CN102585006A - 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 and the test kit that is used to detect Xin Meisu, amikacin and paromycin that can discern Xin Meisu, amikacin and paromycin.
Background technology
Aminoglycoside (AMGs) microbiotic is by extracting in streptomycete or the micromonospora nutrient solution, or is the semi-synthetic one type of water-soluble alkaline microbiotic that obtains of producing of raw material with natural article.What veterinary clinic was commonly used at present has: Streptomycin sulphate, qingfengmeisu qiong, Xin Meisu, kantlex, amikacin etc.It shows the strong disinfecting effect to most gram negative bacillis, and gram-positive bacteria is also had effect, and main sensitive organism is the enterobacteria sensitive strain, and is effective to suis, hepatitis coccus, Clostridium, Rickettsiae.Yet to be prone to accumulate the mikrobe persister that causes ototoxicity and renal toxicity and produced by the aminoglycoside deactivating enzyme at renal cortex and inner ear perilymph comparatively serious owing to AMGs, so its detection paid more and more attention in animal derived food.In view of the toxic side effect of AMGs, each country has all made strict regulation to the maximum residue limit of AMGs.(enzyme-linked immunosorbent assay ELISA) is widely used in the rapid detection field of AMGs to enzyme-linked immune detection method gradually because having advantage such as simple, quick, sensitive, special.But present ELISA method mainly concentrates on single residue detection aspect, with the ELISA method of a kind of antibody AMGs that detection architecture is different simultaneously seldom.Therefore, the foundation ELISA method that can detect multiple AMGs has great importance for the rapid detection of improving such medicine.
Application number is that 200610171540.1 patent of invention discloses a kind of enzyme linked immunological kit that detects Xin Meisu, and this patent adopts carbodiimide (EDC) method to carry out coupling Xin Meisu and human serum albumin and obtains immunogen; The employing albumin rabbit serum is a carrier, and Xin Meisu is a haptin, obtains the Xin Meisu coating antigen with ethyloic azanol-carbodlimide method coupling, and prepared polyclonal antibody and monoclonal antibody can only be discerned Xin Meisu equally, and the detection time of sample is longer.Application number is that 200710064347 patent of invention discloses a kind of enzyme linked immunological kit and method that detects neomycin drug; This patent adopts direct activation proteic method coupling Xin Meisu and bovine serum albumin (bovine serum albu minute; BSA) obtain immunogen; Same method obtains coating antigen with Xin Meisu and thyroprotein coupling, and prepared monoclonal antibody equally only can the specific recognition Xin Meisu, and sample preparation time and reagent are not optimized.
Summary of the invention
First purpose of the present invention provides a kind of monoclonal antibody that can discern Xin Meisu, amikacin and paromycin simultaneously.
Second purpose of the present invention is to utilize this monoclonal antibody, sets up a kind of enzyme-linked immunoassay method that can be used for Xin Meisu, amikacin and paromycin detection.
The 3rd purpose of the present invention provides a kind of test kit that Xin Meisu, amikacin and paromycin detect that is used for.
The 4th purpose of the present invention provides the application of said monoclonal antibody in the enzyme linked immunological kit of preparation detection Xin Meisu, amikacin and paromycin.
The 5th purpose of the present invention provides the application of test kit in animal tissues's Xin Meisu, amikacin and paromycin residue detection that contains said monoclonal antibody.
The present invention realizes through following technical scheme:
A kind of monoclonal antibody that can discern Xin Meisu, amikacin and paromycin, it is to be that the hybridoma EDC/5G04 of CCTCC NO:C201144 is secreted by preserving number.
Above-mentioned hybridoma EDC/5G04 is deposited in the Chinese typical culture collection center (CCTCC) that is positioned at Wuhan City, Hubei Province Wuhan University, and its preserving number is CCTCC NO:C201144.
Used immunogen is by haptin Xin Meisu and bovine serum albumin coupling preparation.
