CN104193637B - One species specificity clenbuterol hapten derivant, clenbuterol artificial antigen and its preparation method and application - Google Patents
One species specificity clenbuterol hapten derivant, clenbuterol artificial antigen and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a species specificity clenbuterol hapten derivant, clenbuterol artificial antigen and its preparation method and application.The specificity clenbuterol hapten derivant structural formula carrying out controlled syntheses according to the structure of clenbuterol is, this derivant structure introduces a carboxyl-reactive structure of functional groups in three methyl one end of clenbuterol, expose the specific phenyl ring accessory structure of clenbuterol to greatest extent, have simultaneously can with the functional group of carrier protein couplet.A kind of clenbuterol artificial antigen
, be that clenbuterol hapten derivant and carrier protein couplet arrived to obtain conjugate, wherein said carrier proteins protein is bovine serum albumin (BSA) or ovalbumin (OVA); Obtain monoclonal antibody and the polyclonal antibody of high specific according to artificial antigen immune animal, artificial antigen and the highly specific monoclonal antibody prepared, can be applicable to colloidal gold method or fluorescence immune chromatography method detection clenbuterol.
Description
Technical field
The present invention relates to biochemical industry technical field of biological, particularly, relate to a kind of clenbuterol haptens and synthesizing artificial antigen and detecting the application in clenbuterol.
Background technology
Along with the development and progress of society, people's living standard constantly improves, and more and more payes attention to health diet.Residue of veterinary drug problem in food-safety problem, particularly food more and more causes the great attention of people.Improve the safety of livestock product, except strictly controlling and effectively managing, the monitoring residual to the toxic chemical substance such as additive and veterinary drug is vital, and therefore, science accurately detection of veterinary drugs in food method is the whether safe important leverage of animal-derived food.
Clenbuterol (Clenbuterol, Clen) is a kind of β of synthetic
2-adrenoceptor agonists, Chang Zuowei bronchodilator is for preventing and treating the bronchospasm caused by the respiratory system diseases such as bronchial asthma, asthmatic chronic bronchitis and pulmonary emphysema.When its consumption reaches therapeutic dose 5 ~ 10 times, Clen can strengthen muscle grow, and reduces fatty deposits, therefore also known as clenbuterol hydrochloride.So Clen often by as fodder additives illegally among the production of meat animals.The more important thing is, because Clen absorbs soon in animal body, distribution is wide, fat-soluble height, has the characteristics such as residual accumulation and long half time.People edible remain the meat of Clen after the poisoning that causes constantly occur.Therefore, national governments' degree forbids Clen as fodder additives among the production of meat animals.The Chinese government also specifies to forbid to add Clen in feed, and has worked out Clen residual quantity standard.So strengthen the residue detection of such medicine in food very necessary with supervision.
At present, in national standard and industry standard, HPLC and GC-MS method is often adopted to the detection of residual of kelengtelu in feed and animal derived food.Although this method high specificity, highly sensitive, accurate, Sample pretreatment complex operation step, cost is high, adds that apparatus expensive, cost are large, operating skill requires high reason and is not suitable for the selective mechanisms of batch samples.Immunoassay (as euzymelinked immunosorbent assay (ELISA), colloidal gold method and fluorescence immune chromatography are sent out), due to the deficiency of the advantage of uniqueness and advantage the compensate for physico-chemical analysis such as analyzing samples easy and simple to handle is large in the qualitative, quantitative of antigen-antibody, play a part more and more important in the residue detection of beta-stimulants.Enzyme-linked immunosorbent assay can detect multiple sample simultaneously, has the advantages such as sensitive, high specificity, but is not suitable for Site Detection.Colloidal gold method and fluorescence immune chromatography method is simple to operate, quick, without the need to large-scale detecting instrument equipment, being more suitable for and on-the-spot detecting in time, is the trend of rapid detection development.
