CN107723278A - One plant of hybridoma cell strain SS0716 for secreting anti-NSC 408735 monoclonal antibody and its application - Google Patents

One plant of hybridoma cell strain SS0716 for secreting anti-NSC 408735 monoclonal antibody and its application Download PDF

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CN107723278A
CN107723278A CN201710999504.2A CN201710999504A CN107723278A CN 107723278 A CN107723278 A CN 107723278A CN 201710999504 A CN201710999504 A CN 201710999504A CN 107723278 A CN107723278 A CN 107723278A
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monoclonal antibody
plant
cell strain
hybridoma cell
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CN107723278B (en
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匡华
陈燕妮
胥传来
徐丽广
马伟
刘丽强
宋珊珊
吴晓玲
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Wuxi Determine Bio Tech Co ltd
Jiangnan University
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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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes

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  • Immunology (AREA)
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  • Urology & Nephrology (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
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  • Biochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract

One plant of hybridoma cell strain SS0716 for secreting anti-NSC 408735 monoclonal antibody and its application, belong to immunochemical technique field.The present invention is prepared for the monoclonal cell strain SS0716 in one plant of mouse source by the cell-fusion techniques of routine, has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCC No.14686.The monoclonal antibody of cell line secretion is determined using Indirect cELISA, to the IC of NSC 40873550For 0.58 μ g/L.To the rate of recovery scope of NSC 408735 is respectively in milk and in honey：98.71%~104.01% and 94.07%~98.68%.The detection remained for NSC 408735 in food provides raw material, has actual application value.