Further, the invention provides a kind of enzyme-linked immune detection method that detects Xin Meisu, amikacin and paromycin simultaneously, this method comprises the step such as processing and detection of preparation and the sample of immunogen, coating antigen, antibody, and is specific as follows:
(1) haptin Xin Meisu and bovine serum albumin (BSA) coupling are obtained immunogen;
(2) haptin Xin Meisu and ovalbumin (OVA) coupling are obtained coating antigen;
(3) utilize the immunogen immune mouse of step (1), obtain the hybridoma EDC/5G04 that preserving number is CCTCC NO:C201144 through cytogamy and screening;
(4) use preserving number to prepare monoclonal antibody as the hybridoma EDC/5G04 of CCTCC NO:C201144;
(5) coating antigen with step (2) encapsulates solid phase carrier (like enzyme plate);
(6) with testing sample with phosphate buffer soln (PBS, 0.1M, pH10～11) extract, hatch, centrifugal and dilution obtains determinand solution;
(7) the determinand solution to step (6) carries out enzyme linked immunosorbent detection,
Wherein:
The 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 with a small amount of ultrapure water dissolving, is settled to 500mL then.
The present invention with said monoclonal antibody and coating antigen as core reagent and other conventional agent combination; Processed the enzyme linked immunological kit that can detect Xin Meisu, amikacin and paromycin; In conjunction with above-mentioned enzyme-linked immunoassay method, realized enzyme linked immunosorbent detection to Xin Meisu, amikacin and paromycin.
Major advantage of the present invention is:
1, monoclonal anti physical efficiency while Xin Meisu, amikacin and the paromycin of the present invention's preparation, and existing patent documentation only can single identification Xin Meisu.
2, the tissue sample treatment process that the present invention relates to is simple, need not expensive instrument, need not organic reagent, the operator is not had Health hazard, and only need 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.
Description of drawings
Fig. 1 is the mass spectrum of the substance assistant laser desorpted method detection of carrier proteins BSA of the present invention.
Fig. 2 is the mass spectrum that the immunogenic substance assistant laser desorpted method of the present invention detects, and is used to explain the coupling effect of haptin Xin Meisu and BSA in conjunction with Fig. 1.
Fig. 3 is the indirect competitive ELISA response curve of monoclonal antibody of the present invention and Xin Meisu (NEO) standard substance; The X axle is Xin Meisu (NEO) concentration of standard solution logarithmic value, and the Y axle is that the OD value of Xin Meisu standard solution is divided by " zero " hole OD value (B/B0).
Embodiment
Through embodiment the present invention is described further below.
The preparation of embodiment 1 immunogen and coating antigen
1.1 Xin Meisu-BSA's is synthetic
Take by weighing Xin Meisu 10.0mg and BSA100.0mg and be dissolved in the 20mL PBS liquid (pH7.4), stir, dropwise adding is dissolved in the 50.0mg EDC in the 1mL pure water, and at room temperature stirring reaction is 8 hours.At last reaction solution is changed in the dialysis tubing, in PBS (pH7.4) liquid, dialysed centrifugal postlyophilization 4 days for 4 ℃.Like Fig. 1, shown in 2, identify the coupling success through substance assistant laser desorpted method (MALDI-TOF-MS), it is subsequent use to put-20 ℃ of preservations.
1.2 Xin Meisu-OVA's is synthetic
Take by weighing Xin Meisu 20.0mg and OVA200.0mg and be dissolved in 20mL PBS liquid (pH7.4), stir, slowly adding is dissolved in the 80.00mgEDC in the 1mL pure water then.At room temperature stirring reaction is 2 hours.At last reaction solution is changed in the dialysis tubing, in PBS (pH7.4) liquid, dialysed 4 days for 4 ℃.Centrifugal postlyophilization, it is subsequent use to put-20 ℃ of preservations.
