CN102585006B - Monoclonal antibody, enzyme-linked immunosorbent assay (ELISA) method and kit for detecting neomycin, amikacin and paromomycin - Google Patents
Monoclonal antibody, enzyme-linked immunosorbent assay (ELISA) method and kit for detecting neomycin, amikacin and paromomycin Download PDFInfo
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Abstract
The invention discloses a specific monoclonal antibody capable of identifying neomycin, amikacin and paromomycin and an enzyme-linked immunosorbent assay (ELISA) method and kit for detecting neomycin, amikacin and paromomycin. The monoclonal antibody is secreted by a hybridoma cell EDC/5G04 and the hybridoma cell is collected in the China Center for Type Culture Collection, with collection number being CCTCC NO:C201144. The ELISA method comprises the steps of preparation of immunogen, coating antigen and the antibody, treatment and detection of samples and the like. Compared with the prior art, the monoclonal antibody, the ELISA method and the kit have the following main advantages that the prepared monoclonal antibody can simultaneously identify neomycin, amikacin and paromomycin, thus improving the detection efficiency of the prior art; the animal tissue sample treatment method is simple and short in time; and the ELISA method and the kit also have the characteristics of high detection sensitivity, good precision and good accuracy.
Description
Technical field
The present invention relates to a kind of monoclonal antibody and a kind of enzyme-linked immunoassay method for detection of Liu Suanyan NEOMYCIN SULPHATE, amikacin and paromycin and the test kit that can identify Liu Suanyan NEOMYCIN SULPHATE, amikacin and paromycin.
Background technology
Aminoglycoside (AMGs) microbiotic is to extract in streptomycete or micromonospora nutrient solution, or take natural product and produce the class water-soluble alkaline microbiotic obtained as raw material is semi-synthetic.What veterinary clinic was commonly used at present has: Streptomycin sulphate, gentamicin, Liu Suanyan NEOMYCIN SULPHATE, kantlex, amikacin etc.It shows the strong disinfecting effect to most gram negative bacillis, and gram-positive bacteria is also had to effect, and main sensitive organism is the enterobacteria sensitive strain, effective to suis, hepatitis coccus, Clostridium, Rickettsiae.Yet due to AMGs easily renal cortex and inner ear perilymph accumulate cause ototoxicity and renal toxicity and the microorganism persister produced by the aminoglycoside deactivating enzyme comparatively serious, so its detection in animal derived food causes people's attention day by day.In view of the toxic side effect of AMGs, every country has all been made strict regulation to the maximum residue limit of AMGs.Enzyme-linked immune detection method (enzyme-linked immunosorbent assay, ELISA) is widely used in the rapid detection field of AMGs gradually because having the advantage such as simple, quick, sensitive, special.But current ELISA method mainly concentrates on single residue detection aspect, by a kind of antibody while detection architecture, the ELISA method of different AMGs seldom.Therefore, the ELISA method that foundation can detect multiple AMGs has great importance for the rapid detection of improving such medicine.
The patent of invention that application number is 200610171540.1 discloses a kind of enzyme linked immunological kit that detects Liu Suanyan NEOMYCIN SULPHATE, and this patent adopts carbodiimide (EDC) method to carry out coupling Liu Suanyan NEOMYCIN SULPHATE and human serum albumin and obtains immunogen; The employing albumin rabbit serum is carrier, and Liu Suanyan NEOMYCIN SULPHATE is haptens, with carboxymethyl azanol-carbodlimide method coupling, obtains the Liu Suanyan NEOMYCIN SULPHATE coating antigen, and prepared polyclonal antibody and monoclonal antibody can only be identified Liu Suanyan NEOMYCIN SULPHATE equally, and the detection time of sample is longer.The patent of invention that application number is 200710064347 discloses a kind of enzyme linked immunological kit and method that detects neomycin drug, this patent adopts method coupling Liu Suanyan NEOMYCIN SULPHATE and bovine serum albumin (the bovine serum albu minute of direct activation albumen, BSA) adaptive immune is former, same method obtains coating antigen by Liu Suanyan NEOMYCIN SULPHATE and thyroprotein coupling, prepared monoclonal antibody equally only can the specific recognition Liu Suanyan NEOMYCIN SULPHATE, and sample preparation time and reagent are not optimized.
