CN102608319B - Monoclonal antibody, enzyme linked immunosorbent assay method and kit for detecting gentamicin and sisomicin - Google Patents
Monoclonal antibody, enzyme linked immunosorbent assay method and kit for detecting gentamicin and sisomicin Download PDFInfo
- Publication number
- CN102608319B CN102608319B CN201210045205.2A CN201210045205A CN102608319B CN 102608319 B CN102608319 B CN 102608319B CN 201210045205 A CN201210045205 A CN 201210045205A CN 102608319 B CN102608319 B CN 102608319B
- Authority
- CN
- China
- Prior art keywords
- gentamicin
- sisomicin
- monoclonal antibody
- kit
- detection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Abstract
The invention discloses a specific monoclonal antibody capable of recognizing gentamicin and sisomicin simultaneously. The monoclonal antibody of the invention is secreted by hybridoma DEC/2D5 which is preserved in China Center for Type Culture Collection with the preservation number of CCTCC NO: C201145. The invention also discloses a preparation method of the specific monoclonal antibody, coating antigen and immunogen as well as an enzyme linked immunosorbent assay kit. Compared with the prior art, the monoclonal antibody prepared according to the invention can recognize gentamicin and sisomicin simultaneously, therefore, the detection efficiency of the prior art is improved. The kit and the method of the invention have the advantages of simplicity and convenience in operation, sensitivity, accuracy and the like.
Description
Technical field
The present invention relates to a kind of monoclonal antibody that can identify gentamicin and Sisomicin and for detection of gentamicin and Sisomicin enzyme-linked immune analytic method and kit.
Background technology
Gentamicin and Sisomicin belong to aminoglycoside antibiotics (AMGs), because this class microbiotic is easily accumulated and caused ototoxicity and renal toxicity at cortex renis and inner ear perilymph, and the microorganism persister being produced by aminoglycoside inactive enzyme is comparatively serious, so its residue detection in animal derived food causes people's attention day by day.Enzyme-linked immuno assay (ELISA) method is due to the advantage such as simple, quick, sensitive, special and be widely used in gradually the quick residue detection field of AMGs.But current ELISA method mainly concentrates on single residue detection aspect, by the ELISA method of a kind of antibody AMGs that detection architecture is different simultaneously seldom.
Application number is that 200710064346.8 patent of invention discloses a kind of enzyme linked immunological kit and method that detects gentamicin medicine, this patent is by gentamicin and carrier protein, carrier protein is: mouse haemocyanin, thyroprotein, bovine serum albumin(BSA), albumin rabbit serum, human serum albumins, ovalbumin or hemocyanin, adopt glutaraldehyde method or carbodlimide method to carry out coupling and obtain immunogene and coating antigen, prepared polyclonal antibody and monoclonal antibody can only be identified gentamicin, and the detection time of its sample is longer.Application number is that 200920246849.1 patent of invention discloses a kind of Gentamicin ELISA checking reagent box, this patent adopts method coupling gentamicin and bovine serum albumin(BSA) (the bovine serum albu minute of direct activation albumen, BSA) adaptive immune is former, same method obtains coating antigen by gentamicin and thyroprotein coupling, prepared monoclonal antibody equally only can be identified gentamicin, and its sample preparation time is longer, reagent is not optimized.
Summary of the invention
First object of the present invention is to provide a kind of monoclonal antibody that can identify gentamicin and Sisomicin.
Second object of the present invention is to utilize this monoclonal antibody, sets up a kind of enzyme-linked immunoassay method that can detect for gentamicin and Sisomicin.
The 3rd object of the present invention is to provide a kind of kit for gentamicin and Sisomicin detection.
The 4th object of the present invention is to provide the application of described monoclonal antibody in the enzyme linked immunological kit of preparation detection gentamicin and Sisomicin.
The 5th object of the present invention is to provide the application of kit in animal tissue's gentamicin and Sisomicin residue detection that contains described monoclonal antibody.
The present invention is achieved through the following technical solutions:
Can identify a monoclonal antibody for gentamicin and Sisomicin, it is to be that the hybridoma EDC/2D5 of CCTCC NO:C201145 is secreted by preserving number.
Above-mentioned hybridoma EDC/2D5, is deposited in the Chinese Typical Representative culture collection center (CCTCC) that is positioned at Wuhan City, Hubei Province Wuhan University, and preserving number is CCTCC NO:C201145.
Immunogene used is prepared by haptens gentamicin and bovine serum albumin(BSA) coupling.
