CN101105492B - Monoclonal antibody and enzyme-linked immunoassay method and reagent kit for detecting tylosin and tilmicosin residue - Google Patents
Monoclonal antibody and enzyme-linked immunoassay method and reagent kit for detecting tylosin and tilmicosin residue Download PDFInfo
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- CN101105492B CN101105492B CN2007100526638A CN200710052663A CN101105492B CN 101105492 B CN101105492 B CN 101105492B CN 2007100526638 A CN2007100526638 A CN 2007100526638A CN 200710052663 A CN200710052663 A CN 200710052663A CN 101105492 B CN101105492 B CN 101105492B
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Abstract
The invention belongs to the veterinary medicine residual analysis and immunity analysis technique field and specifically relates to an enzyme-linked immunity method and the reagent kit thereof which can discern specificity monoclonal antibodies of tylosin and tilmicosin and detect the residuals of tylosin and tilmicosin at the same time; the monoclonal antibodies in the invention is secreted from hybridoma cell strain P3C4 established by the applicant; the hybridoma cell strain is preserved in China Center of Type Culture Collection; the number of preservation of the hybridoma cell strain is CCTCC No: C200719; the invention discloses the preparation method and enzyme-linked immunity detection method of the monoclonal antibody, coating antigen and immunogen. Compared with the prior art, the monoclonal antibody prepared in the invention can discern tylosin and tilmicosin at the same time and add the detecting object in the prior art. The reagent kit and the method in the invention has the advantages of simple, convenience, swiftness, sensitivity and accuracy; furthermore, the reagent kit and method in the invention can detect the residuals of tylosin and tilmicosin in an animal edibility tissue at the same time.
Description
Technical field
The invention belongs to residue of veterinary drug analysis and immunological technique field.Be specifically related to a kind ofly can discern the MONOCLONAL ANTIBODIES SPECIFIC FOR of tylosin and Tilmicosin simultaneously, detect the development of the foundation of tylosin and the how residual enzyme-linked immune analysis method of Tilmicosin and kit simultaneously.
Background technology
Tylosin and Tilmicosin belong to macrolides animal specific microbiotic, are widely used in pneumonia, mammitis, general septicemia that the treatment gram-positive bacteria causes on veterinary clinic, especially for the treatment livestock and poultry mycoplasma infection.Tylosin also is widely used as feed addictive in addition.
Residual in animal tissue of tylosin and Tilmicosin can threaten food security, and human health is worked the mischief.Tylosin and Tilmicosin easily induce bacterium to produce drug resistance, and drug resistance can shift between bacterium.Given this, European Union forbade in 1999 tylosin as feed addictive (Council Regulation2821/98,1998), No. 235, The Ministry of Agriculture of the People's Republic of China, MOA bulletin " animal food herbal medicine maximum residue limit(MRL) " to tylosin and Tilmicosin the residual maximum residue limit(MRL) of also having formulated in animal food.
The method of existing detection tylosin and tilmicosin residue amount mainly comprises microbial process, instrument analytical method and immunochemical analyses method etc.Though microbial method shows good performance on residual screening high flux, detect length consuming time, lack specificity, and used bacterium there are differences to different types of antibacterials susceptibility, easily cause false negative and false positive to produce.The sample pre-treatments complexity of instrument analytical method, the cost height, complex operation is applicable to the conclusive evidence analysis of sample, and is not suitable for the screening and on-the-spot detection of batch samples.Immunochemical analyses method particularly Enzyme Linked Immunoadsorbent Assay (ELISA) technology has advantages such as quick, highly sensitive, simple to operate, that adaptability is strong, is fit to the screening of high flux sample.Therefore for residual in animal tissue of fast detecting tylosin and Tilmicosin, the ELISA method has more advantage.
For the foundation of micromolecular compound ELISA detection method, the Antibody Preparation of micromolecular compound is the core, and the appropriate design haptens is crucial.The tiny difference of haptenic chemical constitution is connected to the difference in the chemical site on the carrier protein, and the difference of crosslinked arm chemical property all is the deciding factors that influence micromolecular compound antibody character.Therefore micromolecular compound is carried out the chemical constitution transformation, the appropriate design haptens from different sites and adopt different crosslinked arms that haptens is connected on the carrier protein, has constituted the core of such invention.
The applicant retrieves the patent documentation relevant with theme of the present invention two pieces, first U.S. publication, the patent No. are 6,506,885, the name of invention is called Monoclonal antibodies to the drug tilmicosin and a method for detecting the same.It two is the Chinese invention patent prospectus, and application number is 200610007286.1, and the name of invention is called: a kind of method and special ELISA reagent kit thereof that detects tylosin, above-mentioned two patent documentations all only relate to the detection to single medicine.The patent No. is 6,506,885 U.S. Patent Publication a kind of derivant with Tilmicosin has obtained a kind of monoclonal antibody of special identification Tilmicosin as the immunogene of haptens preparation.It is any about its creationary biologic test data that the document does not provide, so that those skilled in the art is difficult to reach this and invents described effect.Application number is 200610007286.1 Chinese invention patent applications, disclose tylosin and p-aminobenzoic acid have been synthesized the tylosin haptens by condensation reaction, adopted the mixed anhydride method coupling to prepare immunogene tylosin haptens and ovalbumin then.Prepared monoclonal antibody only can be discerned tylosin, this application does not specifically disclose the core technology of its invention simultaneously, promptly as the main core reagent of kit such as the detailed preparation process of immunogene and coating antigen, make those skilled in the art be difficult to carry out this invention and reach the alleged effect of this invention.
Summary of the invention
The objective of the invention is to overcome the defective that prior art exists, prepare a kind of monoclonal antibody that can discern tylosin and Tilmicosin simultaneously.