Summary of the invention
First purpose of the present invention is to provide a kind of monoclonal antibody that can simultaneously identify Liu Suanyan NEOMYCIN SULPHATE, amikacin and paromycin.
Second purpose of the present invention is to utilize this monoclonal antibody, sets up a kind of enzyme-linked immunoassay method that can detect for Liu Suanyan NEOMYCIN SULPHATE, amikacin and paromycin.
The 3rd purpose of the present invention is to provide a kind of test kit detected for Liu Suanyan NEOMYCIN SULPHATE, amikacin and paromycin.
The 4th purpose of the present invention is to provide the application of described monoclonal antibody in the enzyme linked immunological kit of preparation detection Liu Suanyan NEOMYCIN SULPHATE, amikacin and paromycin.
The 5th purpose of the present invention is to provide the application of test kit in animal tissues's Liu Suanyan NEOMYCIN SULPHATE, amikacin and paromycin residue detection that contains described monoclonal antibody.
The present invention is achieved through the following technical solutions:
A kind of monoclonal antibody that can identify Liu Suanyan NEOMYCIN SULPHATE, amikacin and paromycin, the hybridoma EDC/5G04 that it is is CCTCC NO:C201144 by preserving number is secreted.
Above-mentioned hybridoma EDC/5G04, be deposited in the Chinese Typical Representative culture collection center (CCTCC) that is positioned at Wuhan City, Hubei Province Wuhan University, and its preserving number is CCTCC NO:C201144.
By haptens Liu Suanyan NEOMYCIN SULPHATE and bovine serum albumin coupling, prepared by immunogen used.
Further, the invention provides a kind of enzyme-linked immune detection method that simultaneously detects Liu Suanyan NEOMYCIN SULPHATE, amikacin and paromycin, the method comprises the steps such as the processing of the preparation of immunogen, coating antigen, antibody and sample and detection, specific as follows:
(1) haptens Liu Suanyan NEOMYCIN SULPHATE and bovine serum albumin (BSA) coupling are obtained to immunogen;
(2) haptens Liu Suanyan NEOMYCIN SULPHATE and ovalbumin (OVA) coupling are obtained to coating antigen;
(3) utilize the immunogen immune mouse of step (1), by cytogamy and screening, obtain the hybridoma EDC/5G04 that preserving number is CCTCC NO:C201144;
(4) the hybridoma EDC/5G04 that is CCTCC NO:C201144 with preserving number prepares monoclonal antibody;
(5) with the coated solid phase carrier (as enzyme plate) of the coating antigen of step (2);
(6) phosphate buffer soln for testing sample (PBS, 0.1M, pH10～11) extracted, hatch, centrifugal and dilution obtains determinand solution;
(7) the determinand solution of step (6) carried out to enzyme linked immunosorbent detection,
Wherein:
Component and the proportioning of phosphate buffer soln (0.1M, pH10～11) are: NaCl 20.0g, KH
2PO
40.4g, Na
2HPO
412H
2O 13.4g, KCl 0.5g, dissolve with a small amount of ultrapure water, then is settled to 500mL.
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 Liu Suanyan NEOMYCIN SULPHATE, amikacin and paromycin, in conjunction with above-mentioned enzyme-linked immunoassay method, realized the enzyme linked immunosorbent detection to Liu Suanyan NEOMYCIN SULPHATE, amikacin and paromycin.
Major advantage of the present invention is:
1, the monoclonal anti physical efficiency that prepared by the present invention is Liu Suanyan NEOMYCIN SULPHATE, amikacin and paromycin simultaneously, and existing patent documentation only can single identification Liu Suanyan NEOMYCIN SULPHATE.
2, the tissue sample treatment process the present invention relates to is simple, without expensive instrument, without organic reagent, to the operator, without Health hazard, and only needs generic centrifuge to get final product, and is adapted at basic unit's operation.
3, compared with prior art, the tissue sample treatment time is short, and detection time is short, and detection efficiency is high.
The accompanying drawing explanation
The mass spectrum that the substance assistant laser desorpted method that Fig. 1 is carrier proteins BSA of the present invention detects.
Fig. 2 is the mass spectrum that the immunogenic substance assistant laser desorpted method of the present invention detects, and is used for illustrating the coupling effect of haptens Liu Suanyan NEOMYCIN SULPHATE and BSA in conjunction with Fig. 1.
