CN104031887A - Monoclonal antibody and application thereof in diphacinone detection - Google Patents
Monoclonal antibody and application thereof in diphacinone detection Download PDFInfo
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Abstract
The invention discloses a monoclonal antibody and an application thereof in diphacinone detection. The accession number of a diphacinone monoclonal antibody hybridoma cell strain DPC provided by the invention is CGMCC No.8953. The monoclonal antibody secreted by the hybridoma cell strain DPC also belongs to the scope of protection of the invention. The invention also discloses applications of the hybridoma cell strain DPC or the monoclonal antibody in preparation of kits used for detecting diphacinone or diphacinone derivatives. The monoclonal antibody is high in sensitivity and high in specificity, can be used in the detection of the diphacinone or the diphacinone derivatives, and satisfies the requirements of practical application. By combination of the monoclonal antibody and an enzyme-linked immunoassay method, the diphacinone or the diphacinone derivatives in animal-source foods can be rapidly sensitively and simple detected, and the requirements of high throughout and a low cost can be satisfied. The monoclonal antibody, the application thereof, the hybridoma cell strain DPC and the applications of the hybridoma cell strain DPC have important economic value and social significance.
Description
Technical field
The present invention relates to a kind of monoclonal antibody and the application in detecting diphacinone thereof.
Background technology
Diphacinone (diphacinone) is a kind of common anti-coagulant rodenticide, belongs to indenes diones, for indandione class represents medicine, often uses its sodium salt, is called diphacinone-Na.Diphacinone-Na is the chronic rodenticide of anticoagulation that uses at present the most general efficient, the low toxicity, safety.This medicine mainly destroys blood clotting function and damages tiny blood vessels, causes the general fatal hemorrhages such as subcutaneous, internal organ, face, causes being poisoned to death.Diphacinone or diphacinone-Na are high drugs, concerning mouse is oral, and the LD of diphacinone
50for 112-196mg/kg, the LD of diphacinone-Na
50for 61.2-100.7mg/kg.Become human oral 0.06-0.25g diphacinone-Na to cause poisoning, 0.5-2.5g can be lethal.In China because wrongly taking, improper use or the event that causes people and livestock to be poisoned to death of having a mind to poison happen occasionally.Therefore, carry out the residue detection work of diphacinone, for ensuring food safety, find out in time, exactly poisoning reason, rescue toxic patient and there is very important practical significance.
The residual common method that detects at present diphacinone or diphacinone-Na mainly contains high performance liquid chromatography, reversed-phased high performace liquid chromatographic, vapor-phase chromatography etc.Such technology required equipment is had relatively high expectations, instrument is expensive, to the having relatively high expectations of operator, be therefore not suitable for the screening of high-throughput sample, be difficult to large-scale popularization.
Summary of the invention
The object of this invention is to provide a kind of monoclonal antibody and the application in detecting diphacinone thereof.
Diphacinone monoclonal antibody hybridoma cell strain DPC provided by the invention (being called for short hybridoma cell strain DPC), on March 13rd, 2014, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.8953.
The monoclonal antibody of hybridoma cell strain DPC secretion also belongs to protection scope of the present invention.
The present invention also protects hybridoma cell strain DPC or the application of described monoclonal antibody in the test kit for the preparation of detection diphacinone or diphacinone derivative.Described diphacinone derivative specifically can be diphacinone-Na.
The present invention also protects the application of described monoclonal antibody in detecting diphacinone or diphacinone derivative.Described diphacinone derivative specifically can be diphacinone-Na.
The present invention also protects compound shown in formula I;
Described carrier proteins specifically can be bovine serum albumin (BSA) or oralbumin (OVA).
The present invention also protects a kind of test kit that detects diphacinone or diphacinone derivative, comprises hybridoma cell strain DPC or described monoclonal antibody.Described test kit also can comprise compound shown in formula I.Described diphacinone derivative specifically can be diphacinone-Na.
