CN106434569A - Monoclonal cell strain C4 capable of secreting monoclonal antibodies for identifying methylene blue and application of monoclonal cell strain C4 - Google Patents

Monoclonal cell strain C4 capable of secreting monoclonal antibodies for identifying methylene blue and application of monoclonal cell strain C4 Download PDF

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CN106434569A
CN106434569A CN201611062737.1A CN201611062737A CN106434569A CN 106434569 A CN106434569 A CN 106434569A CN 201611062737 A CN201611062737 A CN 201611062737A CN 106434569 A CN106434569 A CN 106434569A
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methylene blue
cell strain
monoclonal
monoclonal cell
monoclonal antibody
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CN106434569B (en
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匡华
彭娟
胥传来
徐丽广
刘丽强
宋珊珊
吴晓玲
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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

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Abstract

The invention provides a monoclonal cell strain C4 capable of secreting monoclonal antibodies for identifying methylene blue and application of the monoclonal cell strain C4 and belongs to the technical field of immunochemistry. The monoclonal cell strain C4 is preserved in China General Microbiological Culture Collection Center and the preservation number is CGMCC No.12026. The monoclonal antibodies secreted by the monoclonal cell strain C4 can be used for specifically identifying the methylene blue; the monoclonal cell strain C4 has good specificity and high sensitivity; the half inhibition concentration (IC50) is 21.13ng/mL; and a methylene blue immunological detection technology can be established by using the monoclonal antibodies.