Further, the invention provides a kind of enzyme-linked immunoassay method for gentamicin and Sisomicin detection, the method comprises the steps such as the preparation of immunogene, coating antigen, antibody and the processing of sample and detection, specific as follows:
(1) haptens gentamicin and bovine serum albumin(BSA) (BSA) coupling are obtained to immunogene;
(2) haptens gentamicin and ovalbumin (OVA) coupling are obtained to coating antigen;
(3) utilize the immunogen immune mouse of step (1), by Fusion of Cells and screening, obtain the hybridoma EDC/2D5 that preserving number is CCTCC NO:C201145;
(4) with the hybridoma EDC/2D5 that preserving number is CCTCC NO:C201145, prepare monoclonal antibody;
(5) with the coating antigen of step (2), be coated with solid phase carrier (as ELISA Plate);
(6) testing sample is processed with 2% trichloroacetic acid, centrifuging and taking supernatant, adjust pH, is determinand solution;
(7) the determinand solution of step (6) is carried out to enzyme linked immunosorbent detection.
In step (6), the component of 2% trichloroacetic acid and proportioning are: accurately take trichloroacetic acid 20.0g, first add a small amount of distilled water and dissolve, then be settled to 1000mL.
The present invention is usingd said monoclonal antibody and coating antigen as core reagent and conventional other agent combination, made the enzyme linked immunological kit that can detect gentamicin and Sisomicin, in conjunction with above-mentioned enzyme-linked immunoassay method, realized the enzyme linked immunosorbent detection to gentamicin and Sisomicin.
Major advantage of the present invention is:
1, the monoclonal anti physical efficiency that prepared by the present invention is identified gentamicin and Sisomicin simultaneously, and prior art only can single identification gentamicin.
2, the ELISA method that the present invention sets up is short detection time, and detection efficiency is high.
3, the tissue sample disposal route the present invention relates to is simple, without expensive instrument, without organic reagent, operator, without health hazard, and is only needed to generic centrifuge, is adapted at basic unit's operation.
Accompanying drawing explanation
Fig. 1 is the mass spectrogram that the substance assistant laser desorpted method of carrier protein BSA in the present invention detects.
Fig. 2 is the mass spectrogram that in the present invention, immunogenic substance assistant laser desorpted method detects, and is used for illustrating the coupling effect of haptens gentamicin and BSA in conjunction with Fig. 1.
Accompanying drawing 3 is the indirect competitive ELISA response curve of monoclonal antibody of the present invention and gentamicin (GEN) standard items, X-axis is gentamicin (GEN) concentration of standard solution logarithm value, and Y-axis is that the optical density value of gentamicin standard solution is divided by " zero " hole optical density value (B/B0).
Embodiment
Below by embodiment, the invention will be further described.
The preparation of embodiment 1 immunogene and coating antigen
1.1 gentamicins-BSA's is synthetic
Take gentamicin 164.0mg and BSA200.0mg and be dissolved in (pH7.4) in 15mL pure water, stir, then dropwise add carbodiimide (EDC) 430.00mg being dissolved in 5mL pure water, under room temperature, stirring reaction is 8 hours.Finally this reactant liquor proceeds in bag filter, in PBS liquid (pH7.4), dialyse 4 days for 4 ℃, and centrifugal postlyophilization.As shown in Figure 1-2, through substance assistant laser desorpted method (MALDI-TOF-MS), identify coupling success, put-20 ℃ and save backup.
1.2 gentamicin-OVA's is synthetic
Take gentamicin 148.0mg and OVA240.0mg and be dissolved in (pH7.4) in 12mL pure water, stir, then dropwise add the EDC430.00mg being dissolved in 4mL pure water, under room temperature, stirring reaction is 8 hours.Finally this reactant liquor proceeds in bag filter, in PBS liquid (pH7.4), dialyses 4 days for 4 ℃.Centrifugal postlyophilization, puts-20 ℃ and saves backup.