The present invention also protects take the antibody that compound shown in formula I obtains as immunogen.
The present invention adopts following methods to prepare immunogen and coating antigen: thus first adopt carboxymethyl hydroxylamine assay to transform and make it have carboxyl active group and [adopt O (carboxymethyl) hydroxylamine assay by ketone group of diphacinone and O (carboxymethyl) azanol reaction diphacinone haptens, generate O-carboxylic first 9 oxime derivate haptens], through condensation reaction, make diphacinone and carrier proteins mixture afterwards.The antibody that the immunogen that the present invention prepares produces possesses high specific.
Monoclonal antibody provided by the invention is highly sensitive, high specificity, can be applicable to the detection of diphacinone or diphacinone derivative, meets actual needs.Adopt monoclonal antibody provided by the invention in conjunction with enzyme-linked immunosorbent assay, can detect quick, sensitive, easily diphacinone or diphacinone derivative in animal derived food, can meet high-throughput and requirement cheaply.The present invention has great economic worth and social effect.
Accompanying drawing explanation
Fig. 1 is the synthetic route schematic diagram of immunogen and coating antigen.
Fig. 2 is that the ultraviolet of immunogen, diphacinone-Na, BSA scans spectrum schematic diagram.
Fig. 3 is that the ultraviolet of coating antigen, diphacinone-Na, OVA scans spectrum schematic diagram.
Fig. 4 is the IC that indirect competitive ELISA is measured monoclonal antibody
50the graphic representation of value.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.
Diphacinone-Na: LGM Pharma company, CAS 42721-99-3.Carboxymethyl azanol half hydrochloride (CMA): lark prestige Science and Technology Ltd., CAS 2921-14-4.N-hydroxy-succinamide (NHS): Sigma company, CAS 6066-82-6.N, N '-dicyclohexylcarbodiimide (DCC): lark prestige Science and Technology Ltd., CAS 538-75-0.1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDCHCl): Sigma company, CAS 25952-53-8.Bovine serum albumin (BSA): Sigma company, CAS 9048-46-8.Chicken ovalbumin (OVA): Sigma company, CAS 9006-50-1.Balb/c mouse: purchased from Beijing Vital River Experimental Animals Technology Co., Ltd..The sheep anti-mouse igg antibody of horseradish peroxidase-labeled: Jackson Immuno Research Laboratories company, production code member 115-035-003.Bromadiolone: Accustandard company, CAS 28772-56-7.Coumatetralyl: Accustandard company, CAS 5836-29-3.Pindone: Accustandard company, CAS 83-26-1.
The structural formula of diphacinone is as follows:
The preparation of embodiment 1, immunogen and coating antigen
Fig. 1 is shown in by preparation flow schematic diagram.
One, immunogenic preparation
1,370mg diphacinone-Na is dissolved in to 5mL dehydrated alcohol, then adds successively 130mg carboxymethyl azanol half hydrochloride and 200 μ L pyridines, stirring at room 16 hours.
2, after completing steps 1, add 10mL4g/100mL NaHCO
3the aqueous solution.
3, after completing steps 2, add 20mL methylene dichloride to extract, collect respectively water and organic phase.
4, get the water that step 3 obtains, drip 1.5-2mL1mol/L aqueous hydrochloric acid (rock on acid adding limit, limit, until solution becomes muddiness).
5, get the turbid solution that step 4 obtains, with twice of dichloromethane extraction (20mL * 2), merge organic phase.
6, the organic phase that organic phase step 3 being obtained and step 5 obtain merges, and with excessive anhydrous sodium sulfate drying (placement is spent the night), then revolves steaming evaporate to dryness, obtains dry-matter.
7, get the dry-matter that 18.5mg step 6 obtains, be dissolved in 1.2mL DMF, add 10.31mg N-hydroxy-succinamide and 13.86mg N, N '-dicyclohexylcarbodiimide, stirring at room 16 hours, then the centrifugal 10min of 5000rpm, gets supernatant liquor.