Embodiment 2 MONOCLONAL ANTIBODIES SPECIFIC FOR
The preparation of hybridoma: with reference to Yang Hanchun " animal immunology "; Immunogen Xin Meisu-BSA immunity Balb/C mouse (available from Disease Prevention Control Center, Hubei Prov's Experimental Animal Center) with embodiment 1 preparation; Immune programme for children is: fundamental immunity with immunogen and isopyknic Freund's complete adjuvant emulsification after; In the subcutaneous multi-point injection of mouse back, later every interval 2 all booster immunizations are once used Freund emulsification instead.At last in merging first three day (be better than most immunity finish the back 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 prepared fresh 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 the centrifugal gap, the water that temperature is bathed, the PEG that temperature is bathed etc. puts into super clean bench.Take out the thieving paper of sterilization then, after emptying to the greatest extent on the centrifuge tube that myeloma cell and immune spleen cell are housed, tip upside down on that the control solid carbon dioxide drips on the thieving paper, rap the pipe end cell is become flexible.Open timing register, draw 0.8mLPEG with the 1mL suction pipe, the hand-held centrifuge tube that cell mixing is housed places it in a moment in the water-bath, and PEG is added drop-wise on the cell mixing slowly, and the limit edged stirs gently, adds in 1 minute, continues to stir 30 seconds.Get the 10mL basal liquid with suction pipe, slowly be added on the fused cell along tube wall, the limit edged shakes (can not blow and beat) gently, adds 1mL respectively in 5 minutes; 2mL, 3mL, 4mL adds basal liquid at last to 40mL; After adding, cover lid is put upside down several times repeatedly, makes the cell mixing.800r/ minute 5 minutes centrifugal, abandons supernatant.The HAT substratum that absorption contains feeder cell stirs the fused cell in the centrifuge tube with suction pipe gently, dropwise splashes near the liquid level in the serum bottle that contains feeder cell, and stirring and evenly mixing, action will gently be stirred cell gently, must not blow and beat.Put upside down mixing.Then with cell inoculation on Tissue Culture Plate, two in every hole places incubator to cultivate.Once merge and to inoculate 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 contains about 104 SP2/0 cells approximately.In 37 ℃, 5%CO
2Cultivate in the incubator.
Counted 0 day the same day of merging, and the preceding 3 days kinetocyte plates of trying not keep the incubator homeostasis.1 HAT perfect medium was added in every hole in the 3rd day; The 5th day every hole sucking-off 1/2 culture supernatant (100 μ L) adds 1 HT perfect medium again; Later on every at a distance from 2 days the same method suction go 1/2～3/4 culture supernatant, after 7 days, change to the HT perfect medium.
Treat that the fused cell colony grows to culture hole 1/10～1/5, screen with indirect ELISA method and the indirect competitive ELISA method set up 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 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, be 100% until clone's positive rate, finishing screen is selected the hybridoma of secretion anti-Xin Meisu, amikacin and paromycin.Dyed body numeration, the karyomit(e) mean number of this cell strain is 102.8.The applicant is this hybridoma called after EDC/5G04, and delivers the Chinese typical 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.
Preparation of ascites monoclonal antibody and evaluation: only got the Balb/c number of mice in preceding 7 days in inoculation, every mouse peritoneal injection 0.5ml Freund's incomplete adjuvant carries out pre-treatment.Use the RPMI-1640 basic medium to suspend, and cell count is transferred to 1 * 10 by the cell of preserving number as the hybridoma EDC/5G04 enlarged culturing of CCTCC NO:C201144
6Individual/mL, every mouse peritoneal inoculation 0.5ml.Treat that mouse web portion obviously expands, spiritual variation, the dying ascites of gathering 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 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 IgG1 hypotype.
The foundation of embodiment 3 Xin Meisu racing ELISA detecting methods
3.1 the preparation of reagent (reagent that present embodiment uses all adopts following method preparation except that other indicates)
Carbonate buffer solution (pH9.6): accurately take by weighing Na
2CO
31.59g, NaHCO
32.93g a small amount of ultrapure water dissolving is settled to 1000mL.
Washings (pH7.4): accurately take by weighing NaCl 8.00g, KH
2PO
40.20g, Na
2HPO
412H
2O 2.90g, KCl0.20g, a small amount of ultrapure water dissolving adds polysorbas20 0.50mL, is settled to 1000mL.