The preparation of embodiment 2 monoclonal antibodies
The preparation of hybridoma: with reference to Yang Han spring < < animal immunology > >, immunogene gentamicin-BSA immunity Balb/C mouse (purchased from Disease Prevention Control Center, Hubei Prov's Experimental Animal Center) with embodiment 1 preparation, immune programme for children is: fundamental immunity is by after immunogene and isopyknic Freund's complete adjuvant emulsification, in the subcutaneous multi-point injection of mouse back, at interval of 2 weeks booster immunizations once, use Freund's incomplete adjuvant emulsification instead later.Last (be better than most immunity finish rear rest and reorganization January) lumbar injection in merging first three day, reinforced immunological, 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 sterilization in 5 minutes in 75% alcohol.Aseptic taking-up mouse spleen, isolate splenocyte, with the SP2/0 myeloma cell (SP2/0 myeloma cell is from this laboratory) of fresh preparation in the ratio of 1-2 * 107 SP2/0 and 108 immune spleen cells (1: 10～1: 15) in 50mL centrifuge tube, with 15mLRPMI-1640 basal liquid re-suspended cell, 1500r/ minute centrifugal 5 minutes, wash cell 1 time.The nutrient culture media that temperature is bathed in centrifugal gap, the water that temperature is bathed, the PEG that temperature is bathed etc. puts into super-clean bench.Then take out the thieving paper of sterilizing, by after being equipped with and emptying to the greatest extent on the centrifuge tube of myeloma cell and immune spleen cell, tip upside down on and on thieving paper, control solid carbon dioxide and drip, rap the pipe end to make cell loosening.Open timer, with 1mL suction pipe, draw 0.8mLPEG, the hand-held centrifuge tube that cell mixing is housed, places it in a moment in water-bath, and PEG is added drop-wise on cell mixing slowly, and limit edged stirs gently, in 1 minute, adds, and continues to stir 30 seconds.With suction pipe, get 10mL basal liquid, along tube wall, be slowly added on fused cell, limit edged shakes (can not blow and beat) gently, in 5 minutes, adds respectively 1mL, 2mL, 3mL, 4mL, finally adds basal liquid to 40mL, after adding, cover lid, puts upside down several times repeatedly, and cell is mixed.800r/ minute 5 minutes centrifugal, abandons supernatant.The HAT nutrient culture media that absorption contains feeder cells, stirs the fused cell in centrifuge tube gently with suction pipe, dropwise splashes into containing in the serum bottle of feeder cells near liquid level, stirs and evenly mixs, and action will gently be stirred cell gently, must not blow and beat.Put upside down and mix.Then cell is seeded on Tissue Culture Plate, two, every hole, is placed in incubator and cultivates.Single cell fusion can be inoculated 4～6 96 orifice plates.Also can plant less as required, generally press the cell number of SP2/0 and calculate, every hole inoculum concentration is approximately containing a 104 left and right SP2/0 cell.In 37 ℃, in 5%CO2 incubator, cultivate.
Counted 0 day the same day of merging, and the first 3 days kinetocyte plates of trying not keep incubator homeostasis.Within the 3rd day, 1 HAT complete medium is added in every hole; The 5th day every hole sucking-off 1/2 culture supernatant (100 μ L), then add 1 HT complete medium; Every 2 days the same methods, suck 1/2～3/4 culture supernatant later, after 7 days, change to HT complete medium.
Treat that fused cell colony grows to culture hole 1/10～1/5, by indirect ELISA method and the indirect competitive ELISA method set up, screen simultaneously.Compare with zero medicine hole, medicine hole OD value can the repressed positive that is judged to.According to inhibiting rate and cell colony upgrowth situation, select the cell hole that only has 1-2 single colony of 2～6 strong positives, adopt limiting dilution method to clone.
Through 3～4 time clonings, until clone's positive rate is 100%, finishing screen is selected the hybridoma of the anti-gentamicin of secretion and Sisomicin, and the chromosome average of this cell line is 101.Applicant is this hybridoma called after EDC/2D5, and on June 29th, 2011, delivers the Chinese Typical Representative culture collection center preservation that is positioned at Wuhan City, Hubei Province Wuhan University, and its preserving number is CCTCC NO:C201145.
The preparation of ascites monoclonal antibody and evaluation: in inoculation, only within first 7 days, get Balb/c number of mice, every mouse peritoneal injection 0.5ml incomplete Freund's adjuvant carries out pre-service.With RPMI 1640 basal mediums, suspending by preserving number is the hybridoma EDC/2D5 expansion cultured cells of CCTCC NO:C201145, and cell number is adjusted to 1 * 10
6individual/mL, every mouse peritoneal inoculation 0.5ml.Treat that mouse web portion obviously expands, spiritual variation, the dying ascites that gathers when motionless.According to literature method (Zhu Liping, Chen Xueqing. immunology common experimental method. Beijing: People's Medical Officer Press, 2000), purifying obtains monoclonal antibody.Employing is carried out hypotype evaluation purchased 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 result is mouse IgG 1 hypotype.
The foundation of embodiment 3 gentamicin racing ELISA detecting methods
The preparation of 3.1 reagent (reagent that the present embodiment is used all adopts following methods preparation except another indicating)
Carbonate buffer solution (pH9.6): accurately take Na
2cO
31.59g, NaHCO
32.93g, a small amount of ultrapure water dissolves, and is settled to 1000mL.