Description

One plant can secrete identification methylene blue monoclonal cell strain C4 of monoclonal antibody and Its application
Technical field
The present invention relates to one plant of monoclonal cell strain C4 of monoclonal antibody that can secrete identification methylene blue and its application, Belong to immunochemical technique field.
Background technology
Methylene blue belongs to thiazin dyes, from malachite green prohibited for aquaculture in after, its substitute The residue problem of methylene blue causes extensive concern and attention.Methylene blue initially starts anti-for urinary tract infection and malaria Control, with research constantly deeply, studies have found that methylene blue can kill tumour cell and have higher parent to tumour cell And property, therefore methylene blue after labelled with radioisotope be used for diagnose, position and treat kinds of tumors.Methylene blue is simultaneously There is oxidisability and reproducibility, clinically can be used for the drug poisoning such as cyanide and nitrite as antidote.Due to Asia Methyl blue is in ionic state in aqueous, and the enzyme system with periphery microorganism is competed hydrogen ion, leads to microbial enzyme to lose Work is lethal.Therefore methylene blue is also used as disinfectant in veterinary applications particularly aquaculture.There are some researches show, methylene blue is also To some fish diseases such as chilodoniasis, unguiculus worm, saprolegniasis etc. has preferable curative effect.Therefore, when some triphenylmethane dyes such as After malachite green and crystal violet are clearly forbidden for aquaculture, product become raisers' pro-gaze to methylene blue as an alternative Object.In addition to disinfectant and fish disease are prevented, methylene blue also can during aquatic product transportation as antifungal agent use with Reduce the death rate.The toxicity of methylene blue be less than malachite green, but in aquaculture process any to methylene blue not Reasonable employment, is all likely to result in the accumulation of methylene blue, in addition with the extensive utilization of dyestuff, as the methylene of the dye of positive ion Base indigo plant enters surrounding body in a large number with waste water, causes environment Methylene Blue concentration over-standard, when human or animal is chronically exposed to When in the methylene blue environment of high concentration, easily cause intoxicating phenomenon, toxic and side effect has mutagenesis, red cell morphology changes or lean Blood, gangrenosum acne abscess etc., can be lethal when serious.Therefore, including the U.S., Japan, European Union does not allow Asia in interior country or tissue Methyl blue is used in aquaculture.In China, methylene blue is cheap due to it, clear curative effect, and raiser is secondary to its poison to be made With understand less, methylene blue substitute as disinfectant and malachite green in aquaculture is widely used, therefore its Residue problem should not be underestimated, and the detection technique research that it is remained has important meaning to the food security of China and the export trade Justice.Although China does not also put into effect relevant laws and regulations, the research of detection technique is the pioneer of supervision work, in the face of international trade Easy technology barriers, the grinding of the research to methylene blue and its Metabolic residue detection technique, especially fast and convenient examination technology Study carefully very urgent.
In the document delivered, the detection technique of methylene blue is remained on instrumental method, such as liquid chromatography technology, Capillary electrophoresis technique and the chromatography of ions, detector is generally mass spectrum or fluorescence detector, and pretreatment technology has liquid-liquid extraction And solid phase extraction techniques.At present, high performance liquid chromatography tandem mass spectrum method is mainly to the main flow detection means of methylene blue, simply As extracted on detector and sample pre-treatments means, the step such as purification is slightly different.To aquatic products Methylene Blue Immuno analytical method research is substantially at blank stage.
Set up the immunoassay technology of methylene blue, first have to obtain the monoclonal antibody of specific recognition methylene blue.
Content of the invention
For the problems referred to above, the invention provides a kind of monoclonal cell strain C4, it can produce specific recognition methylene The blue monoclonal antibody of base, can be used in setting up the immunoassay technology of methylene blue.
Technical scheme：Monoclonal cell strain C4 of one plant of monoclonal antibody that can secrete identification methylene blue, It has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No. 12026.
A kind of monoclonal antibody specific of identification methylene blue, its list for CGMCC No.12026 by deposit number Clonal cell line C4 secretion produces.
The application of the described monoclonal antibody specific of identification methylene blue, in the immune work of preparation detection methylene blue Application in tool.
The application of the described monoclonal antibody specific of identification methylene blue, in answering of detection flesh of fish Methylene Blue With.
Beneficial effects of the present invention：The monoclonal antibody specific being produced by monoclonal cell strain C4 being capable of specific recognition Methylene blue；And sensitivity is good, half-inhibition concentration（IC50）For 21.13 ng/mL；For developing the detection of food nitrite methyl blue Immune instrument provides raw material, such as Enzyme-multiplied immune technique and immuno-chromatographic test paper strip.
Biological material specimens preservation：The hybridoma cell strain C4 that the present invention provides, Classification And Nomenclature is monoclonal cell strain, should Cell line has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, abbreviation CGMCC, and address is north Jing Shi Chaoyang District North Star West Road 1 institute 3, Institute of Microorganism, Academia Sinica, preservation date on January 20th, 2016, Deposit number is CGMCC No.12026.
Brief description
The calibration curve of Fig. 1 methylene blue monoclonal antibody.
The ultraviolet qualification result of Fig. 2 comlete antigen.
Specific embodiment
The invention provides the monoclonal antibody specific of the anti-methylene blue of monoclonal cell strain C4 and its secretion.Below In conjunction with specific embodiment, the present invention is described further, is conventional method in embodiment if no special instructions.
The preparation of embodiment 1 monoclonal cell strain C4
1st, mouse immune and antiserum screening
Select haptens（MB-HS）By active ester method respectively with hemocyanin（KLH）And ovalbumin（OVA）It is coupled, formed Immunogene（MB-HS-KLH）And coating antigen（MB-HS-OVA）.
Head exempts from using Freund's complete adjuvant and immunogene 11 volume mixture, with subcutaneous inoculation mode immunity BALB/c mouse, immunity 4 weeks interval times, six exempt from rear 7-10d, with coating antigen coated elisa plate, measure serum using Indirect cELISA Potency and suppression, select potency high and mouse that is having suppressed is merged.
Wherein, haptens is methylene blue derivatives thing, and haptenic structure is：
2nd, cell fusion
The 21st day after mouse resisting anteserum evaluation, satisfactory mouse is selected to carry out abdominal cavity spurt immunity.Detailed process is as follows：Will Immunogene normal saline dilution exempts from the half of the dosage that dosage is last booster immunization to 200 μ L volumes, punching, will be dilute The immunogene released injects 1 mL syringe, and on the left of mouse, abdominal cavity is slowly injected in mouse body.