Second purpose of the present invention is to utilize this monoclonal antibody, sets up a kind of ELISA method that can detect tylosin and tilmicosin residue simultaneously.
The 3rd purpose of the present invention is the application of this monoclonal antibody in preparation tylosin and tilmicosin residue detection kit.
The 4th purpose of the present invention is the application of kit in tylosin and tilmicosin residue detection that the present invention prepares.
The present invention is achieved through the following technical solutions:
In order to realize task of the present invention, the inventor has prepared a kind of monoclonal antibody that can discern tylosin and Tilmicosin simultaneously, and it is to be that the hybridoma cell strain P3C4 of CCTCC NO:C200719 is secreted by preserving number.
Above-mentioned hybridoma cell strain P3C4 is deposited in Chinese typical culture collection center, and its preserving number is CCTCC NO:C200719.
Used immunogene be by haptens go the mycaminose tylosin (Desmycosin, DES) with to hydrazino-benzoic acid or adipyl dihydrazide or O-ethyloic azanol again with the compound preparation of bovine serum albumin(BSA) coupling.
Further, the invention provides a kind of enzyme linked immunological (ELISA) method that detects tylosin and tilmicosin residue simultaneously, this method comprises the step of the pre-treatment of the preparation of immunogene, coating antigen and antibody and sample, and it also comprises following concrete steps:
(1) haptens is removed the mycaminose tylosin and hydrazino-benzoic acid or adipyl dihydrazide or O-ethyloic azanol are obtained immunogene with the bovine serum albumin(BSA) coupling again;
(2) haptens is removed the mycaminose tylosin and hydrazino-benzoic acid or adipyl dihydrazide or O-ethyloic azanol are obtained coating antigen with the ovalbumin coupling again;
(3) obtaining preserving number with the immunogen preparing of step (1) is the secreted monoclonal antibody of CCTCC NO:C200719 hybridoma cell strain P3C4;
(4) use the coating antigen bag of step (2) by solid phase carrier (for example ELISA Plate);
(5) the testing sample percent by volume is dissolved again for the 20-30% acetonitrile solution extracts (being applicable to from the sample preparation of animal's liver and animal tissue), ethyl acetate extraction, nitrogen dries up with sample diluting liquid obtain determinand;
(6) determinand to step (5) detects.
Wherein: the component and the proportioning of the sample diluting liquid in the step (5) are: NaCl8.0g, KH
2PO
40.2g, Na
2HPO
412H
2O2.9g, KCl0.2g, thimerosal 0.01g adds distilled water to 1000mL.
The inventor dresses up the ELISA kit that can detect tylosin and Tilmicosin simultaneously with said monoclonal antibody and coating antigen as core reagent and other conventional reagent set, the ELISA method is verified, realize task of the present invention, thereby finished the present invention.
More detailed technical scheme is as described in the embodiment.
Major advantage of the present invention is:
1, the monoclonal anti physical efficiency of the present invention's preparation is discerned tylosin and Tilmicosin simultaneously, and the antibody of existing patent documentation only can single identification tylosin or Tilmicosin.
2, the present invention adopts and goes the mycaminose tylosin as haptens when Antibody Preparation, and this haptens has kept tylosin and the common chemical constitution of Tilmicosin, can discern tylosin and Tilmicosin simultaneously by the antibody of this haptens preparation.
3, the ELISA method and the kit of the present invention's foundation can detect tylosin and Tilmicosin simultaneously, once measure and to detect residual in edibility animal tissue of tylosin and Tilmicosin, and 200610007286.1 patented claims only can detect tylosin, for residual can't the resolution whether of Tilmicosin in the tissue, need to adopt other method to detect, therefore ELISA kit of the present invention has tangible improvement on the kind of detection of drugs, can save analysis times, have better economic worth sample.
4, the suitable detection tissue of kit of the present invention comprises animal muscle and liver, the tissue sample disposal route that the present invention relates to is simple, easy to operate, the used main organic reagent of sample preparation is acetonitrile and ethyl acetate, with respect to the methenyl choloride that 200610007286.1 patented claim sample preparation are adopted, less to the healthy harm of operator.
Description of drawings
Fig. 1 is technology path figure of the present invention.
Fig. 2 is the immunogene UV scanning collection of illustrative plates of one of them embodiment of the present invention, shown as haptens go the mycaminose tylosin (Desmycosin, DES), bovine serum albumin(BSA) (BSA) and remove mycaminose tylosin-to the characteristic ultraviolet absorption of hydrazino-benzoic acid-bovine serum albumin(BSA) (DES-HBA-BSA) conjugate.
Fig. 3 has shown the characteristic ultraviolet absorption that removes mycaminose tylosin (DES), bovine serum albumin(BSA) (BSA) and remove mycaminose tylosin-adipyl dihydrazide-bovine serum albumin(BSA) (DES-ADH-BSA) conjugate as haptenic for the immunogene UV scanning collection of illustrative plates of one of them embodiment of the present invention.
Fig. 4 has shown the characteristic ultraviolet absorption that removes mycaminose tylosin (DES), bovine serum albumin(BSA) (BSA) and remove mycaminose tylosin-O-ethyloic azanol-bovine serum albumin(BSA) (DES-AOAA-BSA) conjugate as haptenic for the immunogene UV scanning collection of illustrative plates of one of them embodiment of the present invention.
Fig. 5 is the indirect competitive ELISA response curve of monoclonal antibody of the present invention and tylosin standard items, and X-axis is tylosin (TYL) concentration of standard solution logarithm value, and Y-axis is that the optical density value of tylosin standard solution is divided by " zero " hole optical density value (B/B
0).