Phosphate buffered saline buffer (pH7.4): accurately take by weighing NaCl 8.00g, KH
2PO
40.20g, Na
2HPO
412H
2O 2.90g, KCl 0.20g, a small amount of ultrapure water dissolving is settled to 1000mL.
Confining liquid: accurately take by weighing ovalbumin 10.00g, add the 1000mL phosphate buffered saline buffer, stirring and evenly mixing dissolves until albumen fully.
Saline water: accurately take by weighing NaCl 8.50g, a small amount of ultrapure water dissolving is settled to 1000mL.
Antibody diluent, enzymic-labelled antibody diluent and substrate solution fly Science and Technology Ltd. far away by Wuhan to be provided.
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 confirmed
It at first is combination through square formation titrating method initial option coating antigen and antibody working concentration.Use carbonate buffer solution that coating antigen Xin Meisu-OVA doubling dilution is become 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, was vertically added enzyme plate in 1: 2000,1: 4000,1: 8000,1: 16000,1: 32000,1: 64000,1: 128000,1: 256000,1: 512000.8000), (1,1: 16000) and (2,1: 32000) the square formation titration results is seen the combination of the following coating antigen concentration of table 1 initial option and antibody working concentration: (1,1:.
The titration of table 1EDC/5G04 monoclonal antibody square formation
3.3 confirming of best coating antigen concentration and antibody working concentration
The best encapsulates concentration and confirms: the concentration that encapsulates so that the square formation titration is selected is done respectively to suppress curve with the antibody dilution combination, and Xin Meisu standard substance concentration is set to 0,1,3,5,7,9 μ g/mL, its zero medicine hole OD value and IC
50Value sees that the ratio of table 2 antigen-antibody is the key that influences its sensitivity, if 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 confirms that it is 1 μ g/mL that the best encapsulates concentration, and antibody dilution is tentatively confirmed as 1: 16000.
Table 2 the best encapsulates concentration optimization
The optimum antibody extent of dilution is confirmed: encapsulating the concentration coated elisa plate with the best, was 5 dilutions of centre concentration equal difference design gradient with antibody with 1: 16000, its zero medicine hole OD value and IC
50Value is seen the reduction of table 3 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, selecting 1: 20000 is 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
Xin Meisu 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 is that the regression equation and the linear dependence index of the indirect competitive ELISA method of standard substance foundation is respectively: y=-0.683x+0.860, r=0.999, IC with the Xin Meisu
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 is with Xin Meisu standard substance IC
50The value contrast obtains cross reacting rate, and the result sees table 4.The 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 Xin Meisu, amikacin, paromycin, and with the equal no cross reaction of other AMGs.
(formula 1)
The specificity of table 4 racing ELISA detecting method of the present invention
| The competition thing | IC 50(μg/L) | Cross reacting rate (%) |
| Xin Meisu | 3.16 | 100.00 |
| Amikacin | 3.91 | 80.90 |
| Paromycin | 2.09 | 151.25 |
| Neomycin A | ＞10000 | ＜0.03 |
| Qingfengmeisu qiong | ＞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 Xin Meisus 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) is coated with the solid phase carrier (enzyme plate) of coating antigen Xin Meisu-OVA;
2) the Xin Meisu 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) Xin Meisu 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: flying scientific & technical corporation far away by Wuhan provides;
8) substrate solution B: flying scientific & technical corporation far away by Wuhan provides;
9) stop buffer: 2mol/L sulphuric acid soln.
4.2 the preparation of enzyme plate
With coating buffer Xin Meisu-OVA is diluted to 1 μ g/mL, every hole adds 100 μ L, and 4 ℃ encapsulated 12～16 hours; The coating buffer that inclines, every hole adds 250 μ L washingss, washs 3 times, claps and does, and every then hole adds confining liquid 250 μ L, hatches 2 hours for 37 ℃; The liquid in the hole that inclines, washings washing 3 times is clapped and is done; Enzyme plate is inverted in 37 ℃ of incubators, leaves standstill oven dry 30 minutes, enzyme plate oven dry back and the siccative aluminium foil bag of packing into together encapsulates with vacuum packaging machine.