The basic reason affecting immunoassay Detection job is specificity and the affinity of antibody, these character depend on again immune haptenic molecular structure, so immune hapten molecule Design and synthesis produces specific antibody and set up small-molecule drug to remain the most basic step with most critical of Fast Detection Technique research.
CN103159852A (2013-6-19) discloses a kind of synthetic method of specific artificial antigen of clenbuterol, but the haptenic molecular structure of immunity used in the method and artificial antigen preparation still have much room for improvement.
Summary of the invention
An object of the present invention is to provide a species specificity clenbuterol hapten derivant.
Two of object of the present invention is to provide the preparation method of a species specificity clenbuterol hapten derivant.
Three of object of the present invention is to provide a species specificity clenbuterol antigen.
Four of object of the present invention is to provide the preparation method of a species specificity clenbuterol antigen.
Five of object of the present invention is to provide described clenbuterol haptens and the application of artificial antigen in clenbuterol immunodetection.
The invention provides a species specificity clenbuterol haptens derivative compound, its chemical structural formula is:
。
Haptens derivative compound of the present invention is the derivative according to the synthesis of clenbuterol structure direction, considers clenbuterol
with salbutamol
structural similar, so select to introduce carboxyl-reactive structure of functional groups with one end design of salbutamol structural similitude, farthest retain and expose clenbuterol and the discrepant and part that immunogenicity is large of salbutamol, having the active group had with carrier protein couplet simultaneously.
The present invention also protects the preparation method of described clenbuterol haptens derivative compound; it is characterized in that, with amino-3, the 5-dichloro bromoacetophenones (CAS:37148-47-3) of existing compound 4-for raw material; prepared by two-step reaction, it is as follows that it prepares path:
As preferably, described reaction conditions is specially the 4-amino-3 at 0.015-0.028mol, Virahol is added as solvent in 5-dichloro bromoacetophenone solid, amino-4 methvl-pentanoic acid of 4-of 0.01-0.03mol are added under ice-water bath condition, normal-temperature reaction 0.8-1.2h, carries out crystallization, filtration, washing drying obtains the first white solid after raw material transforms completely;
Described first white solid is dissolved in ethanol, adds the KBH of 0.001-0.003mol 92-98% successively
40.002-0.003mol ZnCl
2, after normal-temperature reaction 12-18h, filter; Filtrate concentrates, and washed with isopropyl alcohol, obtains the second white solid and be specificity clenbuterol hapten derivant.
Described first white solid is 4-{ [2-(4-amino-3,5-dichlorophenyl)-2-oxoethyl] is amino }-4-methylvaleric acids; Described second white solid is clenbuterol haptens derivative compound.
A kind of specific artificial antigen of clenbuterol, it is characterized in that described clenbuterol artificial antigen is prepared by described specificity clenbuterol haptens derivative compound, its chemical structural formula is:
Described protein and carrier proteins are bovine serum albumin or ovalbumin.
Utilize clenbuterol haptens derivative compound of the present invention to prepare a method for clenbuterol artificial antigen, it is characterized in that utilizing clenbuterol haptens derivative compound and carrier protein couplet described in carbodlimide method to obtain, comprise the following steps:
P1, described clenbuterol haptens derivative compound is dissolved in DMSO or DMF obtains derivative compound solution;
P2, in P1, add EDC(1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride) add NHS (N-hydroxy-succinamide) room temperature again, stir lower Homogeneous phase mixing after reaction 15 ~ 60 minutes; Described EDC, NHS and clenbuterol haptens derivative compound mole dosage ratio are 1 ~ 2:1 ~ 1.5:1 ~ 2;
P3, under 25 ~ 30 degree, to join the reaction solution obtained in P2 containing carrier proteins solution in stir, carry out linked reaction 1 ~ 5 hour, wherein said carrier proteins is bovine serum albumin or ovalbumin;
When described carrier proteins is bovine serum albumin, the mol ratio of derivative compound and bovine serum albumin is 40 ~ 60:1, and the artificial antigen of synthesis is clenbuterol immunogen; When carrier proteins is ovalbumin, the mol ratio of derivative compound and ovalbumin is 30 ~ 50:1, and the artificial antigen of synthesis is coating antigen;
P4, reaction solution described in P3 to be dialysed, collect dialyzate and be clenbuterol artificial antigen.
