CN102020713A - Immune colloidal gold test strip used for detecting aureomycin residue and preparation method thereof - Google Patents
Immune colloidal gold test strip used for detecting aureomycin residue and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of veterinary drug residue immunology rapid detection, and in particular to an immune colloidal gold test strip used for detecting aureomycin (CTC) residue and a preparation method thereof. The test strip comprises a manual control area seal film, a backing plate, an absorbent pad, a cellulose nitrate film, a combination pad, a sample pad and a sample end seal film, and is characterized in that the backing plate is sequentially adhered with the manual control area seal film, the absorbent pad, the cellulose nitrate film, the combination pad, the sample pad and the sample end seal film; the quality control line on the cellulose nitrate film is coated with one rabbit-anti-rat IgG, and the detection line is coated with an aureomycin-bovine serum albumin conjugate (CTC-BSA); and the sample pad is coated with an anti-aureomycin drug monoclonal antibody-colloidal gold marker. The test strip has the obvious advantages of being simple, fast, sensitive and accurate and the like, and is suitable for detecting CTC residue in animal edible tissues.
Description
Technical field
The invention belongs to residue of veterinary drug immunology rapid detection technical field.Be specifically related to residual immunity colloidal gold test paper strip of duomycin and preparation method thereof, be applicable to the residual rapid detection of (muscle, liver etc.) duomycin medicine in the edibility animal tissues.
Background technology
Duomycin (CTC) is one of tetracycline medication member, and the derivative for many ring tetracene Carboxylamide parent nucleus has broad-spectrum antibacterial action, and many kinds of gram-positive microorganisms and Gram-negative bacteria are all had inhibition or killing action.Because these medicines have certain prevention and result of treatment, low price is used very extensive at veterinary clinic.In real work, owing to arbitrarily strengthen using dosage and, cause the duomycin medicine residual in animal tissues not in accordance with the off-drug period.Duomycin medicine extended residual causes resistant organism to be propagated between the mankind in animal tissues, the grievous injury HUMAN HEALTH.A lot of countries and international organization control this drug residue problem by setting up effective detection method of animal food veterinary drug residue.As, China and European Union make explicit provisions to CTC residual maximum residue limit(MRL) (MRL) in animal tissues: the MRL of muscle, liver and kidney is respectively 100 μ g/kg, 300 μ g/kg and 600 μ g/kg.In order to control residue of veterinary drug, ensure human health, be necessary to set up a kind of method for detecting residue fast and effectively to its residual monitoring.
The analysing and detecting method of duomycin has much in animal food at present, comprises high performance liquid chromatography, tlc, liquid chromatograph mass spectrography method, microbial method, euzymelinked immunosorbent assay (ELISA).But there is long, shortage specificity detection time in microbial process, causes false positive and false negative to produce easily.Instrument detecting mainly exist Instrument purchase expense costliness, sample pre-treatments complexity, program loaded down with trivial details time-consuming, testing cost is high, can not execute-in-place etc. defective, be restricted so use aborning.The ELISA method is highly sensitive, cost is low, uses also very extensively in drug residue detects, but this method needs laboratory equipments such as microplate reader, and analysis time is long, and limitation is arranged in application.In recent years, colloidal gold immunochromatographimethod technology (GICA) is compared with the labelled immune analytical technology that generally adopts at present, have easy to be quick, high specificity, susceptibility height, naked eyes are judged, experimental result is easily preserved, and need not advantages such as special instruments and equipment, so the GICA technology has obtained widespread use at the drug residue detection range.At present also not at the immunity colloidal gold test paper strip of duomycin residue detection and preparation method thereof report.
Applied immunology technology screening of the present invention goes out the monoclonal antibody of anti-duomycin, by the preparation of Radioactive colloidal gold and the exploratory development of colloid gold label condition, set up anti-duomycin monoclonal antibody quick detection test paper bar in anti-duomycin monoclonal antibody of application and GICA technical study, and its performance is measured.Test strip sensing range broad, false positive is low, detects sensitivity, accurate, reliable, is applicable to the residual detection of duomycin in the tissues such as edible animal tissue such as muscle, liver.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, prepare a kind of monoclonal antibody hybridoma cell line that can secrete anti-duomycin.Utilize this monoclonal antibody, set up a kind of high specificity, highly sensitive and simple to operate, can detect the residual immunity colloidal gold test paper strip of duomycin the sample simple process.Assemble a kind of colloidal gold kit of duomycin drug residue simultaneously, as application to the immunity colloidal gold test paper strip of duomycin drug residue in the edibility animal tissues.