After mouse punching exempts from 3 days, put to death mouse by the way of disconnected neck, and put into rapidly in 100 mL 75% alcohol, mouse Transfer to superclean bench after soaking 5-10 min in alcohol and take out spleen, be placed on 200 mesh stainless steel mesh, use 5 mL Plunger is lightly ground, and is constantly rinsed with RPMI1640 culture medium, by Spleen cell suspensions collect 50 mL from In heart pipe, 1200 r/min are centrifuged 8 min, abandon supernatant, and gently piping and druming centrifugation bottom of the tube, with cell dispersion, adds RPMI1640 training Foster basic weight hangs splenocyte, and transfers in new 50mL centrifuge tube.10 mL RPMI1640 cultures are added after repeated centrifugation three times Base, mixes, and taking-up 100 μ L therein dilute 1000 times and counted, remaining standby.After splenocyte gathers, start to collect It is in the oncocyte of exponential phase.Oncocyte and splenocyte mix than 1 2-10 ratio in counting, 1200 r/min from The heart 8 min, abandons supernatant, with forefinger gently bullet beat centrifugation bottom of the tube so that cell is uniformly dispersed, prepare merge：In 1st min, will be pre- 1 mL PEG of heat is slowly adherent to be at the uniform velocity added drop-wise in cell mixing, and side edged is shaken gently for centrifuge tube；In 2nd min, will be from Heart pipe is held in the hand, standing；3-4 min, is slowly added dropwise the RPMI 1640 of 2 mL with the speed of 1 mL/min, and side edged shakes Dynamic centrifuge tube；In 5-6 min, it is slowly added dropwise the RPMI 1640 of 4 mL with the speed of 1 mL/30s, side edged shakes；7th min Interior, RPMI 1640 is added with the speed of 1 mL/10s.It is eventually adding RPMI 1640 to 20 mL, centrifuge tube sealed membrane is sealed Be put into well in incubator and stand 5 min, 800 r/min be centrifuged 8 min, abandon supernatant, with forefinger gently bullet beat centrifugation bottom of the tube make Cell spreads out, and is slowly added to HAT Selective agar medium, transfers in 96 porocyte plates by 200 μ L/ holes after gently mixing（Paving Depending on plate quantity is according to cell quantity）, it is placed on CO2Cultivate in incubator.
Carry out HAT Selective agar medium and partly change liquid within the 3rd day after cell fusion, carry out HAT culture medium within the 6th day and entirely change liquid, the 7th day Cell conditioned medium is taken to carry out cell screening.
The 7th day after fusion, the potency according to mouse resisting anteserum and suppression measurement result, determine being coated needed for cell screening Concentration, carries out wrapper sheet closing.Carry out the mensure of cell positive first, further take out the cell conditioned medium in positive hole, carry out suppression and measure, Suppression need to carry out suitable dilution when measuring make the OD value in all holes as far as possible all 1.5 about, is separately added into one in 96 orifice plates Determine methylene blue standard items and the standard dilutions of concentration, 50 μ L/ holes, add, by 50 μ L/ holes, the cell conditioned medium having diluted, 37 DEG C of baking ovens incubate 30min, more routinely ELISA method of operating wash plate add two anti-etc. until measuring light absorption value.According to detection As a result, select the preferably positive 4-6 higher hole of suppression to carry out being subcloned for the first time.After three subclones, select effect good List group RPMI 1640 culture medium containing 10% hyclone for the cell be enlarged cultivate.Treat cell confluent cultures bottom of bottle portion and When cell is in exponential phase, collects cell and carry out frozen and preparation ascites.
The preparation of embodiment 2 monoclonal antibody specific, purifying
1st, the preparation of monoclonal antibody
Prepare ascites using inducing ascites tumor method in vivo.Paraffin oil is sterilized stand-by.Select the good BALB/c mouse of state, every Mouse peritoneal injection enters 0.5 mL sterile paraffin oil, carries out pre-stimulation.Injection paraffin oil is because that paraffin oil can reduce mouse Immunity, weakens the rejection of the hybridoma to injection for the mouse it is also possible to stimulate mouse to produce interleukins, for note The hybridoma penetrated provides good growing environment, is conducive to the generation of ascites.After 7-10 days, collect the hybridization of Amplification Culture Oncocyte, washes through RPMI 1640 culture medium after three times and is injected into mouse peritoneal by 0.5 mL/ pipe.After 7-10 days, mouse starts meeting Produce ascites tumor, observe the state of mouse at any time, after its abdominal cavity swelling, extract abdomen from mouse peritoneal with 5 mL syringes Water, is dispensed in 1.5 mL centrifuge tubes, 8000 r/min centrifugation 10min, draws supernatant, dispenses in 1.5 mL sterile tube, and -20 DEG C save backup.
2nd, monoclonal antibody-purified
Ascites is purified using octanoic acid-ammonium sulfate precipitation method.Take 1 mL ascites in centrifuge tube, add isopyknic sodium acetate molten Liquid（0.01 M, pH 4.0）, mix back vibration side and be slowly added to 33 μ L caprylic acids, sealed membrane is sealed, place normal temperature on shaking table Vibrate 30 min.Subsequent 8000 r/min are centrifuged 10 min, take supernatant to 1.5 new mL centrifuge tubes, add isopyknic full And ammonium sulfate.4 DEG C of refrigerators stand overnight, after fully precipitating, 8000 r/min are centrifuged 15 min, abandon supernatant.Heavy The PBS containing sylvite forming sediment with 1 mL（0.01 M, pH 7.4）Redissolve, put into water and boil in sterilized bag filter, in 0.01 M PBS Dialyse 3 days in dislysate, change dislysate every 12h.After end, it is sub-packed in the sterile tube of 1.5 mL, and preserve in -20 DEG C Standby.
Embodiment 3 antibody performance evaluation
1st, hypotype measures
Hypotype using the monoclonal antibody of the hypotype identification kit detection preparation bought.Detailed process is as follows：It is being coated Add the antibody of the 0.2 μ g/mL having diluted, 50 μ L/ holes in the ELISA Plate of 0.1 μ g/mL coating antigen, incubate in 37 DEG C of baking ovens 30 min, wash after plate pats dry, are separately added into IgM, the ELIAS secondary antibody type such as IgG1, IgG2a, IgG2b, IgG3, IgA, 100 μ L/ hole, incubates 30 min in 37 DEG C of baking ovens, washes colour developing after plate pats dry and terminates, wherein the ELIAS secondary antibody type corresponding to colour developing hole is The hypotype of the antibody by being identified.
2nd, cross reaction（CR）
Interpolation concentration is the reddish black A of 10 μ g/mL respectively, reddish black B, aC, and malachite green and crystal violet are entered to C4 secretory antibody Row cross reaction is tested, and result is as shown in the table, and C4 secretory antibody, to other structures analog no cross reaction, has highly special The opposite sex.
The cross reaction of the anti-methylene blue monoclonal antibody of table 1
Compound IC50（ng/mL） CR(%) Compound IC50（ng/mL） CR(%)
MB 21.13 100 AC >10000 <0/01
Reddish black A >10000 <0/01 Malachite green >10000 <0/01
Reddish black B >10000 <0/01 Crystal violet >10000 <0/01
3rd, sensitivity determination
Use half-inhibition concentration（IC50）Represent the sensitivity of antibody.IC20-IC80The range of linearity of method for expressing
Conjugate（MB-HS-KLH）And conjugate（MB-HS-OVA）All can be obtained by active ester method.
Present invention methylene blue-succinyl oxide haptens with sodium dithionite reduction synthesis, then pass through active ester method It is coupled with hemocyanin and is used as immunogene, thus obtaining the monoclonal antibody of specific recognition methylene blue.
Being coated original content is 0.1 μ g/mL, and the protein concentration of antibody is 0.3 μ g/mL, using origin software to data It is fitted obtaining the calibration curve of methylene blue monoclonal antibody, as shown in figure 1, IC50For 21.13 ng/mL, the range of linearity （IC20-IC80）For 4.26-104.83 ng/mL, coefficient correlation is 0.997.