Preferably, the damping fluid of carrier protein solution described in P3 is the NaHCO of 0.1mol/L
3damping fluid; Dialyzate described in P4 is the phosphoric acid buffer (PH is 7.2 ~ 7.9) of 0.010.1mol/L.
NaHCO
3damping fluid: take 8.4g NaHCO
3, be dissolved in and be settled in 1000ml ultrapure water, regulate PH to 7.4; Phosphoric acid buffer: take NaH
2pO
4h
2o:0.3g, NaCL: 9g, Na
2hPO
412H
2o: 0.3g is settled to 1000ml, regulates PH to be 7.1 ~ 7.8.
The present invention also protects the application of clenbuterol artificial antigen described above in the preparation of clenbuterol specific antibody; Described antibody is monoclonal antibody or polyclonal antibody.
The present invention also protect a kind of utilize clenbuterol artificial antigen described above for raw material clenbuterol immunoassay detect in application, described immunoassay is colloidal gold method or fluorescence immune chromatography method.
Further, described clenbuterol artificial antigen detects the application in clenbuterol in fluorescence immune chromatography method, and its step is as follows:
P1. described clenbuterol artificial antigen immune animal Dispersal risk is utilized;
P2. cytogamy and screening;
P3. ascites preparation and antibody purification;
P4. by the antibody obtained in P3 with anti-rabbit IgG bis-is anti-carries out fluorochrome label respectively, and detection analytic liquid is mixed with;
P5. detection line and nature controlling line are prepared in the rear and front end (close thieving paper end is front end, is rear end near sample pad) described clenbuterol coating antigen and rabbit igg antibody are sprayed at respectively on nitrocellulose filter bar;
P6. the nitrocellulose filter containing detection line and nature controlling line described in glass fiber sample pad, P5, thieving paper and PVC black floor are assembled into immuno-chromatographic test paper strip;
P7. the cutting of test strip described in P6 proper width is loaded in plastics cartridges, and detect analytic liquid described in P4 and support the use and detect on fluorescence immune chromatography detector.
Advantage of the present invention and feature;
The invention provides a kind of clenbuterol derivative compound of controlled syntheses, clenbuterol according to Fig. 3 and the constitutional features of salbutamol, one end (alkyl end) orientation identical in its structure derives active function groups structure (carboxyl), farthest retains the specificity structure exposing the two and the strong structure (i.e. the structure division of phenyl ring end) of immunity; Based on method for preparing artificial antigen of the present invention, clenbuterol derivative compound described above is utilized to prepare specific clenbuterol artificial antigen; Described specificity artificial antigen is prepared specific antibody, and is used successfully in the detection of clenbuterol fluorescence immune chromatography method.
Antigen of the present invention and antibody starting material all belong to self-control, and raw materials cost reduces greatly, and antigen and antibodies specific are by force, and combined with fluorescent immunochromatography technique sets up high specific and highly sensitive clenbuterol method for quick.
Accompanying drawing explanation
Fig. 1 is clenbuterol derivative compound structural formula;
Fig. 2 is clenbuterol artificial antigen structural formula;
Fig. 3 is clenbuterol and salbutamol structural formula comparison schematic diagram;
Fig. 4 is clenbuterol derivative compound
1 h-NMRcollection of illustrative plates;
Fig. 5 is the SDS-PAGE figure of clenbuterol artificial antigen (immunogen and coating antigen);
Fig. 6 is the structure of the clenbuterol fluorescence immune chromatography test card of being unkitted plastic clip;
Fig. 7 is the clenbuterol fluorescence immune chromatography test card structure with plastics cartridge;
Fig. 8 is that fluorescence immune chromatography method detects clenbuterol standard substance suppression graphic representation;
Fig. 9 is that clenbuterol fluorescence immune chromatography detects negative findings collection of illustrative plates;
Figure 10 is that clenbuterol fluorescence immune chromatography detects positive findings collection of illustrative plates.