8, adopt BSA as solute, adopt the carbonate buffer solution of pH9.6,0.05M as solvent, prepare BSA solution (containing 50mg BSA in every 5mL BSA solution).
9, supernatant liquor step 7 being obtained is dropwise added drop-wise in the BSA solution that 5mL step 8 obtains, stirring at room 16 hours.
10, after completing steps 9, pack product into dialysis tubing, in the PBS of pH7.4,0.01M damping fluid, dialyse (4 ℃, every day change liquid 2 times, dialysis 3 days), the solution obtaining is immunogen solution.
11, get the immunogen solution that step 10 obtains, adopt ultraviolet spectrophotometer to carry out full wavelength scanner.Take diphacinone-Na, each 1mg of BSA, be dissolved in respectively in the PBS damping fluid of pH7.4,0.01M, the identical PBS damping fluid of take is blank, does respectively spectral scan.Ultraviolet scanning spectrum figure is shown in Fig. 2.Result shows, three's absorption peak does not all overlap, coupling success.
Two, the preparation of coating antigen
1, get the dry-matter that 6 of 13.8mg step 1 obtains, be dissolved in 1mL DMF.
2, adopt OVA as solute, adopt the carbonate buffer solution of pH9.6,0.05M as solvent, prepare OVA solution (containing 50mg OVA in every 5mL OVA solution).
3, solution step 1 being obtained is dropwise added drop-wise in the OVA solution that 5mL step 2 obtains, and adds 15.4mg N-hydroxy-succinamide and 12.8mg1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate, stirring at room 16 hours.
4, after completing steps 3, pack product into dialysis tubing, in the PBS of pH7.4,0.01M damping fluid, dialyse (4 ℃, every day change liquid 2 times, dialysis 3 days), the solution obtaining is coating antigen solution.
5, get the coating antigen solution that step 4 obtains, adopt ultraviolet spectrophotometer to carry out full wavelength scanner.Take diphacinone-Na, each 1mg of OVA, be dissolved in respectively in the PBS damping fluid of pH7.4,0.01M, the identical PBS damping fluid of take is blank, does respectively spectral scan.Ultraviolet scanning spectrum figure is shown in Fig. 3.Result shows, three's absorption peak does not all overlap, coupling success.
The structural formula of immunogen and coating antigen is as follows:
The acquisition of embodiment 2, hybridoma and preservation
One, the preparation of splenocyte
1, get 8 6-8 Balb/c female mice in age in week, by following program immunity:
Within the 0th day, carry out initial immunity, every following material of the subcutaneous multi-point injection of mouse back: the immunogen solution of embodiment 1 preparation and physiological saline are mixed, obtain mixed solution, 100 μ L mixed solutions (are contained to 100 μ g immunogens, in protein content) mix emulsification with 100 μ L complete Freund's adjuvants;
Within the 14th day, the 28th day, the 42nd day and the 56th day, respectively carry out one time booster immunization, each every following material of the subcutaneous multi-point injection of mouse back: the immunogen solution of embodiment 1 preparation and physiological saline are mixed, obtain mixed solution, 100 μ L mixed solutions (are contained to 50 μ g immunogens, in protein content) mix emulsification with 100 μ L incomplete Freund's adjuvants;
The 63rd day, eye socket venous blood collection, centrifuging and taking serum, adopts indirect elisa method to detect serum titer, chooses the mouse that serum titer is high.
The 70th day, every mouse peritoneal was injected following material: the immunogen solution of embodiment 1 preparation and physiological saline are mixed, obtain 500 μ L mixed solutions (containing 150 μ g immunogens, in protein content).
2, get the highest mouse of serum titer in step 1, extract eyeball, lethal after collection blood, in 75% alcohol, after sterilization, get spleen, remove reticular tissue, prepare splenocyte suspension, transfer in 50mL centrifuge tube, add the incomplete nutrient solution of DMEM to 30mL, the centrifugal supernatant of abandoning, 30mL DMEM liquid again pressure-vaccum for lower floor, until there is not tissue on glass dropper, finally add the incomplete nutrient solution 30mL of DMEM, counting, gets 1 * 10
8individual splenocyte is stand-by.