The mensuration program of embodiment 5 enzyme linked immunological kits of the present invention
5.1 the preparation of reagent
1) washings: the washings that provides in the test kit is used with after 10 times of the ultrapure water dilutions.
2) substrate mixed solution:,, at present join existing usefulness with substrate solution A and by volume 1: 100 mixing of substrate solution B of preparation according to each institute expense.
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 with a small amount of ultrapure water dissolving, is settled to 500mL then.
5.2 tissue sample pre-treatment
The pre-treatment of pig muscle tissue: take by weighing the even quality sample 1.00 ± 0.01g of muscle in the 50mL centrifuge tube, add phosphate buffer soln (0.1M, pH10～11) 10mL; Fully the vortex mixing is 1～2 minute, 60 ℃ hatch 30 minutes after, take out and be cooled to room temperature and shake up; 4000r/ minute, centrifugal 10 minutes of room temperature was got supernatant; With phosphate buffer soln (0.1M, the pH10～11) dilution of 4 times of volumes, get appearance on the 50 μ L.
Annotate: present method is 50 to the extension rate of pig muscle.
5.3 determination step
1) application of sample: in the enzyme plate micropore, add Xin Meisu series concentration standardized solution or sample solution 50 μ L, add Xin Meisu monoclonal antibody working fluid 50 μ L then, place wet box, 37 ℃ of constant temperature were hatched 30 minutes;
2) washing: pour out the liquid in the hole, add washings 250 μ L in every hole, leave standstill 30 seconds after, wash 3 times and claps dried;
3) add ELIAS secondary antibody: add ELIAS secondary antibody working fluid 100 μ L in every hole, constant temperature was hatched 30 minutes in 37 ℃ of wet boxes;
4) washing: pour out the liquid in the hole, add washings 250 μ L in every hole, leave standstill 30 seconds after, wash 3 times and claps dried;
5) add substrate: add substrate mixed solution 100 μ L in every hole, hatched 15 minutes in 37 ℃ of wet boxes;
6) add stop buffer: add stop buffer 50 μ L in every hole;
7) measure: the OD value (OD value) of measuring every hole with ELIASA at the 450nm place.
5.4 the result judges
With the standard substance OD value measured divided by " zero " hole OD value (B/B
0) be ordinate zou, the logarithmic value of Xin Meisu 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, with inhibiting rate substitution regression equation, calculate mensuration concentration, multiply by corresponding extension rate, be the residual concentration of Xin Meisu in the sample, amikacin and paromycin.
Sensitivity, precision and the 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 LDL (LOD) as detection kit of the present invention.The Xin Meisu 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, are got the IC that measures for 5 times
50MV.LOD is through the following steps decision; Measure the OD value of the muscle tissue of 20 parts of blank pigs; Regression equation calculation according to typical curve goes out corresponding Xin Meisu concentration; Calculate the MV
and the standard deviation (SD) of Xin Meisu concentration then,
calculates the LDL in the tissue according to formula.IC of the present invention
50Value is 3.16 ± 0.42 μ g/L, and the lowest detection of Xin Meisu in pig muscle is limited to 15.90 μ g/L.
6.2 the precision of test kit of the present invention
The Xin Meisu 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 Xin Meisu standardized solution, calculates in the plate and the variation coefficient between plate, and the result sees table 5.
The precision of table 5 test kit of the present invention
6.3 the accuracy of test kit of the present invention
In the homogenate pig muscle tissue of 1g, add the Xin Meisu standardized solution, 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 Xin Meisu, amikacin and paromycin in the tissue, calculate recovery rate is examined 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 repeated result see table 6, table 7 and table 8, show that this test kit has reliable accuracy, and be little with interassay coefficient of variation in batch, good reproducibility.
Xin Meisu adds the recovery and the variation coefficient in table 6 pig muscle
Paromycin adds the recovery and the variation coefficient in table 7 pig muscle
Amikacin adds the recovery and the variation coefficient in table 8 pig muscle
Claims (7)
1. the monoclonal antibody that can discern Xin Meisu, amikacin and paromycin is characterized in that, it is that CCTCC NO:C201144 hybridoma EDC/5G04 is secreted by preserving number.