Embodiment
Here further describes the present invention by reference to the accompanying drawings.Except specified otherwise, the present invention in an embodiment used kit raw material is conventional reagent.
Embodiment 1
The first directional synthesis method of clenbuterol haptens derivative compound:
S1. 4-amino-3,5-dichloro bromoacetophenone (domestic CAS:37148-47-3) is taken
Solid 5.65g(0.02mol) in 50ml there-necked flask, add 20ml Virahol as solvent, 2.63g(0.02mol is added under ice-water bath condition) amino-4 methvl-pentanoic acid (import brands of 4-, CAS:3235-46-9) after, normal-temperature reaction 1h(TLC follows the tracks of reaction, raw material transforms completely), crystallization, filtration, washing drying obtain white solid, that is: 4-{ [2-(4-amino-3,5-dichlorophenyl)-2-oxoethyl] is amino }-4-methylvaleric acids.
S2. the white solid 0.66g(0.002mol will obtained in S1) be dissolved in 20ml total ethanol, add 0.11g(0.002mol successively) KBH of 95%
4, 0.27g(0.002mol) and ZnCl
2, after normal-temperature reaction 15h, filter.Filtrate concentrates, and washed with isopropyl alcohol, obtains the white solid 0.58g shown in Fig. 1, that is: be clenbuterol derivative compound.The derivative compound of synthesis is carried out
1 h-NMRqualification, its result as shown in Figure 4.
The second directional synthesis method of clenbuterol haptens derivative compound:
At the 4-amino-3 of 0.015mol, 15ml Virahol is added as solvent in 5-dichloro bromoacetophenone solid, amino-4 methvl-pentanoic acid of 4-of 0.01mol are added under ice-water bath condition, normal-temperature reaction 0.8h, carries out crystallization, filtration, washing drying obtains the first white solid after raw material transforms completely;
First white solid is dissolved in 15ml ethanol, adds the KBH of 0.001mol 92% successively
40.002mol ZnCl
2, after normal-temperature reaction 12h, filter; Filtrate concentrates, and washed with isopropyl alcohol, obtains the second white solid and be specificity clenbuterol hapten derivant.
The third directional synthesis method of clenbuterol haptens derivative compound:
At the 4-amino-3 of 0.028mol, 30ml Virahol is added as solvent in 5-dichloro bromoacetophenone solid, amino-4 methvl-pentanoic acid of 4-of 0.03mol are added under ice-water bath condition, normal-temperature reaction 1.2h, carries out crystallization, filtration, washing drying obtains the first white solid after raw material transforms completely;
First white solid is dissolved in 30ml ethanol, adds the KBH of 0.003mol 98% successively
40.003mol ZnCl
2, after normal-temperature reaction 18h, filter; Filtrate concentrates, and washed with isopropyl alcohol, obtains the second white solid and be specificity clenbuterol hapten derivant.
Embodiment 2
The first synthetic method of clenbuterol artificial antigen (immunogen and coating antigen):
Take 40mg clenbuterol derivative compound, be dissolved in 2ml DMF, form the final concentration of 20mg/ml, get wherein 90 μ L and 6 μ L EDC(100mg/ml) mix, add 10ul NHS(50mg/ml again), react 15 ~ 60min under agitation after Homogeneous phase mixing.
To join after in 1ml 6mg/mlBSA solution, (or joining in 1ml 4.5mg/ml OVA solution) reacts 1 ~ 5h under stirring at room temperature condition after centrifugal for above-mentioned reaction mixture (1600rmp), dialyse 4 times with phosphoric acid buffer, 12h changes a not good liquor.Collect dialyzate, the concentration measuring immunogen and coating antigen with the Quick Start Bradford Protein Assay Kit of U.S. Bole (BIO-RAD) company is respectively 5.4mg/ml and 3.8mg/ml.Structural formula as shown in Figure 2 for the clenbuterol artificial antigen (immunogen and coating antigen) obtained.In Fig. 2, protein is BSA(bovine serum albumin) or OVA(ovalbumin).