Two, the preparation of feeder cell
A Balb/c female mice that does not carry out any immunity is extractd to eyeball, bloodletting is lethal, be soaked in 75% alcohol 3min, in Bechtop, cut off skin of abdomen, expose peritonaeum, after alcohol disinfecting, inject 5mL containing the DMEM perfect medium of HAT, rub gently the abdominal cavity 3min of mouse thigh root, liquid in abdominal cavity is once drawn back in suction afterwards repeatedly, and inject the plate contain 30mL HAT perfect medium, after mixing with volley of rifle fire pressure-vaccum, add in (100 μ L/ hole) in 96 porocyte plates, be placed in 37 ℃ of CO
2incubator is cultivated.
Three, myeloma cell's preparation.
Get (viable count >95%) the SP2/0 myeloma cell that growth conditions is good, in Bechtop, with connector bend dropping tube, it is blown down completely, transfer in 50mL centrifuge tube, add DMEM nutrient solution to the centrifugal supernatant of abandoning of 20mL, with counting after the dilution of 30mL DMEM nutrient solution, get 2 * 10
7individual cell is stand-by.
Four, cytogamy
By quantity than splenocyte: myeloma cell=5:1 mixes two kinds of cells, add the incomplete nutrient solution of DMEM to 30mL, the centrifugal 5min of 1000rpm, abandon supernatant, sedimentation cell bullet becomes pasty state, put 37 ℃ of water-bath uniform rotation test tube preheating 1min, in 1min, add the polyoxyethylene glycol fusogen of 37 ℃ of preheatings, stir gently afterwards 30s, standing 30s, adds 1 dropper, 2min2 dropper, 3min3 dropper, the incomplete nutrient solution of 4min4 dropper DMEM in back to back 1min, finally with DMEM, is settled to 35mL, the centrifugal 7min of 800rpm, abandons supernatant.
Five, cell cultures
Gently that the cell bullet after merging is even, add 55-60mL HAT nutrient solution, by cell gently pressure-vaccum mix, join in preprepared feeder cell plate 37 ℃, CO
2incubator is cultivated, is observed; Generally in 5-6 days, change liquid, according to cell mass, be less than greatly and merge the rear ELISA detection of carrying out for 7-10 days.
Six, the screening of hybridoma.
After fusion, when hybridoma colony grows into certain size, nutrient solution starts flavescence, starts to carry out the screening of hybridoma.Generally all cells hole was detected in 7-10 days after merging, first with the positive hole of indirect elisa method preliminary screening, then screens for positive hole by indirect competitive ELISA method, obtains the cell hole of secreting specificity antibody.
Seven, the cloning of hybridoma is cultivated.
Hybridoma in the positive hole of energy secreting specificity antibody is made to suspension, with limiting dilution assay, it is cloned, size according to cell colony, in clone after within about the 8th day, detect antibody, select the hole of specific secretion monoclonal antibody, clone, until continuous 3 times reach 100% for mono-clonal hole and antibody positive rate.To meeting the clone that above requirement and Growth of Cells are good, carry out enlarged culturing, building is to preserve.
Eight, the preservation of hybridoma
By the hybridoma called after DPC of a strain stably excreting monoclonal antibody.Diphacinone monoclonal antibody hybridoma cell strain DPC is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on March 13rd, 2014 and (is called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.8953.
The preparation of embodiment 3, monoclonal antibody
One, cell culture method
The preparation method of cell culture medium (7.4): add calf serum and sodium bicarbonate in RPMI-1640 substratum, the final concentration of calf serum is 20% (quality percentage composition), and the final concentration of sodium bicarbonate is 0.2% (quality percentage composition).