The indirect competitive ELISA response curve that Fig. 3 is monoclonal antibody of the present invention and Liu Suanyan NEOMYCIN SULPHATE (NEO) standard substance, X-axis is Liu Suanyan NEOMYCIN SULPHATE (NEO) concentration of standard solution logarithmic value, and the optical density value that Y-axis is the Liu Suanyan NEOMYCIN SULPHATE 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 immunogen and coating antigen
1.1 Liu Suanyan NEOMYCIN SULPHATE-BSA's is synthetic
Take Liu Suanyan NEOMYCIN SULPHATE 10.0mg and BSA100.0mg and be dissolved in 20mL PBS liquid (pH7.4), stir, dropwise add the 50.0mg EDC be dissolved in the 1mL pure water, at room temperature stirring reaction is 8 hours.Finally reaction solution is proceeded in dialysis tubing, dialyse 4 days for 4 ℃ in PBS (pH7.4) liquid, centrifugal postlyophilization.As shown in Figure 1, 2, through substance assistant laser desorpted method (MALDI-TOF-MS), identify the coupling success, put-20 ℃ and save backup.
1.2 Liu Suanyan NEOMYCIN SULPHATE-OVA's is synthetic
Take Liu Suanyan NEOMYCIN SULPHATE 20.0mg and OVA200.0mg and be dissolved in 20mL PBS liquid (pH7.4), stir, then slowly add the 80.00mgEDC be dissolved in the 1mL pure water.At room temperature stirring reaction is 2 hours.Finally reaction solution is proceeded in dialysis tubing, dialyse 4 days for 4 ℃ in PBS (pH7.4) liquid.Centrifugal postlyophilization, put-20 ℃ and save backup.
The preparation of embodiment 2 monoclonal antibodies
The preparation of hybridoma: with reference to Yang Hanchun " animal immunology ", the immunogen Liu Suanyan NEOMYCIN SULPHATE prepared with embodiment 1-BSA immunity Balb/C mouse (purchased from Disease Prevention Control Center, Hubei Prov's Experimental Animal Center), immune programme for children is: fundamental immunity is by after immunogen 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 Freunds incomplete adjuvant emulsification instead later.Finally in merging first three day (be better than most after immunity finishes and rest and reorganize January) abdominal 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 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 the 50mL centrifuge tube, with 15mLRPMI-1640 basal liquid re-suspended cell, 1500r/ minute centrifugal 5 minutes, wash cell 1 time.The substratum 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 on the centrifuge tube of myeloma cell and immune spleen cell and emptying to the greatest extent, tip upside down on control solid carbon dioxide on thieving paper and drip, rap the pipe end to make cell loosening.Open timing register, with the 1mL suction pipe, draw 0.8mLPEG, the hand-held centrifuge tube that cell mixing is housed, place it in a moment in water-bath, and PEG is added drop-wise on cell mixing slowly, and the limit edged stirs gently, in 1 minute, adds, and continues to stir 30 seconds.Get the 10mL basal liquid with suction pipe, slowly be added on fused cell along tube wall, the limit edged shakes (can not blow and beat) gently, in 5 minutes, adds respectively 1mL, 2mL, 3mL, 4mL, finally add basal liquid to 40mL, after adding, cover lid, put upside down several times repeatedly, and cell is mixed.800r/ minute 5 minutes centrifugal, abandons supernatant.The HAT substratum that absorption contains feeder cell, stir the fused cell in centrifuge tube gently with suction pipe, near liquid level, dropwise splashes into containing in the serum bottle of feeder cell, and stirring and evenly mixing, 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, be placed in incubator and cultivate.Single cell fusion can be inoculated 4～6 96 orifice plates.Also can plant less as required, generally press the cell count of SP2/0 and calculate, every hole inoculum size is approximately containing 104 a left and right SP2/0 cell.In 37 ℃, 5%CO
2In incubator, cultivate.
Counted 0 day the same day of merging, and the first 3 days kinetocyte plates of trying not keep the incubator homeostasis.Within the 3rd day, 1 HAT perfect medium is added in every hole; The 5th day every hole sucking-off 1/2 culture supernatant (100 μ L), then add 1 HT perfect medium; Suck 1/2～3/4 culture supernatant every 2 days the same methods later, changed to the HT perfect medium after 7 days.