Described BSA solution (or OVA solution) compound method: take 60mg BSA(or 45mg OVA) be dissolved in the NaHCO of the 0.1mol/L of 10ml
3damping fluid.
Described NaHCO
3damping fluid preparation method: take 8.4g NaHCO
3, be dissolved in and be settled in 1000ml ultrapure water, regulate PH to 7.1 ~ 7.9;
Described phosphoric acid buffer liquid and preparation method thereof: take NaH
2pO
42H
2o:0.3g, NaCL: 9g, Na
2hPO
412H
2o: 0.3g is settled to 1000ml, regulates PH to be 7.3 ~ 7.8.
The SDS-PAGE figure of clenbuterol immunogen (Clen-BSA), BSA is shown in shown in accompanying drawing 5, M:marker; 1:BSA; 2:Clen-BSA; 3:Clen-OVA; 4:OVA, the molecular weight being 66kDa, part Clen-BSA by the molecular weight of the visible BSA of accompanying drawing is greater than BSA, so electrophoretic mobility is less than BSA, coupling success is described.Equally, the molecular weight of Clen-OVA is also greater than OVA, coupling success.
The second synthetic method of clenbuterol artificial antigen (immunogen and coating antigen):
P1, clenbuterol haptens derivative compound is dissolved in DMSO obtains derivative compound solution;
P2, in P1, add EDC(1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride) add NHS (N-hydroxy-succinamide) room temperature again, stir lower Homogeneous phase mixing after reaction 15 minutes; EDC, NHS and clenbuterol haptens derivative compound mole dosage are than being 1:1 ~ 1:1;
P3, to join the reaction solution obtained in P2 at 25 degrees c containing carrier proteins solution in stir, carry out linked reaction 1 hour, wherein said carrier proteins is bovine serum albumin or ovalbumin;
When described carrier proteins is bovine serum albumin, the mol ratio of derivative compound and bovine serum albumin is 60:1, and the artificial antigen of synthesis is clenbuterol immunogen; When carrier proteins is ovalbumin, the mol ratio of derivative compound and ovalbumin is 30:1, and the artificial antigen of synthesis is coating antigen;
P4, reaction solution described in P3 to be dialysed, collect dialyzate and be clenbuterol artificial antigen.
The third synthetic method of clenbuterol artificial antigen (immunogen and coating antigen):
P1, clenbuterol haptens derivative compound is dissolved in DMF obtains derivative compound solution;
P2, in P1, add EDC(1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride) add NHS (N-hydroxy-succinamide) room temperature again, stir lower Homogeneous phase mixing after reaction 15 minutes; Described EDC, NHS and clenbuterol haptens derivative compound mole dosage are than being 2:1.5:1;
P3, to join the reaction solution obtained in P2 at 25 degrees c containing carrier proteins solution in stir, carry out linked reaction 1 hour, wherein said carrier proteins is bovine serum albumin or ovalbumin;
When described carrier proteins is bovine serum albumin, the mol ratio of derivative compound and bovine serum albumin is 40:1, and the artificial antigen of synthesis is clenbuterol immunogen; When carrier proteins is ovalbumin, the mol ratio of derivative compound and ovalbumin is 50:1, and the artificial antigen of synthesis is coating antigen;
P4, reaction solution described in P3 to be dialysed, collect dialyzate and be clenbuterol artificial antigen.