Cleansing solution (pH7.4): accurately take NaCl 8.00g, KH
2pO
40.20g, Na
2hPO
412H
2o 2.90g, KCl 0.20g, a small amount of ultrapure water dissolves, and adds Tween20 0.50mL, is settled to 1000mL.
Phosphate buffer (PBS) is (pH7.4): accurately take NaCl8.00g, KH
2pO
40.20g, Na
2hPO
412H
2o 2.90g, KCl0.20g, a small amount of ultrapure water dissolves, and is settled to 1000mL.
Confining liquid: accurately take ovalbumin 10.00g, add 1000mL phosphate buffer, stir and evenly mix until albumen dissolves completely.
Physiological saline: accurately take NaCl 8.50g, a small amount of ultrapure water dissolves, and is settled to 1000mL.
Antibody diluent, enzymic-labelled antibody dilution and substrate solution provide by Wuhan Fei Yuan Science and Technology Ltd..
Stop buffer: accurately measure concentrated sulphuric acid 100mL, be slowly added drop-wise in 800mL ultrapure water.
The preliminary of 3.2 coating antigen concentration and antibody working concentration determined
First be by the combination of method initial option coating antigen and the antibody working concentration of square formation titration.Use carbonate buffer solution that GEN-OVA coating antigen doubling dilution is become to the horizontal coated elisa plate of 32,16,8,4,2,1,0.5,0.25 μ g/mL; EDC/2D05 monoclonal antibody is used phosphate buffer doubling dilution to become 1: 500,1: 1000,1: 2000,1: 4000,1: 8000,1: 16000,1: 32000,1: 64000,1: 128000,1: 512000 etc. is longitudinally added ELISA Plate.16000) and (8,1: 32000) square formation titration results is in Table 1, the following coating antigen concentration of initial option and the combination of antibody working concentration: (8,1:.
Determining of 3.3 best coating antigen concentration and antibody working concentration
Best coated concentration is determined: the coated concentration of selecting with square formation titration and antibody dilution combination are done respectively to suppress curve, and gentamicin standard items concentration is set to 0,5,10,20,40,80 μ g/mL, its zero medicine hole and IC
50value is in Table 2.The ratio of antigen-antibody is the key that affects its sensitivity, if there is antigen or antibody excess, all will cause IC
50higher, comprehensive IC
50the linearly dependent coefficient of value, zero hole OD value and inhibition curve, best coated concentration is 8 μ g/mL, antibody dilution is tentatively defined as 1: 32000.
The titration of table 1EDC/2D5 monoclonal antibody square formation
The best coated concentration optimization of table 2
| Coating antigen concentration (μ g/mL) | Antibody dilution multiple (1: X) | 0 hole OD value | IC 50Value (μ g/L) |
| 8 | 16000 | 2.81 | 27.20 |
| 8 | 32000 | 2.44 | 18.55 |
Optimum antibody dilutability is determined: with the coated concentration coated elisa plate of the best, by antibody 7 dilution gradients of concentration equal difference design centered by 1: 40000, its zero medicine hole and IC
50value is in Table 3.Along with the increase of antibody dilution, IC
50value reduces, and zero medicine hole value also reduces.Comprehensive IC
50the linearly dependent coefficient of value, zero hole OD value and inhibition curve, selects 1: 48000 for optimum antibody dilutability.
Table 3 optimum antibody dilutability is optimized
| Antibody coefficient multiple (1: X) | 0 hole OD value | IC50 value (μ g/L) |
| 16000 | 2.81 | 27.20 |
| 32000 | 2.44 | 18.55 |
| 36000 | 2.3 | 17.14 |
| 40000 | 2.25 | 17.7 |
| 44000 | 2.27 | 17.12 |
| 48000 | 2.02 | 13.09 |
| 52000 | 2.45 | 17.01 |
The foundation of 3.4 typical curves
Gentamicin standard items concentration is set to 6 concentration such as 0,5,10,20,40,80 μ g/L, measures drawing standard curve above according to definite indirect competitive ELISA method.As shown in Figure 3, the gentamicin that the present invention sets up and regression equation and the linear dependence index of the residual indirect competitive ELISA method of Sisomicin are respectively: y=-0.547x+1.166, r=0.996, IC
50 valuesbe 16.33 ± 1.24 μ g/L (n=5).The range of linearity is 5～80 μ g/L.