Treat that the fused cell colony grows to culture hole 1/10～1/5, screened by indirect ELISA method and the indirect competitive ELISA method set up simultaneously.With zero medicine hole, compare, 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 1-2 single colony only arranged of 2～6 strong positives, adopt the limiting dilution method to carry out cloning,
Through 3～4 time clonings, until clone's positive rate is 100%, finishing screen is selected the hybridoma of secretion anti-Liu Suanyan NEOMYCIN SULPHATE, amikacin and paromycin.Dyed body numeration, the karyomit(e) mean number of this cell strain is 102.8.The applicant was this hybridoma called after EDC/5G04, and delivered the Chinese Typical Representative culture collection center preservation that is positioned at Wuhan City, Hubei Province Wuhan University on June 29th, 2011, and its preserving number is CCTCC NO:C201144.
The preparation of ascites monoclonal antibody and evaluation: only within first 7 days, get the Balb/c number of mice in inoculation, every mouse peritoneal injection 0.5ml Freund's incomplete adjuvant carries out pre-treatment.The cell of the hybridoma EDC/5G04 enlarged culturing that is CCTCC NO:C201144 by preserving number with the suspension of RPMI-1640 basic medium, and cell count 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 the 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 Liu Suanyan NEOMYCIN SULPHATE racing ELISA detecting methods
3.1 the preparation of reagent (reagent that the present embodiment is used all adopts the 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.
Washings (pH7.4): accurately take NaCl 8.00g, KH
2PO
40.20g, Na
2HPO
412H
2O 2.90g, KCl0.20g, a small amount of ultrapure water dissolves, and adds polysorbas20 0.50mL, is settled to 1000mL.
Phosphate buffered saline buffer (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 is settled to 1000mL.
Confining liquid: accurately take ovalbumin 10.00g, add the 1000mL phosphate buffered saline buffer, stirring and evenly mixing until albumen dissolve fully.
Physiological saline: accurately take NaCl 8.50g, a small amount of ultrapure water dissolves, and is settled to 1000mL.
Antibody diluent, enzymic-labelled antibody diluent and substrate solution provide by Wuhan Fei Yuan Science and Technology Ltd..
Stop buffer: accurately measure vitriol oil 100mL, slowly be added drop-wise in the 800mL ultrapure water.
3.2 the preliminary of coating antigen concentration and antibody working concentration determined
At 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 coating antigen Liu Suanyan NEOMYCIN SULPHATE-OVA doubling dilution is become to the horizontal coated elisa plate of 8,4,2,1,0.5,0.25,0.125,0.0625 μ g/mL; The EDC/5G04 monoclonal antibody is used the phosphate buffered saline buffer doubling dilution to become 1: 1000, within 1: 2000,1: 4000,1: 8000,1: 16000,1: 32000,1: 64000,1: 128000,1: 256000,1: 512000, vertically add enzyme plate.8000), (1,1: 16000) and (2,1: 32000) the square formation titration results is in Table the combination of the following coating antigen concentration of 1 initial option and antibody working concentration: (1,1:.
The titration of table 1EDC/5G04 monoclonal antibody square formation
3.3 determining of best coating antigen concentration and antibody working concentration
Best coated concentration is determined: the coated concentration of selecting with the square formation titration and antibody dilution combination are done respectively to suppress curve, and Liu Suanyan NEOMYCIN SULPHATE standard substance concentration is set to 0,1,3,5,7,9 μ g/mL, its zero medicine hole OD value and IC
50Value is the keys that affect its sensitivity in Table the ratio of 2 antigen-antibodies, if there is antigen or antibody excess, all will cause IC
50Higher.According to IC
50The linearly dependent coefficient of value, zero medicine hole OD value and inhibition curve, determine that best coated concentration is 1 μ g/mL, and antibody dilution tentatively is defined as 1: 16000.