Fig. 6 is the indirect competitive ELISA response curve of monoclonal antibody of the present invention and Tilmicosin standard items, and X-axis is Tilmicosin (TIL) concentration of standard solution logarithm value, and Y-axis is that the optical density value of Tilmicosin standard solution is divided by " zero " hole optical density value (B/B
0).
Embodiment
The invention will be further described below by embodiment, but do not limit the present invention.
The preparation of embodiment 1 immunogene and coating antigen
1.1 haptens goes the synthetic of mycaminose tylosin (DES)
Take by weighing Tylosin Tartrate 10g, be dissolved in the 200mL sulfuric acid solution (pH2) backflow 2h.Reaction finishes the back with saturated sodium carbonate solution adjusting pH to 8.8, changes in the separating funnel, adds methylene chloride 60mL extraction, repeats above the operation 1 time.The combined dichloromethane layer, standing over night after the adding anhydrous sodium sulfate 5g jolting.After the filtration, get 60 ℃ of decompression rotations of dichloromethane layer evaporate to dryness, obtain product and remove the mycaminose tylosin.
1.2 remove mycaminose tylosin-to the preparation of hydrazino-benzoic acid-bovine serum albumin(BSA)/ovalbumin conjugate (DES-HBA-BSA/OVA)
Take by weighing mycaminose tylosin (DES) 770mg, hydrazino-benzoic acid (HBA) 150mg is joined in the 12mL absolute ethyl alcohol, room temperature lower magnetic force stirring reaction 5h.60 ℃ of decompression rotation evaporates to dryness obtain product D ES-HBA.Take by weighing DES-HBA46.2mg, N, N-dicyclohexylcarbodiimide (DCC) 12.4mg, N-hydroxy-succinamide (NHS) 6.9mg are dissolved in 2mL N, and in the dinethylformamide (DMF), room temperature lower magnetic force stirring reaction 12h is called A liquid after reaction finishes.Take by weighing bovine serum albumin(BSA) (BSA) 165.5mg, be dissolved in the 18mL0.1mol/L phosphate buffer (take by weighing sodium hydrogen phosphate 13.45g, sodium dihydrogen phosphate 0.64g is settled to 1000mL), be called B liquid.A liquid is dropwise joined in the B liquid, and the limit edged stirs, and puts magnetic agitation reaction 10h in the ice-water bath.Get supernatant after centrifugal, the 3d that dialyses in 4 ℃ of physiological saline changes dislysate every day 2 times, promptly gets conjugate DES-HBA-BSA.Get the supernatant freeze drying after centrifugal, put-20 ℃ of preservations, use as immunogene.
In above-mentioned steps, change BSA into OVA, promptly get conjugate DES-HBA-OVA, this conjugate is used as coating antigen.
1.3 go the preparation of mycaminose tylosin-adipyl dihydrazide-bovine serum albumin(BSA)/ovalbumin conjugate (DES-ADH-BSA/OVA)
Take by weighing adipyl dihydrazide (ADH) 334mg, bovine serum albumin(BSA) (BSA) 80mg and 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC) 80mg and be dissolved in the 16mLMES damping fluid [2-of 0.1mol/L (N-morphine quinoline) ethyl sulfonic acid], room temperature magnetic agitation reaction 1.5h.Get supernatant after 2000rpm is centrifugal, the 3d that dialyses in 4 ℃ of physiological saline changes dislysate every day 2 times.2000rpm is centrifugal in the dialysis back, gets supernatant, adds DMF2mL and removes mycaminose tylosin (DES) 8mg, reacts 12h in the ice-water bath.After reaction finishes,, change dislysate every day 2 times, promptly get conjugate DES-ADH-BSA with 4 ℃ of normal saline dialysis 3d.Get the supernatant freeze drying after centrifugal, put-20 ℃ of preservations, use as immunogene.
In above-mentioned steps, change bovine serum albumin(BSA) into ovalbumin (OVA), promptly get conjugate DES-ADH-OVA, this conjugate is used as coating antigen.
1.4 go the preparation of mycaminose tylosin-O-ethyloic azanol-bovine serum albumin(BSA)/ovalbumin conjugate (DES-AOAA-BSA/OVA)
Take by weighing mycaminose tylosin (DES) 150mg and be dissolved in 4mL methyl alcohol, be called A liquid.Take by weighing O-ethyloic azanol half hydrochloride (AOAA) 22mg, sodium bicarbonate 8.4mg and be dissolved in the 4mL water, be called B liquid.B liquid is dropwise joined A liquid, room temperature magnetic agitation reaction 2.5h.After reaction finished, 60 ℃ of decompression rotation evaporates to dryness obtained product DES-AOAA.
Take by weighing DES-AOAA44mg, DCC12.4mg, NHS6.9mg is dissolved among the 3mL DMF, room temperature lower magnetic force stirring reaction 12h, reaction is called A liquid after finishing.Take by weighing BSA110mg, be dissolved in the 17mL0.1mol/L sodium phosphate buffer (take by weighing sodium hydrogen phosphate 13.45g, sodium dihydrogen phosphate 0.64g is settled to 1000mL), be called B liquid.A liquid is dropwise joined in the B liquid, and the limit edged stirs, and puts room temperature lower magnetic force stirring reaction 12h.Get supernatant after centrifugal, the 3d that dialyses in 4 ℃ of physiological saline changes dislysate every day 2 times, promptly gets conjugate DES-AOAA-BSA.Get the supernatant freeze drying after 2000rpm is centrifugal, put-20 ℃ of preservations, use for immunity.