Technological line figure of the present invention sees shown in Figure 1.
Technical scheme of the present invention is:
A kind of immunity colloidal gold test paper strip of duomycin drug residue comprises that the structure of sample detection test paper comprises manual district: manual district envelope film (1), backboard (2), absorbent pad (3); Test zone: nitrocellulose filter (4), nature controlling line (5), detection line (6); Sample end: pad (7), sample pad (8) and sample end envelope film (9).Its structure is: adhere to manual district envelope film (1), absorbent pad (3), nitrocellulose filter (4), nature controlling line (5), detection line (6), pad (7), sample pad (8) and sample end envelope film (9) on backboard (2) in order successively; On nature controlling line (5), be coated with one of rabbit anti-mouse igg; Be coated with one in duomycin-bovine serum albumin conjugate (CTC-BSA) on the detection line (6); Be coated with described anti-duomycin anti-drug monoclonal antibody-colloid gold label thing on the sample pad (8).
A kind of preparation method who detects the residual immunity colloidal gold test paper strip of duomycin that is applicable to, its step is as follows:
1) with after the haptens duomycin employing Huffman elimination reaction, again with bovine serum albumin coupling synthesis of coupling thing (CTC-BSA);
2) with step 1) synthetic conjugate (CTC-BSA) immunogen immune mouse, obtain secreting the cell strain of the monoclonal antibody of anti-duomycin medicine by cytogamy screening, cell strain is injected mouse peritoneal, obtain anti-duomycin monoclonal antibody;
3) with trisodium citrate and hydrochloro-auric acid prepared in reaction Radioactive colloidal gold;
4) extract mouse IgG immune health rabbit, obtain rabbit anti-mouse igg antibody
5) with step 2) the anti-duomycin monoclonal antibody made adds in the Radioactive colloidal gold of step 3) preparation, obtains anti-duomycin monoclonal antibody and-colloid gold label thing;
6) will resist duomycin monoclonal antibody and-colloid gold label thing to be coated on the backboard;
7) synthetic duomycin-bovine serum albumin (BSA) the coupling synthesis of coupling thing (CTC-BSA) of step 1) is coated on nitrocellulose filter (4) and obtains detection line (6);
8) the step 4) rabbit anti-mouse igg is coated on nitrocellulose filter (4) and obtains nature controlling line (5);
9) manual control district envelope film (1), absorbent pad (3), nitrocellulose filter (4) pad (7), sample pad (8) and sample end envelope film (9) are sticked on the backboard (2) successively.
The present invention selects for use pad (7) bag of sample area by the good anti-CTC monoclonal antibody of colloid gold label, and the detection line of test zone (6) is CTC-BSA, and control line is a rabbit anti-mouse igg.With detection line and the control line of sample flow to the test zone, if there is not CTC to exist in the sample, the CTC-BSA of test zone just can be in conjunction with CTC gold mark monoclonal antibody because of capillarity for gold mark CTC monoclonal antibody, and it is red that detection line and control line all show, and shows negative findings.If there is CTC to exist in the sample, CTC just and the CTC-BSA of test zone competition ground in conjunction with CTC gold mark monoclonal antibody, when existing as if the CTC that capacity is arranged, just stop the combination of CTC gold mark monoclonal antibody and CTC-BSA competitively, detection line does not develop the color, and it is red that control line shows, and shows positive findings.If detection line and control line do not develop the color, the incorrect or test strip inefficacy of the method that expression detects must be reused new test strip and detect.The CTC test strip can be used for the CTC of test sample.Get the sample that is no more than 80 μ l and add on 96 orifice plates, under the room temperature test strip sample end is immersed sample 10-20 and take out horizontal positioned after second, have only the control line colour developing to show the positive.Detection line and control line all develop the color and show feminine gender.
Description of drawings
Fig. 1: general technical route map of the present invention;
Fig. 2: antigens c TC-BSA synthetic technology route map of the present invention;
Fig. 3: the assembling synoptic diagram of test strip of the present invention;
(1) is that manual district envelope film, (2) are that absorbent pad, (4) are that sample pad and (9) be sample end envelope film for detection line, (7) for pad, (8) for nature controlling line, (6) for nitrocellulose filter, (5) for backboard, (3) among the figure.