Diphacinone monoclonal antibody hybridoma cell strain DPC is placed in to cell culture medium, cultivates 2 days for 37 ℃, adopt sad-saturated ammonium sulphate method that the cell culture supernatant obtaining is carried out to purifying, obtain monoclonal antibody solution (20 ℃ of preservations).
Protein concn in monoclonal antibody solution (mg/ml)=1.45 * OD
280-0.74 * OD
260.
Protein concn=6.8mg/ml in monoclonal antibody solution.
Two, ascites preparation method
Get the male young mouse of Balb/c, every injection 500 μ L whiteruss sensitization, every mouse peritoneal injection 0.5mL cell suspension (1-2 * 10
6individual diphacinone monoclonal antibody hybridoma cell strain DPC/mL, with the incomplete nutrient solution of DMEM, adjust concentration), inactive and the abdominal cavity enlargement of visible mouse state after one week, now with asepsis injector, in mouse abdominal cavity, gather ascites (gather once every 1-2d, so repeatedly gather until mouse natural death), the centrifugal 10min of 4000rpm, remove upper strata fat and lower sediment thing, collect middle level, get sample segment mensuration and tire, rest part is frozen standby.
Embodiment 4, detect the tiring of monoclonal antibody, IC
50value and specificity
One, indirect elisa method is measured tiring of monoclonal antibody
Adopt the coating antigen solution of embodiment 1 preparation to be coated with, coated concentration is 1.5 μ g/mL (in protein content).Monoclonal antibody solution prepared by the step 1 of the phosphate buffered saline buffer gradient dilution embodiment 3 of employing pH7.4,0.01M (adopting the serum of the Balb/c female mice that does not carry out any immunity as the negative control of monoclonal antibody solution diluent).Adopt the sheep anti-mouse igg antibody of horseradish peroxidase-labeled as ELIAS secondary antibody.In microplate reader, in 450nm, measure each hole OD value, what the maximum dilution multiple of P/N value >=2.1 was this monoclonal antibody tires that (P represents the absorbance of test holes; N represents the absorbance of negative control hole).
Tiring as 1:500000 of monoclonal antibody solution prepared by the step 1 of embodiment 3.
Two, indirect competitive ELISA method is measured IC
50value
1, get the coating antigen solution of embodiment 1 preparation, carbonate buffer solution dilution with pH9.6,0.05M, obtaining coating antigen concentration is the coating buffer of 1.5 μ g/mL (in protein content), coating buffer is added to enzyme plate, 100 μ L/ holes, hatch 2h for 37 ℃, then abandon supernatant, with PBST solution washing one time, pat dry.
2, add confining liquid (containing the PBS damping fluid of 0.25% casein, 5% calf serum), 150 μ L/ holes, hatch 1h for 37 ℃, abandon supernatant, pat dry.
3, test holes adds 50 μ L diphacinone-Na solution (solvent is the phosphate buffered saline buffer of pH7.4,0.01M, and the concentration of diphacinone-Na is followed successively by 0.014ng/mL, 0.042ng/mL, 0.125ng/mL, 0.25ng/mL, 0.5ng/mL, 1ng/mL, 2ng/mL, 4ng/mL, 8ng/mL or 24ng/mL) and 50 μ L monoclonal antibody diluents (monoclonal antibody solution that the phosphate buffered saline buffer of employing pH7.4,0.01M obtains the step 1 of embodiment 3 is diluted to 100000 times of volumes); The negative control hole that replaces 50 μ L diphacinone-Na solution with the phosphate buffered saline buffer of 50 μ L pH7.4,0.01M is set; Hatch 1h for 4 ℃, abandon supernatant, use PBST solution washing 4 times, pat dry.
4, the sheep anti-mouse igg antibody (2000 times of dilutions) that adds horseradish peroxidase-labeled, 100 μ L/ holes, hatch 30min for 37 ℃, abandon supernatant, use PBST solution washing 4 times, pat dry.