Claims (4)

1. monoclonal cell strain C4 of one plant of monoclonal antibody that can secrete identification methylene blue, has been preserved in China Microbiological bacterium Plant preservation administration committee common micro-organisms center, deposit number is CGMCC No. 12026.
2. a kind of identification methylene blue monoclonal antibody specific it is characterised in that：It is CGMCC by deposit number The monoclonal cell strain C4 secretion of No.12026 produces.
3. described in claim 2 identification methylene blue monoclonal antibody specific application it is characterised in that：In preparation inspection Survey the application in the immune instrument of methylene blue.
4. according to claim 3 identification methylene blue monoclonal antibody specific application it is characterised in that：In inspection Survey the application of flesh of fish Methylene Blue.
CN201611062737.1A 2016-11-28 2016-11-28 One plant of monoclonal cell strain C4 that can secrete the monoclonal antibody for identifying methylene blue and its application Active CN106434569B (en)

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PCT/CN2017/111098 WO2018095254A1 (en) 2016-11-28 2017-11-15 Monoclonal cell line c4 capable of secreting monoclonal antibody recognizing methylene blue and application thereof

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106282124A (en) * 2016-08-24 2017-01-04 江南大学 The mass selection monoclonal antibody of a kind of monoclonal cell strain C4 and generation thereof and application
WO2018095254A1 (en) * 2016-11-28 2018-05-31 江南大学 Monoclonal cell line c4 capable of secreting monoclonal antibody recognizing methylene blue and application thereof
WO2019136335A1 (en) * 2018-01-05 2019-07-11 Gencyte Therapeutics, Inc. Precision molecular adaptor system for car-t immunotherapy

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103983703A (en) * 2014-03-07 2014-08-13 浙江省海洋水产研究所 High-performance-liquid-chromatography detection method for methylene blue in aquaculture water

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106434569B (en) * 2016-11-28 2018-08-07 江南大学 One plant of monoclonal cell strain C4 that can secrete the monoclonal antibody for identifying methylene blue and its application

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103983703A (en) * 2014-03-07 2014-08-13 浙江省海洋水产研究所 High-performance-liquid-chromatography detection method for methylene blue in aquaculture water

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
XU，Y.J.等: "Simultaneous Determination of Malachite Green， Crystal Violet， Methylene blue and the Metabolite Residues in Aquatic Products by Ultra-Performance Liquid Chromatography with Electrospray Ionization Tandem Mass Spectrometry", 《JOURNAL OF CHROMATOGRAPHIC SCIENCE》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106282124A (en) * 2016-08-24 2017-01-04 江南大学 The mass selection monoclonal antibody of a kind of monoclonal cell strain C4 and generation thereof and application
WO2018095254A1 (en) * 2016-11-28 2018-05-31 江南大学 Monoclonal cell line c4 capable of secreting monoclonal antibody recognizing methylene blue and application thereof
WO2019136335A1 (en) * 2018-01-05 2019-07-11 Gencyte Therapeutics, Inc. Precision molecular adaptor system for car-t immunotherapy

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