The best coated concentration optimization of table 2
The optimum antibody extent of dilution is determined: with the coated concentration coated elisa plate of the best, by antibody 5 dilution gradients of concentration equal difference design centered by 1: 16000, its zero medicine hole OD value and IC
50Value is in Table 3 reductions along with antibody dilution, IC
50Value raises, and the OD value in zero medicine hole also raises; Along with the increase of antibody dilution, IC
50Value reduces, and zero medicine OD hole value also reduces.Comprehensive IC
50The linearly dependent coefficient of value, zero hole OD value and inhibition curve, select 1: 20000 for the optimum antibody extent of dilution.
Table 3 optimum antibody extent of dilution is optimized
| Antibody coefficient multiple (1: X) | Zero medicine hole OD value | IC 50Value (μ g/L) |
| 8000 | 2.623 | 7.69 |
| 12000 | 2.462 | 3.51 |
| 16000 | 2.291 | 3.35 |
| 20000 | 2.097 | 3.36 |
| 24000 | 2.064 | 2.50 |
3.4 the foundation of typical curve
Liu Suanyan NEOMYCIN SULPHATE standard substance concentration is set to 6 concentration gradients such as 0,1,3,5,7,9 μ g/L, measures the drawing standard curve according to top definite indirect competitive ELISA method.As shown in Figure 3, the present invention be take Liu Suanyan NEOMYCIN SULPHATE and is that regression equation and the linear dependence index of the indirect competitive ELISA method that standard substance are set up are respectively: y=-0.683x+0.860, r=0.999, IC
50Value is 3.16 ± 0.42 μ g/L (n=5).Linearity range is 1～9 μ g/L.
3.5 specificity
Become concentration gradient to carry out indirect competitive ELISA various AMGs doubling dilutions commonly used respectively, calculate IC
50Value, with Liu Suanyan NEOMYCIN SULPHATE standard substance IC
50The value 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 100.00%, 80.90%, 151.25% to the cross reacting rate of Liu Suanyan NEOMYCIN SULPHATE, amikacin, paromycin, and with the equal no cross reaction of other AMGs.
The specificity of table 4 racing ELISA detecting method of the present invention
| The competition thing | IC 50(μg/L) | Cross reacting rate (%) |
| Liu Suanyan NEOMYCIN SULPHATE | 3.16 | 100.00 |
| Amikacin | 3.91 | 80.90 |
| Paromycin | 2.09 | 151.25 |
| Neomycin A | ＞10000 | ＜0.03 |
| Gentamicin | ＞10000 | ＜0.03 |
| Kantlex | ＞10000 | ＜0.03 |
| Streptomycin sulphate | ＞10000 | ＜0.03 |
| Ribostamycin | ＞10000 | ＜0.03 |
| Spectinomycin | ＞10000 | ＜0.03 |
| Apramycin | ＞10000 | ＜0.03 |
| Tobramycin | ＞10000 | ＜0.03 |
| Sisomicin | ＞10000 | ＜0.03 |
| Totomycin | ＞10000 | ＜0.03 |
| Kasugamycin | ＞10000 | ＜0.03 |
The assembling of embodiment 4 Liu Suanyan NEOMYCIN SULPHATE of the present invention, amikacin and many residue detection of paromycin ELISA test kit
4.1 the composition of ELISA test kit of the present invention
1) be coated with the solid phase carrier (enzyme plate) of coating antigen Liu Suanyan NEOMYCIN SULPHATE-OVA;
2) the Liu Suanyan NEOMYCIN SULPHATE standardized solution is 6 bottles, and concentration is respectively 0 μ g/L, 1 μ g/L, 3 μ g/L, 5 μ g/L, 7 μ g/L, 9 μ g/L;
3) Liu Suanyan NEOMYCIN SULPHATE 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, add 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, add ultrapure water to 1000mL;
7) substrate solution A: by Wuhan, Fei Yuan scientific & technical corporation provides;
8) substrate solution B: by Wuhan, Fei Yuan scientific & technical corporation provides;
9) stop buffer: 2mol/L sulphuric acid soln.
4.2 the preparation of enzyme plate
With coating buffer, Liu Suanyan NEOMYCIN SULPHATE-OVA is diluted to 1 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 washingss, 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, washings washing 3 times, pat dry; Enzyme plate is inverted in to 37 ℃ of incubators, standing oven dry 30 minutes, enzyme plate is dried the rear aluminium foil bag of packing into together with siccative, with vacuum packaging machine, encapsulates.