Embodiment 3
Clenbuterol artificial antigen detects the application in clenbuterol at fluorescence immune chromatography:
P1. immune animal
The clenbuterol immunogen (Clen-BSA) the first synthetic method in embodiment 2 obtained is diluted to 0.2mg/ml, get after 500ul immunogen mixes with isopyknic Freund's complete adjuvant, emulsification is complete, immune BALB/c mouse, and at mouse back, subcutaneous and foot carries out injecting immune.First time immunity complete Freund's adjuvant, cannot be used up full freund's adjuvant afterwards.After 4th immunity, one week eye socket gets blood, separation of serum, surveys tiring of anti-clenbuterol antibody.Detect through ELISA, the antibody titer after mouse four immunity is 1:322,000.
P2. cytogamy and screening
Mouse after four immunity, through abdominal injection about 100 μ g immunogen booster immunization again, after 3 days, gets this mouse spleen for merging.Get SP2/0 cell to mix with splenocyte, add serum-free medium, centrifugal (1500rpm, 3min), gets sedimentation cell, dropwise adds 1ml 50% Macrogol 4000, leaves standstill 90 seconds.Then dropwise add the serum-free medium 10ml of 37 DEG C of pre-temperature, leave standstill 5min.Cell suspension centrifugal (1000rpm, 3min) after merging, with complete culture solution, is inoculated in and is added with in 96 orifice plates of feeder layer cells, every hole 2 × 10
4/ ml myeloma cell.Put 37 DEG C, 5%CO
2cultivate two days in incubator, add the complete culture solution of 2 × HAT, make final concentration in hole be 1 × HAT.When treating that hybridoma colonies grows to 1/10-1/5 area at the bottom of hole, namely with ELISA method screening fused cell antibody positive wells.
P3. ascites preparation and antibody purification
Get BALB/c mouse, in abdominal injection 0.5ml paraffin oil, after 7 days, abdominal injection 0.5ml 1 × 10
6positive hybridoma cell.Observe mouse growth situation, about 7 days visible belly protuberances, gather ascites in time.Utilize affinity chromatography technology (Protein G Resin affinity purification) purifying to obtain highly purified monoclonal antibody, protein content is 4mg.
P4. analytic liquid preparation is detected
A. the monoclonal antibody obtained in the above-mentioned P3 of fluorescent mark respectively and anti-rabbit IgG antibody.
B. with the phosphoric acid buffer containing BSA, the antibody dilution after fluorescent mark is mixed with detection analytic liquid.
P5. the blocking of clenbuterol fluorescence immune chromatography test paper is standby
A. be buffered with bag clenbuterol coating antigen (Clen-OVA) that liquid (phosphoric acid buffer) just obtains in embodiment 2 respectively and rabbit IgG antibody is diluted to proper concn (0.25 mg/ml).At the temperature of 25 ± 5 DEG C, the Clen-OVA after dilution and rabbit IgG antibody are evenly sprayed on nitrocellulose membrane and (form detection line and nature controlling line respectively), under the humidity condition of 12%-30%, dry 5 ~ 8 hours, kept dry is for subsequent use;
B. the bag that gluing steps a obtains successively respectively on black PVC substrate is formed test card (Fig. 6) by good nitrocellulose filter, glass fiber paper and thieving paper, cuts into proper width as requested.In Fig. 6,1: black PVC substrate; 2: thieving paper: 3: nitrocellulose filter; 4: glass fibre; 5: Quality Control tape (C line); 6: detect tape (T line);
C. the test card obtained by step b loads in the lower cover of cartridge, covers upper cover, forms complete band cartridge test card (Fig. 7).In Fig. 7,1: lower cover; 2: upper cover; 3: application of sample window; 4: glass fibre; 5: detection window; 6: Quality Control tape (C line); 7: detect tape (T line); 8: nitrocellulose filter; 9: clenbuterol Item Mark (Clen); 10: bar code recognition region;
P6. detect
After getting the detection analytic liquid Homogeneous phase mixing of 100ul sample and 100ul, get the application of sample window that 100ul joins test card, after reaction 5-10min, detect, according to T line signal value and C line signal ratio (T/C value) and the Cutof value contrast judgement yin and yang attribute result of sample FCR fluorescence immunity analyzer (being produced by SeaBird bio tech ltd, Huzhou) is upper.