Fig. 4: test strip result of the present invention judges synoptic diagram;
In the way: A is negative; B is the weak positive; C is positive; D, E were for losing efficacy.
Embodiment
The invention will be further described below by embodiment, but do not limit the present invention.
The preparation preparation of embodiment 1 duomycin (CTC)-carrier protein couplet thing
Coating antigen of the present invention (CTC-BSA) is synthetic according to technology synthetic route shown in the accompanying drawing 2.Accurately take by weighing duomycin 0.096g (0.2mmol) and be dissolved in the 3mL ultrapure water, then 4mL bromine water alkali lye is added in the duomycin solution reacting by heating 2h, 4 ℃ of precoolings.Under 4 ℃ of lucifuge conditions, in above-mentioned reaction system, slowly drip the hydrochloric acid 0.6mL (transferring pH to 2) of 1mol/L, in ice-water bath, dropwise add the 1mol/LNaNO that now joins while stirring
20.3mL, add and be placed on 4 ℃ of refrigerator shady places and hatch 6h.According to 20: 1 mol ratios, with 340mg BSA (5 * 10
-6Mol) be dissolved in 5mLPBS, azo CTC is slowly splashed in the BSA solution, add the back 4 ℃ of refrigerator shady place overnight incubation.The centrifugal precipitation of removing is got supernatant liquor phosphoric acid buffer (PBS) dialysis 3d, and every 6h changes dialyzate, and is with the products therefrom lyophilized, standby in-20 ℃ of preservations.
2.1 animal immune
With the immunogen (CTC-BSA) of embodiment 1 preparation female Balb/c mouse in immune respectively 6 ages in week, the immunizing dose of every mouse is 100 μ g/0.2mL.First immunisation, former with aseptic 0.01mol/LpH7.4PBS lytic immunity, mix emulsification fully, the subcutaneous branch 2 of nape portion～3 injections again with the equivalent Freund's complete adjuvant; Booster immunization mixes with the equivalent Freund's incomplete adjuvant with the 0.01mol/LpH7.4PBS lytic immunity is former, and is fully emulsified, the mouse peritoneal injection.Each 14～21d at interval, the 3rd time immunity back 7～10d begins the immune mouse tail vein blood, collects serum, detects mice serum with ELISA and tires.More than 4 weeks of interval, 3～4d before cytogamy, abdominal injection CTC-BSA antigen 1 00 μ g/0.2mL/ only inject the back and note observing every day after the last immunity, and it is in good condition that preceding mouse is merged in assurance.
2.2 duomycin Monoclonal Antibody
The splenocyte of separating immune mouse, and carry out homogenate and prepare immune Pi cell.Get the Balb/c mouse of 1 immunity, as negative serum, put to death from eye socket bloodletting separation of serum.Mouse carries out overall disinfection with soaking 5min in 75% alcohol.The mouse four limbs are fixed, clamped mouse abdomen skin with tweezers then, cut off an osculum, tear skin with tweezers again, expose peritonaeum, on peritonaeum, cut an osculum (in belly central authorities) with scissors (wanting transducer set tweezers and scissors during operation).Cut off peritonaeum (changing 1 cover tweezers and scissors), expose spleen, clamp spleen (changing 1 cover apparatus again), the spleen adventitia is broken, put into the homogenizer of prior sterilization then with scissors with tweezers.Add an amount of basic medium (RPMI-1640) in homogenizer, grind, squeeze out splenocyte, take out the homogenate rod of homogenizer, add an amount of basic medium (RPMI-1640) again, leave standstill 2min, after the upper strata cell suspension is drawn, put into the peritoneal macrophage centrifuge tube, repeat aforesaid operations 1 time.The centrifugal 10min of 1200r/min removes supernatant.With 10
8Individual immune spleen cell and 1～2 * 10
7Individual SP2/0 myeloma cell adds in the 50mL centrifuge tube according to the ratio of 1: 10 or 1: 5, carries out mixing, then in the centrifugal 10min of 1500r/min level, supernatant discarded.Centrifuge tube is tipped upside down on the thieving paper of sterilization, liquid in the pipe is blotted.Knock the pipe end gently with finger or desktop, make sedimentary cell loosening, again centrifuge tube is placed 37 ℃ of water-baths.Slowly 50%PEG 0.8mL is splashed in the centrifuge tube in 1min, the limit edged stirs sedimentation cell with pipette tip gently.After continuing again to stir 30sec, leave standstill 1min, slowly add 40mL 1640 basic mediums (carrying out 37 ℃ of pre-temperature in