The mensuration program of embodiment 5 enzyme linked immunological kits of the present invention
5.1 the preparation of reagent
1) washings: the washings provided in test kit is used afterwards with 10 times of ultrapure water dilutions.
2) substrate mixed solution: according to each institute expense, substrate solution A and the substrate solution B of preparation are mixed in by volume 1: 100, now with the current.
3) component and the proportioning of phosphate buffer soln (0.1M, pH10～11) are: NaCl 20.0g, KH
2PO
40.4g, Na
2HPO
412H
2O 13.4g, KCl 0.5g, dissolve with a small amount of ultrapure water, then is settled to 500mL.
5.2 tissue sample pre-treatment
The pre-treatment of pig muscle tissue: take the homogeneous sample 1.00 ± 0.01g of muscle in the 50mL centrifuge tube, add phosphate buffer soln (0.1M, pH10～11) 10mL, fully vortex mixes 1～2 minute, 60 ℃ hatch 30 minutes after, taking-up is cooled to room temperature and shakes up, 4000r/ minute, centrifugal 10 minutes of room temperature, get supernatant liquor, with phosphate buffer soln (0.1M, the pH10～11) dilution of 4 times of volumes, get 50 μ L loadings.
Annotate: present method is 50 to the extension rate of pig muscle.
5.3 determination step
1) application of sample: add Liu Suanyan NEOMYCIN SULPHATE series concentration standardized solution or sample solution 50 μ L in the enzyme plate micropore, then add Liu Suanyan NEOMYCIN SULPHATE 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 washings 250 μ L in every hole, after standing 30 seconds, wash 3 times and pat dry;
3) add ELIAS secondary antibody: add ELIAS secondary antibody working fluid 100 μ L in every hole, in 37 ℃ of wet boxes, constant-temperature incubation is 30 minutes;
4) washing: pour out the liquid in hole, add washings 250 μ L in every hole, after standing 30 seconds, wash 3 times and pat dry;
5) add substrate: add substrate mixed solution 100 μ L in every hole, hatch 15 minutes in 37 ℃ of wet boxes;
6) add stop buffer: add stop buffer 50 μ L in every hole;
7) measure: measure the optical density value (OD value) in every hole at the 450nm place by microplate reader.
5.4 result judgement
With the standard substance OD value measured divided by " zero " hole OD value (B/B
0) be ordinate zou, the logarithmic value of Liu Suanyan NEOMYCIN SULPHATE concentration is that X-coordinate 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 1 calculation sample, by inhibiting rate substitution regression equation, calculate mensuration concentration, be multiplied by corresponding extension rate, be the residual concentration of Liu Suanyan NEOMYCIN SULPHATE in sample, amikacin and paromycin.
(formula 1)
Sensitivity, the preci-sion and accuracy of embodiment 6 test kits of the present invention
6.1 the sensitivity of test 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 Liu Suanyan NEOMYCIN SULPHATE standard substance are diluted to 0 μ g/L, 1 μ g/L, 3 μ g/L, 5 μ g/L, 7 μ g/L, 9 a μ g/L6 concentration, and 5 repeating holes of each concentration, according to indirect competitive ELISA method replication 5 times, get the IC measured for 5 times
50Mean value.LOD determines by following steps, measures the OD value of the muscle tissue of 20 parts of blank pigs, according to the regression equation calculation of typical curve, goes out corresponding Liu Suanyan NEOMYCIN SULPHATE concentration, then calculates the mean value of Liu Suanyan NEOMYCIN SULPHATE concentration
And standard deviation (SD), according to formula
Calculate the lowest detectable limit in tissue.IC of the present invention
50Value is 3.16 ± 0.42 μ g/L, and the lowest detection of Liu Suanyan NEOMYCIN SULPHATE in pig muscle is limited to 15.90 μ g/L.
6.2 the precision of test kit of the present invention
The Liu Suanyan NEOMYCIN SULPHATE standard substance are diluted to 0 μ g/L, 1 μ g/L, 3 μ g/L, 5 μ g/L, 7 μ g/L, 9 a μ g/L6 concentration, 5 repetitions 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 Liu Suanyan NEOMYCIN SULPHATE standardized solution, the variation coefficient in computing board and between plate, the results are shown in Table 5.