In above-mentioned steps, change BSA into OVA, promptly get conjugate DES-AOAA-OVA, this conjugate is used as coating antigen.
2.1 anti-tylosin and Tilmicosin MONOCLONAL ANTIBODIES SPECIFIC FOR:
With reference to the method in Xue Qingshan " philosophy and technique of in vitro culture " the Science Press calendar year 2001 version: utilize the inventor place animal doctor's pharmacology prepared in laboratory go mycaminose tylosin-O-ethyloic azanol-bovine serum albumin(BSA) (DES-AOAA-BSA) conjugate immunity Balb/C mouse (available from Hubei Prov. Academy of Medical Sciences's Experimental Animal Center).Immune programme for children is after getting the protein solution and isopyknic Freund's complete adjuvant (available from sigma company) emulsification that contains DES-AOAA-BSA conjugate 50～100 μ g, in the subcutaneous multi-point injection of mouse back.2 weeks of later every interval are strengthened once, use Freund (available from sigma company) emulsification instead.At last in merging first three day (preferably and immunity last time be separated by more than 4 weeks), lumbar injection, reinforced immunological, the antigen amount doubles, and does not add adjuvant.During fusion, one of the Balb/C mouse of the last reinforced immunological of learning from else's experience, eye socket sacrificed by exsanguination (collect serum, be positive serum) is soaked the 5min sterilization in 75% alcohol.Aseptic taking-up mouse spleen is isolated splenocyte, with SP2/0 myeloma cell's (SP2/0 myeloma cell is available from Ministry of Public Health's Wuhan institute of Biological Products) of prepared fresh by 1～2 * 10
7Individual SP2/0 and 10
8Individual immunocyte (ratio mixing in the 50mL centrifuge tube of 1:10～1:15), 1500r/m, centrifugal 10min.Evacuation supernatant (filter paper of available sterilization blots) knocks the pipe end gently, makes cell precipitation loosening slightly.The centrifuge tube that cell mixture is housed is put in 37 ℃ of water-baths.Slowly splash into 50% polyglycol (PEG) 0.8mL (available from sigma company product) of pre-temperature to 37 ℃ then in 1min, the limit edged stirs with pipette tip gently, continues to stir 1min.1640 (available from the commercial nutrient culture media of Hyclone company) the basal liquid 10mL that slowly adds 37 ℃ of pre-temperature then.Concrete grammar is; Dropwise splashed into 1mL in first minute, added 1mL in second minute, added 3mL on the 3rd～4 minute, added remaining 5mL on the 5th minute, each added-time needs slowly to add, and constantly stirs lightly.Add 30mL1640 liquid at last, also need slowly to add.The centrifugal 5min of 800r/m removes supernatant, places 5～8min in 37 ℃.Suspend with HAT (available from Sigma company) nutrient culture media, simultaneously also with the HAT nutrient culture media suspend the raising splenocyte for preparing and with merge after mixing with cells, add an amount of HAT nutrient culture media as required, divide and plant in 96 well culture plates, about 250 μ L/ holes.Once merge and to inoculate 4～8 96 orifice plates.Also can plant less as required, generally press the cell number of SP2/0 and calculate, every hole inoculum concentration contains 10 approximately
4About SP2/0 cell.In 37 ℃, 5%CO
2Cultivate in the incubator.Merging to begin in back second day to observe had pollution-freely, added 1 HAT nutrient culture media in the 4th day, and suction in the 8th～10 day goes 100 μ L nutrient culture media to change HT (available from sigma company) nutrient culture media 100 μ L.Treat that the fused cell colony grows to culture hole 1/4, when nutrient culture media omits flavescence, carry out antibody test.Adopt the inventor place animal doctor's pharmacology prepared in laboratory go mycaminose tylosin-O-ethyloic azanol-ovalbumin as screening antigen, utilize the ELISA method to filter out the positive hole of the anti-tylosin antibody of secretion.Use limiting dilution assay (with reference to Xue Qingshan " philosophy and technique of in vitro culture " Science Press calendar year 2001 version) to clone, screen at once to the positive hole that screens.Through 3～4 time clonings, finishing screen is selected the monoclonal hybridoma strain of the anti-tylosin antibody of secretion.To this monoclonal hybridoma strain that filters out, the applicant is its called after P3C4, and send Chinese typical culture collection center (CCTCC) preservation on March 28th, 2007, and deposit number is CCTCC NO:C200719.This clone has been carried out chromosome counting, and the result shows that the chromosomal average of SP2/0 is 58, and splenocyte chromosome is 40, and the chromosome number of hybridoma all is higher than the chromosome number of two parent's cells between 90～95.The chromosome number of hybridoma illustrates the cell of SP2/0 really of fused cell and the hybridization product of splenocyte obviously more than the chromosome of myeloma cell SP2/0.With this cell line through lumbar injection Balb/C mouse, manufacture order clonal antibody.Employing is carried out the hypotype evaluation available from the mouse source monoclonal antibody hypotype identification kit (Mouse Mab Isotyping Test Kit) of ROCKLAND company to the resulting monoclonal antibody of the present invention, and the result is a mouse IgG2a hypotype.