5, add DAB nitrite ion (100 μ L/ hole), hatch 15min for 37 ℃, then add 2M sulphuric acid soln (50 μ L/ hole), then in microplate reader, in 450nm, measure each hole OD value.
The OD value in each hole the results are shown in Table 1.
The OD value result in each hole of table 1
Adopt Origin8.5 mapping, the sodium diphacinone concentration denary logarithm of take is X-coordinate, take OD value as ordinate zou, and curve plotting, is shown in Fig. 4.
Inhibiting rate (%)=[(B
0-B)/B
0] * 100%.The concentration of diphacinone-Na when inhibiting rate is 50% is the IC of this monoclonal antibody
50value.IC
50＝0.808ng/mL。
Three, specificity identification.
With the analog (bromadiolone, coumatetralyl and pindone) of diphacinone, replace diphacinone-Na, other same step 2.
Cross reacting rate (%)=IC
50(diphacinone)/IC
50(analog) * 100%
Cross reacting rate the results are shown in Table 2.The cross reacting rate of analog is less than 0.074%, illustrates that the monoclonal antibody specificity that utilizes the present invention to prepare is good.
The IC of table 2 analog
50value and cross reacting rate
| Title | IC 50 | Cross reacting rate |
| Bromadiolone | Be greater than 1 μ g | Be less than 0.074% |
| Coumatetralyl | Be greater than 1 μ g | Be less than 0.074% |
| Pindone | Be greater than 1 μ g | Be less than 0.074% |
Claims (8)
1. diphacinone monoclonal antibody hybridoma cell strain DPC, its deposit number is CGMCC No.8953.
2. the monoclonal antibody that described in claim 1, hybridoma cell strain is secreted.
3. the application of monoclonal antibody in the test kit for the preparation of detection diphacinone or diphacinone derivative described in hybridoma cell strain DPC or claim 2 described in claim 1.
4. the application of monoclonal antibody in detecting diphacinone or diphacinone derivative described in claim 2.
5. compound shown in formula I;
6. detect a test kit for diphacinone or diphacinone derivative, comprise described in claim 1 monoclonal antibody described in hybridoma cell strain DPC or claim 2.
7. test kit as claimed in claim 6, is characterized in that: described test kit also comprises compound described in claim 5.
8. take the antibody that compound obtains as immunogen described in claim 5.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109897025A (en) * | 2019-02-28 | 2019-06-18 | 中国农业大学 | Anticoagulation raticide haptens and artificial antigen and the preparation method and application thereof |
| CN111443202A (en) * | 2020-04-13 | 2020-07-24 | 北京维德维康生物技术有限公司 | Enzyme linked immunosorbent assay kit for detecting anticoagulant rodenticide and preparation and application thereof |
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| CN102297891A (en) * | 2010-06-23 | 2011-12-28 | 中国科学院大连化学物理研究所 | Method for rapidly detecting diphacinone or diphacinone sodium salt in beverage |
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| CN101865845A (en) * | 2010-06-11 | 2010-10-20 | 宁波市疾病预防控制中心 | Simple fluorescence analysis method for determining diphacinone and chlorophacinone |
| CN102297891A (en) * | 2010-06-23 | 2011-12-28 | 中国科学院大连化学物理研究所 | Method for rapidly detecting diphacinone or diphacinone sodium salt in beverage |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109897025A (en) * | 2019-02-28 | 2019-06-18 | 中国农业大学 | Anticoagulation raticide haptens and artificial antigen and the preparation method and application thereof |
| CN111443202A (en) * | 2020-04-13 | 2020-07-24 | 北京维德维康生物技术有限公司 | Enzyme linked immunosorbent assay kit for detecting anticoagulant rodenticide and preparation and application thereof |
| CN111443202B (en) * | 2020-04-13 | 2024-04-02 | 北京维德维康生物技术有限公司 | ELISA kit for detecting anticoagulant rodenticide, preparation and application thereof |
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