3.5 specificity
Become gradient concentration to carry out indirect competitive ELISA various conventional aminoglycoside antibiotics doubling dilutions respectively, calculate IC
50value, with gentamicin standard items IC
50value contrast obtains cross reacting rate, the results are shown in Table 4.Result shows, the indirect competitive ELISA method that the present invention sets up is respectively 100% and 33.80% to the cross reacting rate of gentamicin and Sisomicin, and with the equal no cross reaction of other aminoglycoside antibiotics.
The specificity of the residual ELISA detection method of table 4 gentamicin and Sisomicin
| Competition thing | IC 50(μg/L) | Cross reacting rate (%) |
| Gentamicin | 16.33 | 100 |
| Sisomicin | 48.31 | 33.80 |
| Neomycin | ＞10000 | ＜0.02 |
| Amikacin | ＞10000 | ＜0.02 |
| Paromomycin | ＞10000 | ＜0.02 |
| Neamine | ＞10000 | ＜0.02 |
| Kanamycins | ＞10000 | ＜0.02 |
| Streptomysin | ＞10000 | ＜0.02 |
| Dihydrostreptomycin | ＞10000 | ＜0.02 |
| Ribostamycin | ＞10000 | ＜0.02 |
| Spectinomycin | ＞10000 | ＜0.02 |
| Apramycin | ＞10000 | ＜0.02 |
| Certomycin | ＞10000 | ＜0.02 |
| Tobramycin | ＞10000 | ＜0.02 |
| Hygromycin | ＞10000 | ＜0.03 |
| Kasugarnycin | ＞10000 | ＜0.03 |
The assembling of embodiment 4 gentamicin of the present invention and the how residual ELISA detection kit of Sisomicin
The composition of 4.1 ELISA kits of the present invention
1) be coated with the solid phase carrier (ELISA Plate) of coating antigen gentamicin-OVA;
2) gentamicin 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) gentamicin monoclonal antibody working fluid;
4) the sheep anti-mouse igg antibody working fluid of horseradish peroxidase (HRP) mark;
5) concentrated phosphoric acid salt buffer: NaCl 80.0g, KH
2pO
42.0g, Na
2hPO
412H
2o 29.0g, KCl 2.0g, adds ultrapure water water to 1000mL;
6) concentrated cleaning solution: NaCl 80.0g, KH
2pO
42.0g, Na
2hPO
412H
2o 29.0g, KCl 2.0g, polysorbas20 5mL, adds ultrapure water to 1000mL;
7) substrate solution A: Fei Yuan scientific & technical corporation provides by Wuhan;
8) substrate solution B: Fei Yuan scientific & technical corporation provides by Wuhan;
9) stop buffer: 2mol/L sulfuric acid solution.
The preparation of 4.2 ELISA Plate
With coating buffer, gentamicin-OVA is diluted to 8 μ g/mL, every hole adds 100 μ L, and 4 ℃ are coated with 12～16 hours; The coating buffer that inclines, every hole adds 250 μ L cleansing solutions, washs 3 times, pats dry, and then every hole adds confining liquid 250 μ L, hatches 2 hours for 37 ℃; The liquid in hole that inclines, cleansing solution washing 3 times, after patting dry, is inverted in 37 ℃ of incubators by ELISA Plate on thieving paper, standing oven dry 30 minutes, ELISA Plate packs aluminium foil bag into after drying together with drying agent, with vacuum packaging machine, encapsulates.
The mensuration program of embodiment 5 kits of the present invention
The preparation of 5.1 reagent
1) cleansing solution: the cleansing solution providing in kit is used afterwards with 10 times of ultrapure water dilutions.
2) substrate mixed liquor: according to each institute expense, substrate solution A and the substrate solution B of preparation are mixed for by volume 1: 100, now with the current.
3) component of 2% trichloroacetic acid and proportioning are: accurately take trichloroacetic acid 20.0g, first add a small amount of distilled water and dissolve, then be settled to 1000mL.
5.2 tissue sample pre-treatments
Qu2g homogeneous structure sample, adds 2% trichloroacetic acid 8mL, vortex 3 minutes, then 4000r/ minute centrifugal 10 minutes, get 1mL supernatant, add 1M NaOH 75 μ l to adjust pH, get 50 μ l stand-by.
Note: this method is 4 to the extension rate of sample.