2.2 Purification of Monoclonal Antibodies
According to literature method (Zhu Liping, Chen Xueqing. immunology common experimental method. Beijing: People's Medical Officer Press, 2000), adopt sad-ammonium sulfate salting-out process monoclonal antibody purification.Step is as follows: (get sodium acetate 3.44g, glacial acetic acid 1.03mL constant volume in 1000mL, pH5.0) 4mL is with the salt acid for adjusting pH value to 4.5 of 0.1mol/L to add the 0.06mol/L acetate buffer in 2mL ascites.Stirring at room, and in 30min, dropwise add sad 66 μ L.4 ℃ leave standstill 2h, and the centrifugal 30min of 15000r/min gets supernatant, add the 0.1mol/L phosphate buffer and (get Na
2HPO
411.50g, NaH
2PO
42.28g, NaCl8.5g is settled to 1000mL, pH7.4) 0.6mL.Add the ammonium sulfate of 0.227g/mL under ice bath, make into 45% saturation degree in 30min, leave standstill more than the 1h, 4 ℃ of centrifugal 30min of following 12000r/min abandon supernatant, sediment is dissolved in an amount of PBS (gets Na
2HPO
42.2g, NaH
2PO
40.2g, NaCl8.5g is settled to 1000mL, pH7.4) in, and with PBS 4 ℃ of dialysis.The back 4 ℃ of centrifugal 30min of following 12000r/min that dialyse abandon precipitation, and supernatant is the monoclonal antibody of purifying.After the monoclonal antibody packing behind the purifying, put-20 ℃ of preservations.
The foundation of embodiment 3 tylosin racing ELISA detecting methods
3.1 the preparation of reagent (reagent that present embodiment uses all adopts following method preparation except that other indicates)
Phosphate buffer: NaCl8.0g, KH
2PO
40.2g, Na
2HPO
412H
2O2.9g, KCl0.2g adds distilled water to 1000mL, regulates pH to 7.4;
Coating buffer: get Na
2CO
31.5g, NaHCO
32.9g, add tri-distilled water to 1000mL, regulate pH value to 9.6;
Cleansing solution: NaCl8.0g, KH
2PO
40.2g, Na
2HPO
412H
2O2.9g, KCl0.2g, Tween200.5mL, thimerosal 0.1g adds distilled water to 1000mL, regulates pH to 7.4;
Confining liquid: ovalbumin 0.1g is dissolved in the 100mL phosphate buffer;
Substrate solution A:3,3 ', 5 ', 5-tetramethyl biphenyl diamines (TMB) 200mg, absolute ethyl alcohol 100mL adds distilled water to 1000mL;
Substrate solution B:Na
2HPO
414.6g, citric acid 9.3g, 0.75% carbamide peroxide 6.4mL adds distilled water to 1000mL;
The substrate mixed liquor: with A liquid and B liquid by volume 1:1 mix promptly, now with the current;
Stop buffer: 2mol/L sulfuric acid solution.
3.2 the preliminary of coating antigen concentration and antibody working concentration determined
Select above-mentionedly synthetic to go mycaminose tylosin-O-ethyloic azanol-ovalbumin (DES-AOAA-OVA) as coating antigen, be diluted to 4mg/L, 2mg/L, 1mg/L, 0.5mg/L, 0.25mg/L, a 0.125mg/L6 concentration with coating buffer, in 96 hole ELISA Plate, from first to the 6th leu adds, and 4 ℃ are spent the night; Wash 3 times, pat dry, add confining liquid 200 μ L, 37 ℃ of sealing 1h; Wash 3 times, pat dry, walking to extension rate that the 8th row adds 100 μ L phosphate buffers dilutions successively in first of ELISA Plate is 4000,8000,16000,32000,64000,128000,256000,512000 monoclonal antibody, hatches 1h for 37 ℃, wash 3 times, pat dry; (be called for short two resists the sheep anti-mouse igg antibody of the horseradish peroxidase-labeled of 1:5000 times of phosphate buffer dilution of each hole adding, the following indication two anti-sheep anti mouse 1gG antibody that are horseradish peroxidase-labeled, available from Wuhan two hawk Bioisystech Co., Ltd) 100 μ L, hatch 1h for 37 ℃, wash 5 times, pat dry; Each hole adds 100 μ L substrate mixed liquors, and lucifuge colour developing 15min adds 50 μ L stop buffers, measures optical density value (OD value) at 450nm wavelength place with automatic microplate reader, the results are shown in Table 1.
The result shows, determines that tentatively the bag of coating antigen DES-AOAA-OVA is 0.5mg/L by concentration, and the antibody working concentration is 1:64000.
The preliminary of table 1 coating antigen concentration and antibody working concentration determined
3.3 determining of best coating antigen concentration and antibody working concentration
With the preliminary coating antigen DES-AOAA-OVA bag of determining by concentration as centre concentration, equal difference is provided with 0.6mg/L, 0.5mg/L, 0.4mg/L, a 0.3mg/L4 concentration coated elisa plate.Tylosin (TYL) is diluted to 320 μ g/L, 160 μ g/L, 80 μ g/L, 40 μ g/L, 20 μ g/L, 10 μ g/L, 5 μ g/L, 0 a μ g/L8 concentration with phosphate buffer, with the monoclonal antibody of 1:32000 phosphate buffer dilution (because when making indirect competitive ELISA, the application of sample volume of monoclonal antibody has reduced half, and correspondingly the monoclonal antibody dilutability reduces half) and above-mentioned tylosin solution respectively add 50 μ L and carry out indirect competitive ELISA.With TYL concentration logarithm value as horizontal ordinate, with the ratio (B/B of OD value Yu " zero " hole OD value of TYL standard solution
0) draw the inhibition curve as ordinate, selection is linear preferable, TYL concentration (IC when generation 50% suppresses
50) junior as the bag by concentration.With best coating antigen concentration coated elisa plate, TYL is diluted to 320 μ g/L, 160 μ g/L, 80 μ g/L, 40 μ g/L, 20 μ g/L, 10 μ g/L, 5 μ g/L, 0 a μ g/L8 concentration, antibody is provided with 4 dilutabilitys with centre concentration 1:32000 equal difference, monoclonal antibody and series concentration TYL standard solution respectively add 50 μ L and carry out indirect competitive ELISA, draw and suppress curve, select linear preferable, IC
50The junior is as the optimum antibody working concentration for value.The results are shown in Table 2.