advance) then.Adding the basic medium method is: dropwise splash into 1mL in the 1min, add 2mL in the 2min, add 3mL in the 3min, add remaining 4mL in the 4min, when adding substratum, need slowly to add at every turn, and stir culture base lightly, at last remaining 1640 substratum are slowly added.The centrifugal 5min of 1000r/min removes supernatant.Use HAT substratum suspension blended cell then, add again and raise splenocyte.Add an amount of HAT substratum as required, mix, the cytogamy drop that will contain feeder cell again is added on the 96 porocyte culture plates, and dripping quantity is about 150 μ L/ holes.Culture plate is placed 37 ℃, 5%CO2 saturated humidity incubator, cultivate.With the indirect ELISA screening positive cell clone of setting up.Select the hole of strong positive colony growth, clone with limiting dilution assay.And to other positive holes, carry out 24 hole enlarged culturing, detect with indirect ELISA and indirect competitive ELISA supernatant liquor the enlarged culturing hole, to indirect ELISA and indirect competitive ELISA all the cell in positive hole carry out liquid nitrogen freezing and preserve.By fusion detection, and carry out obtaining hybridoma cell strain behind 3 subclones.Hybridoma cell strain through repeatedly go down to posterity, frozen, recovery, the hybridoma secretory antibody is stable.Carry out the chromosomal counting of hybridoma, every strain of hybridoma is selected 20 cells at random, carry out the counting of cell chromosome bar number, calculate the mean value of cell chromosome bar number again.The mouse boosting cell chromosome number is 40, and the chromosome number mean number of SP2/0 cell is 62～68, and the 20 strain of hybridoma chromosome numbers that this test obtains are all between 92～103,97.8 of average out to.This hybridoma chromosome number is higher than the chromosome number of two parent's cells, and explanation is the hybridization product of two kinds of cells.Get the culture supernatant of cell strain emiocytosis, after carrying out diluting at 1: 10, measure antibody subtype with sandwich ELISA method, this cell strain excretory antibody subtype is IgG1.Adopt sad-ammonium sulfate method that mouse ascites is carried out purifying.This monoclonal antibody can be used for preparing Radioactive colloidal gold.
2.3 the preparation of rabbit anti-mouse igg antibody
With Balb/C mouse IgG immune health new zealand white rabbit, the high rabbit anti-mouse igg hyper-immune serum of tiring of preparation adopts the saturated ammonium sulphate method that serum is slightly carried, and obtains highly purified rabbit anti-mouse igg after G-200 crosses post.Utilize rabbit anti-mouse igg as nature controlling line.
The preparation of embodiment 3 anti-duomycin monoclonal antibody-colloid gold label things
3.1 the preparation of Radioactive colloidal gold
Measure deionized water 100mL, move in the Florence flask of 500mL, flask is placed in the heating jacket of magnetic stirring apparatus, open and stir knob and heating knob, be heated to boiling.Add 1%HAuCl4 solution 1.0mL, continue heating 2min, by disposable rapid adding 1% citric acid three sodium solution of volume shown in the table 5-1, continue heating then.Treat solution become shiny red or orange red after, continue heated and stirred 15min again.Turn off the heating turn-knob, be cooled to room temperature, filter standby.
3.2 the preparation and the purifying of anti-duomycin monoclonal antibody-colloid gold label thing
In the small beaker of 50mL, add the Radioactive colloidal gold of 30mL, transfer pH to reach optimum mark pH value in right amount, under the state that stirs, slowly add the anti-duomycin monoclonal antibody of the good 0.1mg/mL of a certain amount of dilution, continue to stir 30min with 0.1mol/L K2CO3; Adding 10%BSA, to make its final concentration be 1%, stirs 30min; Behind 4 ℃ of placement 2h,,, get supernatant, discard precipitation with the centrifugal 15min of 2000r/min with the packing of above colloid gold label thing; With the centrifugal 30min of 10000r/min, abandon supernatant, add the mark washings to original volume; With the centrifugal 30min of 10000r/min, abandon supernatant once more, add the mark washings to original volume; With the centrifugal 30min of 10000r/min, abandon supernatant, precipitation is carried out resuspended with 1.0mL mark washings, put 4 ℃ of refrigerators then.