5.3 determination step
1) application of sample: add gentamicin series concentration standard solution or sample solution 50 μ L in ELISA Plate micropore, then add gentamicin monoclonal antibody working fluid 50 μ L, be placed in wet box, 37 ℃ of constant-temperature incubations 30 minutes;
2) washing: pour out the liquid in hole, add cleansing solution 250 μ L in every hole, after standing 30 seconds, wash 3 times and pat dry;
3) add the sheep anti-mouse igg antibody working fluid of horseradish peroxidase (HRP) mark: in every hole, add the sheep anti-mouse igg antibody working fluid 100 μ L of horseradish peroxidase (HRP) mark, in 37 ℃ of wet boxes, constant-temperature incubation is 30 minutes;
4) washing: pour out the liquid in hole, add cleansing solution 250 μ L in every hole, after standing 30 seconds, wash 3 times and pat dry;
5) add substrate: in every hole, add substrate mixed liquor 100 μ L, in 37 ℃ of wet boxes, hatch 15 minutes;
6) add stop buffer: in every hole, add stop buffer 50 μ L;
7) measure: by microplate reader, at 450nm place, measure the optical density value (OD value) in every hole.
5.4 result judgements
With the standard items OD value measured divided by " zero " hole OD value (B/B
0) be ordinate, the logarithm value of gentamicin concentration is that horizontal ordinate is made typical curve, the line linearity of going forward side by side returns, and provides regression equation.According to the inhibiting rate of formula calculation sample, by inhibiting rate substitution regression equation, calculate mensuration concentration, be multiplied by corresponding extension rate, be the residual concentration of gentamicin and Sisomicin in sample.
Sensitivity, the preci-sion and accuracy of embodiment 6 kits of the present invention
The sensitivity of 6.1 kits of the present invention
IC with typical curve
50value and organize lowest detectable limit (LOD) as the sensitivity index of detection kit of the present invention.Gentamicin standard items are diluted to 6 concentration such as 0 μ g/L, 5 μ g/L, 10 μ g/L, 20 μ g/L, 40 μ g/L, 80 μ g/L, 5 repeating holes of each concentration, according to indirect competitive ELISA method replication 5 times, get the IC measuring for 5 times
50mean value.LOD determines by following steps, measures the OD value of the musculature of 20 parts of blank chickens, according to the regression equation calculation of typical curve, goes out corresponding gentamicin concentration, then calculates the mean value of gentamicin concentration
and standard deviation (SD), according to formula
calculate the lowest detectable limit in tissue.IC of the present invention
50value is 16.33 ± 1.24 μ g/L, and the lowest detection of gentamicin in chicken muscle is limited to 34.09 μ g/L.
The precision of 6.2 kits of the present invention
Respectively its typical curve equations of OD value substitution corresponding to gentamicin standard items concentration such as 5,10,20,40,80 μ g/L are obtained to the measured value that ELISA detects, with the coefficient of variation between the plate inner panel of standard items concentration determination value calculating indirect competitive ELISA typical curve, the results are shown in Table 5.Result shows, the equal < 15% of the coefficient of variation in the plate of typical curve and between plate, illustrates that indirect competitive ELISA method of the present invention has good precision.
The coefficient of variation in the plate of table 5 typical curve and between plate
The accuracy of 6.3 kits of the present invention
In the homogenate chicken muscle tissue of 2g, add gentamicin standard solution, make its final concentration be respectively 200 μ g/kg, 100 μ g/kg and 50 μ g/kg; Add Sisomicin standard solution, make its final concentration be respectively 200 μ g/kg, 100 μ g/kg and 50 μ g/kg; 5 repetitions of each concentration, replication 3 times.Gentamicin in mensuration interpolation tissue and the concentration of Sisomicin, calculate recovery rate, examines the accuracy of kit according to the following equation; Calculate batch interior and interassay coefficient of variation, the repeatability of examination kit.Accuracy and repeatability the results are shown in Table 6 and table 7, show that this kit has reliable accuracy, in batch and interassay coefficient of variation little, reproducible.
In table 6 chicken muscle, gentamicin adds the recovery and the coefficient of variation
In table 7 chicken muscle, Sisomicin adds the recovery and the coefficient of variation
Claims (7)
1. can identify a monoclonal antibody for gentamicin and Sisomicin, it is characterized in that, it is to be that the hybridoma cell strain EDC/2D5 of CCTCC NO:C201145 is secreted by preserving number.
2. the hybridoma cell strain EDC/2D5 described in claim 1, is deposited in Chinese Typical Representative culture collection center, and its preserving number is CCTCC NO:C201145.
3. the kit that comprises monoclonal antibody described in claim 1.
4. kit according to claim 3, this kit is the enzyme linked immunological kit that detects gentamicin and Sisomicin.