Determining of best coating antigen concentration of table 2 and antibody working concentration
The result shows, along with the reduction of coating antigen concentration, IC
50Reduce gradually, but consider that coating antigen crosses when low, " zero " hole OD value is on the low side, thus adopt 0.4mg/L as the best bag by concentration.Along with the dilution increase of monoclonal antibody, IC
50Reduce gradually, but when considering that antibody dilution is excessive, " zero " hole OD value is on the low side, so adopts 1:40000 as the optimum antibody working concentration.
3.4 determining of best competition time and two anti-incubation times
TYL is diluted to 0,5,10,20,40,80,160,320 μ g/L with phosphate buffer, and monoclonal anti body and function phosphate buffer of the present invention is pressed the 1:40000 dilution.Fixing two anti-incubation times is 1h, is set is 30,40,50 the competition time, 60min, and indirect competitive ELISA is made in 3 multiple holes of each time point, and the drawing standard curve calculates IC
50Value.After having obtained the best competition time, two anti-incubation times be set be 30,40,50,60min, indirect competitive ELISA is made in 3 multiple holes of each time point, and the drawing standard curve calculates IC
50Value.With " zero " hole OD value and IC
50Value is judged best competition time and two anti-incubation times as investigating index.The results are shown in Table 3.
Determining of best competition time of table 3 and two anti-incubation times
The result shows that along with the increase of competition time, " zero " hole OD value changes little, IC
50Value increases bigger.Behind the antigen-antibody competitive reaction 30min, IC
50Therefore be worth minimumly, select 30min to compete the time as the best.Two anti-hatch 50min, and " zero " hole OD value meets the demands, and IC
50Therefore be worth minimumly, select 50min as best two anti-incubation times.
3.5 the foundation of typical curve
Tylosin is mixed with 0 μ g/L, 5 μ g/L, 10 μ g/L, 20 μ g/L, 40 μ g/L, 80 a μ g/L6 series concentration with phosphate buffer, and each concentration repeats 3 holes, measures replication 5 times according to the indirect competitive ELISA method.Logarithm value with the tylosin solution concentration is a horizontal ordinate, B/B
0For ordinate drawing standard curve, obtain IC
50The IC of this kit
50Value is 19.14 ± 2.4 μ g/L.
3.6 cross reaction test
Macrocyclolactone lactone kind medicine is mixed with debita spissitudo with phosphate buffer, measures the IC of each medicine with the ELISA method of setting up
50Value, the multiple holes of 3 of each medicines are 100% with monoclonal antibody to the cross reacting rate of tylosin, utilize formula 1 to calculate the cross reacting rate of monoclonal antibody to each medicine, the results are shown in Table 4.
Table 4 kit of the present invention is to the cross reacting rate of various Macrocyclolactone lactone kind medicines
The result shows that monoclonal antibody has higher cross reacting rate to tylosin and Tilmicosin, can be used for the foundation of tylosin and tilmicosin residue ELISA detection method in the animal tissue.
The assembling of embodiment 4 tylosins of the present invention and many residue detection of Tilmicosin ELISA kit
4.1 ELISA kit of the present invention is made up of following part:
1) is coated with the solid phase carrier (ELISA Plate) of coating antigen DES-AOAA-OVA;
2) the tylosin standard solution is 6 bottles, and concentration is respectively 0 μ g/L, 5 μ g/L, 10 μ g/L, 20 μ g/L, 40 μ g/L, 80 μ g/L;
3) preserving number is the tylosin monoclonal antibody working fluid of CCTCC NO:C200719;
4) the sheep anti-mouse igg antibody working fluid of horseradish peroxidase (HRP) mark;
5) concentrated phosphoric acid salt buffer: NaCl80.0g, KH
2PO
42.0g, Na
2HPO
412H
2O29.0g, KCl2.0g, thimerosal 0.1g adds distilled water to 1000mL;
6) concentrated cleaning solution: NaCl80.0g, KH
2PO
42.0g, Na
2HPO
412H
2O29.0g, KCl2.0g, Tween205mL, thimerosal 0.1g adds distilled water to 1000mL;
7) substrate solution A:3,3 ', 5 ', 5-tetramethyl biphenyl diamines (TMB) 200mg, absolute ethyl alcohol 100mL adds distilled water to 1000mL;
8) substrate solution B:Na
2HPO
414.6g, citric acid 9.3g, 0.75% hydrogen peroxide urea 6.4mL adds distilled water to 1000mL, regulates pH to 5.0～5.4;
9) stop buffer: 2mol/L sulfuric acid solution.
4.2 the preparation of ELISA Plate
With coating buffer DES-AOAA-OVA is diluted to 0.4mg/L, every hole adds 100 μ L, and 4 ℃ are spent the night, and coating buffer inclines, every hole adds 210 μ L cleansing solutions washing 3 times, pat dry, every then hole adds confining liquid 200 μ L, hatches 1h for 37 ℃, liquid in the hole inclines, cleansing solution washing 3 times, clap in, preserve with masking foil vacuum seal.
The mensuration program of embodiment 5 enzyme linked immunological kits of the present invention
5.1 the preparation of reagent
1) sample diluting liquid: use with after 10 times of the tri-distilled water dilutions according to the concentrated phosphoric acid salt buffer that will provide in the kit.
2) cleansing solution: the cleansing solution that provides in the kit is used with after 10 times of the tri-distilled water dilutions.
3) substrate mixed liquor:,, now with the current with the substrate solution A and the substrate solution B 1:1 mixing by volume of preparation according to each institute expense.