3.3 the preparation of gold mark pad
The clip specification is the glass fibre cotton of 20 * 4mm respectively, then golden labeling antibody complex solution evenly is sprayed on the glass fibre cotton with ZX1000 specking plateform system, places 37 ℃ of dry 1h, adds the siccative sealing and preserves, and is placed on 4 ℃ of refrigerators.
3.4 the preparation of nitrocellulose filter (NC film)
The NC film is placed on the ZX1000 specking plateform system, is that the complete antigen CTC-BSA of 0.1mg/mL is put in storage pool A with concentration, and concentration is that the GaMIgG of 0.5mg/mL is put in storage pool B.The NC film is flattened, put press strip, nature controlling line and detection line are positioned at the intermediate phase of film apart from 0.5cm, and its back gauge apart from film is 0.75cm.After the start, the specking plateform system with complete antigen CTC-BSA and rabbit anti-mouse igg respectively fixed fire on the NC film, form detection line and nature controlling line, put natural drying at room temperature after, add the siccative sealing, place 4 ℃ of preservations, standby.
3.5 the assembling of test strip
Backboard (2), sample end envelope film (9), pad (7), nitrocellulose filter (4), absorbent pad (3) and manual district's envelope film (1) are assembled into together by certain technology.The work in-process test strip of assembling is put into test strip cutting trough cuts, will prepare then finished product the dated production time of test strip and batch, put 4 ℃ of preservations (sealing).
4.1 tissue sample pre-treatment
With tissue sample homogenate to be checked such as pig muscle, livers, get 2g and add 4mL ethyl acetate vibration 2min, the centrifugal 5min of room temperature 3000r/min, get 300 μ L supernatant liquids, 80 ℃ of water bath methods (approximately 30min) or under nitrogen gas stream, dry up in the 1.5mL centrifuge tube, with 150 μ L diluent dissolution residual substances.
4.2 detected result is judged
As shown in Figure 4, sample solution is added on the test strip sample pad, leave standstill 10min, the result judges according to visual inspection test colour developing, along with CTC concentration increase detection line color in the sample is thin out gradually, its detection line color and negative control detection line do not have notable difference when CTC content in the tissue sample is lower than 20 μ g/L, result of determination negative (-); When CTC content during in 20～100 μ g/L scopes, its detection line color obviously is shallower than the negative control detection line, is judged to the weak positive (±); When CTC content during greater than 100 μ g/L scopes, its detection line does not have color and occurs, and is judged to the positive (+), meets the residual limit standard of we national CTC (100 μ g/L).
The application of embodiment 5 duomycin drug testing test strip of the present invention
5.1CTC the sensitivity determination of residue detection test strip
CTC is made into the standard substance that concentration is respectively 0,5,10,20,40,80,100 μ g/L, be added to respectively on the sample pad of test strip, test repeats 8 times, leave standstill test strip reaction 10min, read the relative optical density value (G/Peak-ROD value) of bar instrument scanning detection line then with BioDot-TSR3000, machine-readablely the results are shown in Table 1.Percentage (B/B0%) with different concns standard substance and zero standard product relative optical density value is an ordinate zou, as X-coordinate, draw the typical curve of test strip with the denary logarithm value of standard substance different concns, ask regression equation, carry out the correlation regression analysis, the results are shown in Table 1.Carry out statistical analysis by G/Peak-ROD value, when the standard substance detection line G/Peak-ROD value (B/B0) of G/Peak-ROD value and 0ng/mL is 80%, be machine-readable sensitivity, determine the detectability of test strip detection line.This test strip is when B/B0%=80%, the CTC concentration that returns typical curve equation correspondence is 10.76 μ g/L, be machine-readable sensitivity, consider error, determine that its machine-readable detection is limited to 11 μ g/L in actual detected requirements of one's work and actual experiment operating aspect.The range estimation detection method is set up: with different concns CTC standard solution, be added on the test strip sample pad, leave standstill 10min, the result judges according to visual inspection test colour developing, along with standard substance concentration increase detection line color is thin out gradually, when the detection line line develops the color the result between two standard substance test strip detection line line colors, show that CTC content is also between these two standard substance concentration in the sample; CTC drug level when detection line obviously is shallower than the standard substance detection line of 0ng/mL is the naked eyes judgement detectability that CTC drug residue test strip detects this kind CTC medicine.This test strip is when CTC standard substance concentration is lower than 10 μ g/L and the standard substance detection line no significant difference of 0 μ g/L.When CTC standard substance concentration was in 20～100 μ g/L scopes, its detection line of visual inspection obviously was shallower than the standard substance detection line of 0 μ g/L.Detection line does not have the color appearance when CTC standard substance concentration is higher than 100 μ g/L.By visual inspection, to residual sxemiquantitative and the qualitative detection of carrying out of CTC, range estimation judges that detection is limited to 20 μ g/L, and its detection line color and negative control detection line do not have notable difference when CTC content is lower than 20 μ g/L, result of determination negative (-); When CTC content during in 20～100 μ g/L scopes, its detection line color obviously is shallower than the negative control detection line, is judged to the weak positive (±); When CTC content during greater than 100 μ g/L scopes, its detection line does not have color and occurs, and is judged to the positive (+), meets the residual limit standard of we national CTC (100 μ g/L).
