691
Nicarbazin monoclonal antibody hybridoma cell strain SS0713 secrete generate.
3. the application of Nicarbazin monoclonal antibody described in claim 2, it is characterised in that: for Buddhist nun's card in food safety detection
Bar remaining analysis detection of piperazine.
4. the preparation method for the comlete antigen that cell strain SS0713 described in claim 1 is used is prepared, it is characterized in that steps are as follows:
(1) derivative of haptens:
Nicarbazin raw medicine are as follows:
Haptens: amino is derived with Nicarbazin raw medicine；
(2) preparation of comlete antigen DNC-BSA: weighing 4.38mg DNC derivative, is dissolved in 300 μ L N, N- dimethyl formyls
In amine DMF, 10min is stirred to react under the conditions of ice-water bath；Under conditions of ice-water bath, 48 μ L HCL are added dropwise and carry out acidification 0.5h；
150mg sodium nitrite is weighed again, after completely dissolution with 500 μ L pure water, 5.56 μ L sodium nitrite solutions is added to DNC derivative
In solution, 0.5-1 h, referred to as A liquid are reacted under the conditions of ice-water bath；6mg BSA, the DNC is taken to be with bovine serum albumin(BSA) BSA molar ratio
60:1 is dissolved, referred to as B liquid with the carbonate buffer solution of 2mL 0.01M, pH=9.0；A liquid is slowly added into B liquid dropwise again,
4h is reacted under the conditions of ice-water bath；Then it is dialysed with 0.01M PBS solution, removes unreacted small haptens, obtained completely
Antigen DNC-BSA, and identified by UV absorption scan method.
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| CN110117575A (en) * | 2019-05-22 | 2019-08-13 | 江南大学 | One plant of pyrimethanil monoclonal antibody hybridoma cell strain HFG and its application |
| CN110256298A (en) * | 2019-06-20 | 2019-09-20 | 中国农业大学 | 4,4 '-dinitro phenylurea haptens and artificial antigen and the preparation method and application thereof |
| CN113684187A (en) * | 2021-09-22 | 2021-11-23 | 江南大学 | Hybridoma cell strain secreting monoclonal antibody to fluazifop-butyl as well as preparation method and application of hybridoma cell strain |
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2018
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110117575A (en) * | 2019-05-22 | 2019-08-13 | 江南大学 | One plant of pyrimethanil monoclonal antibody hybridoma cell strain HFG and its application |
| CN110117575B (en) * | 2019-05-22 | 2022-03-15 | 江南大学 | Pyrimidinemonoclonal antibody hybridoma cell strain HFG and application thereof |
| CN110256298A (en) * | 2019-06-20 | 2019-09-20 | 中国农业大学 | 4,4 '-dinitro phenylurea haptens and artificial antigen and the preparation method and application thereof |
| CN113684187A (en) * | 2021-09-22 | 2021-11-23 | 江南大学 | Hybridoma cell strain secreting monoclonal antibody to fluazifop-butyl as well as preparation method and application of hybridoma cell strain |
| CN113684187B (en) * | 2021-09-22 | 2023-07-18 | 江南大学 | Hybridoma cell strain secreting fluazinam monoclonal antibody as well as preparation method and application thereof |
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