The precision of table 5 test kit of the present invention
6.3 the accuracy of test kit of the present invention
Add the Liu Suanyan NEOMYCIN SULPHATE standardized solution in the homogenate pig muscle tissue of 1g, make its final concentration be respectively 200 μ g/kg, 100 μ g/kg and 50 μ g/kg; Add the amikacin standardized solution, make its final concentration be respectively 200 μ g/kg, 100 μ g/kg and 50 μ g/kg; Add the paromycin standardized 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.Measure the concentration of adding Liu Suanyan NEOMYCIN SULPHATE, amikacin and paromycin in tissue, calculate recovery rate, examine the accuracy of test kit according to the following equation; Calculate batch interior and interassay coefficient of variation, the repeatability of examination test kit.Accuracy and repeatability the results are shown in Table 6, table 7 and table 8, show that this test kit has reliable accuracy, in batch and interassay coefficient of variation little, reproducible.
In table 6 pig muscle, Liu Suanyan NEOMYCIN SULPHATE adds the rate of recovery and the variation coefficient
In table 7 pig muscle, paromycin adds the rate of recovery and the variation coefficient
In table 8 pig muscle, amikacin adds the rate of recovery and the variation coefficient
Claims (7)
1. the monoclonal antibody that can identify Liu Suanyan NEOMYCIN SULPHATE, amikacin and paromycin, is characterized in that, it is that CCTCC NO:C201144 hybridoma EDC/5G04 is secreted by preserving number.
2. the hybridoma EDC/5G04 described in claim 1, be deposited in Chinese Typical Representative culture collection center, and its preserving number is CCTCC NO:C201144.
3. the test kit that comprises monoclonal antibody claimed in claim 1.
4. test kit according to claim 3, this test kit is the enzyme linked immunological kit for detection of Liu Suanyan NEOMYCIN SULPHATE, amikacin and paromycin.
5. the enzyme-linked immunoassay method for detection of Liu Suanyan NEOMYCIN SULPHATE, amikacin and paromycin, comprise the preparation of coating antigen, antibody and processing and the detection of sample, and its step is as follows:
(1) haptens Liu Suanyan NEOMYCIN SULPHATE and ovalbumin coupling are obtained to coating antigen;
(2) the hybridoma EDC/5G04 that is CCTCC NO:C201144 with preserving number prepares monoclonal antibody;
(3) with the coated solid phase carrier of the coating antigen of step (1);
(4) phosphate buffer soln of 0.1M, pH10～11 for testing sample extracted, hatch, centrifugal and dilution obtains determinand solution;
(5) the determinand solution of step (4) 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 Liu Suanyan NEOMYCIN SULPHATE, amikacin and paromycin.
7. the application of the described test kit of claim 3 or 4 in animal tissues's Liu Suanyan NEOMYCIN SULPHATE, amikacin and paromycin residue detection.
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| CN103232358B (en) * | 2012-11-05 | 2015-07-29 | 南京工业大学 | For detecting the vesica probe of Liu Suanyan NEOMYCIN SULPHATE, purposes and preparation method |
| 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 |
| 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 |
| CN107058241B (en) * | 2017-03-20 | 2019-08-13 | 江南大学 | The anti-amikacin monoclonal antibody hybridoma cell strain AB2 of one plant of secretion and its application |
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| CN1996022A (en) * | 2006-12-30 | 2007-07-11 | 中国兽医药品监察所 | Enzyme-linked immunologic kit for detecting neomycin |
| CN101021536A (en) * | 2007-03-12 | 2007-08-22 | 北京望尔康泰生物技术有限公司 | Enzyme-linked immunological kit for detecting neomycin drug and method |
| CN102778563A (en) * | 2012-05-31 | 2012-11-14 | 华中农业大学 | Colloidal gold test strip for detecting neomycin residual and use method and application thereof |
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| CN1996022A (en) * | 2006-12-30 | 2007-07-11 | 中国兽医药品监察所 | Enzyme-linked immunologic kit for detecting neomycin |
| CN101021536A (en) * | 2007-03-12 | 2007-08-22 | 北京望尔康泰生物技术有限公司 | Enzyme-linked immunological kit for detecting neomycin drug and method |
| CN102778563A (en) * | 2012-05-31 | 2012-11-14 | 华中农业大学 | Colloidal gold test strip for detecting neomycin residual and use method and application thereof |
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