Table 1 test strip sensitivity test
5.2CTC the specific assay of residue detection test strip
The test of table 2 test strip specificity
The CTC standardized solution is respectively 0,20,40,80,100,200,1000 μ g/L totally 7 concentration with PBS solution dilution to final concentration, respectively compete the standard solution of thing with test strip mensuration at different concns, the cross reactivity of test paper bar, 8 repetitions of each concentration determination, the specificity test-results of test strip sees Table 2.
5.3 sample detection
(1) experimentation on animals
The healthy piglet of the two-way cross castration of growing up about 9 15kg of this test and Selection is divided into 2 groups, i.e. control group and test group at random.Control group fed does not contain the feed of any antibacterials, and test group is fed and contained the feed of 100mg/kg duomycin.Duration of test, free choice feeding is freely drunk water.Feed continuously behind the 7d, test group stops the medicated feed of feeding.0d, 2d, 4d difference slaughter experiment group pig are 2 after drug withdrawal, and control group is butchered 1 at drug withdrawal 0d, 2d, 4d respectively, adopts muscle and liver, uses test strip method and liquid phase (HPLC) method to measure synchronously simultaneously.Measurement result sees Table 3.Two kinds of methods detect with NF recombination rate be 100%, test-results shows that test strip has higher sensitivity, the duomycin that can be used in the detection by quantitative animal food is residual.
Table 3 test strip method of the present invention and HPLC method are to duomycin drug residue test in pork, the pig liver relatively
Annotate: negative-; Positive+
(2) detection of sample in the market of farm produce and the supermarket
In March, 2010 and April, gather 100 parts of pig muscle samples and 100 parts of chicken muscle samples in the market of farm produce and the supermarket in certain city, sample preparation detects the HPLC method according to embodiment 4 usefulness CTC drug residues and the test strip method detects, and experimental result sees Table 4.HPLC method detected result: contain CTC in 2 duplicate samples in 100 parts of pig muscle samples, be positive, positive rate is 2%; Contain CTC in 2 duplicate samples in 100 parts of chicken muscle samples, be positive, positive rate is 2%.The test strip detected result: contain the CTC medicine in 4 duplicate samples in 100 parts of pig muscle samples, wherein 1 part is weak positive, and recall rate is 1%; Other 3 parts of positive samples, positive rate is 3%; Contain CTC in 4 duplicate samples in 100 parts of chicken muscle samples, wherein 2 parts is weak positive, and recall rate is 2%, other 2 parts of positive samples, and positive rate is 2%.The recombination rate of HPLC method and test strip method is 99%.
Table 4HPLC and test strip are to the clinical sample detected result
Claims (7)
1. the monoclonal antibody that can discern the duomycin medicine specially.
2. the test strip that comprises the described monoclonal antibody of claim 1.
3. the described test strip of claim 2 is characterized in that, described test strip is to detect the residual test strip of duomycin.
4. the test strip of the described detection duomycin of claim 3 drug residue, it comprises that manual district envelope film (1), backboard (2), absorbent pad (3), acid cellulose film (4), pad (7), sample pad (8) and sample end seal film (9).It is characterized in that on backboard (2), adhering to successively in order manual district envelope film (1), absorbent pad (3), nitrocellulose filter (4), pad (7), sample pad (8) and sample end envelope film (9); Be coated with on the nature controlling line (5) on the nitrocellulose filter (4) and be coated with one in duomycin-bovine serum albumin conjugate (CTC-BSA) on one of rabbit anti-mouse igg, the detection line (4); Be coated with described anti-duomycin anti-drug monoclonal antibody-colloid gold label thing on the sample pad (8).