5. detect an enzyme-linked immunoassay method for gentamicin and Sisomicin, comprise the preparation of coating antigen, antibody and the processing of sample and detection, its step is as follows:
(1) haptens gentamicin and ovalbumin coupling are obtained to coating antigen;
(2) with the hybridoma cell strain EDC/2D5 that preserving number is CCTCC NO:C201145, prepare monoclonal antibody;
(3) with the coating antigen of step (1), be coated with solid phase carrier;
(4) testing sample is processed with 2% trichloroacetic acid, centrifuging and taking supernatant, adjust pH, obtains determinand solution;
(5) the determinand solution of step (4) is carried out to enzyme linked immunosorbent detection.
6. the application of monoclonal antibody claimed in claim 1 in the enzyme linked immunological kit of preparation detection gentamicin and Sisomicin.
7. the application of the kit described in claim 3 or 4 in animal tissue's gentamicin and Sisomicin residue detection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210045205.2A CN102608319B (en) | 2012-02-27 | 2012-02-27 | Monoclonal antibody, enzyme linked immunosorbent assay method and kit for detecting gentamicin and sisomicin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210045205.2A CN102608319B (en) | 2012-02-27 | 2012-02-27 | Monoclonal antibody, enzyme linked immunosorbent assay method and kit for detecting gentamicin and sisomicin |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102608319A CN102608319A (en) | 2012-07-25 |
| CN102608319B true CN102608319B (en) | 2014-02-05 |
Family
ID=46525858
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210045205.2A Expired - Fee Related CN102608319B (en) | 2012-02-27 | 2012-02-27 | Monoclonal antibody, enzyme linked immunosorbent assay method and kit for detecting gentamicin and sisomicin |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102608319B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104569325B (en) * | 2014-12-26 | 2016-11-23 | 华中农业大学 | For detecting antibody chip test kit and the method for aminoglycoside antibiotics residual in food |
| CN106525835A (en) * | 2016-11-09 | 2017-03-22 | 百奥森（江苏）食品安全科技有限公司 | Detection kit for gentamycin in food |
| CN106520705B (en) * | 2016-12-06 | 2019-04-30 | 得利斯集团有限公司 | The anti-paromomycin monoclonal antibody hybridoma cell strain C1 of one plant of secretion and its application |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101021535A (en) * | 2007-03-12 | 2007-08-22 | 北京望尔康泰生物技术有限公司 | Enzyme-linked immunalogical kit for detecting gentamicin medicine and method |
| CN101429242A (en) * | 2008-12-04 | 2009-05-13 | 浙江大学 | Gentamicin and carrier protein couplet product, method for producing gentamicin antibody and uses thereof |
| CN201535773U (en) * | 2009-11-11 | 2010-07-28 | 北京望尔康泰生物技术有限公司 | Gentamicin ELISA checking reagent box |
| CN101793893A (en) * | 2009-02-03 | 2010-08-04 | 王武康 | Direct competitive enzyme-linked immunoassay kit for detecting gentamicin |
| CN101907623A (en) * | 2010-08-03 | 2010-12-08 | 中国农业大学 | Method for detecting gentamicin and/or micronomicin and special quantum dot fluorescence immunoassay kit thereof |
-
2012
- 2012-02-27 CN CN201210045205.2A patent/CN102608319B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101021535A (en) * | 2007-03-12 | 2007-08-22 | 北京望尔康泰生物技术有限公司 | Enzyme-linked immunalogical kit for detecting gentamicin medicine and method |
| CN101429242A (en) * | 2008-12-04 | 2009-05-13 | 浙江大学 | Gentamicin and carrier protein couplet product, method for producing gentamicin antibody and uses thereof |
| CN101793893A (en) * | 2009-02-03 | 2010-08-04 | 王武康 | Direct competitive enzyme-linked immunoassay kit for detecting gentamicin |
| CN201535773U (en) * | 2009-11-11 | 2010-07-28 | 北京望尔康泰生物技术有限公司 | Gentamicin ELISA checking reagent box |
| CN101907623A (en) * | 2010-08-03 | 2010-12-08 | 中国农业大学 | Method for detecting gentamicin and/or micronomicin and special quantum dot fluorescence immunoassay kit thereof |
Non-Patent Citations (10)
| Title |
|---|
| Development of an enzyme-linked immunoassay for the detection of gentamicin in swine tissues;Yiqiang Chen et al;《Food Chemistry》;20080501;第108卷;304-309页 * |
| Development of ELISA and Immunochromatographic Assay for the Detection of Gentamicin;Yong Jin et al;《J.Agric.Food Chem.》;20050908;第53卷(第20期);7639-7643页 * |