2. the described hybridoma EDC/5G04 of claim 1 is deposited in Chinese typical culture collection center, and its preserving number is CCTCC NO:C201144.
3. the test kit that comprises the described monoclonal antibody of claim 1.
4. test kit according to claim 3, this test kit are the enzyme linked immunological kits that is used to detect Xin Meisu, amikacin and paromycin.
5. an enzyme-linked immunoassay method that is used to detect Xin Meisu, amikacin and paromycin comprises the preparation of immunogen, coating antigen, antibody and the processing and the detection of sample, and its step is following:
(1) haptin Xin Meisu and bovine serum albumin coupling are obtained immunogen;
(2) haptin Xin Meisu and ovalbumin coupling are obtained coating antigen;
(3) utilize the immunogen immune mouse of step (1), obtain the hybridoma EDC/5G04 that preserving number is CCTCC NO:C201144 through cytogamy and screening;
(4) use preserving number to prepare monoclonal antibody as the hybridoma EDC/5G04 of CCTCC NO:C201144;
(5) coating antigen with step (2) encapsulates solid phase carrier;
(6) with testing sample with the phosphate buffer soln of 0.1M, pH10～11 extract, hatch, centrifugal and dilution obtains determinand solution;
(7) the determinand solution to step (6) carries out enzyme linked immunosorbent detection.
6. the application of the described monoclonal antibody of claim 1 in the enzyme linked immunological kit of preparation detection Xin Meisu, amikacin and paromycin.
7. claim 3 or the application of 4 described test kits in animal tissues's Xin Meisu, amikacin and paromycin residue detection.
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| CN2012100452029A Expired - Fee Related CN102585006B (en) | 2012-02-27 | 2012-02-27 | Monoclonal antibody, enzyme-linked immunosorbent assay (ELISA) method and kit for detecting neomycin, amikacin and paromomycin |
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| CN (1) | CN102585006B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103232358A (en) * | 2012-11-05 | 2013-08-07 | 南京工业大学 | Vesicle probe used for detecting neomycin, and application and preparation method thereof |
| CN104569325A (en) * | 2014-12-26 | 2015-04-29 | 华中农业大学 | Antibody microarray kit and method for detecting residue of aminoglycoside antibiotics in food |
| CN106520705A (en) * | 2016-12-06 | 2017-03-22 | 得利斯集团有限公司 | Hybridoma cell line C1 capable of secreting anti-paromomycin monoclonal antibody and application of hybridoma cell line C1 |
| CN107058241A (en) * | 2017-03-20 | 2017-08-18 | 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 |
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| 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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| WO2001045742A1 (en) * | 1999-12-22 | 2001-06-28 | Samyang Corporation | Liquid composition of biodegradable block copolymer for drug delivery system and process for the preparation thereof |
| 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 |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103232358A (en) * | 2012-11-05 | 2013-08-07 | 南京工业大学 | Vesicle probe used for detecting neomycin, and application and preparation method thereof |
| CN104569325A (en) * | 2014-12-26 | 2015-04-29 | 华中农业大学 | Antibody microarray kit and method for detecting residue of aminoglycoside antibiotics in food |
| 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 |
| CN106520705A (en) * | 2016-12-06 | 2017-03-22 | 得利斯集团有限公司 | Hybridoma cell line C1 capable of secreting anti-paromomycin monoclonal antibody and application of hybridoma cell line C1 |
| WO2018103630A1 (en) * | 2016-12-06 | 2018-06-14 | 得利斯集团有限公司 | Hybridoma cell strain c1 for secreting anti-paromomycin monoclonal antibody and use thereof |
| CN107058241A (en) * | 2017-03-20 | 2017-08-18 | 江南大学 | The anti-amikacin monoclonal antibody hybridoma cell strain AB2 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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| CN102585006B (en) | 2013-12-04 |
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