5. the described a kind of preparation method who is applicable to the immunity colloidal gold test paper strip that detects the duomycin drug residue of claim 4, its step comprises:
1) with after the haptens duomycin employing Huffman elimination reaction, again with bovine serum albumin coupling synthesis of coupling thing (CTC-BSA);
2) with synthetic conjugate (CTC-BSA) immunogen immune mouse, obtain secreting the cell strain of the monoclonal antibody of anti-duomycin medicine by cytogamy screening, cell strain is injected mouse peritoneal, obtain anti-duomycin monoclonal antibody;
3) with trisodium citrate and hydrochloro-auric acid prepared in reaction Radioactive colloidal gold;
4) extract mouse IgG immune health rabbit, obtain rabbit anti-mouse igg antibody
5) with step 2) the anti-duomycin monoclonal antibody made adds in the Radioactive colloidal gold of step 3) preparation, obtains anti-duomycin monoclonal antibody and-colloid gold label thing;
6) will resist duomycin monoclonal antibody and-colloid gold label thing to be coated on the backboard;
7) synthetic duomycin-bovine serum albumin (BSA) the coupling synthesis of coupling thing (CTC-BSA) of step 1) is coated on nitrocellulose filter (4) and obtains detection line (6);
7) rabbit anti-mouse igg that step 4) is made is coated on nitrocellulose filter (4) and obtains nature controlling line (5);
8) manual control district envelope film (1), absorbent pad (3), nitrocellulose filter (4) pad (7), sample pad (8) and sample end envelope film (9) are sticked on the backboard (2) successively.
6. the application of the described monoclonal antibody of claim 1 in preparation duomycin drug residue test strip.
7. the application of each described test strip of claim 2-6 in detecting the duomycin drug residue.
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| CN102590498A (en) * | 2012-01-13 | 2012-07-18 | 重庆市科学技术研究院 | Immune colloidal gold test paper for detecting quinoxaline-2-carboxylic acid residue and preparation method of immune colloidal gold test paper |
| CN102590516A (en) * | 2012-01-17 | 2012-07-18 | 重庆师范大学 | Immune colloidal gold test strip for simultaneously carrying out residue detection and analysis on Tylosin and Tilmicosin and preparation method thereof |
| CN103353525A (en) * | 2012-12-12 | 2013-10-16 | 河南省农业科学院 | Test strip for detecting aureomycin residue, and test card |
| CN103675270A (en) * | 2013-11-29 | 2014-03-26 | 中山鼎晟生物科技有限公司 | Method for quickly screening aflatoxin |
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| CN201518027U (en) * | 2009-07-24 | 2010-06-30 | 杭州南开日新生物技术有限公司 | Tetracycline immune colloidal gold quick test reagent board |
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| CN1403815A (en) * | 2002-01-10 | 2003-03-19 | 河南省农业科学院生物技术研究所 | Fast detection test paper strip for medicine residue in animal body and product |
| CN101429244A (en) * | 2008-12-04 | 2009-05-13 | 浙江大学 | Tetracycline and carrier protein couplet product, method for producing tetracycline antibiotic antibody uses thereof |
| CN201518027U (en) * | 2009-07-24 | 2010-06-30 | 杭州南开日新生物技术有限公司 | Tetracycline immune colloidal gold quick test reagent board |
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| CN102297970A (en) * | 2011-05-27 | 2011-12-28 | 重庆市科学技术研究院 | Two-joint detection test strip for rapidly detecting residue of cyromazine (CA) and melamine (MA) and preparation method thereof |
| CN102590498A (en) * | 2012-01-13 | 2012-07-18 | 重庆市科学技术研究院 | Immune colloidal gold test paper for detecting quinoxaline-2-carboxylic acid residue and preparation method of immune colloidal gold test paper |
| CN102590516A (en) * | 2012-01-17 | 2012-07-18 | 重庆师范大学 | Immune colloidal gold test strip for simultaneously carrying out residue detection and analysis on Tylosin and Tilmicosin and preparation method thereof |
| CN103353525A (en) * | 2012-12-12 | 2013-10-16 | 河南省农业科学院 | Test strip for detecting aureomycin residue, and test card |
| CN103675270A (en) * | 2013-11-29 | 2014-03-26 | 中山鼎晟生物科技有限公司 | Method for quickly screening aflatoxin |
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