Description

The hybridoma cell strain SS0716 of one plant of anti-NSC 408735 monoclonal antibody of secretion and It is applied
Technical field
The present invention relates to the hybridoma cell strain SS0716 in one plant of mouse source and its monoclonal that can identify NSC 408735 of secretion Antibody, the detection remained available for NSC 408735 in food, belongs to immunochemical technique field.
Background technology
NSC 408735 is a kind of conventional sulfanilamide (SN) Trimethoprim, and diaminopyrimidine chemical synthesis is belonged in classification Antimicrobial, and be animal specific.Early stage research, which finds that the medicine shares with sulfa antibiotics, can make sulfa drugs antibacterial Spectrum expands, antibacterial activity greatly enhances, or even the bacteriostasis of sulfa antibiotics is become bactericidal action, so being known as " trimethoprim (TMP) ".NSC 408735 has antibacterial activity in itself, is clinically mainly compounded with sulfa drugs for treating Bacterium infection and the coccidium infections such as the intestines and stomach of livestock and poultry, respiratory tract, urethra, in recent years, there are some researches show the compound can be with The effect of strengthening Multiple Classes of Antibiotics class medicine and plurality of Chinese.But the long-term use of the medicine, even abuse, abuse, meeting Cause the medicament residue in animal food.The appearance of part bacterial drug resistance is also resulted in simultaneously.At present, only Japan provides NSC 408735 is in edible chicken tissues（Muscle, liver, kidney and fat）In highest residual quantity be 50 μ g/kg, it is other each State and international organization do not formulate food security standard to NSC 408735.Used for NSC 408735 in animal farming industry Supervision, and to the residue detection of NSC 408735 in animal food and exercise supervision particularly significant.
At present, Instrumental Analysis, such as liquid chromatogram etc. mainly are relied on for the detection method of NSC 408735.But this A little methods are needed by expensive instrument and equipment, and detection time is long, it is necessary to which the personnel of specialty operate.Enzyme-linked immune analytic method （ELISA）It is a kind of quick and sensitive high-flux detection method based on monoclonal antibody, it is residual for the medicine in food Stay detection significant.Currently without the report on anti-NSC 408735 monoclonal antibody.
The content of the invention
The purpose of the present invention is monoclonal antibody of the exploitation for NSC 408735, and establishes ELISA side on this basis Method.This content of the invention includes two aspects：The preparation of monoclonal antibody and its application.
Anti- NSC 408735 monoclonal antibody is obtained by mouse immune NSC 408735 immunogene.Immunogene and coating Original is synthesized by glutaraldehyde method.After 5th time immune, the high mouse suppressed of screening seropositivity takes spleen, and spleen is thin Born of the same parents are merged with Sp2/0 oncocytes.Cell conditioned medium is detected with indirect ELISA method after fusion, is screened to positive height and to dimethoxy The cell that benzyl pyridine has suppressed is subcloned, and carries out obtaining pure cell line after being subcloned three times.After cell line is expanded into culture The intraperitoneal preparation ascites for the mouse for playing paraffin oil is injected into, the ascites of acquisition is purified with octanoic acid-ammonium sulfate method.Survey Half-inhibition concentration (IC of the order clonal antibody to NSC 40873550), and the IC to its analogue50, calculate and intersect Reactivity.NSC 408735 medicine is added into milk, sample addition recovery experiment is carried out, calculates the rate of recovery.
Technical scheme：The hybridoma cell strain SS0716 of one plant of anti-NSC 408735 monoclonal antibody of secretion, China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved in, deposit number is CGMCC No.14686.
Anti- NSC 408735 monoclonal antibody, by the hybridoma that the deposit number is CGMCC No.14686 SS0716 secretions produce, its IC50For 0.58 μ g/L.
The application of described anti-NSC 408735 monoclonal antibody, for the detection remained to NSC 408735 in food.
Beneficial effects of the present invention：It is right using the monoclonal antibody of indirect competitive ELISA measure cell line SS0716 secretions The IC of NSC 40873550For 0.58 μ g/L.To the rate of recovery scope of NSC 408735 in milk and honey for 98.71%~ 104.01% and 94.07%~98.68%.The immune detection remained for NSC 408735 in animal food provides raw material, has Actual application value.
Biological material specimens preservation：Monoclonal cell strain SS0716, Chinese microorganism strain preservation management committee has been preserved in it Member's meeting common micro-organisms center, abbreviation CGMCC, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the micro- life of the Chinese Academy of Sciences Thing research institute, deposit number are CGMCC No.14686, preservation date September in 2017 5 days.
Brief description of the drawings
Standard suppression curve of the monoclonal antibody of Fig. 1 SS0716 secretions to NSC 408735.
Embodiment
Further explanation of the following examples of the present invention only as present invention, it is impossible to the restriction as the present invention Inside perhaps scope.Below by embodiment, the invention will be further described.
Embodiment 1：Immunogene and coating antigen synthesis
Immunogene and coating antigen are synthesized by glutaraldehyde method：5 mg NSC 408735s are dissolved in 2 mL methanol solutions, PH to 4 is adjusted with 1M HCL.The glutaraldehyde water solutions of 5.67 μ L 25% are added in diaveridine solution, activation 30 is stirred at room temperature min.20 mg hemocyanin are dissolved in 4 mL 0.01M PBS, and the NSC 408735 activated is slowly added into protein solution In, reaction 4h is stirred at room temperature.After reaction terminates, reactant is dialysed three days with 0.01M PBS solution, obtained immunogene packing It is stored in -20 DEG C.The synthesis of coating antigen is identical with the synthetic method of immunogene, changes hemocyanin into oralbumin.
Embodiment 2：Mouse immune
Immunogene is emulsified with normal saline dilution to suitable concentration, then with the Freund's complete adjuvant of equivalent, in 6-8 week old The back of BALB/c mouse carries out multi-point injection.First immunisation is Freund's complete adjuvant, and immunizing dose is 100 μ g.Below Booster immunization in, immunogene and incomplete Freund's adjuvant emulsify, and immunizing dose be 50 μ g, and immunization interval is 21 days.For the third time After immune, serum is detected by the tail vein blood of mouse, and by indirect ELISA.5th time it is immune after to screen potency high The mouse suppressed carries out abdominal cavity spurt and is immunized, and mouse spleen is taken out after three days and carries out cell fusion.
Embodiment 3：Cell fusion and screening
Mouse boosting cell and oncocyte merge in the presence of PEG 1500 forms hybridoma.Examined within the 7th day after fusion Survey, screening potency is high and the cell line good to NSC 408735 identity is subcloned, and by that analogy, carries out 3 subclones Obtain pure cell line.
Embodiment 4：The purifying of monoclonal antibody
After the cell line that filters out expands culture, it is injected into the mouse peritoneal for having played paraffin oil, can be from after 7 to 14 days Mouse peritoneal extracts ascites.Ascites is purified with octanoic acid-ammonium sulfate method, is stored in -20 DEG C.
Embodiment 5：The property of monoclonal antibody
The sensitivity and specificity of antibody are determined by indirect ELISA.
1st, the cross reacting rate of monoclonal antibody
The optium concentration of envelope antigen and monoclonal antibody reactive is determined by chessboard method first.Surveyed again with indirect ELISA method The cross reacting rate of order clonal antibody.Concretely comprise the following steps：Coating antigen is diluted with the carbonate buffer solutions of 0.05M pH 9.6, 100 μ L/ holes, 37 DEG C of reaction 2h.Solution in plate is inclined, dried, and washed 3 times with cleaning solution PBST, each 3min.Pat dry Afterwards, 200 μ L/ holes confining liquids, 37 DEG C of reaction 2h are added.It is dried for standby after washing.50 μ L reference materials are added in every hole of ELISA Plate Solution and 50 μ L antibody, are cultivated to wash after 30 min at 37 DEG C and pat dry afterwards three times, and 1 ︰ 3000 dilution secondary antibodies 100 μ is added per hole L, cultivate at 37 DEG C and patted dry after washing 4 times after 30 min, add the μ L of TMB nitrite ions 100 prepared, 37 DEG C of cultures 15min, it is last that 50 μ L 2M sulfuric acid solution terminating reactions are added per hole.The light absorption value at 450nm is determined with ELIASA.
Determine IC of the monoclonal antibody to other synergist50：TMP, Ormetoprim and Baquiloprim.Intersect anti- Should rate be analog IC50With NSC 408735 IC50Ratio.Its cross-over experiment result is as shown in table 1.
The monoclonal antibody SS0716 of table 1. intersection
2nd, recovery experiment is added
Add the NSC 408735 of various concentrations respectively into negative milk and honey, indirect competitive ELISA determines addition sample Target concentration in this.5 times of milk Sample Dilution, honey Sample Dilution 10 exclude matrix interference again, calculate the rate of recovery.Add Add-back receives experimental result and is shown in Table 2.
Table 2. adds recovery experiment
Solution is prepared：
Carbonate buffer solution（CBS）：Weigh Na2CO31.59g NaHCO32.93g mixed after being dissolved in a small amount of distilled water respectively, Add distilled water to be mixed to about 800 mL, adjust pH to 9.6, add distilled water to be settled to 1000 mL, 4 DEG C of storages are standby；
Phosphate buffer（PBS）：8.00 g NaCl, 0.2g KCl, 0.2 g KH2PO4, 2.9 g Na2HPO4·12 H2O, It is dissolved in 800 mL pure water, adjusts pH to 7.2~7.4 with NaOH or HCl, be settled to 1000 mL；
PBST：PBS containing 0.05% polysorbas20；
TMB nitrite ions：A liquid：Na2HPO4·12H2O 18.43g, citric acid 9.33g, pure water are settled to 1000 mL；B liquid：60 Mg TMB are dissolved in 100 mL ethylene glycol.A, B liquid 1 ︰ 5 mixing by volume is TMB nitrite ions, current existing mixed.
It is only presently preferred embodiments of the present invention in summary, is not used for limiting the practical range of the present invention.It is i.e. all The equivalent changes and modifications made according to the content of the present patent application scope, it all should be the technology category of the present invention.