5.2 tissue sample pre-treatment
1, the pre-treatment of pig muscle tissue:
1) take by weighing and organize equal pledge 2.0g in the 50mL centrifuge tube, add 20% acetonitrile solution 10mL, behind the thermal agitation 5min, the centrifugal 5min of room temperature 4800rpm;
2) get supernatant 3mL, add 1mol/L NaOH solution 30 μ L, behind the mixing, add ethyl acetate 5mL, behind the thermal agitation 2min, the centrifugal 5min of room temperature 6000rpm;
3) draw ethyl acetate layer, 45 ℃ of nitrogen dry up, and add sample dilution 1.2mL, and fully the dissolving back is for kit measurement, and this processing is 2 to the dilution factor of tissue sample.
2, the pre-treatment of pig liver tissue:
1) take by weighing and organize equal pledge 1.0g in the 50mL centrifuge tube, add 30% acetonitrile solution 11mL, behind the thermal agitation 5min, 4 ℃ of centrifugal 10min of 10000rpm;
2) get supernatant 3mL in another container, add 1mol/L NaOH solution 30 μ L, behind the mixing, add ethyl acetate 4mL, behind the thermal agitation 2min, the centrifugal 5min of room temperature 6000rpm;
3) draw ethyl acetate layer, 45 ℃ of nitrogen dry up, and add sample dilution 1mL, and fully the dissolving back is for kit measurement, and this processing is 4 to the dilution factor of tissue sample.
5.3 determination step
1) application of sample: add tylosin series concentration standard solution or sample solution 50 μ L in the ELISA Plate micropore, add macrolides monoclonal antibody working fluid 50 μ L then, place wet box, 37 ℃ of constant temperature are hatched 1h;
2) washing: pour out the liquid in the hole, add cleansing solution 210 μ L in every hole and wash 3 times and pat dry;
3) add ELIAS secondary antibody: add ELIAS secondary antibody working fluid 100 μ L in every hole, place wet box, 37 ℃ of constant temperature are hatched 1h;
4) washing: pour out the liquid in the hole, add cleansing solution 210 μ L in every hole and wash 5 times and pat dry;
5) add substrate: add substrate mixed liquor 100 μ L in every hole, place wet box, 37 ℃ of constant temperature are hatched 15min;
6) add stop buffer: add stop buffer 50 μ L in every hole;
7) measure: the optical density value (OD value) of measuring every hole with microplate reader at the 450nm place.
5.4 the result judges
Typical curve:
With the standard items OD value measured divided by " zero " hole OD value (B/B
0) be ordinate, the logarithm value of tylosin concentration is that horizontal ordinate is made typical curve, the line linearity of going forward side by side returns, and provides regression equation.
Tylosin or Tilmicosin concentration are calculated in the muscle:
The inhibiting rate of calculation sample (the OD value of the sample that is obtained is divided by " zero " hole OD value) in the regression equation of substitution typical curve, and multiply by dilution factor 2, calculates TYL concentration (C in the muscle
TYL, μ g/kg), be converted to Tilmicosin concentration (C according to formula 2
TIL, μ g/kg).
Tylosin or Tilmicosin concentration are calculated in the liver:
The inhibiting rate of calculation sample (the OD value of the sample that is obtained is divided by " zero " hole OD value) in the regression equation of substitution typical curve, and multiply by dilution factor 4, calculates tylosin concentration (C in the liver
TYL, μ g/kg), be converted to Tilmicosin concentration (C according to formula 2
TIL, μ g/kg).
Sensitivity, precision, accuracy, the replica test of embodiment 6 kits of the present invention
6.1 the sensitivity test of kit of the present invention
IC with typical curve
50Value and organize the sensitivity index of lowest detectable limit (LOD) as detection kit of the present invention.The tylosin standard items are diluted to 0 μ g/L, 5 μ g/L, 10 μ g/L, 20 μ g/L, 40 μ g/L, 80 a μ g/L6 concentration, and the IC that measures for 5 times according to indirect competitive ELISA method replication 5 times, is got in the multiple holes of 3 of each concentration
50Mean value.LOD determines by following steps, measure the OD value of 20 parts of blank livers and musculature, regression equation calculation according to typical curve goes out corresponding tylosin concentration, calculate the mean value (X) and the standard deviation (SD) of tylosin concentration then, calculate lowest detectable limit in the tissue according to formula LOD=X+3SD.IC of the present invention
50Value is 19.14 ± 2.4 μ g/L, and the lowest detectable limit of tylosin in muscle and in the liver is respectively 4.3 μ g/L, 6.1 μ g/L.
6.2 the precision of kit of the present invention test
The tylosin standard items are diluted to 0 μ g/L, 5 μ g/L, 10 μ g/L, 20 μ g/L, 40 μ g/L, 80 a μ g/L6 concentration, 3 multiple holes of every concentration, according to indirect competitive ELISA method replication 5 times, the regression equation calculation of application standard curve goes out the measured value of each concentration tylosin standard solution, calculate in the plate and the coefficient of variation between plate, the results are shown in Table 5.