P7. fluorescence immune chromatography method detects the sensitivity of clenbuterol
By adding clenbuterol standard substance (purchased from German DR company) in blank diaper, be configured to 0ng/ml, 0.25ng/ml, 0.5ng/ml, 1ng/ml, 2ng/ml, 4ng/ml, 8ng/ml series concentration sample.Above-mentioned series concentration sample is detected according to the step in P6, each sample repeats 3 times, the experimental result detected is as shown in table 1, according to table 1 data with Clen concentration for X-coordinate, T/C value be ordinate zou drawing standard curve (four parametric regression methods recurrence) as shown in Figure 8.The equation that in Fig. 8, curve is corresponding is as shown in table 2, calculates IC
50=0.86ng/ml.
By 0ng/ml sample duplicate detection 6 times, calculate the mean value (X) of its T/C value, standard deviation (SD) and precision (CV value) respectively.The account form of sensitivity is, the Clen concentration value of T/C value corresponding in the typical curve of figure (8) of X-2*SD.
The T/C value of the X-2*SD in table 3 data is substituted into (equation: y=(A-D)/[1+(x/C) in equation corresponding to Fig. 8 typical curve as y value
b]+D) and the x value arrived be 0.16ng/ml, namely sensitivity is 0.16ng/ml.
P8. clenbuterol fluorescence immune chromatography Test paper card Cleaning Principle
Employing competition law detects, and clenbuterol antigen (coating antigen) competition binding on the clenbuterol antigen in sample and detection line (T line) detects the fluorescently-labeled anti-clenbuterol antibody in analytic liquid.When antigen concentration in sample is very low, the fluorescence antibody that detection line combines increases, and then fluorescent signal on detection line is just strong, and therefore, the ratio (T/C value) of detection line (T line) fluorescent signal and the upper fluorescent signal of nature controlling line (C line) is with regard to greatly; Otherwise when in sample, clenbuterol antigen concentration is higher, T/C value is just very little.So clenbuterol content is higher in sample, T/C value is lower.According to the yin and yang attribute of the multilevel iudge detected result of T/C value and Cut of value.
As shown in Figures 9 and 10, be respectively clenbuterol fluorescence immune chromatography method detect feminine gender and positive findings collection of illustrative plates.As seen from the figure, compare with negative sample detected result collection of illustrative plates, in positive sample detected result collection of illustrative plates, T line peak value is far smaller than the peak value of negative sample.Negative and positive sample is distinguished obviously.
P9. fluorescence immune chromatography method detects the cross reaction of clenbuterol
Common beta 2 receptor agonist is configured to respectively the sample of 30ng/ml with negative pig urine, carries out fluorescence immune chromatography detection, all present negative findings compared with normal negative urine sample and Cutof value.Detected result is as shown in table 2:
The fluorescence immune chromatography detected result of the common beta 2 receptor agonist of table 2
Note: the T/C value of Cut off value corresponding to 1ng/ml clenbuterol adds 2 times of SD scopes.That is: 0.47 ~ 0.86.
Claims (9)
1. a species specificity clenbuterol hapten derivant, it is characterized in that described derivative is applicable to prepare clenbuterol artificial antigen, its chemical structural formula is:
。
2. prepare a method for specificity clenbuterol hapten derivant as claimed in claim 1, it is characterized in that, with amino-3, the 5-dichloro bromoacetophenones of compound 4-for raw material, prepared by two-step reaction, it is as follows that it prepares path:
。
3. prepare the method for specificity clenbuterol hapten derivant as claimed in claim 2 for one kind, it is characterized in that, described reaction conditions is specially the 4-amino-3 at 0.015-0.028mol, Virahol is added as solvent in 5-dichloro bromoacetophenone solid, amino-4 methvl-pentanoic acid of 4-of 0.01-0.03mol are added under ice-water bath condition, normal-temperature reaction 0.8-1.2h, carries out crystallization, filtration, washing drying obtains the first white solid after raw material transforms completely;
Described first white solid is dissolved in ethanol, adds the KBH of 0.001-0.003mol 92-98% successively
4, 0.002-0.003mol ZnCl
2, after normal-temperature reaction 12-18h, filter; Filtrate concentrates, and washed with isopropyl alcohol, obtains the second white solid and be specificity clenbuterol hapten derivant.