Claims (3)

1. the hybridoma cell strain SS0716 of one plant of anti-NSC 408735 monoclonal antibody of secretion, has been preserved in China Microbiological bacterium Kind preservation administration committee common micro-organisms center, deposit number is CGMCC No.14686.
2. anti-NSC 408735 monoclonal antibody, it is characterised in that the deposit number as described in claim 1 is CGMCC No.14686 hybridoma cell strain SS0716 secretions produce, its IC50For 0.58 μ g/L.
3. the application of the anti-NSC 408735 monoclonal antibody described in claim 2, it is characterised in that for diformazan in food The detection of oxygen benzyl pyridine residual.
CN201710999504.2A 2017-10-24 2017-10-24 One plant of hybridoma cell strain SS0716 for secreting anti-NSC 408735 monoclonal antibody and its application Active CN107723278B (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108251381A (en) * 2018-04-04 2018-07-06 江南大学 A kind of paraquat monoclonal antibody hybridoma cell strain and its application
CN108410824A (en) * 2018-03-14 2018-08-17 江南大学 One plant of stresnil monoclonal antibody hybridoma cell strain and its application
US20200040106A1 (en) * 2018-08-03 2020-02-06 Jiangnan University Hybridoma cell line of secreting meloxicam monoclonal antibodies and application thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101776685A (en) * 2009-11-11 2010-07-14 北京望尔康泰生物技术有限公司 Enzyme linked immunosorbent assay kit for detecting trimethoprim medicament and application thereof
CN103524427A (en) * 2012-07-03 2014-01-22 北京勤邦生物技术有限公司 Preparation method as well as application of trimethoprem hapten
CN104341357A (en) * 2014-09-29 2015-02-11 江南大学 Method for preparing complete antigen from trimethoprim hapten T2 and application of complete antigen
CN104370829A (en) * 2014-09-29 2015-02-25 江南大学 Method for preparing complete antigen from trimethoprim semiantigen compound T1 and use of complete antigen

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101776685A (en) * 2009-11-11 2010-07-14 北京望尔康泰生物技术有限公司 Enzyme linked immunosorbent assay kit for detecting trimethoprim medicament and application thereof
CN103524427A (en) * 2012-07-03 2014-01-22 北京勤邦生物技术有限公司 Preparation method as well as application of trimethoprem hapten
CN104341357A (en) * 2014-09-29 2015-02-11 江南大学 Method for preparing complete antigen from trimethoprim hapten T2 and application of complete antigen
CN104370829A (en) * 2014-09-29 2015-02-25 江南大学 Method for preparing complete antigen from trimethoprim semiantigen compound T1 and use of complete antigen

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108410824A (en) * 2018-03-14 2018-08-17 江南大学 One plant of stresnil monoclonal antibody hybridoma cell strain and its application
CN108251381A (en) * 2018-04-04 2018-07-06 江南大学 A kind of paraquat monoclonal antibody hybridoma cell strain and its application
CN108251381B (en) * 2018-04-04 2020-09-04 江南大学 Paraquat monoclonal antibody hybridoma cell strain and application thereof
US20200040106A1 (en) * 2018-08-03 2020-02-06 Jiangnan University Hybridoma cell line of secreting meloxicam monoclonal antibodies and application thereof
US10562979B1 (en) * 2018-08-03 2020-02-18 Jiangnan University Hybridoma cell line of secreting meloxicam monoclonal antibodies and application thereof

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