Reach error between plate in the plate of table 5 typical curve
6.3 the accuracy of kit of the present invention, replica test
In the homogenate musculature of 2g, add the tylosin standard solution, make its final concentration be respectively 20 μ g/kg, 100 μ g/kg, 200 μ g/kg, 400 μ g/kg, add the Tilmicosin standard solution, make its final concentration be respectively 30 μ g/kg, 50 μ g/kg, 100 μ g/kg, 200 μ g/kg; In 1g liver homogenate tissue, add the tylosin standard solution, make its final concentration be respectively 30 μ g/kg, 100 μ g/kg, 200 μ g/kg, 400 μ g/kg; Add the Tilmicosin standard solution, make its final concentration be respectively 30 μ g/kg, 750 μ g/kg, 1500 μ g/kg, 3000 μ g/kg.5 repetitions of each concentration, replication 3 times.The tylosin in the mensuration interpolation tissue and the concentration of Tilmicosin, according to formula 3 calculate recovery rates, the accuracy of examination kit; Calculate batch interior and interassay coefficient of variation, the repeatability of examination kit.Accuracy and repeatability the results are shown in Table 6, table 7, table 8, table 9, show that this kit has reliable accuracy, in batch and interassay coefficient of variation little, good reproducibility.
Table 6 tylosin adds the recovery and the coefficient of variation in muscle
Table 7 Tilmicosin adds the recovery and the coefficient of variation in muscle
Table 8 tylosin adds the recovery and the coefficient of variation in liver
Table 9 Tilmicosin adds the recovery and the coefficient of variation in liver
Claims (7)
1. the monoclonal antibody that can discern tylosin and Tilmicosin simultaneously is characterized in that, it is to be that the hybridoma cell strain P3C4 of CCTCC NO:C200719 is secreted by preserving number.
2. the described hybridoma cell strain P3C4 of claim 1 is deposited in Chinese typical culture collection center, and its preserving number is CCTCC NO:C200719.
3. the kit that comprises the described monoclonal antibody of claim 1.
4. kit according to claim 3, this kit are the enzyme linked immunological kits that detects tylosin and Tilmicosin simultaneously.
5. an enzyme-linked immunoassay method that detects tylosin and tilmicosin residue simultaneously comprises the preparation of immunogene, coating antigen and antibody and the pre-treatment of sample, and its step is as follows:
(1) haptens is removed the mycaminose tylosin and hydrazino-benzoic acid or adipyl dihydrazide or O-ethyloic azanol are obtained immunogene with the bovine serum albumin(BSA) coupling again;
(2) haptens is removed the mycaminose tylosin and hydrazino-benzoic acid or adipyl dihydrazide or O-ethyloic azanol are obtained coating antigen with the ovalbumin coupling again;
(3) be that CCTCC NO:C200719 hybridoma cell strain P3C4 prepares monoclonal antibody with the described preserving number of claim 1;
(4) use the coating antigen bag of step (2) by solid phase carrier;
(5) be the extraction of 20-30% acetonitrile solution, ethyl acetate extraction with the testing sample percent by volume, nitrogen dries up with sample diluting liquid dissolves again and obtains determinand;
(6) determinand to step (5) detects;
Wherein:
The component of sample diluting liquid and proportioning are: NaCl 8.0g, KH
2PO
40.2g, Na
2HPO
412H
2O 2.9g, KCl 0.2g, thimerosal 0.01g adds distilled water to 1000mL.
6. the application of the described monoclonal antibody of claim 1 in preparing the enzyme linked immunological kit that detects tylosin and Tilmicosin simultaneously.
7. claim 3 or the 4 described kits application in tylosin and tilmicosin residue detection.
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| CN102590516A (en) * | 2012-01-17 | 2012-07-18 | 重庆师范大学 | Immune colloidal gold test strip for simultaneously carrying out residue detection and analysis on Tylosin and Tilmicosin and preparation method thereof |
| CN103288959A (en) * | 2012-02-29 | 2013-09-11 | 华中农业大学 | Tylosin and tilmicosin gene engineering antibody, and preparation method and application |
| CN103389379B (en) * | 2012-05-08 | 2016-12-14 | 北京勤邦生物技术有限公司 | A kind of detect tylosin and the test strips of tilmicosin and method |
| CN102707059A (en) * | 2012-05-31 | 2012-10-03 | 华中农业大学 | Colloidal gold test paper strip for residue detection of tylosin and tilmicosin, method for using colloidal gold test paper strip and application of colloidal gold test paper strip |
| CN102768279B (en) * | 2012-05-31 | 2014-06-25 | 华中农业大学 | Colloidal gold test strip for detecting residue of sulfonamides, usage method and application thereof |
| CN102830232A (en) * | 2012-09-20 | 2012-12-19 | 重庆市科学技术研究院 | Time resolution fluoroimmunoassay kit for detecting tylosin and tilmicosin and detection method of kit |
| CN104698180A (en) * | 2015-03-18 | 2015-06-10 | 天津农学院 | Immunoaffinity stir bar for adsorbing macrolide antibiotics, and preparation method and application thereof |
| CN106610433B (en) * | 2016-12-07 | 2018-08-10 | 北京勤邦生物技术有限公司 | Detect enzyme linked immunological kit and its application of Tilmicosin |
| CN108490166B (en) * | 2018-02-28 | 2020-10-09 | 广州市丰华生物工程有限公司 | Improved experiment buffer solution and application thereof |
| CN109180760B (en) * | 2018-08-30 | 2021-09-17 | 华中农业大学 | Ivermectin derivative, monoclonal antibody of anti-avermectin drug and application of monoclonal antibody |
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| US6506885B1 (en) * | 2001-09-27 | 2003-01-14 | The United States Of America As Represented By The Secretary Of Agriculture | Monoclonal antibodies to the drug tilmicosin and a method for detecting the same |
| CN1811442A (en) * | 2006-02-17 | 2006-08-02 | 中国农业大学 | Method for detecting tylosin and special enzyme-linked immune reagent kit thereof |
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| CN1811442A (en) * | 2006-02-17 | 2006-08-02 | 中国农业大学 | Method for detecting tylosin and special enzyme-linked immune reagent kit thereof |
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