| Elma E.M.G.Loomans et al.Neamin as an Immunogen for the Development of a Generic ELISA Detecting Gentamicin, Kanamycin, and Neomycin in Milk.《J.Agric.Food Chem》.2003,第51卷(第3期),587-593页. |
| Neamin as an Immunogen for the Development of a Generic ELISA Detecting Gentamicin, Kanamycin, and Neomycin in Milk;Elma E.M.G.Loomans et al;《J.Agric.Food Chem》;20030103;第51卷(第3期);587-593页 * |
| Yiqiang Chen et al.Development of an enzyme-linked immunoassay for the detection of gentamicin in swine tissues.《Food Chemistry》.2008,第108卷304-309页. |
| Yong Jin et al.Development of ELISA and Immunochromatographic Assay for the Detection of Gentamicin.《J.Agric.Food Chem.》.2005,第53卷(第20期),7639-7643页. |
| 庆大霉素单克隆抗体的制备及ELISA检测方法研究;金涌等;《中国兽医杂志》;20091231;第45卷(第8期);9-12页 * |
| 抗庆大霉素单克隆抗体的制备及其初步应用;职爱民等;《中国农业科学》;20101231;第43卷(第12期);2584-2589页 * |
| 职爱民等.抗庆大霉素单克隆抗体的制备及其初步应用.《中国农业科学》.2010,第43卷(第12期),2584-2589页. |
| 金涌等.庆大霉素单克隆抗体的制备及ELISA检测方法研究.《中国兽医杂志》.2009,第45卷(第8期),9-12页. |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102608319A (en) | 2012-07-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101105492B (en) | Monoclonal antibody and enzyme-linked immunoassay method and reagent kit for detecting tylosin and tilmicosin residue | |
| CN102323416A (en) | Kit for rapid detection of staphylococcus aureus in sample and detection method thereof | |
| CN102608319B (en) | Monoclonal antibody, enzyme linked immunosorbent assay method and kit for detecting gentamicin and sisomicin | |
| CN102585005B (en) | Monoclonal antibody and ELLSA (Enzyme Linked Immunosorbent Assay) method for detection of methyl-3-quinoxaline-2-carboxylic acid (MQCA), and kit | |
| CN102585006B (en) | Monoclonal antibody, enzyme-linked immunosorbent assay (ELISA) method and kit for detecting neomycin, amikacin and paromomycin | |
| CN101339195A (en) | Elisa kit for checking soluble PD-1 protein and checking method | |
| CN102766208A (en) | Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting T-2 and HT-2 toxin | |
| CN103288872A (en) | Methyl parathion hapten, and preparation method and application thereof | |
| CN105566493B (en) | Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting florfenicol | |
| CN102766212A (en) | Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting residues of fluoroquinolones | |
| CN102766213A (en) | Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting furaltadone residue marker AMOZ | |
| CN104558171B (en) | Monoclonal antibody and enzyme-linked immunoassay method and kit for detecting methyltestosterone | |
| CN104558184B (en) | Monoclonal antibody and enzyme-linked immunoassay method and kit for detecting nitroimidazoles medicine | |
| CN101162230A (en) | Kit for quantitative determining enrofloxacin content in food product and testing method thereof | |
| CN1979169A (en) | Diarrhea sheufish-poison competitive enzyme-linked immune quantitative detection reagent box, its preparation and use | |
| CN102766211B (en) | Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting arsanilic acid, nitarsone and Carbarsone | |
| CN104558176B (en) | Monoclonal antibody and enzyme-linked immunoassay method and kit for detecting androgens medicine | |
| CN105566494A (en) | Monoclonal antibody and enzyme-linked immunosorbent assay method for detecting aflatoxin M1 | |
| CN104388392A (en) | Enrofloxacin monoclonal antibody as well as preparation method and application thereof | |
| CN104558187B (en) | For detecting the monoclonal antibody and enzyme-linked immunoassay method and kit of cephalosporins | |
| CN102608318B (en) | Monoclonal antibody, enzyme linked immunosorbent assay method and kit for detecting sulfonamides | |
| CN102608320B (en) | Monoclonal antibody, ELISA method and kit used for detecting malachite green, leuco malachite green, and leuco crystal violet | |
| CN107677807A (en) | A kind of kitasamycin magnetic immunochemiluminescence detection kit | |
| CN105524168B (en) | Monoclonal antibody, enzyme linked immunosorbent assay method and kit for detecting aflatoxin | |
| CN106565846B (en) | For detecting the monoclonal antibody and enzyme linked immunological kit of amantadine and Rimantadine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140205 Termination date: 20200227 |