4. the specific artificial antigen of clenbuterol prepared by specificity clenbuterol hapten derivant described in claim 1, is characterized in that its chemical structural formula is:
Described protein and carrier proteins are bovine serum albumin or ovalbumin.
5. prepare a method for specific artificial antigen of clenbuterol as claimed in claim 4, it is characterized in that utilizing carbodlimide method described specificity clenbuterol hapten derivant compound and carrier protein couplet to be obtained, comprise the following steps:
P1, described clenbuterol haptens derivative compound is dissolved in DMSO or DMF obtains derivative compound solution;
P2, in P1, add EDCHCl(1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride) add NHS (N-hydroxy-succinamide) room temperature again, stir lower Homogeneous phase mixing after reaction 15 ~ 60 minutes; Described EDC, NHS and clenbuterol haptens derivative compound mole dosage ratio are 1 ~ 2:1 ~ 1.5:1 ~ 2;
P3, under 25 ~ 30 degree, to join the reaction solution obtained in P2 containing carrier proteins solution in stir, carry out linked reaction 1 ~ 5 hour, wherein said carrier proteins is bovine serum albumin or ovalbumin;
When described carrier proteins is bovine serum albumin, the mol ratio of derivative compound and bovine serum albumin is 40 ~ 60:1, and the artificial antigen of synthesis is clenbuterol immunogen; When carrier proteins is ovalbumin, the mol ratio of derivative compound and ovalbumin is 30 ~ 50:1, and the artificial antigen of synthesis is coating antigen;
P4, reaction solution described in P3 to be dialysed, collect dialyzate and be clenbuterol artificial antigen.
6. the preparation method of specific artificial antigen of clenbuterol according to claim 5, it is characterized in that, the damping fluid of carrier protein solution described in P3 is the NaHCO of 0.1mol/L
3damping fluid; Dialyzate described in P4 to be 0.01mol/L, PH be 7.2 ~ 7.9 phosphoric acid buffer.
7. specific artificial antigen of clenbuterol according to claim 4, it is characterized in that, can be used for preparing antibody of clenbuteral, described antibody is monoclonal antibody or polyclonal antibody.
8. the application of specific artificial antigen of clenbuterol according to claim 4, is characterized in that, can be used for colloidal gold method or fluorescence immune chromatography method detects in clenbuterol.
9. the application of specific artificial antigen of clenbuterol according to claim 8, is characterized in that, described clenbuterol artificial antigen detects the application in clenbuterol in fluorescence immune chromatography method, and its step is as follows:
P1. described clenbuterol artificial antigen immune animal Dispersal risk is utilized;
P2. cytogamy and screening;
P3. ascites preparation and antibody purification;
P4. by the antibody obtained in P3 with anti-rabbit IgG bis-is anti-carries out fluorochrome label respectively, and detection analytic liquid is mixed with;
P5. clenbuterol coating antigen and the rabbit igg antibody rear and front end be sprayed at respectively on nitrocellulose filter bar are prepared detection line and nature controlling line, wherein near thieving paper end be front end, near sample pad be rear end;
P6. the nitrocellulose filter containing detection line and nature controlling line described in glass fiber sample pad, P5, thieving paper and PVC black floor are assembled into immuno-chromatographic test paper strip;
P7. the cutting of test strip described in P6 is loaded in plastics cartridges, and detect analytic liquid described in P4 and support the use and detect on fluorescence immune chromatography detector.
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| CN1811445A (en) * | 2006-01-25 | 2006-08-02 | 扬州大学 | Enzyme-linked immunosorbent analytical technique with specificity to clenbuterol and its detecting reagent kit |
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